U.S. patent application number 17/064404 was filed with the patent office on 2021-10-28 for microorganisms and methods for improving product yields on methanol using acetyl-coa synthesis.
The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Stefan Andrae, Anthony P. Burgard, Robin E. Osterhout, Priti Pharkya.
Application Number | 20210332393 17/064404 |
Document ID | / |
Family ID | 1000005684828 |
Filed Date | 2021-10-28 |
United States Patent
Application |
20210332393 |
Kind Code |
A1 |
Osterhout; Robin E. ; et
al. |
October 28, 2021 |
MICROORGANISMS AND METHODS FOR IMPROVING PRODUCT YIELDS ON METHANOL
USING ACETYL-COA SYNTHESIS
Abstract
The invention provides non-naturally occurring microbial
organisms containing enzymatic pathways and/or metabolic
modifications for enhancing carbon flux through acetyl-CoA. In some
embodiments, the microbial organisms of the invention having such
pathways also include pathways for generating reducing equivalents,
formaldehyde fixation and/or formate assimilation. The enhanced
carbon flux through acetyl-CoA, in combination with pathways for
generating reducing equivalents, formaldehyde fixation and/or
formate assimilation can, in some embodiments, be used for
production of a bioderived compound. Accordingly, in some
embodiments, the microbial organisms of the invention can include a
pathway capable of producing a bioderived compound of the
invention. The invention still further provides a bioderived
compound produced by a microbial organism of the invention, culture
medium having the bioderived compound of the invention,
compositions having the bioderived compound of the invention, a
biobased product comprising the bioderived compound of the
invention, and a process for producing a bioderived compound of the
invention.
Inventors: |
Osterhout; Robin E.; (San
Diego, CA) ; Burgard; Anthony P.; (Elizabeth, PA)
; Pharkya; Priti; (San Diego, CA) ; Andrae;
Stefan; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
1000005684828 |
Appl. No.: |
17/064404 |
Filed: |
October 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15039221 |
May 25, 2016 |
10808262 |
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PCT/US2014/067287 |
Nov 25, 2014 |
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17064404 |
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61911414 |
Dec 3, 2013 |
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61945056 |
Feb 26, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02E 50/30 20130101;
C12P 13/001 20130101; C12P 7/42 20130101; C12P 7/18 20130101; C12P
7/44 20130101; C12N 15/70 20130101; C12N 1/14 20130101; C12N 1/20
20130101; C12P 7/04 20130101; C12N 15/81 20130101; C12P 7/02
20130101; C12P 17/10 20130101; C12P 5/026 20130101; C12P 13/005
20130101; C12P 7/40 20130101 |
International
Class: |
C12P 5/02 20060101
C12P005/02; C12P 7/18 20060101 C12P007/18; C12N 1/14 20060101
C12N001/14; C12N 1/20 20060101 C12N001/20; C12P 7/42 20060101
C12P007/42; C12P 7/02 20060101 C12P007/02; C12P 7/40 20060101
C12P007/40; C12N 15/70 20060101 C12N015/70; C12N 15/81 20060101
C12N015/81; C12P 7/04 20060101 C12P007/04; C12P 7/44 20060101
C12P007/44; C12P 13/00 20060101 C12P013/00; C12P 17/10 20060101
C12P017/10 |
Claims
1. A non-naturally occurring microbial organism having a methanol
metabolic pathway and an acetyl-CoA pathway, wherein said methanol
metabolic pathway comprises 2A or 2J, wherein 2A is a methanol
methyltransferase, wherein 2J is a methanol dehydrogenase, wherein
said acetyl-CoA pathway (3) 1U and 1V; or (4) 1U, 1W, and 1X;
wherein 1U is a xylulose-5-phosphate phosphoketolase, wherein 1V is
a phosphotransacetylase, wherein 1X is an acetyl-CoA transferase,
an acetyl-CoA synthetase, or an acetyl-CoA ligase, wherein an
enzyme of the methanol metabolic pathway or the acetyl-CoA pathway
is encoded by at least one exogenous nucleic acid and is expressed
in a sufficient amount to enhance carbon flux through
acetyl-CoA.
2. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism further comprises a formaldehyde
fixation pathway, wherein said formaldehyde fixation pathway
comprises: (1) 1D and 1Z; (2) 1D; or (3) 1B and 1C, wherein 1B is a
3-hexulose-6-phosphate synthase, wherein 1C is a
6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone
synthase, wherein 1Z is a fructose-6-phosphate aldolase.
3. The non-naturally occurring microbial organism of claim 2,
wherein an enzyme of the formaldehyde fixation pathway is encoded
by at least one exogenous nucleic acid and is expressed in a
sufficient amount to enhance carbon flux through acetyl-CoA.
4. The non-naturally occurring microbial organism of claim 1,
wherein said methanol metabolic pathway comprises a pathway
selected from: (1) 2A and 2B; (2) 2A, 2B and 2C; (3) 2J, 2K and 2C;
(4) 2J, 2M, and 2N; (5) 2J and 2L; (6) 2J, 2L, and 2G; (7) 2J, 2L,
and 2I; (8) 2A, 2B, 2C, 2D, and 2E; (9) 2A, 2B, 2C, 2D, and 2F;
(10) 2J, 2K, 2C, 2D, and 2E; (11) 2J, 2K, 2C, 2D, and 2F; (12) 2J,
2M, 2N, and 2O; (13) 2A, 2B, 2C, 2D, 2E, and 2G; (14) 2A, 2B, 2C,
2D, 2F, and 2G; (15) 2J, 2K, 2C, 2D, 2E, and 2G; (16) 2J, 2K, 2C,
2D, 2F, and 2G; (17) 2J, 2M, 2N, 2O, and 2G; (18) 2A, 2B, 2C, 2D,
2E, and 2I; (19) 2A, 2B, 2C, 2D, 2F, and 2I; (20) 2J, 2K, 2C, 2D,
2E, and 2I; (21) 2J, 2K, 2C, 2D, 2F, and 2I; and (22) 2J, 2M, 2N,
2O, and 2I, wherein 2A is a methanol methyltransferase, wherein 2B
is a methylenetetrahydrofolate reductase, wherein 2C is a
methylenetetrahydrofolate dehydrogenase, wherein 2D is a
methenyltetrahydrofolate cyclohydrolase, wherein 2E is a
formyltetrahydrofolate deformylase, wherein 2F is a
formyltetrahydrofolate synthetase, wherein 2G is a formate hydrogen
lyase, wherein 2I is a formate dehydrogenase, wherein 2J is a
methanol dehydrogenase, wherein 2K is a formaldehyde activating
enzyme or spontaneous, wherein 2L is a formaldehyde dehydrogenase,
wherein 2M is a S-(hydroxymethyl)glutathione synthase or
spontaneous, wherein 2N is a glutathione-dependent formaldehyde
dehydrogenase, wherein 20 is a S-formylglutathione hydrolase,
5. The non-naturally occurring microbial organism of claim 4,
wherein said microbial organism comprises: (a) one, two, three,
four, five, or six exogenous nucleic acids each encoding a methanol
metabolic pathway enzyme; or (b) exogenous nucleic acids encoding
each of the enzymes of at least one of the pathways selected from
(1)-(22).
6. (canceled)
7. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism comprises: (a) one, two, or three
exogenous nucleic acids each encoding an acetyl-CoA pathway enzyme;
or (b) exogenous nucleic acids encoding each of the enzymes of at
least one of the pathways selected from (1)-(4).
8. (canceled)
9. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism further comprises a formate
assimilation pathway, wherein said formate assimilation pathway
comprises a pathway selected from: (1) 1E; (2) 1F, and 1G; (3) 1H,
1I, 1J, and 1K; (4) 1H, 1I, 1J, 1L, 1M, and 1N; (5) 1E, 1H, 1I, 1J,
1L, 1M, and 1N; (6) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1K, 1H,
1I, 1J, 1L, 1M, and 1N; and (8) 1H, 1I, 1J, 1O, and 1P, wherein 1E
is a formate reductase, 1F is a formate ligase, a formate
transferase, or a formate synthetase, wherein 1G is a formyl-CoA
reductase, wherein 1H is a formyltetrahydrofolate synthetase,
wherein 1I is a methenyltetrahydrofolate cyclohydrolase, wherein 1J
is a methylenetetrahydrofolate dehydrogenase, wherein 1K is a
formaldehyde-forming enzyme or spontaneous, wherein 1L is a glycine
cleavage system, wherein 1M is a serine hydroxymethyltransferase,
wherein 1N is a serine deaminase, wherein 1O is a
methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA
synthase.
10. The non-naturally occurring microbial organism of claim 9,
wherein an enzyme of the formate assimilation pathway is encoded by
at least one exogenous nucleic acid and is expressed in a
sufficient amount to enhance carbon flux through acetyl-CoA.
11. A non-naturally occurring microbial organism having a
formaldehyde fixation pathway, a formate assimilation pathway and
an acetyl-CoA pathway, wherein said formaldehyde fixation pathway
comprises: (1) 1D and 1Z; (2) 1D; or (3) 1B and 1C, wherein 1B is a
3-hexulose-6-phosphate synthase, wherein 1C is a
6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone
synthase, wherein 1Z is a fructose-6-phosphate aldolase, wherein
said formate assimilation pathway comprises a pathway selected
from: (4) 1E; (5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7) 1H, 1I,
1J, 1L, 1M, and 1N; (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9) 1F, 1G,
1H, 1I, 1J, 1L, 1M, and 1N; (10) 1K, 1H, 1I, 1J, 1L, 1M, and 1N;
and (11) 1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate reductase,
1F is a formate ligase, a formate transferase, or a formate
synthetase, wherein 1G is a formyl-CoA reductase, wherein 1H is a
formyltetrahydrofolate synthetase, wherein 1I is a
methenyltetrahydrofolate cyclohydrolase, wherein 1J is a
methylenetetrahydrofolate dehydrogenase, wherein 1K is a
formaldehyde-forming enzyme or spontaneous, wherein 1L is a glycine
cleavage system, wherein 1M is a serine hydroxymethyltransferase,
wherein 1N is a serine deaminase, wherein 1O is a
methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA
synthase, wherein said acetyl-CoA pathway comprises a pathway
selected from: (12) 1T and 1V; (13) 1T, 1W, and 1X; (14) 1U and 1V;
and (15) 1U, 1W, and 1X, wherein 1T is a fructose-6-phosphate
phosphoketolase, wherein 1U is a xylulose-5-phosphate
phosphoketolase, wherein 1V is a phosphotransacetylase, wherein 1W
is an acetate kinase, wherein 1X is an acetyl-CoA transferase, an
acetyl-CoA synthetase, or an acetyl-CoA ligase, wherein an enzyme
of the formaldehyde fixation pathway, the formate assimilation
pathway, or the acetyl-CoA pathway is encoded by at least one
exogenous nucleic acid and is expressed in a sufficient amount to
enhance carbon flux through acetyl-CoA.
12-23. (canceled)
24. The non-naturally occurring microbial organism of claim 1,
wherein said non-naturally occurring microbial organism further
comprises a pathway capable of producing a bioderived compound.
25. The non-naturally occurring microbial organism of claim 24,
wherein said bioderived compound is an alcohol, a glycol, an
organic acid, an alkene, a diene, an organic amine, an organic
aldehyde, a vitamin, a nutraceutical or a pharmaceutical.
26-29. (canceled)
30. The non-naturally occurring microbial organism of claim 25,
wherein said bioderived compounds is selected from the group
consisting of: (i) 1,4-butanediol or an intermediate thereto,
wherein said intermediate is optionally 4-hydroxybutanoic acid
(4-HB); (ii) butadiene (1,3-butadiene) or an intermediate thereto,
wherein said intermediate is optionally 1,4-butanediol,
1,3-butanediol, 2,3-butanediol, crotyl alcohol, 3-buten-2-ol
(methyl vinyl carbinol) or 3-buten-1-ol; (iii) 1,3-butanediol or an
intermediate thereto, wherein said intermediate is optionally
3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotyl alcohol or
3-buten-1-ol; (iv) adipate, 6-aminocaproic acid, caprolactam,
hexamethylenediamine, levulinic acid or an intermediate thereto,
wherein said intermediate is optionally adipyl-CoA or
4-aminobutyryl-CoA; (v) methacrylic acid or an ester thereof,
3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate
thereto, wherein said ester is optionally methyl methacrylate or
poly(methyl methacrylate); (vi) 1,2-propanediol (propylene glycol),
1,3-propanediol, glycerol, ethylene glycol, diethylene glycol,
triethylene glycol, dipropylene glycol, tripropylene glycol,
neopentyl glycol, bisphenol A or an intermediate thereto; (vii)
succinic acid or an intermediate thereto; and (viii) a fatty
alcohol, a fatty aldehyde or a fatty acid comprising C4 to C27
carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms, or
C12 to C14 carbon atoms, wherein said fatty alcohol is optionally
dodecanol (C12; lauryl alcohol), tridecyl alcohol (C13;
1-tridecanol, tridecanol, isotridecanol), myristyl alcohol (C14;
1-tetradecanol), pentadecyl alcohol (C15; 1-pentadecanol,
pentadecanol), cetyl alcohol (C16; 1-hexadecanol), heptadecyl
alcohol (C17; 1-n-heptadecanol, heptadecanol) and stearyl alcohol
(C18; 1-octadecanol) or palmitoleyl alcohol (C16 unsaturated;
cis-9-hexadecen-1-ol).
31-42. (canceled)
43. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism is a species of bacteria, yeast, or
fungus.
44. A method for producing a bioderived compound, comprising
culturing the non-naturally occurring microbial organism of any
claim 24 under conditions and for a sufficient period of time to
produce said bioderived compound.
45. The method of claim 44, wherein said method further comprises
separating the bioderived compound from other components in the
culture.
46. The method of claim 45, wherein the separating comprises
extraction, continuous liquid-liquid extraction, pervaporation,
membrane filtration, membrane separation, reverse osmosis,
electrodialysis, distillation, crystallization, centrifugation,
extractive filtration, ion exchange chromatography, absorption
chromatography, or ultrafiltration.
47-59. (canceled)
60. The non-naturally occurring microbial organism of claim 1,
wherein said acetyl-CoA pathway comprises 1U and 1V 1T and 1V.
61. The non-naturally occurring microbial organism of claim 60,
wherein said formaldehyde fixation pathway comprises 1D and 1Z.
62. The non-naturally occurring microbial organism of claim 60,
wherein said formaldehyde fixation pathway comprises 1B and 1C.
63. The non-naturally occurring microbial organism of claim 1,
wherein said microbial organism further comprises: (a) attenuation
of one or more endogenous enzymes selected from DHA kinase,
methanol oxidase, PQQ-dependent methanol dehydrogenase, DHA
synthase or any combination thereof; or (b) a gene disruption of
one or more endogenous nucleic acids encoding enzymes selected from
DHA kinase, methanol oxidase, PQQ-dependent methanol dehydrogenase,
DHA synthase or any combination thereof.
64-88. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/039,221, which is a United States National
Stage Application under 35 U.S.C. .sctn. 371 of International
Patent Application No. PCT/US2014/067287, filed Nov. 25, 2014,
which claims the benefit of priority of U.S. Provisional
Application Ser. No. 61/945,056, filed Feb. 26, 2014, and
61/911,414, filed Dec. 3, 2013, the entire contents of which are
each incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to biosynthetic
processes, and more specifically to organisms having pathways for
enhanced carbon flux through acetyl-CoA.
[0003] 1,3-butanediol (1,3-BDO) is a four carbon diol traditionally
produced from acetylene via its hydration. The resulting
acetaldehyde is then converted to 3-hydroxybutyraldehdye which is
subsequently reduced to form 1,3-BDO. More recently, acetylene has
been replaced by the less expensive ethylene as a source of
acetaldehyde. 1,3-BDO is commonly used as an organic solvent for
food flavoring agents. It is also used as a co-monomer for
polyurethane and polyester resins and is widely employed as a
hypoglycemic agent. Optically active 1,3-BDO is a useful starting
material for the synthesis of biologically active compounds and
liquid crystals. Another use of 1,3-butanediol is that its
dehydration affords 1,3-butadiene (Ichikawa et al. Journal of
Molecular Catalysis A--Chemical 256:106-112 (2006); Ichikawa et al.
Journal of Molecular Catalysis A--Chemical 231:181-189 (2005),
which is useful in the manufacture synthetic rubbers (e.g., tires),
latex, and resins. The reliance on petroleum based feedstocks for
either acetylene or ethylene warrants the development of a
renewable feedstock based route to 1,3-butanediol and to
butadiene.
[0004] 1,4-butanediol (1,4-BDO) is a valuable chemical for the
production of high performance polymers, solvents, and fine
chemicals. It is the basis for producing other high value chemicals
such as tetrahydrofuran (THF) and gamma-butyrolactone (GBL). The
value chain is comprised of three main segments including: (1)
polymers, (2) THF derivatives, and (3) GBL derivatives. In the case
of polymers, 1,4-BDO is a comonomer for polybutylene terephthalate
(PBT) production. PBT is a medium performance engineering
thermoplastic used in automotive, electrical, water systems, and
small appliance applications. Conversion to THF, and subsequently
to polytetramethylene ether glycol (PTMEG), provides an
intermediate used to manufacture spandex products such as
LYCRA.RTM. fibers. PTMEG is also combined with 1,4-BDO in the
production of specialty polyester ethers (COPE). COPEs are high
modulus elastomers with excellent mechanical properties and of
resistance, allowing them to operate at high and low temperature
extremes. PTMEG and 1,4-BDO also make thermoplastic polyurethanes
processed on standard thermoplastic extrusion, calendaring, and
molding equipment, and are characterized by their outstanding
toughness and abrasion resistance. The GBL produced from 1,4-BDO
provides the feedstock for making pyrrolidones, as well as serving
the agrochemical market. The pyrrolidones are used as high
performance solvents for extraction processes of increasing use,
including for example, in the electronics industry and in
pharmaceutical production.
[0005] 1,4-BDO is produced by two main petrochemical routes with a
few additional routes also in commercial operation. One route
involves reacting acetylene with formaldehyde, followed by
hydrogenation. More recently 1,4-BDO processes involving butane or
butadiene oxidation to maleic anhydride, followed by hydrogenation
have been introduced. 1,4-BDO is used almost exclusively as an
intermediate to synthesize other chemicals and polymers.
[0006] Over 25 billion pounds of butadiene (1,3-butadiene, BD) are
produced annually and is applied in the manufacture of polymers
such as synthetic rubbers and ABS resins, and chemicals such as
hexamethylenediamine and 1,4-butanediol. For example, butadiene can
be reacted with numerous other chemicals, such as other alkenes,
e.g. styrene, to manufacture numerous copolymers, e.g.
acrylonitrile 1,3-butadiene styrene (ABS), styrene-1,3-butadiene
(SBR) rubber, styrene-1,3-butadiene latex. These materials are used
in rubber, plastic, insulation, fiberglass, pipes, automobile and
boat parts, food containers, and carpet backing Butadiene is
typically produced as a by-product of the steam cracking process
for conversion of petroleum feedstocks such as naphtha, liquefied
petroleum gas, ethane or natural gas to ethylene and other olefins.
The ability to manufacture butadiene from alternative and/or
renewable feedstocks would represent a major advance in the quest
for more sustainable chemical production processes.
[0007] Crotyl alcohol, also referred to as 2-buten-1-ol, is a
valuable chemical intermediate. It serves as a precursor to crotyl
halides, esters, and ethers, which in turn are chemical
intermediates in the production of monomers, fine chemicals,
agricultural chemicals, and pharmaceuticals. Exemplary fine
chemical products include sorbic acid, trimethylhydroquinone,
crotonic acid and 3-methoxybutanol. Crotyl alcohol is also a
precursor to 1,3-butadiene. Crotyl alcohol is currently produced
exclusively from petroleum feedstocks. For example Japanese Patent
47-013009 and U.S. Pat. Nos. 3,090,815, 3,090,816, and 3,542,883
describe a method of producing crotyl alcohol by isomerization of
1,2-epoxybutane. The ability to manufacture crotyl alcohol from
alternative and/or renewable feedstocks would represent a major
advance in the quest for more sustainable chemical production
processes.
[0008] 3-Buten-2-ol (also referenced to as methyl vinyl carbinol
(MVC)) is an intermediate that can be used to produce butadiene.
There are significant advantages to use of 3-buten-2-ol over
1,3-BDO because there are fewer separation steps and only one
dehydration step. 3-Buten-2-ol can also be used as a solvent, a
monomer for polymer production, or a precursor to fine chemicals.
Accordingly, the ability to manufacture 3-buten-2-ol from
alternative and/or renewable feedstock would again present a
significant advantage for sustainable chemical production
processes.
[0009] Adipic acid, a dicarboxylic acid, has a molecular weight of
146.14. It can be used is to produce nylon 6,6, a linear polyamide
made by condensing adipic acid with hexamethylenediamine. This is
employed for manufacturing different kinds of fibers. Other uses of
adipic acid include its use in plasticizers, unsaturated
polyesters, and polyester polyols. Additional uses include for
production of polyurethane, lubricant components, and as a food
ingredient as a flavorant and gelling aid.
[0010] Historically, adipic acid was prepared from various fats
using oxidation. Some current processes for adipic acid synthesis
rely on the oxidation of KA oil, a mixture of cyclohexanone, the
ketone or K component, and cyclohexanol, the alcohol or A
component, or of pure cyclohexanol using an excess of strong nitric
acid. There are several variations of this theme which differ in
the routes for production of KA or cyclohexanol. For example,
phenol is an alternative raw material in KA oil production, and the
process for the synthesis of adipic acid from phenol has been
described. The other versions of this process tend to use oxidizing
agents other than nitric acid, such as hydrogen peroxide, air or
oxygen.
[0011] In addition to hexamethylenediamine (HMDA) being used in the
production of nylon-6,6 as described above, it is also utilized to
make hexamethylene diisocyanate, a monomer feedstock used in the
production of polyurethane. The diamine also serves as a
cross-linking agent in epoxy resins. HMDA is presently produced by
the hydrogenation of adiponitrile.
[0012] Caprolactam is an organic compound which is a lactam of
6-aminohexanoic acid (.epsilon.-aminohexanoic acid, 6-aminocaproic
acid). It can alternatively be considered cyclic amide of caproic
acid. One use of caprolactam is as a monomer in the production of
nylon-6. Caprolactam can be synthesized from cyclohexanone via an
oximation process using hydroxylammonium sulfate followed by
catalytic rearrangement using the Beckmann rearrangement process
step.
[0013] Methylacrylic acid (MAA) is a key precursor of methyl
methacrylate (MMA), a chemical intermediate with a global demand in
excess of 4.5 billion pounds per year, much of which is converted
to polyacrylates. The conventional process for synthesizing methyl
methacrylate (i.e., the acetone cyanohydrin route) involves the
conversion of hydrogen cyanide (HCN) and acetone to acetone
cyanohydrin which then undergoes acid assisted hydrolysis and
esterification with methanol to give MAA. Difficulties in handling
potentially deadly HCN along with the high costs of byproduct
disposal (1.2 tons of ammonium bisulfate are formed per ton of MAA)
have sparked a great deal of research aimed at cleaner and more
economical processes. As a starting material, MAA can easily be
converted into MAA via esterification with methanol.
[0014] Thus, there exists a need for the development of methods for
effectively producing commercial quantities of compounds such as
fatty alcohols, 1,3-butanediol, 1,4-butanediol, butadiene, crotyl
alcohol, 3-buten-2-ol, adipate, 6-aminocaproate, caprolactam,
hexamethylenediamine and methacylic acid. The present invention
satisfies this need and provides related advantages as well.
SUMMARY OF INVENTION
[0015] The invention provides non-naturally occurring microbial
organisms containing enzymatic pathways for enhancing carbon flux
through acetyl-CoA. In some embodiments, the microbial organisms of
the invention having such pathways also include pathways for
generating reducing equivalents, formaldehyde fixation and/or
formate assimilation. The enhanced carbon flux through acetyl-CoA,
in combination with pathways for generating reducing equivalents,
formaldehyde fixation and/or formate assimilation can, in some
embodiments, be used for production of a bioderived compound of the
invention. Accordingly, in some embodiments, the microbial
organisms of the invention can include a pathway capable of
producing a bioderived compound of the invention. Bioderived
compounds of the invention include alcohols, glycols, organic
acids, alkenes, dienes, organic amines, organic aldehydes,
vitamins, nutraceuticals and pharmaceuticals. In some embodiments,
the bioderived compound is 1,3-butanediol, crotyl alcohol,
butadiene, 3-buten-2-ol, 1,4-butanediol, adipate, 6-aminocaproate,
caprolactam, hexamethylenediamine, methacrylic acid,
2-hydroxyisobutyric acid, or an intermediate thereto.
[0016] In some embodiments, a non-naturally occurring microbial
organism of the invention includes a methanol metabolic pathway as
depicted in FIG. 2 and an acetyl-CoA pathway as depicted in FIG. 1
or 3. In some aspects the microbial organism can further includes a
formaldehyde fixation pathway and/or formate assimilation pathway
as depicted in FIG. 1. Alternatively, in some embodiments, the
non-naturally occurring microbial organism of the invention
includes a formaldehyde fixation pathway as depicted in FIG. 1, a
formate assimilation pathway as depicted in FIG. 1, and/or an
acetyl-CoA pathway as depicted in FIG. 1 or 3.
[0017] In one aspect, the formaldehyde fixation pathway, formate
assimilation pathway, and/or a methanol metabolic pathway present
in the microbial organisms of the invention enhances the
availability of substrates and/or pathway intermediates, such as
acetyl-CoA, and/or reducing equivalents, which can be utilized for
bioderived compound production through one or more bioderived
compound pathways of the invention. For example, in some
embodiments, a non-naturally occurring microbial organism of the
invention that includes a methanol metabolic pathway can enhance
the availability of reducing equivalents in the presence of
methanol and/or convert methanol to formaldehyde, a substrate for
the formaldehyde fixation pathway. Likewise, a non-naturally
occurring microbial organism of the invention having a formate
assimilation pathway can reutilize formate to generate substrates
and pathway intermediates such as formaldehyde, pyruvate and/or
acetyl-CoA. In another embodiment, a non-naturally occurring
microbial organism of the invention can include a pathway for
producing acetyl-CoA and/or succinyl-CoA by a pathway depicted in
FIG. 4. Such substrates, intermediates and reducing equivalents can
be used to increase the yield of a bioderived compound produced by
the microbial organism.
[0018] In some embodiments, the invention provides a non-naturally
occurring microbial organism containing an acetyl-CoA pathway, a
methanol oxidation pathway, a hydrogenase and/or a carbon monoxide
dehydrogenase. Accordingly, in some embodiments, the invention
provides a non-naturally occurring microbial organism having an
acetyl-CoA pathway and at least one exogenous nucleic acid encoding
an acetyl-CoA pathway enzyme expressed in a sufficient amount to
produce or enhance carbon flux through acetyl-CoA, wherein the
acetyl-CoA pathway includes a pathway shown in FIG. 3. In some
embodiments, the invention provides a non-naturally occurring
microbial organism having a methanol oxidation pathway enzyme
expressed in a sufficient amount to produce formaldehyde in the
presence of methanol. An exemplary methanol oxidation pathway
enzyme is a methanol dehydrogenase as depicted in FIG. 1, Step A.
In some embodiments, the invention provides a non-naturally
occurring microbial organism having a hydrogenase and/or a carbon
monoxide dehydrogenase for generating reducing equivalents as
depicted in FIGS. 2 and 3.
[0019] The invention further provides non-naturally occurring
microbial organisms that have elevated or enhanced synthesis or
yield of acetyl-CoA (e.g. intracellular) or bioderived compound
including alcohols, diols, fatty acids, glycols, organic acids,
alkenes, dienes, organic amines, organic aldehydes, vitamins,
nutraceuticals and pharmaceuticals and methods of using those
non-naturally occurring organisms to produce such biosynthetic
products. The enhanced synthesis of intracellular acetyl-CoA
enables enhanced production of bioderived compounds for which
acetyl-CoA is an intermediate and further, may have been
rate-limiting.
[0020] In some embodiments, the invention provides a non-naturally
occurring microbial organism having attenuation of one or more
endogenous enzymes, which enhances carbon flux through acetyl-CoA,
or a gene disruption of one or more endogenous nucleic acids
encoding such enzymes. For example, in some aspects, the endogenous
enzyme can be selected from DHA kinase, methanol oxidase,
PQQ-dependent methanol dehydrogenase, DHA synthase or any
combination thereof.
[0021] In some embodiments, the invention provides a non-naturally
occurring microbial organism having attenuation of one or more
endogenous enzymes of a competing formaldehyde assimilation or
dissimilation pathway or a gene disruption of one or more
endogenous nucleic acids encoding enzymes of a competing
formaldehyde assimilation or dissimilation pathway. Examples of
these endogenous enzymes are described herein.
[0022] The invention still further provides a bioderived compound
produced by a microbial organism of the invention, culture medium
having the bioderived compound of the invention, compositions
having the bioderived compound of the invention, a biobased product
comprising the bioderived compound of the invention, and a process
for producing a bioderived compound of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows exemplary metabolic pathways enabling the
conversion of CO2, formate, formaldehyde (Fald), methanol (MeOH),
glycerol, xylose (XYL) and glucose (GLC) to acetyl-CoA (ACCOA) and
exemplary endogenous enzyme targets for optional attenuation or
disruption. The exemplary pathways can be combined with bioderived
compound pathways, including the pathways depicted herein that
utilize ACCOA, such as those depicted in FIGS. 4-10. The enzyme
targets are indicated by arrows having A'' markings. The endogenous
enzyme targets include DHA kinase, methanol oxidase (AOX),
PQQ-dependent methanol dehydrogenase (PQQ) and/or DHA synthase. The
enzymatic transformations shown are carried out by the following
enzymes: A) methanol dehydrogenase, B) 3-hexulose-6-phosphate
synthase, C) 6-phospho-3-hexuloisomerase, D) dihydroxyacetone
synthase, E) formate reductase, F) formate ligase, formate
transferase, or formate synthetase, G) formyl-CoA reductase, H)
formyltetmhydrofolate synthetase, I) methenyltetrahydrofolate
cyclohydrolase, J) methylenetetrahydrofolate dehydrogenase, K)
spontaneous or formaldehyde-forming enzyme, L) glycine cleavage
system, M) serine hydroxymethyltransferase, N) serine deaminase, O)
methylenetetrahydrofolate reductase, P) acetyl-CoA synthase, Q)
pyruvate formate lyase, R) pyruvate dehydrogenase, pyruvate
ferredoxin oxidoreductase, or pyruvate:NADP+ oxidoreductase, S)
formate dehydrogenase, T) fructose-6-phosphate phosphoketolase, U)
xylulose-5-phosphate phosphoketolase, V) phosphotransacetylase, W)
acetate kinase, X) acetyl-CoA transferase, synthetase, or ligase,
Y) lower glycolysis including glyceraldehyde-3-phosphate
dehydrogenase, Z) fructose-6-phosphate aldolase. See abbreviation
list below for compound names.
[0024] FIG. 2 shows exemplary metabolic pathways that provide the
extraction of reducing equivalents from methanol, hydrogen, or
carbon monoxide. Enzymes are: A) methanol methyltransferase, B)
methylenetetrahydrofolate reductase, C) methylenetetrahydrofolate
dehydrogenase, D) methenyltetrahydrofolate cyclohydrolase, E)
formyltetrahydrofolate deformylase, F) formyltetmhydrofolate
synthetase, G) formate hydrogen lyase, H) hydrogenase, I) formate
dehydrogenase, J) methanol dehydrogenase, K) spontaneous or
formaldehyde activating enzyme, L) formaldehyde dehydrogenase, M)
spontaneous or S-(hydroxymethyl)glutathione synthase, N)
Glutathione-Dependent Formaldehyde Dehydrogenase, O)
S-formylglutathione hydrolase, P) carbon monoxide dehydrogenase.
See abbreviation list below for compound names.
[0025] FIG. 3 shows exemplary pathways which can be used to
increase carbon flux through acetyl-CoA from carbohydrates when
reducing equivalents produced by a methanol or hydrogen oxidation
pathway provided herein are available. The enzymatic
transformations shown are carried out by the following enzymes: T)
fructose-6-phosphate phosphoketolase, U) xylulose-5-phosphate
phosphoketolase, V) phosphotransacetylase, W) acetate kinase, X)
acetyl-CoA transferase, synthetase, or ligase, and H) hydrogenase.
See abbreviation list below for compound names.
[0026] FIG. 4 shows exemplary metabolic pathways enabling the
conversion of the glycolysis intermediate glyceraldehye-3-phosphate
(G3P) to acetyl-CoA (ACCOA) and/or succinyl-CoA (SUCCOA). The
enzymatic transformations shown are carried out by the following
enzymes: A) PEP carboxylase or PEP carboxykinase, B) malate
dehydrogenase, C) fumarase, D) fumarate reductase, E) succinyl-CoA
synthetase or transferase, F) pyruvate kinase or PTS-dependent
substrate import, G) pyruvate dehydrogenase, pyruvate formate
lyase, or pyruvate:ferredoxin oxidoreductase, H) citrate synthase,
I) aconitase, J) isocitrate dehydrogenase, K) alpha-ketoglutamte
dehydrogenase, L) pyruvate carboxylase, M) malic enzyme, N)
isocitrate lyase and malate synthase. See abbreviation list below
for compound names.
[0027] FIG. 5 shows exemplary pathways enabling production of
1,3-butanediol, crotyl alcohol, and butadiene from acetyl-CoA.
1,3-butanediol, crotyl alcohol, and butadiene production is carried
out by the following enzymes: A) acetyl-CoA carboxylase, B) an
acetoacetyl-CoA synthase, C) an acetyl-CoA:acetyl-CoA
acyltransferase, D) an acetoacetyl-CoA reductase (ketone reducing),
E) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), F) a
3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, G) a
3-hydroxybutyrate reductase, H) a 3-hydroxybutyraldehyde reductase,
I) chemical dehydration or FIG. 6, J) a 3-hydroxybutyryl-CoA
dehydratase, K) a crotonyl-CoA reductase (aldehyde forming), L) a
crotonyl-CoA hydrolase, transferase or synthetase, M) a crotonate
reductase, N) a crotonaldehyde reductase, O) a crotyl alcohol
kinase, P) a 2-butenyl-4-phosphate kinase, Q) a butadiene synthase,
R) a crotyl alcohol diphosphokinase, S) chemical dehydration or a
crotyl alcohol dehydratase, T) a butadiene synthase
(monophosphate), T) a butadiene synthase (monophosphate), U) a
crotonyl-CoA reductase (alcohol forming), and V) a
3-hydroxybutyryl-CoA reductase (alcohol forming). See abbreviation
list below for compound names.
[0028] FIG. 6 shows exemplary pathways for converting
1,3-butanediol to 3-buten-2-ol and/or butadiene. 3-Buten-2-ol and
butadiene production is carried out by the following enzymes: A.
1,3-butanediol kinase, B. 3-hydroxybutyrylphosphate kinase, C.
3-hydroxybutyryldiphosphate lyase, D. 1,3-butanediol
diphosphokinase, E. 1,3-butanediol dehydratase, F.
3-hydroxybutyrylphosphate lyase, G. 3-buten-2-ol dehydratase or
chemical dehydration.
[0029] FIG. 7 shows exemplary pathways enabling production of
1,4-butanediol from succinyl-CoA. 1,4-Butanediol production is
carried out by the following enzymes: A) a succinyl-CoA transferase
or a succinyl-CoA synthetase, B) a succinyl-CoA reductase (aldehyde
forming), C) a 4-HB dehydrogenase, D) a 4-HB kinase, E) a
phosphotrans-4-hydroxybutyrylase, F) a 4-hydroxybutyryl-CoA
reductase (aldehyde forming), G) a 1,4-butanediol dehydrogenase, H)
a succinate reductase, I) a succinyl-CoA reductase (alcohol
forming), J) a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, K) a 4-HB reductase, L) a
4-hydroxybutyryl-phosphate reductase, and M) a 4-hydroxybutyryl-CoA
reductase (alcohol forming).
[0030] FIG. 8 shows exemplary pathways enabling production of
adipate, 6-aminocaproic acid, caprolactam, and hexamethylenediamine
from succinyl-CoA and acetyl-CoA. Adipate, 6-aminocaproic acid,
caprolactam, and hexamethylenediamine production is carried out by
the following enzymes: A) 3-oxoadipyl-CoA thiolase, B)
3-oxoadipyl-CoA reductase, C) 3-hydroxyadipyl-CoA dehydratase, D)
5-carboxy-2-pentenoyl-CoA reductase, E) adipyl-CoA reductase
(aldehyde forming), F) 6-aminocaproate transaminase or
6-aminocaproate dehydrogenase, G) 6-aminocaproyl-CoA/acyl-CoA
transferase or 6-aminocaproyl-CoA synthase, H) amidohydrolase, I)
spontaneous cyclization, J) 6-aminocaproyl-CoA reductase (aldehyde
forming), K) HMDA transaminase or HMDA dehydrogenase, L) Adipyl-CoA
hydrolase, adipyl-CoA ligase, adipyl-CoA transferase, or
phosphotransadipylase/adipate kinase.
[0031] FIG. 9 shows exemplary pathways enabling production of
3-hydroxyisobutyrate and methacrylic acid from succinyl-CoA.
3-Hydroxyisobutyrate and methacrylic acid production are carried
out by the following enzymes: A) Methylmalonyl-CoA mutase, B)
Methylmalonyl-CoA epimerase, C) Methylmalonyl-CoA reductase
(aldehyde forming), D) Methylmalonate semialdehyde reductase, E)
3-hydroxyisobutyrate dehydratase, F) Methylmalonyl-CoA reductase
(alcohol forming).
[0032] FIG. 10 shows exemplary pathways enabling production of
2-hydroxyisobutyrate and methacrylic acid from acetyl-CoA.
2-Hydroxyisobutyrate and methacrylic acid production are carried
out by the following enzymes: A) acetyl-CoA:acetyl-CoA
acyltransferase, B) acetoacetyl-CoA reductase (ketone reducing), C)
3-hydroxybutyrl-CoA mutase, D) 2-hydroxyisobutyryl-CoA dehydratase,
E) methacrylyl-CoA synthetase, hydrolase, or transferase, F)
2-hydroxyisobutyryl-CoA synthetase, hydrolase, or transferase.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention is directed to metabolic and
biosynthetic processes and microbial organisms capable of enhancing
carbon flux through acetyl-CoA. The invention disclosed herein is
based, at least in part, on non-naturally occurring microbial
organisms capable of synthesizing a bioderived compound using an
acetyl-CoA pathway, methanol metabolic pathway, a formaldehyde
fixation pathway, and/or a formate assimilation pathway in
combination with a bioderived compound pathway. Additionally, in
some embodiments, the non-naturally occurring microbial organisms
can further include a methanol oxidation pathway, a hydrogenase
and/or a carbon monoxide dehydrogenase.
[0034] The following is a list of abbreviations and their
corresponding compound or composition names. These abbreviations,
which are used throughout the disclosure and the figures. It is
understood that one of ordinary skill in the art can readily
identify these compounds/compositions by such nomenclature: MeOH or
MEOH=methanol; Fald=formaldehyde; GLC=glucose;
G6P=glucose-6-phosphate; H6P=hexulose-6-phosphate;
F6P=fructose-6-phosphate; FDP=fructose diphosphate or
fructose-1,6-diphosphate; DHA=dihydroxyacetone;
DHAP=dihydroxyacetone phosphate; G3P=glyceraldehyde-3-phosphate;
PYR=pyruvate; ACTP=acetyl-phosphate; ACCOA=acetyl-CoA;
AACOA=acetoacetyl-CoA; MALCOA=malonyl-CoA;
FTHF=formyltetrahydrofolate; THF=tetrahydrofolate;
E4P=erythrose-4-phosphate: Xu5P=xyulose-5-phosphate;
Ru5P=ribulose-5-phosphate; S7P=sedoheptulose-7-phosphate:
R5P=ribose-5-phosphate; XYL=xylose; TCA=tricarboxylic acid;
PEP=Phosphoenolpyruvate; OAA=Oxaloacetate; MAL=malate; CIT=citrate;
ICIT=isocitrate; AKG=alpha-ketoglutarate; FUM=Fumamte;
SUCC=Succinate; SUCCOA=Succinyl-CoA; 3HBCOA=3-hydroxybutyryl-CoA;
3-HB=3-hydroxybutyrate; 3HBALD=3-hydroxybutyraldehyde;
13BDO=1,3-butanediol; CROTCOA=crotonyl-CoA; CROT=crotonate;
CROTALD=crotonaldehyde; CROTALC=crotyl alcohol; CROT-Pi=crotyl
phosphate; CROT-PPi=crotyl diphosphate or
2-butenyl-4-diphosphate.
[0035] It is also understood that association of multiple steps in
a pathway can be indicated by linking their step identifiers with
or without spaces or punctuation; for example, the following are
equivalent to describe the 4-step pathway comprising Step W, Step
X, Step Y and Step Z: steps WXYZ or W,X,Y,Z or W;X:X;Z or W-X-Y-Z.
One of ordinary skill can readily distinguish a single step
designator of "AA" or "AB" or "AD" from a multiple step pathway
description based on context and use in the description and figures
herein.
[0036] As used herein, the term "non-naturally occurring" when used
in reference to a microbial organism or microorganism of the
invention is intended to mean that the microbial organism has at
least one genetic alteration not normally found in a naturally
occurring strain of the referenced species, including wild-type
strains of the referenced species. Genetic alterations include, for
example, modifications introducing expressible nucleic acids
encoding metabolic polypeptides, other nucleic acid additions,
nucleic acid deletions and/or other functional disruption of the
microbial organism's genetic material. Such modifications include,
for example, coding regions and functional fragments thereof, for
heterologous, homologous or both heterologous and homologous
polypeptides for the referenced species. Additional modifications
include, for example, non-coding regulatory regions in which the
modifications alter expression of a gene or operon. Exemplary
metabolic polypeptides include enzymes or proteins within an
acetyl-CoA or bioderived compound biosynthetic pathway.
[0037] A metabolic modification refers to a biochemical reaction
that is altered from its naturally occurring state. Therefore,
non-naturally occurring microorganisms can have genetic
modifications to nucleic acids encoding metabolic polypeptides, or
functional fragments thereof. Exemplary metabolic modifications are
disclosed herein.
[0038] As used herein, the term "isolated" when used in reference
to a microbial organism is intended to mean an organism that is
substantially free of at least one component as the referenced
microbial organism is found in nature. The term includes a
microbial organism that is removed from some or all components as
it is found in its natural environment. The term also includes a
microbial organism that is removed from some or all components as
the microbial organism is found in non-naturally occurring
environments. Therefore, an isolated microbial organism is partly
or completely separated from other substances as it is found in
nature or as it is grown, stored or subsisted in non-naturally
occurring environments. Specific examples of isolated microbial
organisms include partially pure microbes, substantially pure
microbes and microbes cultured in a medium that is non-naturally
occurring.
[0039] As used herein, the terms "microbial," "microbial organism"
or "microorganism" are intended to mean any organism that exists as
a microscopic cell that is included within the domains of archaea,
bacteria or eukarya. Therefore, the term is intended to encompass
prokaryotic or eukaryotic cells or organisms having a microscopic
size and includes bacteria, archaea and eubacteria of all species
as well as eukaryotic microorganisms such as yeast and fungi. The
term also includes cell cultures of any species that can be
cultured for the production of a biochemical.
[0040] As used herein, the term "CoA" or "coenzyme A" is intended
to mean an organic cofactor or prosthetic group (nonprotein portion
of an enzyme) whose presence is required for the activity of many
enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A
functions in certain condensing enzymes, acts in acetyl or other
acyl group transfer and in fatty acid synthesis and oxidation,
pyruvate oxidation and in other acetylation.
[0041] As used herein, the term "substantially anaerobic" when used
in reference to a culture or growth condition is intended to mean
that the amount of oxygen is less than about 10% of saturation for
dissolved oxygen in liquid media. The term also is intended to
include sealed chambers of liquid or solid medium maintained with
an atmosphere of less than about 1% oxygen.
[0042] "Exogenous" as it is used herein is intended to mean that
the referenced molecule or the referenced activity is introduced
into the host microbial organism. The molecule can be introduced,
for example, by introduction of an encoding nucleic acid into the
host genetic material such as by integration into a host chromosome
or as non-chromosomal genetic material such as a plasmid.
Therefore, the term as it is used in reference to expression of an
encoding nucleic acid refers to introduction of the encoding
nucleic acid in an expressible form into the microbial organism.
When used in reference to a biosynthetic activity, the term refers
to an activity that is introduced into the host reference organism.
The source can be, for example, a homologous or heterologous
encoding nucleic acid that expresses the referenced activity
following introduction into the host microbial organism. Therefore,
the term "endogenous" refers to a referenced molecule or activity
that is present in the host. Similarly, the term when used in
reference to expression of an encoding nucleic acid refers to
expression of an encoding nucleic acid contained within the
microbial organism. The term "heterologous" refers to a molecule or
activity derived from a source other than the referenced species
whereas "homologous" refers to a molecule or activity derived from
the host microbial organism. Accordingly, exogenous expression of
an encoding nucleic acid of the invention can utilize either or
both a heterologous or homologous encoding nucleic acid.
[0043] It is understood that when more than one exogenous nucleic
acid is included in a microbial organism that the more than one
exogenous nucleic acids refers to the referenced encoding nucleic
acid or biosynthetic activity, as discussed above. It is further
understood, as disclosed herein, that such more than one exogenous
nucleic acids can be introduced into the host microbial organism on
separate nucleic acid molecules, on polycistronic nucleic acid
molecules, or a combination thereof, and still be considered as
more than one exogenous nucleic acid. For example, as disclosed
herein a microbial organism can be engineered to express two or
more exogenous nucleic acids encoding a desired pathway enzyme or
protein. In the case where two exogenous nucleic acids encoding a
desired activity are introduced into a host microbial organism, it
is understood that the two exogenous nucleic acids can be
introduced as a single nucleic acid, for example, on a single
plasmid, on separate plasmids, can be integrated into the host
chromosome at a single site or multiple sites, and still be
considered as two exogenous nucleic acids. Similarly, it is
understood that more than two exogenous nucleic acids can be
introduced into a host organism in any desired combination, for
example, on a single plasmid, on separate plasmids, can be
integrated into the host chromosome at a single site or multiple
sites, and still be considered as two or more exogenous nucleic
acids, for example three exogenous nucleic acids. Thus, the number
of referenced exogenous nucleic acids or biosynthetic activities
refers to the number of encoding nucleic acids or the number of
biosynthetic activities, not the number of separate nucleic acids
introduced into the host organism.
[0044] As used herein, the phrase "enhance carbon flux" is intended
to mean to intensify, increase, or further improve the extent or
flow of metabolic carbon through or to a desired pathway, pathway
product, intermediate, or bioderived compound. The intensity,
increase or improvement can be relative to a predetermined baseline
of a pathway product, intermediate or bioderived compound. For
example, an increased yield of acetyl-CoA can be achieved per mole
of methanol with a phosphoketolase enzyme described herein (see,
e.g., FIG. 1) than in the absence of a phosphoketolase enzyme.
Similarly, an increased yield of acetyl-CoA can be achieved per
mole of methanol with the formate assimilation enzymes (see, e.g.,
FIG. 1) than in the absence of the enzymes. Since an increased
yield of acetyl-CoA can be achieved, a higher yield of acetyl-CoA
derived compounds, such as 1,3-butanediol, crotyl alcohol,
butadiene, 3-buten-2-ol, 1,4-butanediol, adipate, 6-aminocaproate,
caprolactam, hexamethylenediamine, methacylic acid and
2-hydroxyisobutyric acid the invention, can also be achieved.
[0045] As used herein, the term "gene disruption," or grammatical
equivalents thereof, is intended to mean a genetic alteration that
renders the encoded gene product inactive or attenuated. The
genetic alteration can be, for example, deletion of the entire
gene, deletion of a regulatory sequence required for transcription
or translation, deletion of a portion of the gene which results in
a truncated gene product, or by any of various mutation strategies
that inactivate or attenuate the encoded gene product, for example,
replacement of a gene's promoter with a weaker promoter,
replacement or insertion of one or more amino acid of the encoded
protein to reduce its activity, stability or concentration, or
inactivation of a gene's transactivating factor such as a
regulatory protein. One particularly useful method of gene
disruption is complete gene deletion because it reduces or
eliminates the occurrence of genetic reversions in the
non-naturally occurring microorganisms of the invention. A gene
disruption also includes a null mutation, which refers to a
mutation within a gene or a region containing a gene that results
in the gene not being transcribed into RNA and/or translated into a
functional gene product. Such a null mutation can arise from many
types of mutations including, for example, inactivating point
mutations, deletion of a portion of a gene, entire gene deletions,
or deletion of chromosomal segments.
[0046] As used herein, the term "growth-coupled" when used in
reference to the production of a biochemical product is intended to
mean that the biosynthesis of the referenced biochemical product is
produced during the growth phase of a microorganism. In a
particular embodiment, the growth-coupled production can be
obligatory, meaning that the biosynthesis of the referenced
biochemical is an obligatory product produced during the growth
phase of a microorganism.
[0047] As used herein, the term "attenuate," or grammatical
equivalents thereof, is intended to mean to weaken, reduce or
diminish the activity or amount of an enzyme or protein.
Attenuation of the activity or amount of an enzyme or protein can
mimic complete disruption if the attenuation causes the activity or
amount to fall below a critical level required for a given pathway
to function. However, the attenuation of the activity or amount of
an enzyme or protein that mimics complete disruption for one
pathway, can still be sufficient for a separate pathway to continue
to function. For example, attenuation of an endogenous enzyme or
protein can be sufficient to mimic the complete disruption of the
same enzyme or protein for production of acetyl-CoA or a bioderived
compound of the invention, but the remaining activity or amount of
enzyme or protein can still be sufficient to maintain other
pathways, such as a pathway that is critical for the host microbial
organism to survive, reproduce or grow. Attenuation of an enzyme or
protein can also be weakening, reducing or diminishing the activity
or amount of the enzyme or protein in an amount that is sufficient
to increase yield of acetyl-CoA or a bioderived compound of the
invention, but does not necessarily mimic complete disruption of
the enzyme or protein.
[0048] As used herein, the term "bioderived" means derived from or
synthesized by a biological organism and can be considered a
renewable resource since it can be generated by a biological
organism. Such a biological organism, in particular the microbial
organisms of the invention, can utilize a variety of carbon sources
described herein including feedstock or biomass, such as, sugars
and carbohydrates obtained from an agricultural, plant, bacterial,
or animal source. Alternatively, the biological organism can
utilize, for example, atmospheric carbon and/or methanol as a
carbon source.
[0049] As used herein, the term "biobased" means a product as
described herein that is composed, in whole or in part, of a
bioderived compound of the invention. A biobased product is in
contrast to a petroleum based product, wherein such a product is
derived from or synthesized from petroleum or a petrochemical
feedstock.
[0050] A "bioderived compound," as used herein, refers to a target
molecule or chemical that is derived from or synthesized by a
biological organism. In the context of the present invention,
engineered microbial organisms are used to produce a bioderived
compound or intermediate thereof via acetyl-CoA, including
optionally further through acetoacetyl-CoA, malonyl-CoA and/or
succinyl-CoA. Bioderived compounds of the invention include, but
are not limited to, alcohols, glycols, organic acids, alkenes,
dienes, organic amines, organic aldehydes, vitamins, nutraceuticals
and pharmaceuticals.
[0051] Alcohols of the invention, including biofuel alcohols,
include primary alcohols, secondary alcohols, diols and triols,
preferably having C3 to C10 carbon atoms. Alcohols include
n-propanol and isopropanol. Biofuel alcohols are preferably C3-C10
and include 1-Propanol, Isopropanol, 1-Butanol, Isobutanol,
1-Pentanol, Isopentenol, 2-Methyl-1-butanol, 3-Methyl-1-butanol,
1-Hexanol, 3-Methyl-1-pentanol, 1-Heptanol, 4-Methyl-1-hexanol, and
5-Methyl-1-hexanol. Diols include propanediols and butanediols,
including 1,4 butanediol, 1,3-butanediol and 2,3-butanediol. Fatty
alcohols include C4-C27 fatty alcohols, including C12-C18,
especially C12-C14, including saturate or unsaturated linear fatty
alcohols.
[0052] Further exemplary bioderived compounds of the invention
include: (a) 1,4-butanediol and intermediates thereto, such as
4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate
(4-HB); (b) butadiene (1,3-butadiene) and intermediates thereto,
such as 1,4-butanediol, 1,3-butanediol, 2,3-butanediol, crotyl
alcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol; (c)
1,3-butanediol and intermediates thereto, such as 3-hydroxybutyrate
(3-HB), 2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol; (d)
adipate, 6-aminocaproic acid (6-ACA), caprolactam,
hexamethylenediamine (HMDA) and levulinic acid and their
intermediates, e.g. adipyl-CoA, 4-aminobutyryl-CoA; (e) methacrylic
acid (2-methyl-2-propenoic acid) and its esters, such as methyl
methacrylate and methyl methacrylate (known collectively as
methacrylates), 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate
and their intermediates; (f) glycols, including 1,2-propanediol
(propylene glycol), 1,3-propanediol, glycerol, ethylene glycol,
diethylene glycol, triethylene glycol, dipropylene glycol,
tripropylene glycol, neopentyl glycol and bisphenol A and their
intermediates; (g) succinic acid and intermediates thereto; and (h)
fatty alcohols, which are aliphatic compounds containing one or
more hydroxyl groups and a chain of 4 or more carbon atoms, or
fatty acids and fatty aldehydes thereof, which are preferably
C4-C27 carbon atoms. Fatty alcohols include saturated fatty
alcohols, unsaturated fatty alcohols and linear saturated fatty
alcohols. Examples fatty alcohols include butyl, pentyl, hexyl,
heptyl, octyl, nonyl, decyl, undecyl and dodecyl alcohols, and
their corresponding oxidized derivatives, i.e. fatty aldehydes or
fatty acids having the same number of carbon atoms. Preferred fatty
alcohols, fatty aldehydes and fatty acids have C8 to C18 carbon
atoms, especially C12-C18, C12-C14, and C16-C18, including C12,
C13, C14, C15, C16, C17, and C18 carbon atoms. Preferred fatty
alcohols include linear unsaturated fatty alcohols, such as
dodecanol (C12; lauryl alcohol), tridecyl alcohol (C13;
1-tridecanol, tridecanol, isotridecanol), myristyl alcohol (C14;
1-tetradecanol), pentadecyl alcohol (C15; 1-pentadecanol,
pentadecanol), cetyl alcohol (C16; 1-hexadecanol), heptadecyl
alcohol (C17; 1-n-heptadecanol, heptadecanol) and stearyl alcohol
(C18; 1-octadecanol) and unsaturated counterparts including
palmitoleyl alcohol (C16 unsaturated; cis-9-hexadecen-1-ol), or
their corresponding fatty aldehydes or fatty acids.
[0053] 1,4-Butanediol and intermediates thereto, such as
4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate,
4-HB), are bioderived compounds that can be made via enzymatic
pathways described herein and in the following publications.
Suitable bioderived compound pathways and enzymes, methods for
screening and methods for isolating are found in: WO2008115840A2
published 25 Sep. 2008 entitled Compositions and Methods for the
Biosynthesis of 1,4-Butanediol and Its Precursors; WO2010141780A1
published 9 Dec. 2010 entitled Process of Separating Components of
A Fermentation Broth; WO2010141920A2 published 9 Dec. 2010 entitled
Microorganisms for the Production of 1,4-Butanediol and Related
Methods; WO2010030711A2 published 18 Mar. 2010 entitled
Microorganisms for the Production of 1,4-Butanediol; WO2010071697A1
published 24 Jun. 2010 Microorganisms and Methods for Conversion of
Syngas and Other Carbon Sources to Useful Products; WO2009094485A1
published 30 Jul. 2009 Methods and Organisms for Utilizing
Synthesis Gas or Other Gaseous Carbon Sources and Methanol;
WO2009023493A1 published 19 Feb. 2009 entitled Methods and
Organisms for the Growth-Coupled Production of 1,4-Butanediol; and
WO2008115840A2 published 25 Sep. 2008 entitled Compositions and
Methods for the Biosynthesis of 1,4-Butanediol and Its Precursors,
which are all incorporated herein by reference.
[0054] Butadiene and intermediates thereto, such as 1,4-butanediol,
2,3-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol
(methyl vinyl carbinol) and 3-buten-1-ol, are bioderived compounds
that can be made via enzymatic pathways described herein and in the
following publications. In addition to direct fermentation to
produce butadiene, 1,3-butanediol, 1,4-butanediol, crotyl alcohol,
3-buten-2-ol (methyl vinyl carbinol) or 3-buten-1-ol can be
separated, purified (for any use), and then chemically dehydrated
to butadiene by metal-based catalysis. Suitable bioderived compound
pathways and enzymes, methods for screening and methods for
isolating are found in: WO2011140171A2 published 10 Nov. 2011
entitled Microorganisms and Methods for the Biosynthesis of
Butadiene; WO2012018624A2 published 9 Feb. 2012 entitled
Microorganisms and Methods for the Biosynthesis of Aromatics,
2,4-Pentadienoate and 1,3-Butadiene; WO2011140171A2 published 10
Nov. 2011 entitled Microorganisms and Methods for the Biosynthesis
of Butadiene; WO2013040383A1 published 21 Mar. 2013 entitled
Microorganisms and Methods for Producing Alkenes; WO2012177710A1
published 27 Dec. 2012 entitled Microorganisms for Producing
Butadiene and Methods Related thereto; WO2012106516A1 published 9
Aug. 2012 entitled Microorganisms and Methods for the Biosynthesis
of Butadiene; and WO2013028519A1 published 28 Feb. 2013 entitled
Microorganisms and Methods for Producing 2,4-Pentadienoate,
Butadiene, Propylene, 1,3-Butanediol and Related Alcohols, which
are all incorporated herein by reference.
[0055] 1,3-Butanediol and intermediates thereto, such as
2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol, are bioderived
compounds that can be made via enzymatic pathways described herein
and in the following publications. Suitable bioderived compound
pathways and enzymes, methods for screening and methods for
isolating are found in: WO2011071682A1 published 16 Jun. 2011
entitled Methods and Organisms for Converting Synthesis Gas or
Other Gaseous Carbon Sources and Methanol to 1,3-Butanediol;
WO2011031897A published 17 Mar. 2011 entitled Microorganisms and
Methods for the Co-Production of Isopropanol with Primary Alcohols,
Diols and Acids; WO2010127319A2 published 4 Nov. 2010 entitled
Organisms for the Production of 1,3-Butanediol; WO2013071226A1
published 16 May 2013 entitled Eukaryotic Organisms and Methods for
Increasing the Availability of Cytosolic Acetyl-CoA, and for
Producing 1,3-Butanediol; WO2013028519A1 published 28 Feb. 2013
entitled Microorganisms and Methods for Producing
2,4-Pentadienoate, Butadiene, Propylene, 1,3-Butanediol and Related
Alcohols; WO2013036764A1 published 14 Mar. 2013 entitled Eukaryotic
Organisms and Methods for Producing 1,3-Butanediol; WO2013012975A1
published 24 Jan. 2013 entitled Methods for Increasing Product
Yields; and WO2012177619A2 published 27 Dec. 2012 entitled
Microorganisms for Producing 1,3-Butanediol and Methods Related
Thereto, which are all incorporated herein by reference.
[0056] Adipate, 6-aminocaproic acid, caprolactam,
hexamethylenediamine and levulinic acid, and their intermediates,
e.g. 4-aminobutyryl-CoA, are bioderived compounds that can be made
via enzymatic pathways described herein and in the following
publications. Suitable bioderived compound pathways and enzymes,
methods for screening and methods for isolating are found in:
WO2010129936A1 published 11 Nov. 2010 entitled Microorganisms and
Methods for the Biosynthesis of Adipate, Hexamethylenediamine and
6-Aminocaproic Acid; WO2013012975A1 published 24 Jan. 2013 entitled
Methods for Increasing Product Yields; WO2012177721A1 published 27
Dec. 2012 entitled Microorganisms for Producing 6-Aminocaproic
Acid; WO2012099621A1 published 26 Jul. 2012 entitled Methods for
Increasing Product Yields; and WO2009151728 published 17 Dec. 2009
entitled Microorganisms for the production of adipic acid and other
compounds, which are all incorporated herein by reference.
[0057] Methacrylic acid (2-methyl-2-propenoic acid) is used in the
preparation of its esters, known collectively as methacrylates (e.g
methyl methacrylate, which is used most notably in the manufacture
of polymers). Methacrylate esters such as methyl methacrylate,
3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and their
intermediates are bioderived compounds that can be made via
enzymatic pathways described herein and in the following
publications. Suitable bioderived compound pathways and enzymes,
methods for screening and methods for isolating are found in:
WO2012135789A2 published 4 Oct. 2012 entitled Microorganisms for
Producing Methacrylic Acid and Methacrylate Esters and Methods
Related Thereto; and WO2009135074A2 published 5 Nov. 2009 entitled
Microorganisms for the Production of Methacrylic Acid, which are
all incorporated herein by reference.
[0058] 1,2-Propanediol (propylene glycol), n-propanol,
1,3-propanediol and glycerol, and their intermediates are
bioderived compounds that can be made via enzymatic pathways
described herein and in the following publications. Suitable
bioderived compound pathways and enzymes, methods for screening and
methods for isolating are found in: WO2009111672A1 published 9 Nov.
2009 entitled Primary Alcohol Producing Organisms; WO2011031897A1
17 Mar. 2011 entitled Microorganisms and Methods for the
Co-Production of Isopropanol with Primary Alcohols, Diols and
Acids; WO2012177599A2 published 27 Dec. 2012 entitled
Microorganisms for Producing N-Propanol 1,3-Propanediol,
1,2-Propanediol or Glycerol and Methods Related Thereto, which are
all incorporated herein by referenced.
[0059] Succinic acid and intermediates thereto, which are useful to
produce products including polymers (e.g. PBS), 1,4-butanediol,
tetrahydrofuran, pyrrolidone, solvents, paints, deicers, plastics,
fuel additives, fabrics, carpets, pigments, and detergents, are
bioderived compounds that can be made via enzymatic pathways
described herein and in the following publication. Suitable
bioderived compound pathways and enzymes, methods for screening and
methods for isolating are found in: EP1937821A2 published 2 Jul.
2008 entitled Methods and Organisms for the Growth-Coupled
Production of Succinate, which is incorporated herein by
reference.
[0060] Primary alcohols and fatty alcohols (also known as long
chain alcohols), including fatty acids and fatty aldehydes thereof,
and intermediates thereto, are bioderived compounds that can be
made via enzymatic pathways in the following publications. Suitable
bioderived compound pathways and enzymes, methods for screening and
methods for isolating are found in: WO2009111672 published 11 Sep.
2009 entitled Primary Alcohol Producing Organisms; WO2012177726
published 27 Dec. 2012 entitled Microorganism for Producing Primary
Alcohols and Related Compounds and Methods Related Thereto, which
are all incorporated herein by reference.
[0061] Further suitable bioderived compounds that the microbial
organisms of the invention can be used to produce via acetyl-CoA,
including optionally further through acetoacetyl-CoA and/or
succinyl-CoA, are included in the invention. Exemplary well known
bioderived compounds, their pathways and enzymes for production,
methods for screening and methods for isolating are found in the
following patents and publications: succinate (U.S. publication
2007/0111294, WO 2007/030830, WO 2013/003432), 3-hydroxypropionic
acid (3-hydroxypropionate) (U.S. publication 2008/0199926, WO
2008/091627, U.S. publication 2010/0021978), 1,4-butanediol (U.S.
Pat. No. 8,067,214, WO 2008/115840, U.S. Pat. No. 7,947,483, WO
2009/023493, U.S. Pat. No. 7,858,350, WO 2010/030711, U.S.
publication 2011/0003355, WO 2010/141780, U.S. Pat. No. 8,129,169,
WO 2010/141920, U.S. publication 2011/0201068, WO 2011/031897, U.S.
Pat. No. 8,377,666, WO 2011/047101, U.S. publication 2011/0217742,
WO 2011/066076, U.S. publication 2013/0034884, WO 2012/177943),
4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate,
4-hydroxybutryate) (U.S. Pat. No. 8,067,214, WO 2008/115840, U.S.
Pat. No. 7,947,483, WO 2009/023493, U.S. Pat. No. 7,858,350, WO
2010/030711, U.S. publication 2011/0003355, WO 2010/141780, U.S.
Pat. No. 8,129,155, WO 2010/071697), .gamma.-butyrolactone (U.S.
Pat. No. 8,067,214, WO 2008/115840, U.S. Pat. No. 7,947,483, WO
2009/023493, U.S. Pat. No. 7,858,350, WO 2010/030711, U.S.
publication 2011/0003355, WO 2010/141780, U.S. publication
2011/0217742, WO 2011/066076), 4-hydroxybutyryl-CoA (U.S.
publication 2011/0003355, WO 2010/141780, U.S. publication
2013/0034884, WO 2012/177943), 4-hydroxybutanal (U.S. publication
2011/0003355, WO 2010/141780, U.S. publication 2013/0034884, WO
2012/177943), putrescine (U.S. publication 2011/0003355, WO
2010/141780, U.S. publication 2013/0034884, WO 2012/177943),
Olefins (such as acrylic acid and acrylate ester) (U.S. Pat. No.
8,026,386, WO 2009/045637), acetyl-CoA (U.S. Pat. No. 8,323,950, WO
2009/094485), methyl tetrahydrofolate (U.S. Pat. No. 8,323,950, WO
2009/094485), ethanol (U.S. Pat. No. 8,129,155, WO 2010/071697),
isopropanol (U.S. Pat. No. 8,129,155, WO 2010/071697, U.S.
publication 2010/0323418, WO 2010/127303, U.S. publication
2011/0201068, WO 2011/031897), n-butanol (U.S. Pat. No. 8,129,155,
WO 2010/071697), isobutanol (U.S. Pat. No. 8,129,155, WO
2010/071697), n-propanol (U.S. publication 2011/0201068, WO
2011/031897), methylacrylic acid (methylacrylate) (U.S. publication
2011/0201068, WO 2011/031897), primary alcohol (U.S. Pat. No.
7,977,084, WO 2009/111672, WO 2012/177726), long chain alcohol
(U.S. Pat. No. 7,977,084, WO 2009/111672, WO 2012/177726), adipate
(adipic acid) (U.S. Pat. No. 8,062,871, WO 2009/151728, U.S. Pat.
No. 8,377,680, WO 2010/129936, WO 2012/177721), 6-aminocaproate
(6-aminocaproic acid) (U.S. Pat. No. 8,062,871, WO 2009/151728,
U.S. Pat. No. 8,377,680, WO 2010/129936, WO 2012/177721),
caprolactam (U.S. Pat. No. 8,062,871, WO 2009/151728, U.S. Pat. No.
8,377,680, WO 2010/129936, WO 2012/177721), hexamethylenediamine
(U.S. Pat. No. 8,377,680, WO 2010/129936, WO 2012/177721),
levulinic acid (U.S. Pat. No. 8,377,680, WO 2010/129936),
2-hydroxyisobutyric acid (2-hydroxyisobutyrate) (U.S. Pat. No.
8,241,877, WO 2009/135074, U.S. publication 2013/0065279, WO
2012/135789), 3-hydroxyisobutyric acid (3-hydroxyisobutyrate) (U.S.
Pat. No. 8,241,877, WO 2009/135074, U.S. publication 2013/0065279,
WO 2012/135789), methacrylic acid (methacrylate) (U.S. Pat. No.
8,241,877, WO 2009/135074, U.S. publication 2013/0065279, WO
2012/135789), methacrylate ester (U.S. publication 2013/0065279, WO
2012/135789), fumarate (fumaric acid) (U.S. Pat. No. 8,129,154, WO
2009/155382), malate (malic acid) (U.S. Pat. No. 8,129,154, WO
2009/155382), acrylate (carboxylic acid) (U.S. Pat. No. 8,129,154,
WO 2009/155382), methyl ethyl ketone (U.S. publication
2010/0184173, WO 2010/057022, U.S. Pat. No. 8,420,375, WO
2010/144746), 2-butanol (U.S. publication 2010/0184173, WO
2010/057022, U.S. Pat. No. 8,420,375, WO 2010/144746),
1,3-butanediol (U.S. publication 2010/0330635, WO 2010/127319, U.S.
publication 2011/0201068, WO 2011/031897, U.S. Pat. No. 8,268,607,
WO 2011/071682, U.S. publication 2013/0109064, WO 2013/028519, U.S.
publication 2013/0066035, WO 2013/036764), cyclohexanone (U.S.
publication 2011/0014668, WO 2010/132845), terephthalate
(terephthalic acid) (U.S. publication 2011/0124911, WO 2011/017560,
U.S. publication 2011/0207185, WO 2011/094131, U.S. publication
2012/0021478, WO 2012/018624), muconate (muconic acid) (U.S.
publication 2011/0124911, WO 2011/017560), aniline (U.S.
publication 2011/0097767, WO 2011/050326), p-toluate (p-toluic
acid) (U.S. publication 2011/0207185, WO 2011/094131, U.S.
publication 2012/0021478, WO 2012/018624),
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (U.S. publication
2011/0207185, WO 2011/094131, U.S. publication 2012/0021478, WO
2012/018624), ethylene glycol (U.S. publication 2011/0312049, WO
2011/130378, WO 2012/177983), propylene (U.S. publication
2011/0269204, WO 2011/137198, U.S. publication 2012/0329119, U.S.
publication 2013/0109064, WO 2013/028519), butadiene
(1,3-butadiene) (U.S. publication 2011/0300597, WO 2011/140171,
U.S. publication 2012/0021478, WO 2012/018624, U.S. publication
2012/0225466, WO 2012/106516, U.S. publication 2013/0011891, WO
2012/177710, U.S. publication 2013/0109064, WO 2013/028519),
toluene (U.S. publication 2012/0021478, WO 2012/018624), benzene
(U.S. publication 2012/0021478, WO 2012/018624),
(2-hydroxy-4-oxobutoxy)phosphonate (U.S. publication 2012/0021478,
WO 2012/018624), benzoate (benzoic acid) (U.S. publication
2012/0021478, WO 2012/018624), styrene (U.S. publication
2012/0021478, WO 2012/018624), 2,4-pentadienoate (U.S. publication
2012/0021478, WO 2012/018624, U.S. publication 2013/0109064, WO
2013/028519), 3-butene-1-ol (U.S. publication 2012/0021478, WO
2012/018624, U.S. publication 2013/0109064, WO 2013/028519),
3-buten-2-ol (U.S. publication 2013/0109064, WO 2013/028519),
1,4-cyclohexanedimethanol (U.S. publication 2012/0156740, WO
2012/082978), crotyl alcohol (U.S. publication 2013/0011891, WO
2012/177710, U.S. publication 2013/0109064, WO 2013/028519), alkene
(U.S. publication 2013/0122563, WO 2013/040383, US 2011/0196180),
hydroxyacid (WO 2012/109176), ketoacid (WO 2012/109176), wax esters
(WO 2007/136762) or caprolactone (U.S. publication 2013/0144029, WO
2013/067432) pathway. The patents and patent application
publications listed above that disclose bioderived compound
pathways are herein incorporated herein by reference.
[0062] Acetyl-CoA is the immediate precursor for the synthesis of
bioderived compounds as shown in FIGS. 5-10. Phosphoketolase
pathways make possible synthesis of acetyl-CoA without requiring
decarboxylation of pyruvate (Bogorad et al, Nature, 2013, published
online 29 Sep. 2013; United States Publication 2006-0040365), which
thereby provides higher yields of bioderived compounds of the
invention from carbohydrates and methanol than the yields
attainable without phosphoketolase enzymes.
[0063] For example, synthesis of an exemplary fatty alcohol,
dodecanol, from methanol using methanol dehydrogenase (step A of
FIG. 1), a formaldehyde assimilation pathway (steps B, C, D of FIG.
1), the pentose phosphate pathway, and glycolysis can provide a
maximum theoretical yield of 0.0556 mole dodecanol/mole
methanol.
18 CH.sub.4O+9 O.sub.2.fwdarw.C.sub.12H.sub.26O+23 H.sub.2O+6
CO.sub.2
However, if these pathways are combined with a phosphoketolase
pathway (steps T, U, V, W, X of FIG. 1), a maximum theoretical
yield of 0.0833 mole dodecanol/mole methanol can be obtained if we
assume that the pathway is not required to provide net generation
of ATP for cell growth and maintenance requirements.
12 CH.sub.4O.fwdarw.C.sub.12H.sub.26O+11 H.sub.2O
[0064] ATP for energetic requirements can be synthesized, at the
expense of lowering the maximum theoretical product yield, by
oxidizing methanol to CO.sub.2 using several combinations of
enzymes depicted in FIG. 2, glycolysis, the TCA cycle, the pentose
phosphate pathway, and oxidative phosphorylation.
[0065] Similarly, synthesis of isopropanol from methanol using
methanol dehydrogenase (step A of FIG. 1), a formaldehyde
assimilation pathway (steps B, C, D of FIG. 1), the pentose
phosphate pathway and glycolysis can provide a maximum theoretical
yield of 0.1667 mole isopropanol/mole methanol.
6 CH.sub.4O+4.5 O.sub.2.fwdarw.C.sub.3H.sub.8O+8 H.sub.2O+3
CO.sub.2
[0066] However, if these pathways are applied in combination with a
phosphoketolase pathway (steps T, U, V, W, X of FIG. 1), a maximum
theoretical yield of 0.250 mole isopropanol/mole methanol can be
obtained.
4 CH.sub.4O+1.5 O.sub.2.fwdarw.C.sub.3H.sub.8O+4
H.sub.2O+CO.sub.2
[0067] The overall pathway is ATP and redox positive enabling
synthesis of both ATP and NAD(P)H from conversion of MeOH to
isopropanol. Additional ATP can be synthesized, at the expense of
lowering the maximum theoretical product yield, by oxidizing
methanol to CO.sub.2 using several combinations of enzymes depicted
in FIG. 2, glycolysis, the TCA cycle, the pentose phosphate
pathway, and oxidative phosphorylation.
[0068] Synthesis of several other products from methanol using
methanol dehydrogenase (step A of FIG. 1), a formaldehyde
assimilation pathway (steps B, C, D of FIG. 1), the pentose
phosphate pathway and glycolysis can provide the following maximum
theoretical yield stoichiometries:
TABLE-US-00001 Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol
6.000 3.500 0.000 --> 1.000 7.000 2.000 Crotyl Alcohol 6.000
3.500 0.000 --> 1.000 8.000 2.000 3-Buten-2-ol 6.000 3.500 0.000
--> 1.000 8.000 2.000 Butadiene 6.000 3.500 0.000 --> 1.000
9.000 2.000 2-Hydroxyisobutyrate 6.000 4.500 0.000 --> 1.000
8.000 2.000 Methacrylate (via 2-hydroxyisobutyrate) 6.000 4.500
0.000 --> 1.000 9.000 2.000 3-Hydroxyisobutyrate (oxidative TCA
cycle) 6.000 4.500 0.000 --> 1.000 8.000 2.000 Methacrylate (via
3-hydroxyisobutyrate) 6.000 4.500 0.000 --> 1.000 9.000 2.000
1,4-Butanediol (oxidative TCA cycle) 6.000 3.500 0.000 --> 1.000
9.000 2.000 Adipate (oxidative TCA cycle) 9.000 7.000 0.000 -->
1.000 13.000 3.000 6-Aminocaproate (oxidative TCA cycle) 9.000
6.000 1.000 --> 1.000 13.000 3.000 Caprolactam (via
6-aminocaproate) 9.000 6.000 1.000 --> 1.000 14.000 3.000
Hexamethylenediamine (oxidative TCA cycle) 9.000 5.000 2.000 -->
1.000 13.000 3.000
[0069] In the products marked "oxidative TCA cycle", the maximum
yield stoichiometries assume that the reductive TCA cycle enzymes
(e.g., malate dehydrogenase, fumarase, fumarate reductase, and
succinyl-CoA ligase) are not utilized for product formation.
Exclusive use of the oxidative TCA cycle for product formation can
be advantageous for succinyl-CoA derived products such as
3-hydroxyisobutyrate, 1,4-butanediol, adipate, 6-aminocaproate, and
hexamethylenediamine because it enables all of the product pathway
flux to originate from alpha-ketoglutarate dehydrogenase--an
irreversible enzyme in vivo.
[0070] However, if these pathways are applied in combination with a
phosphoketolase pathway (steps T, U, V, W, X of FIG. 1), an
increased maximum theoretical yield can be obtained as shown
below:
TABLE-US-00002 Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol
4.000 0.500 0.000 --> 1.000 3.000 0.000 Crotyl Alcohol 4.000
0.500 0.000 --> 1.000 4.000 0.000 3-Buten-2-ol 4.000 0.500 0.000
--> 1.000 4.000 0.000 Butadiene 4.000 0.500 0.000 --> 1.000
5.000 0.000 2-Hydroxyisobutyrate 4.000 1.500 0.000 --> 1.000
4.000 0.000 Methacrylate (via 2-hydroxyisobutyrate) 4.000 1.500
0.000 --> 1.000 5.000 0.000 3-Hydroxyisobutyrate (oxidative TCA
cycle) 5.000 3.000 0.000 --> 1.000 6.000 1.000 Methacrylate (via
3-hydroxyisobutyrate) 5.000 3.000 0.000 --> 1.000 7.000 1.000
1,4-Butanediol (oxidative TCA cycle) 5.000 2.000 0.000 --> 1.000
5.000 1.000 Adipate (oxidative TCA cycle) 7.000 4.000 0.000 -->
1.000 9.000 1.000 6-Aminocaproate (oxidative TCA cycle) 7.000 3.000
1.000 --> 1.000 9.000 1.000 Caprolactam (via 6-aminocaproate)
7.000 3.000 1.000 --> 1.000 10.000 1.000 Hexamethylenediamine
(oxidative TCA cycle) 7.000 2.000 2.000 --> 1.000 9.000
1.000
[0071] The theoretical yield of bioderived compounds of the
invention from carbohydrates including but not limited to glucose,
glycerol, sucrose, fructose, xylose, arabinose, and galactose, can
also be enhanced by phosphoketolase enzymes, particularly when
reducing equivalents are provided by an exogenous source such as
hydrogen or methanol. This is because phosphoketolase enzymes
provide acetyl-CoA synthesis with 100% carbon conversion efficiency
(e.g., 3 acetyl-CoA's per glucose, 2.5 acetyl-CoA's per xylose, 1.5
acetyl-CoA's per glycerol).
[0072] For example, synthesis of an exemplary fatty alcohol,
dodecanol, from glucose in the absence of phosphoketolase enzymes
can reach a maximum theoretical dodecanol yield of 0.3333 mole
dodecanol/mole glucose.
3 C.sub.6H.sub.12O.sub.6.fwdarw.C.sub.12H.sub.26O+5 H.sub.2O+6
CO.sub.2
[0073] However, if enzyme steps T, U, V, W, X of FIG. 1 are applied
in combination with glycolysis, the pentose phosphate pathway, and
an external redox source (e.g., methanol, hydrogen) using the
pathways shown in FIG. 2, the maximum theoretical yield can be
increased to 0.5000 mole dodecanol/mole glucose.
2 C.sub.6H.sub.12O.sub.6+4 CH.sub.4O.fwdarw.C.sub.12H.sub.26O+7
H.sub.2O+4 CO.sub.2
[0074] This assumes that the pathway is not required to provide net
generation of ATP for cell growth and maintenance requirements. ATP
for energetic requirements can be synthesized by oxidizing
additional methanol to CO.sub.2 using several combinations of
enzymes depicted in FIG. 2.
[0075] Similarly, synthesis of isopropanol from glucose in the
absence of phosphoketolase enzymes can achieve a maximum
theoretical isopropanol yield of 1.000 mole isopropanol/mole
glucose.
C.sub.6H.sub.12O.sub.6+1.5 O.sub.2.fwdarw.C.sub.3H.sub.8O+2
H.sub.2O+3 CO.sub.2
[0076] However, if enzyme steps T, U, V, W, X of FIG. 1 are applied
in combination with glycolysis and the pentose phosphate pathway,
the maximum theoretical yield can be increased to 1.333 mole
isopropanol/mole glucose.
C.sub.6H.sub.12O.sub.6.fwdarw.1.333 C.sub.3H.sub.8O+0.667
H.sub.2O+2 CO.sub.2
[0077] If enzyme steps T, U, V, W, X of FIG. 1 are applied in
combination with glycolysis, the pentose phosphate pathway, and
external redox source (e.g., methanol, hydrogen) using the pathways
shown in FIG. 2, the maximum theoretical yield can be increased to
1.500 mole isopropanol/mole glucose.
C.sub.6H.sub.12O.sub.6+0.5 CH.sub.4O.fwdarw.1.5
C.sub.3H.sub.8O+H.sub.2O+2 CO.sub.2
[0078] In the absence of phosphoketolase activity, synthesis of
several other products from a carbohydrate source (e.g., glucose)
can provide the following maximum theoretical yield stoichiometries
using glycolysis, pentose phosphate pathway, and TCA cycle
reactions to build the pathway precursors.
TABLE-US-00003 Product C6H12O6 O2 NH3 Product H2O CO2
1,3-Butanediol 1.000 0.500 0.000 .fwdarw. 1.000 1.000 2.000 Crotyl
Alcohol 1.000 0.500 0.000 .fwdarw. 1.000 2.000 2.000 3-Buten-2-ol
1.000 0.500 0.000 .fwdarw. 1.000 2.000 2.000 Butadiene 1.000 0.500
0.000 .fwdarw. 1.000 3.000 2.000 2-Hydroxyisobutyrate 1.000 1.500
0.000 .fwdarw. 1.000 2.000 2.000 Methacrylate
(via2-hydroxyisobutyrate) 1.000 1.500 0.000 .fwdarw. 1.000 3.000
2.000 3-Hydroxyisobutyrate (oxidative TCA cycle) 1.000 1.500 0.000
.fwdarw. 1.000 2.000 2.000 Methacrylate (via 3-hydroxyisobutyrate)
1.000 1.500 0.000 .fwdarw. 1.000 3.000 2.000 1,4-Butanediol
(oxidative TCA cycle) 1.000 0.500 0.000 .fwdarw. 1.000 1.000 2.000
Adipate (oxidative TCA cycle) 1.000 1.667 0.000 .fwdarw. 0.667
2.667 2.000 6-Aminocaproate (oxidative TCA cycle) 1.000 1.000 0.667
.fwdarw. 0.667 2.667 2.000 Caprolactam (via 6-aminocaproate) 1.000
1.000 0.667 .fwdarw. 0.667 3.333 2.000 Hexamethylenediamine
(oxidative TCA cycle) 1.000 0.333 1.333 .fwdarw. 0.667 2.667
2.000
[0079] In the products marked "oxidative TCA cycle", the maximum
yield stoichiometries assume that the TCA cycle enzymes (e.g.,
malate dehydrogenase, fumarase, fumarase reductase, and
succinyl-CoA ligase) are not utilized for product formation in the
reductive direction. Exclusive use of the oxidative TCA cycle for
product formation can be advantageous for succinyl-CoA derived
products such as 3-hydroxyisobutyrate, 1,4-butanediol, adipate,
6-aminocaproate, and hexamethylenediamine because it enables all of
the product pathway flux to originate from alpha-ketoglutarate
dehydrogenase--an irreversible enzyme in vivo.
[0080] Notably, when these product pathways are applied in
combination with a phosphoketolase pathway (steps T, U, V, W, X of
FIG. 1), an increased maximum theoretical yield can be obtained as
shown below:
TABLE-US-00004 Product C6H12O6 O2 NH3 Product H2O CO2
1,3-Butanediol 1.000 0.000 0.000 .fwdarw. 1.091 0.545 1.636 Crotyl
Alcohol 1.000 0.000 0.000 .fwdarw. 1.091 1.636 1.636 3-Buten-2-ol
1.000 0.000 0.000 .fwdarw. 1.091 1.636 1.636 Butadiene 1.000 0.107
0.000 .fwdarw. 1.071 2.786 1.714 2-Hydroxyisobutyrate 1.000 0.014
0.000 .fwdarw. 1.330 0.679 0.679 Methacrylate
(via2-hydroxyisobutyrate) 1.000 0.014 0.000 .fwdarw. 1.330 2.009
0.679 3-Hydroxyisobutyrate (oxidative TCA cycle) 1.000 0.600 0.000
.fwdarw. 1.200 1.200 1.200 Methacrylate (via 3-hydroxyisobutyrate)
1.000 0.600 0.000 .fwdarw. 1.200 2.400 1.200 1,4-Butanediol
(oxidative TCA cycle) 1.000 0.124 0.000 .fwdarw. 1.068 0.658 1.727
Adipate (oxidative TCA cycle) 1.000 0.429 0.000 .fwdarw. 0.857
1.714 0.857 6-Aminocaproate (oxidative TCA cycle) 1.000 0.000 0.800
.fwdarw. 0.800 2.000 1.200 Caprolactam (via 6-aminocaproate) 1.000
0.000 0.800 .fwdarw. 0.800 2.800 1.200 Hexamethylenediamine
(oxidative TCA cycle) 1.000 0.064 1.397 .fwdarw. 0.698 2.508
1.810
[0081] As with glucose, a similar yield increase can occur with use
of a phosphoketolase enzyme on other carbohydrates such as
glycerol, sucrose, fructose, xylose, arabinose and galactose.
[0082] Also provided herein are methanol metabolic pathways and a
methanol oxidation pathway to improve that availability of reducing
equivalents and/or substrates for production of a compound of the
invention. Because methanol is a relatively inexpensive organic
feedstock that can be used as a redox, energy, and carbon source
for the production of bioderived compounds of the invention, and
their intermediates, it is a desirable substrate for the
non-naturally occurring microbial organisms of the invention.
Employing one or more methanol metabolic enzymes as described
herein, for example as shown in FIGS. 1 and 2, methanol can enter
central metabolism in most production hosts by employing methanol
dehydrogenase (FIG. 1, step A) along with a pathway for
formaldehyde assimilation. One exemplary formaldehyde assimilation
pathway that can utilize formaldehyde produced from the oxidation
of methanol is shown in FIG. 1, which involves condensation of
formaldehyde and D-ribulose-5-phosphate to form
hexulose-6-phosphate (H6P) by hexulose-6-phosphate synthase (FIG.
1, step B). The enzyme can use Mg.sup.2+ or Mn.sup.2+ for maximal
activity, although other metal ions are useful, and even
non-metal-ion-dependent mechanisms are contemplated. H6P is
converted into fructose-6-phosphate by 6-phospho-3-hexuloisomerase
(FIG. 1, step C). Another exemplary pathway that involves the
detoxification and assimilation of formaldehyde produced from the
oxidation of methanol proceeds through dihydroxyacetone.
Dihydroxyacetone synthase (FIG. 1, step D) is a transketolase that
first transfers a glycoaldehyde group from xylulose-5-phosphate to
formaldehyde, resulting in the formation of dihydroxyacetone (DHA)
and glyceraldehyde-3-phosphate (G3P), which is an intermediate in
glycolysis. The DHA obtained from DHA synthase can be then further
phosphorylated to form DHA phosphate by a DHA kinase. DHAP can be
assimilated into glycolysis, e.g. via isomerization to G3P, and
several other pathways. Alternatively, DHA and G3P can be converted
by fructose-6-phosphate aldolase to form fructose-6-phosphate (F6P)
(FIG. 1, step Z).
[0083] By combining the pathways for methanol oxidation (FIG. 1,
step A) and formaldehyde fixation (FIG. 1, Steps B and C or Step
D), molar yields of 0.333 mol acetyl-CoA/mol methanol can be
achieved for production of a bioderived compound and their
intermediates. The following maximum theoretical yield
stoichiometries for a fatty alcohol (e.g., a C12), a fatty acid
(e.g., a C12), a fatty aldehyde (e.g., a C12), isopropanol,
1,3-butanediol, crotyl alcohol, butadiene, 3-buten-2-ol,
1,4-butanediol, adipate, 6-aminocaproate, caprolactam,
hexamethylenediamine, methacylic acid and 2-hydroxyisobutyric acid
are thus made possible by combining the steps for methanol
oxidation, formaldehyde fixation, and product synthesis.
18 CH.sub.4O+9 O.sub.2.fwdarw.C.sub.12H.sub.26O+6 CO.sub.2+23
H.sub.2O (Fatty Alcohol on MeOH)
18 CH.sub.4O+10 O.sub.2.fwdarw.C.sub.12H.sub.24O.sub.2+6
CO.sub.2+24 H.sub.2O (Fatty Acid on MeOH)
18 CH.sub.4O+9.5 O.sub.2.fwdarw.C.sub.12H.sub.24O+6 CO.sub.2+24
H.sub.2O (Fatty Aldehyde on MeOH)
6 CH.sub.4O+4.5 O.sub.2.fwdarw.C3H.sub.8O+3 CO.sub.2+8 H.sub.2O
(Isopropanol on MeOH)
[0084] Additional stoichiometries are shown in the table below:
TABLE-US-00005 Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol
6.000 3.500 0.000 --> 1.000 7.000 2.000 Crotyl Alcohol 6.000
3.500 0.000 --> 1.000 8.000 2.000 3-Buten-2-ol 6.000 3.500 0.000
--> 1.000 8.000 2.000 Butadiene 6.000 3.500 0.000 --> 1.000
9.000 2.000 2-Hydroxyisobutyrate 6.000 4.500 0.000 --> 1.000
8.000 2.000 Methacrylate (via 2-hydroxyisobutyrate) 6.000 4.500
0.000 --> 1.000 9.000 2.000 3-Hydroxyisobutyrate (oxidative TCA
cycle) 6.000 4.500 0.000 --> 1.000 8.000 2.000 Methacrylate (via
3-hydroxyisobutyrate) 6.000 4.500 0.000 --> 1.000 9.000 2.000
1,4-Butanediol (oxidative TCA cycle) 6.000 3.500 0.000 --> 1.000
9.000 2.000 Adipate (oxidative TCA cycle) 9.000 7.000 0.000 -->
1.000 13.000 3.000 6-Aminocaproate (oxidative TCA cycle) 9.000
6.000 1.000 --> 1.000 13.000 3.000 Caprolactam (via
6-aminocaproate) 9.000 6.000 1.000 --> 1.000 14.000 3.000
Hexamethylenediamine (oxidative TCA cycle) 9.000 5.000 2.000 -->
1.000 13.000 3.000
[0085] In the products marked "oxidative TCA cycle", the maximum
yield stoichiometries assume that the reductive TCA cycle enzymes
(e.g., malate dehydrogenase, fumarase, fumarate reductase, and
succinyl-CoA ligase) are not utilized for product formation.
Exclusive use of the oxidative TCA cycle for product formation can
be advantageous for succinyl-CoA derived products such as
3-hydroxyisobutyrate, 1,4-butanediol, adipate, 6-aminocaproate, and
hexamethylenediamine because it enables all of the product pathway
flux to originate from alpha-ketoglutarate dehydrogenase--an
irreversible enzyme in vivo.
[0086] The yield on several substrates, including methanol, can be
further increased by capturing some of the carbon lost from the
conversion of pathway intermediates, e.g. pyruvate to acetyl-CoA,
using one of the formate reutilization pathways shown in FIG. 1.
For example, the CO.sub.2 generated by conversion of pyruvate to
acetyl-CoA (FIG. 1, step R) can be converted to formate via formate
dehydrogenase (FIG. 1, step S). Alternatively, pyruvate formate
lyase, which forms formate directly instead of CO.sub.2, can be
used to convert pyruvate to acetyl-CoA (FIG. 1, step Q). Formate
can be converted to formaldehyde by using: 1) formate reductase
(FIG. 1, step E), 2) a formyl-CoA synthetase, transferase, or
ligase along with formyl-CoA reductase (FIG. 1, steps F-G), or 3)
formyltetrahydrofolate synthetase, methenyltetrahydrofolate
cyclohydrolase, methylenetetrahydrofolate dehydrogenase, and
formaldehyde-forming enzyme (FIG. 1, steps H-I-J-K). Conversion of
methylene-THF to formaldehyde alternatively will occur
spontaneously. Alternatively, formate can be reutilized by
converting it to pyruvate or acetyl-CoA using FIG. 1, steps
H-I-J-L-M-N or FIG. 1, steps H-I-J-O-P, respectively. Formate
reutilization is also useful when formate is an external carbon
source. For example, formate can be obtained from organocatalytic,
electrochemical, or photoelectrochemical conversion of CO2 to
formate. An alternative source of methanol for use in the present
methods is organocatalytic, electrochemical, or
photoelectrochemical conversion of CO.sub.2 to methanol.
[0087] By combining the pathways for methanol oxidation (FIG. 1,
step A), formaldehyde fixation (FIG. 1, Steps B and C or Step D),
and formate reutilization, molar yields as high as 0.500 mol
acetyl-CoA/mol methanol can be achieved for production of a
bioderived compound and their intermediates. Thus, for example, the
following maximum theoretical yield stoichiometries for a fatty
alcohol (e.g., a C12), a fatty acid (e.g., a C12), a fatty aldehyde
(e.g., a C12), isopropanol, 1,3-butanediol, crotyl alcohol,
butadiene, 3-buten-2-ol, 1,4-butanediol, adipate, 6-aminocaproate,
caprolactam, hexamethylenediamine, methacylic acid and
2-hydroxyisobutyric acid are thus made possible by combining the
steps for methanol oxidation, formaldehyde fixation, formate
reutilization, and product synthesis.
12 CH.sub.4O.fwdarw.C.sub.12H.sub.26O+11 H.sub.2O (Fatty Alcohol on
MeOH)
12 CH.sub.4O+O.sub.2.fwdarw.C.sub.12H.sub.24O.sub.2+12 H.sub.2O
(Fatty Acid on MeOH)
12 CH.sub.4O+0.5 O.sub.2.fwdarw.C.sub.12H.sub.24O+12 H.sub.2O
(Fatty Aldehyde on MeOH)
4 CH.sub.4O+1.5 O.sub.2.fwdarw.C.sub.3H.sub.8O+4 H.sub.2O+CO.sub.2
(Isopropanol on MeOH)
[0088] Additional enhanced maximum yield stoichiometnes can be
found in the table below. These stoichiometnes assume that the
carbon generated from converting pyruvate to acetyl-CoA is recycled
back into product and not emitted as CO2.
TABLE-US-00006 Product CH4O O2 NH3 Product H2O CO2 1,3-Butanediol
4.000 0.500 0.000 --> 1.000 3.000 0.000 Crotyl Alcohol 4.000
0.500 0.000 --> 1.000 4.000 0.000 3-Buten-2-ol 4.000 0.500 0.000
--> 1.000 4.000 0.000 Butadiene 4.000 0.500 0.000 --> 1.000
5.000 0.000 2-Hydroxyisobutyrate 4.000 1.500 0.000 --> 1.000
4.000 0.000 Methacrylate (via 2-hydroxyisobutyrate) 4.000 1.500
0.000 --> 1.000 5.000 0.000 3-Hydroxyisobutyrate (oxidative TCA
cycle) 5.000 3.000 0.000 --> 1.000 6.000 1.000 Methacrylate (via
3-hydroxyisobutyrate) 5.000 3.000 0.000 --> 1.000 7.000 1.000
1,4-Butanediol (oxidative TCA cycle) 5.000 2.000 0.000 --> 1.000
5.000 1.000 Adipate (oxidative TCA cycle) 7.000 4.000 0.000 -->
1.000 9.000 1.000 6-Aminocaproate (oxidative TCA cycle) 7.000 3.000
1.000 --> 1.000 9.000 1.000 Caprolactam (via 6-aminocaproate)
7.000 3.000 1.000 --> 1.000 10.000 1.000 Hexamethylenediamine
(oxidative TCA cycle) 7.000 2.000 2.000 --> 1.000 9.000
1.000
[0089] By combining pathways for formaldehyde fixation and formate
reutilization, yield increases on additional substrates are also
available including but not limited to glucose, glycerol, sucrose,
fructose, xylose, arabinose and galactose. For example, the
following maximum theoretical yield stoichiometries for a fatty
alcohol (e.g., a C12), a fatty acid (e.g., a C12), a fatty aldehyde
(e.g., a C12) and isopropanol on glucose are made possible by
combining the steps for formaldehyde fixation, formate
reutilization, and compound synthesis.
3 C.sub.6H.sub.12O.sub.6.fwdarw.C.sub.12H.sub.26O+5 H.sub.2O+6
CO.sub.2 (Fatty Alcohol on glucose)
3 C.sub.6H.sub.12O.sub.6.fwdarw.1.0588
C.sub.12H.sub.24O.sub.2+5.2941 H.sub.2O+5.2941 CO.sub.2 (Fatty Acid
on glucose)
3 C.sub.6H.sub.12O.sub.6.fwdarw.1.0286 C.sub.12H.sub.24O+5.6571
H.sub.2O+5.6571 CO.sub.2 (Fatty Aldehyde on glucose)
C.sub.6H.sub.12O.sub.6.fwdarw.1.3333 C.sub.3H.sub.8O+0.6667
H.sub.2O+2 CO.sub.2 (Isopropanol on glucose)
[0090] Similar yield increases are observed for 1,3-butanediol,
crotyl alcohol, butadiene, 3-buten-2-ol, 1,4-butanediol, adipate,
6-aminocaproate, caprolactam, hexamethylenediamine, methacylic
acid, 3-hydroxyisobutric acid and 2-hydroxyisobutyric acid when
formaldehyde fixation and formate reutilization pathways are used
in conjunction with glycolysis, the TCA cycle, and the pentose
phosphate pathway.
TABLE-US-00007 Product C6H12O6 O2 NH3 Product H2O CO2
1,3-Butanediol 1.000 0.000 0.000 .fwdarw. 1.091 0.545 1.636 Crotyl
Alcohol 1.000 0.000 0.000 .fwdarw. 1.091 1.636 1.636 3-Buten-2-ol
1.000 0.000 0.000 .fwdarw. 1.091 1.636 1.636 Butadiene 1.000 0.107
0.000 .fwdarw. 1.071 2.786 1.714 2-Hydroxyisobutyrate 1.000 0.000
0.000 .fwdarw. 1.333 0.667 0.667 Methacrylate (via
2-hydroxyisobutyrate) 1.000 0.000 0.000 .fwdarw. 1.333 2.000 0.667
3-Hydroxyisobutyrate (oxidative TCA cycle) 1.000 0.214 0.000
.fwdarw. 1.286 0.857 0.857 Methacrylate (via 3-hydroxyisobutyrate)
1.000 0.214 0.000 .fwdarw. 1.286 2.143 0.857 1,4-Butanediol
(oxidative TCA cycle) 1.000 0.107 0.000 .fwdarw. 1.071 0.643 1.714
Adipate (oxidative TCA cycle) 1.000 0.168 0.000 .fwdarw. 0.897
1.514 0.617 6-Aminocaproate (oxidative TCA cycle) 1.000 0.000 0.800
.fwdarw. 0.800 2.000 1.200 Caprolactam (via 6-aminocaproate) 1.000
0.000 0.800 .fwdarw. 0.800 2.800 1.200 Hexamethylenediamine
(oxidative TCA cycle) 1.000 0.050 1.400 .fwdarw. 0.700 2.500
1.800
[0091] Similarly, the maximum theoretical yield of a bioderived
compound from glycerol can be increased by enabling fixation of
formaldehyde from generation and utilization of formate. The
following maximum theoretical yield stoichiometries for a fatty
alcohol (e.g., a C12), a fatty acid (e.g., a C12), a fatty aldehyde
(e.g., a C12) and isopropanol on glycerol are thus made possible by
combining the steps for formaldehyde fixation, formate
reutilization, and product synthesis.
6 C.sub.3H.sub.8O.sub.3.fwdarw.1.1667 C.sub.12H.sub.26O+8.8333
H.sub.2O+4 CO.sub.2 (Fatty Alcohol on glycerol)
6 C.sub.3H.sub.8O.sub.3.fwdarw.1.2353
C.sub.12H.sub.24O.sub.2+9.1765 H.sub.2O+3.1765 CO.sub.2 (Fatty Acid
on glycerol)
6 C.sub.3H.sub.8O.sub.3.fwdarw.1.2000 C.sub.12H.sub.24O+9.6000
H.sub.2O+3.6000 CO.sub.2 (Fatty Aldehyde on glycerol)
C.sub.3H.sub.8O.sub.3.fwdarw.0.7778 C.sub.3H.sub.8O+0.8889
H.sub.2O+0.6667 CO.sub.2 (Isopropanol on glycerol)
[0092] In numerous engineered pathways, product yields based on
carbohydrate feedstock are hampered by insufficient reducing
equivalents or by loss of reducing equivalents to byproducts.
Methanol is a relatively inexpensive organic feedstock that can be
used to generate reducing equivalents by employing one or more
methanol metabolic enzymes as shown in FIG. 2. Reducing equivalents
can also be extracted from hydrogen and carbon monoxide by
employing hydrogenase and carbon monoxide dehydrogenase enzymes,
respectively, as shown in FIG. 2. The reducing equivalents are then
passed to acceptors such as oxidized ferredoxins, oxidized
quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen
peroxide to form reduced ferredoxin, reduced quinones, reduced
cytochromes, NAD(P)H, H.sub.2, or water, respectively. Reduced
ferredoxin, reduced quinones and NAD(P)H are particularly useful as
they can serve as redox carriers for various Wood-Ljungdahl
pathway, reductive TCA cycle, or product pathway enzymes.
[0093] The reducing equivalents produced by the metabolism of
methanol, hydrogen, and carbon monoxide can be used to power
several bioderived compound production pathways. For example, the
maximum theoretical yield of a fatty alcohol, a fatty acid, a fatty
aldehyde, isopropanol, 1,3-butanediol, crotyl alcohol, butadiene,
3-buten-2-ol, 1,4-butanediol, adipate, 6-aminocaproate,
caprolactam, hexamethylenediamine, methacylic acid or
2-hydroxyisobutyric acid from glucose and glycerol can be increased
by enabling fixation of formaldehyde, formate reutilization, and
extraction of reducing equivalents from an external source such as
hydrogen. In fact, by combining pathways for formaldehyde fixation,
formate reutilization, reducing equivalent extraction, and product
synthesis, the following maximum theoretical yield stoichiometries
for fatty alcohol, a fatty acid, a fatty aldehyde, and isopropanol
on glucose and glycerol are made possible.
2 C.sub.6H.sub.12O.sub.6+12 H.sub.2.fwdarw.C.sub.12H.sub.26O+11
H.sub.2O (Fatty Alcohol on glucose+external redox)
2 C.sub.6H.sub.12O.sub.6+10
H.sub.2.fwdarw.C.sub.12H.sub.24O.sub.2+10 H.sub.2O (Fatty Acid on
glucose+external redox)
2 C.sub.6H.sub.12O.sub.6+11 H.sub.2.fwdarw.C.sub.12H.sub.24O+11
H.sub.2O (Fatty Aldehyde on glucose+external redox)
C.sub.6H.sub.12O.sub.6+6 H.sub.2.fwdarw.2 C.sub.3H.sub.8O+4
H.sub.2O (Isopropanol on glucose+external redox)
4 C.sub.3H.sub.8O.sub.3+8 H.sub.2.fwdarw.C.sub.12H.sub.26O+11
H.sub.2O (Fatty Alcohol on glycerol+external redox)
4 C.sub.3H.sub.8O.sub.3+6 H.sub.2.fwdarw.C.sub.12H.sub.24O.sub.2+10
H.sub.2O (Fatty Acid on glycerol+external redox)
4 C.sub.3H.sub.8O.sub.3+7 H.sub.2.fwdarw.C.sub.12H.sub.24O+11
H.sub.2O (Fatty Aldehyde on glycerol+external redox)
C.sub.3H.sub.8O.sub.3+2 H.sub.2.fwdarw.C.sub.3H.sub.8O+2 H.sub.2O
(Isopropanol on glycerol+external redox)
[0094] In most instances, achieving such maximum yield
stoichiometries may require some oxidation of reducing equivalents
(e.g., H.sub.2+1/2O.sub.2.fwdarw.H.sub.2O,
CO+1/2O.sub.2.fwdarw.CO.sub.2, CH.sub.4+1.5
O.sub.2.fwdarw.CO.sub.2+2 H.sub.2O, C.sub.6H.sub.12O.sub.6+6
O.sub.2.fwdarw.6 CO.sub.2+6 H.sub.2O) to provide sufficient energy
for the substrate to product pathways to operate. Nevertheless, if
sufficient reducing equivalents are available, enabling pathways
for fixation of formaldehyde, formate reutilization, extraction of
reducing equivalents, and product synthesis can even lead to
production of a fatty alcohol, a fatty acid, a fatty aldehyde,
isopropanol, and their intermediates, directly from CO.sub.2.
[0095] Pathways identified herein, and particularly pathways
exemplified in specific combinations presented herein, are superior
over other pathways based in part on the applicant's ranking of
pathways based on attributes including maximum theoretical compound
yield, maximal carbon flux, maximal production of reducing
equivalents, minimal production of CO.sub.2, pathway length, number
of non-native steps, thermodynamic feasibility, number of enzymes
active on pathway substrates or structurally similar substrates,
and having steps with currently characterized enzymes, and
furthermore, the latter pathways are even more favored by having in
addition at least the fewest number of non-native steps required,
the most enzymes known active on pathway substrates or structurally
similar substrates, and the fewest total number of steps from
central metabolism.
[0096] The non-naturally occurring microbial organisms of the
invention can contain stable genetic alterations, which refers to
microorganisms that can be cultured for greater than five
generations without loss of the alteration. Generally, stable
genetic alterations include modifications that persist greater than
10 generations, particularly stable modifications will persist more
than about 25 generations, and more particularly, stable genetic
modifications will be greater than 50 generations, including
indefinitely.
[0097] In the case of gene disruptions, a particularly useful
stable genetic alteration is a gene deletion. The use of a gene
deletion to introduce a stable genetic alteration is particularly
useful to reduce the likelihood of a reversion to a phenotype prior
to the genetic alteration. For example, stable growth-coupled
production of a biochemical can be achieved, for example, by
deletion of a gene encoding an enzyme catalyzing one or more
reactions within a set of metabolic modifications. The stability of
growth-coupled production of a biochemical can be further enhanced
through multiple deletions, significantly reducing the likelihood
of multiple compensatory reversions occurring for each disrupted
activity.
[0098] Those skilled in the art will understand that the genetic
alterations, including metabolic modifications exemplified herein,
are described with reference to a suitable host organism such as E.
coli and their corresponding metabolic reactions or a suitable
source organism for desired genetic material such as genes for a
desired metabolic pathway. However, given the complete genome
sequencing of a wide variety of organisms and the high level of
skill in the area of genomics, those skilled in the art will
readily be able to apply the teachings and guidance provided herein
to essentially all other organisms. For example, the E. coli
metabolic alterations exemplified herein can readily be applied to
other species by incorporating the same or analogous encoding
nucleic acid from species other than the referenced species. Such
genetic alterations include, for example, genetic alterations of
species homologs, in general, and in particular, orthologs,
paralogs or nonorthologous gene displacements.
[0099] An ortholog is a gene or genes that are related by vertical
descent and are responsible for substantially the same or identical
functions in different organisms. For example, mouse epoxide
hydrolase and human epoxide hydrolase can be considered orthologs
for the biological function of hydrolysis of epoxides. Genes are
related by vertical descent when, for example, they share sequence
similarity of sufficient amount to indicate they are homologous, or
related by evolution from a common ancestor. Genes can also be
considered orthologs if they share three-dimensional structure but
not necessarily sequence similarity, of a sufficient amount to
indicate that they have evolved from a common ancestor to the
extent that the primary sequence similarity is not identifiable.
Genes that are orthologous can encode proteins with sequence
similarity of about 25% to 100% amino acid sequence identity. Genes
encoding proteins sharing an amino acid similarity less that 25%
can also be considered to have arisen by vertical descent if their
three-dimensional structure also shows similarities. Members of the
serine protease family of enzymes, including tissue plasminogen
activator and elastase, are considered to have arisen by vertical
descent from a common ancestor.
[0100] Orthologs include genes or their encoded gene products that
through, for example, evolution, have diverged in structure or
overall activity. For example, where one species encodes a gene
product exhibiting two functions and where such functions have been
separated into distinct genes in a second species, the three genes
and their corresponding products are considered to be orthologs.
For the production of a biochemical product, those skilled in the
art will understand that the orthologous gene harboring the
metabolic activity to be introduced or disrupted is to be chosen
for construction of the non-naturally occurring microorganism. An
example of orthologs exhibiting separable activities is where
distinct activities have been separated into distinct gene products
between two or more species or within a single species. A specific
example is the separation of elastase proteolysis and plasminogen
proteolysis, two types of serine protease activity, into distinct
molecules as plasminogen activator and elastase. A second example
is the separation of Mycoplasma 5'-3' exonuclease and Drosophila
DNA polymerase III activity. The DNA polymerase from the first
species can be considered an ortholog to either or both of the
exonuclease or the polymerase from the second species and vice
versa.
[0101] In contrast, paralogs are homologs related by, for example,
duplication followed by evolutionary divergence and have similar or
common, but not identical functions. Paralogs can originate or
derive from, for example, the same species or from a different
species. For example, microsomal epoxide hydrolase (epoxide
hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II)
can be considered paralogs because they represent two distinct
enzymes, co-evolved from a common ancestor, that catalyze distinct
reactions and have distinct functions in the same species. Paralogs
are proteins from the same species with significant sequence
similarity to each other suggesting that they are homologous, or
related through co-evolution from a common ancestor. Groups of
paralogous protein families include HipA homologs, luciferase
genes, peptidases, and others.
[0102] A nonorthologous gene displacement is a nonorthologous gene
from one species that can substitute for a referenced gene function
in a different species. Substitution includes, for example, being
able to perform substantially the same or a similar function in the
species of origin compared to the referenced function in the
different species. Although generally, a nonorthologous gene
displacement will be identifiable as structurally related to a
known gene encoding the referenced function, less structurally
related but functionally similar genes and their corresponding gene
products nevertheless will still fall within the meaning of the
term as it is used herein. Functional similarity requires, for
example, at least some structural similarity in the active site or
binding region of a nonorthologous gene product compared to a gene
encoding the function sought to be substituted. Therefore, a
nonorthologous gene includes, for example, a paralog or an
unrelated gene.
[0103] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having acetyl-CoA or
a bioderived compound biosynthetic capability, those skilled in the
art will understand with applying the teaching and guidance
provided herein to a particular species that the identification of
metabolic modifications can include identification and inclusion or
inactivation of orthologs. To the extent that paralogs and/or
nonorthologous gene displacements are present in the referenced
microorganism that encode an enzyme catalyzing a similar or
substantially similar metabolic reaction, those skilled in the art
also can utilize these evolutionally related genes. Similarly for a
gene disruption, evolutionally related genes can also be disrupted
or deleted in a host microbial organism to reduce or eliminate
functional redundancy of enzymatic activities targeted for
disruption.
[0104] Orthologs, paralogs and nonorthologous gene displacements
can be determined by methods well known to those skilled in the
art. For example, inspection of nucleic acid or amino acid
sequences for two polypeptides will reveal sequence identity and
similarities between the compared sequences. Based on such
similarities, one skilled in the art can determine if the
similarity is sufficiently high to indicate the proteins are
related through evolution from a common ancestor. Algorithms well
known to those skilled in the art, such as Align, BLAST, Clustal W
and others compare and determine a raw sequence similarity or
identity, and also determine the presence or significance of gaps
in the sequence which can be assigned a weight or score. Such
algorithms also are known in the art and are similarly applicable
for determining nucleotide sequence similarity or identity.
Parameters for sufficient similarity to determine relatedness are
computed based on well known methods for calculating statistical
similarity, or the chance of finding a similar match in a random
polypeptide, and the significance of the match determined. A
computer comparison of two or more sequences can, if desired, also
be optimized visually by those skilled in the art. Related gene
products or proteins can be expected to have a high similarity, for
example, 25% to 100% sequence identity. Proteins that are unrelated
can have an identity which is essentially the same as would be
expected to occur by chance, if a database of sufficient size is
scanned (about 5%). Sequences between 5% and 24% may or may not
represent sufficient homology to conclude that the compared
sequences are related. Additional statistical analysis to determine
the significance of such matches given the size of the data set can
be carried out to determine the relevance of these sequences.
[0105] Exemplary parameters for determining relatedness of two or
more sequences using the BLAST algorithm, for example, can be as
set forth below. Briefly, amino acid sequence alignments can be
performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the
following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap
extension: 1; x_dropoff 50; expect: 10.0; wordsize: 3; filter on.
Nucleic acid sequence alignments can be performed using BLASTN
version 2.0.6 (Sep. 16, 1998) and the following parameters: Match:
1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff 50;
expect: 10.0; wordsize: 11; filter off. Those skilled in the art
will know what modifications can be made to the above parameters to
either increase or decrease the stringency of the comparison, for
example, and determine the relatedness of two or more
sequences.
[0106] In some embodiments, the invention provides a non-naturally
occurring microbial organism having a methanol metabolic pathway
and an acetyl-CoA pathway as depicted in FIGS. 1 and 2. In some
embodiments, the methanol metabolic pathway comprises 2A or 2J,
wherein 2A is a methanol methyltransferase and 2J is a methanol
dehydrogenase. In some embodiments, the methanol metabolic pathway
comprises 2A. In some embodiments, the methanol metabolic pathway
comprises 2J. In some embodiments, the acetyl-CoA pathway comprises
a pathway selected from: (1) 1T and 1V; (2) 1T, 1W, and 1X; (3) 1U
and 1V; (4) 1U, 1W, and 1X; wherein 1T is a fructose-6-phosphate
phosphoketolase, wherein 1U is a xylulose-5-phosphate
phosphoketolase, wherein 1V is a phosphotransacetylase, wherein 1W
is an acetate kinase, wherein 1X is an acetyl-CoA transferase, an
acetyl-CoA synthetase, or an acetyl-CoA ligase. In some
embodiments, the acetyl-CoA pathway comprises (1) 1T and 1V. In
some embodiments, the acetyl-CoA pathway comprises (2) 1T, 1W, and
1X. In some embodiments, the acetyl-CoA pathway comprises (3) 1U
and 1V. In some embodiments, the acetyl-CoA pathway comprises (4)
1U, 1W, and 1X. In some embodiments, an enzyme of the methanol
metabolic pathway or the acetyl-CoA pathway is encoded by at least
one exogenous nucleic acid and is expressed in a sufficient amount
to enhance carbon flux through acetyl-CoA.
[0107] In some embodiments, the invention provides a non-naturally
occurring microbial organism as described herein further comprising
a formaldehyde fixation pathway as depicted in FIG. 1. In some
embodiments, the formaldehyde fixation pathway comprises: (1) 1D
and 1Z; (2) 1D; or (3) 1B and 1C, wherein 1B is a
3-hexulose-6-phosphate synthase, wherein 1C is a
6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone
synthase, wherein 1Z is a fructose-6-phosphate aldolase. In some
embodiments, the formaldehyde fixation pathway comprises (1) 1D and
1Z. In some embodiments, the formaldehyde fixation pathway
comprises (2) 1D. In some embodiments, the formaldehyde fixation
pathway comprises (3) 1B and 1C. In some embodiments, an enzyme of
the formaldehyde fixation pathway is encoded by at least one
exogenous nucleic acid and is expressed in a sufficient amount to
enhance carbon flux through acetyl-CoA.
[0108] In some embodiments, the invention provides a non-naturally
occurring microbial organism as described herein further comprising
a methanol metabolic pathway selected from: (1) 2A and 2B; (2) 2A,
2B and 2C; (3) 2J, 2K and 2C; (4) 2J, 2M, and 2N; (5) 2J and 2L;
(6) 2J, 2L, and 2G; (7) 2J, 2L, and 2I; (8) 2A, 2B, 2C, 2D, and 2E;
(9) 2A, 2B, 2C, 2D, and 2F; (10) 2J, 2K, 2C, 2D, and 2E; (11) 2J,
2K, 2C, 2D, and 2F; (12) 2J, 2M, 2N, and 2O; (13) 2A, 2B, 2C, 2D,
2E, and 2G; (14) 2A, 2B, 2C, 2D, 2F, and 2G; (15) 2J, 2K, 2C, 2D,
2E, and 2G; (16) 2J, 2K, 2C, 2D, 2F, and 2G; (17) 2J, 2M, 2N, 2O,
and 2G; (18) 2A, 2B, 2C, 2D, 2E, and 2I; (19) 2A, 2B, 2C, 2D, 2F,
and 2I; (20) 2J, 2K, 2C, 2D, 2E, and 2I; (21) 2J, 2K, 2C, 2D, 2F,
and 2I; and (22) 2J, 2M, 2N, 2O, and 2I, wherein 2A is a methanol
methyltransferase, wherein 2B is a methylenetetrahydrofolate
reductase, wherein 2C is a methylenetetrahydrofolate dehydrogenase,
wherein 2D is a methenyltetrahydrofolate cyclohydrolase, wherein 2E
is a formyltetrahydrofolate deformylase, wherein 2F is a
formyltetrahydrofolate synthetase, wherein 2G is a formate hydrogen
lyase, wherein 2I is a formate dehydrogenase, wherein 2J is a
methanol dehydrogenase, wherein 2K is a formaldehyde activating
enzyme or spontaneous, wherein 2L is a formaldehyde dehydrogenase,
wherein 2M is a S-(hydroxymethyl)glutathione synthase or
spontaneous, wherein 2N is a glutathione-dependent formaldehyde
dehydrogenase, and wherein 2O is a S-formylglutathione hydrolase.
In some embodiments, the methanol metabolic pathway comprises (1)
2A and 2B. In some embodiments, the methanol metabolic pathway
comprises (2) 2A, 2B and 2C. In some embodiments, the methanol
metabolic pathway comprises (3) 2J, 2K and 2C. In some embodiments,
the methanol metabolic pathway comprises (4) 2J, 2M, and 2N. In
some embodiments, the methanol metabolic pathway comprises (5) 2J
and 2L. In some embodiments, the methanol metabolic pathway
comprises (6) 2J, 2L, and 2G. In some embodiments, the methanol
metabolic pathway comprises (7) 2J, 2L, and 2I. In some
embodiments, the methanol metabolic pathway comprises (8) 2A, 2B,
2C, 2D, and 2E. In some embodiments, the methanol metabolic pathway
comprises (9) 2A, 2B, 2C, 2D, and 2F. In some embodiments, the
methanol metabolic pathway comprises (10) 2J, 2K, 2C, 2D, and 2E.
In some embodiments, the methanol metabolic pathway comprises (11)
2J, 2K, 2C, 2D, and 2F. In some embodiments, the methanol metabolic
pathway comprises (12) 2J, 2M, 2N, and 2O. In some embodiments, the
methanol metabolic pathway comprises (13) 2A, 2B, 2C, 2D, 2E, and
2G; (14) 2A, 2B, 2C, 2D, 2F, and 2G. In some embodiments, the
methanol metabolic pathway comprises (15) 2J, 2K, 2C, 2D, 2E, and
2G. In some embodiments, the methanol metabolic pathway comprises
(16) 2J, 2K, 2C, 2D, 2F, and 2G. In some embodiments, the methanol
metabolic pathway comprises (17) 2J, 2M, 2N, 2O, and 2G. In some
embodiments, the methanol metabolic pathway comprises (18) 2A, 2B,
2C, 2D, 2E, and 2I. In some embodiments, the methanol metabolic
pathway comprises (19) 2A, 2B, 2C, 2D, 2F, and 2I. In some
embodiments, the methanol metabolic pathway comprises (20) 2J, 2K,
2C, 2D, 2E, and 2I. In some embodiments, the methanol metabolic
pathway comprises (21) 2J, 2K, 2C, 2D, 2F, and 2I. In some
embodiments, the methanol metabolic pathway comprises (22) 2J, 2M,
2N, 2O, and 2I.
[0109] In some embodiments, the non-naturally occurring microbial
organism described herein comprises one, two, three, four, five, or
six exogenous nucleic acids each encoding a methanol metabolic
pathway enzyme. In some embodiments, the non-naturally occurring
microbial organism described herein comprises exogenous nucleic
acids encoding each of the enzymes of at least one of the pathways
selected from (1)-(22) as describe above. In some embodiments, the
non-naturally occurring microbial organism described herein
comprises one, two, or three exogenous nucleic acids each encoding
an acetyl-CoA pathway enzyme. In some embodiments, the
non-naturally occurring microbial organism described herein
comprises exogenous nucleic acids encoding each of the enzymes of
at least one of the acetyl-CoA pathway selected from (1)-(4)
described above.
[0110] In some embodiments, the invention provides a non-naturally
occurring microbial organism as described herein further comprising
a formate assimilation pathway as depicted in FIG. 1. In some
embodiments, the formate assimilation pathway comprises a pathway
selected from: (1) 1E; (2) 1F, and 1G; (3) 1H, 1I, 1J, and 1K; (4)
1H, 1I, 1J, 1L, 1M, and 1N; (5) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (6)
1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1K, 1H, 1I, 1J, 1L, 1M, and
1N; and (8) 1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate
reductase, 1F is a formate ligase, a formate transferase, or a
formate synthetase, wherein 1G is a formyl-CoA reductase, wherein
1H is a formyltetrahydrofolate synthetase, wherein 1I is a
methenyltetrahydrofolate cyclohydrolase, wherein 1J is a
methylenetetrahydrofolate dehydrogenase, wherein 1K is a
formaldehyde-forming enzyme or spontaneous, wherein 1L is a glycine
cleavage system, wherein 1M is a serine hydroxymethyltransferase,
wherein 1N is a serine deaminase, wherein 1O is a
methylenetetrahydrofolate reductase, and wherein 1P is an
acetyl-CoA synthase. In some embodiments, the formate assimilation
pathway comprises (1) 1E. In some embodiments, the formate
assimilation pathway comprises (2) IF, and 1G. In some embodiments,
the formate assimilation pathway comprises (3) 1H, 1I, 1J, and 1K.
In some embodiments, the formate assimilation pathway comprises (4)
1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, the formate
assimilation pathway comprises (5) 1E, 1H, 1I, 1J, 1L, 1M, and 1N.
In some embodiments, the formate assimilation pathway comprises (6)
1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, the
formate assimilation pathway comprises (7) 1K, 1H, 1I, 1J, 1L, 1M,
and 1N. In some embodiments, the formate assimilation pathway
comprises (8) 1H, 1I, 1J, 1O, and 1P. In some embodiments, an
enzyme of the formate assimilation pathway is encoded by at least
one exogenous nucleic acid and is expressed in a sufficient amount
to enhance carbon flux through acetyl-CoA.
[0111] In some embodiments, the invention provides a non-naturally
occurring microbial organism having a formaldehyde fixation
pathway, a formate assimilation pathway and/or an acetyl-CoA
pathway as described here. Accordingly, in some embodiments, the
non-naturally occurring microbial organism of the invention can
have an acetyl-CoA pathway as depicted in FIG. 3. In some
embodiments, the non-naturally occurring microbial organism of the
invention can have a formaldehyde fixation pathway and an
acetyl-CoA pathway as depicted in FIG. 1. In some embodiments, the
non-naturally occurring microbial organism of the invention can
have a formate assimilation pathway and an acetyl-CoA pathway as
depicted in FIG. 1. In some embodiments, the non-naturally
occurring microbial organism of the invention can have a
formaldehyde fixation pathway, a formate assimilation pathway and
an acetyl-CoA pathway as depicted in FIG. 1. In some embodiments,
the formaldehyde fixation pathway comprises: (1) 1D and 1Z; (2) 1D;
or (3) 1B and 1C, wherein 1B is a 3-hexulose-6-phosphate synthase,
wherein 1C is a 6-phospho-3-hexuloisomerase, wherein 1D is a
dihydroxyacetone synthase, wherein 1Z is a fructose-6-phosphate
aldolase. In some embodiments, the formaldehyde fixation pathway
comprises (1) 1D and 1Z. In some embodiments, the formaldehyde
fixation pathway comprises (2) 1D. In some embodiments, the
formaldehyde fixation pathway comprises (3) 1B and 1C. In some
embodiment, the formate assimilation pathway comprises a pathway
selected from: (4) 1E; (5) 1F, and 1G; (6) 1H, 1I, 1J, and 1K; (7)
1H, 1I, 1J, 1L, 1M, and 1N; (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (9)
1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (10) 1K, 1H, 1I, 1J, 1L, 1M,
and 1N; and (11) 1H, 1I, 1J, 1O, and 1P, wherein 1E is a formate
reductase, 1F is a formate ligase, a formate transferase, or a
formate synthetase, wherein 1G is a formyl-CoA reductase, wherein
1H is a formyltetrahydrofolate synthetase, wherein 1I is a
methenyltetrahydrofolate cyclohydrolase, wherein 1J is a
methylenetetrahydrofolate dehydrogenase, wherein 1K is a
formaldehyde-forming enzyme or spontaneous, wherein 1L is a glycine
cleavage system, wherein 1M is a serine hydroxymethyltransfemse,
wherein 1N is a serine deaminase, wherein 1O is a
methylenetetrahydrofolate reductase, and wherein 1P is an
acetyl-CoA synthase. In some embodiments, the formate assimilation
pathway comprises (4) 1E. In some embodiments, the formate
assimilation pathway comprises (5) 1F, and 1G. In some embodiments,
the formate assimilation pathway comprises (6) 1H, 1I, 1J, and 1K.
In some embodiments, the formate assimilation pathway comprises (7)
1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, the formate
assimilation pathway comprises (8) 1E, 1H, 1I, 1J, 1L, 1M, and 1N.
In some embodiments, the formate assimilation pathway comprises (9)
1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N. In some embodiments, the
formate assimilation pathway comprises (10) 1K, 1H, 1I, 1J, 1L, 1M,
and 1N. In some embodiments, the formate assimilation pathway
comprises (11) 1H, 1I, 1J, 1O, and 1P. In some embodiments,
acetyl-CoA pathway comprises a pathway selected from: (12) 1T and
1V; (13) 1T, 1W, and 1X; (14) 1U and 1V; and (15) 1U, 1W, and 1X,
wherein 1T is a fructose-6-phosphate phosphoketolase, wherein 1U is
a xylulose-5-phosphate phosphoketolase, wherein 1V is a
phosphotransacetylase, wherein 1W is an acetate kinase, wherein 1X
is an acetyl-CoA transferase, an acetyl-CoA synthetase, or an
acetyl-CoA ligase. In some embodiments, acetyl-CoA pathway
comprises (12) 1T and 1V. In some embodiments, acetyl-CoA pathway
comprises (13) 1T, 1W, and 1X. In some embodiments, acetyl-CoA
pathway comprises (14) 1U and 1V. In some embodiments, acetyl-CoA
pathway comprises (15) 1U, 1W, and 1X. In some embodiments, the
non-naturally occurring microbial organism described herein
comprises an acetyl-CoA pathway that comprises 1T and 1V and a
formaldehyde fixation pathway that comprises 1D and 1Z. In some
embodiments, the non-naturally occurring microbial organism
described herein comprises an acetyl-CoA pathway that comprises 1T
and 1V and a formaldehyde fixation pathway comprises 1B and 1C. In
some embodiments, an enzyme of the formaldehyde fixation pathway,
the formate assimilation pathway, and/or the acetyl-CoA pathway is
encoded by at least one exogenous nucleic acid and is expressed in
a sufficient amount to enhance carbon flux through acetyl-CoA.
[0112] In some embodiments, the non-naturally occurring microbial
organism described herein comprises one or two exogenous nucleic
acids each encoding an formaldehyde fixation pathway enzyme. In
some embodiments, the non-naturally occurring microbial organism
described herein comprises one, two, three, four, five, six, seven
or eight exogenous nucleic acids each encoding a formate
assimilation pathway enzyme. In some embodiments, the non-naturally
occurring microbial organism described herein comprises one, two,
or three exogenous nucleic acids each encoding an acetyl-CoA
pathway enzyme. In some embodiments, the non-naturally occurring
microbial organism described herein comprises exogenous nucleic
acids encoding each of the enzymes of at least one of the pathways
selected from (1)-(15) as described above.
[0113] In some embodiments, the invention further provides a
non-naturally occurring microbial organism described herein that
has a formate assimilation pathway further comprises: (1) 1Q; (2)
1R and 1S, (3) 1Y and 1Q; or (4) 1Y, 1R, and 1S, as depicted in
FIG. 1, wherein 1Q is a pyruvate formate lyase, wherein 1R is a
pyruvate dehydrogenase, a pyruvate ferredoxin oxidoreductase, or a
pyruvate:NADP+ oxidoreductase, wherein 1S is a formate
dehydrogenase, wherein 1Y is a glyceraldehyde-3-phosphate
dehydrogenase or an enzyme of lower glycolysis. In some
embodiments, the formate assimilation pathway further comprises (1)
1Q. In some embodiments, the formate assimilation pathway further
comprises (2) 1R and 1S. In some embodiments, the formate
assimilation pathway further comprises (3) 1Y and 1. In some
embodiments, the formate assimilation pathway further comprises (4)
1Y, 1R, and 1S.
[0114] In some embodiments, a non-naturally occurring microbial
organism of the invention includes a methanol oxidation pathway.
Such a pathway can include at least one exogenous nucleic acid
encoding a methanol oxidation pathway enzyme expressed in a
sufficient amount to produce formaldehyde in the presence of
methanol. An exemplary methanol oxidation pathway enzyme is a
methanol dehydrogenase. Accordingly, in some embodiments, a
non-naturally occurring microbial organism of the invention
includes at least one exogenous nucleic acid encoding a methanol
dehydrogenase expressed in a sufficient amount to produce
formaldehyde in the presence of methanol.
[0115] In some embodiments, the exogenous nucleic acid encoding an
methanol dehydrogenase is expressed in a sufficient amount to
produce an amount of formaldehyde greater than or equal to 1 .mu.M,
10 .mu.M, 20 .mu.M, or 50 .mu.M, or a range thereof, in culture
medium or intracellularly. In other embodiments, the exogenous
nucleic acid encoding an methanol dehydrogenase is capable of
producing an amount of formaldehyde greater than or equal to 1
.mu.M, 10 .mu.M, 20 .mu.M, or 50 .mu.M, or a range thereof, in
culture medium or intracellularly. In some embodiments, the range
is from 1 .mu.M to 50 .mu.M or greater. In other embodiments, the
range is from 10 .mu.M to 50 .mu.M or greater. In other
embodiments, the range is from 20 .mu.M to 50 .mu.M or greater. In
other embodiments, the amount of formaldehyde production is 50
.mu.M or greater. In specific embodiments, the amount of
formaldehyde production is in excess of, or as compared to, that of
a negative control, e.g., the same species of organism that does
not comprise the exogenous nucleic acid, such as a wild-type
microbial organism or a control microbial organism thereof. In
certain embodiments, the methanol dehydrogenase is selected from
those provided herein, e.g., as exemplified in Example II (see FIG.
1, Step A, or FIG. 10, Step J). In certain embodiments, the amount
of formaldehyde production is determined by a whole cell assay,
such as that provided in Example II (see FIG. 1, Step A, or FIG.
10, Step J), or by another assay provided herein or otherwise known
in the art. In certain embodiments, formaldehyde utilization
activity is absent in the whole cell.
[0116] In certain embodiments, the exogenous nucleic acid encoding
an methanol dehydrogenase is expressed in a sufficient amount to
produce at least 1.times., 2.times., 3.times., 4.times., 5.times.,
6.times., 7.times., 8.times., 9.times., 10.times., 15.times.,
20.times., 30.times., 40.times., 50.times., 100.times. or more
formaldehyde in culture medium or intracellularly. In other
embodiments, the exogenous nucleic acid encoding an methanol
dehydrogenase is capable of producing an amount of formaldehyde at
least 1.times., 2.times., 3.times., 4.times., 5.times., 6.times.,
7.times., 8.times., 9.times., 10.times., 15.times., 20.times.,
30.times., 40.times., 50.times., 100.times., or a range thereof, in
culture medium or intracellularly. In some embodiments, the range
is from 1.times. to 100.times.. In other embodiments, the range is
from 2.times. to 100.times.. In other embodiments, the range is
from 5.times. to 100.times.. In other embodiments, the range is
from 10.times. to 100.times.. In other embodiments, the range is
from 50.times. to 100.times.. In some embodiments, the amount of
formaldehyde production is at least 20.times.. In other
embodiments, the amount of formaldehyde production is at least
50.times.. In specific embodiments, the amount of formaldehyde
production is in excess of, or as compared to, that of a negative
control, e.g, the same species of organism that does not comprise
the exogenous nucleic acid, such as a wild-type microbial organism
or a control microbial organism thereof. In certain embodiments,
the methanol dehydrogenase is selected from those provided herein,
e.g, as exemplified herein (see FIG. 1, Step A, or FIG. 2, Step J).
In certain embodiments, the amount of formaldehyde production is
determined by a whole cell assay, such as that provided herein (see
FIG. 1, Step A, or FIG. 2, Step J), or by another assay provided
herein or otherwise known in the art. In certain embodiments,
formaldehyde utilization activity is absent in the whole cell.
[0117] In some embodiments, a non-naturally occurring microbial
organism of the invention includes one or more enzymes for
generating reducing equivalents. For example, the microbial
organism can further include a hydrogenase and/or a carbon monoxide
dehydrogenase. In some aspects, the organism comprises an exogenous
nucleic acid encoding the hydrogenase or the carbon monoxide
dehydrogenase.
[0118] A reducing equivalent can also be readily obtained from a
glycolysis intermediate by any of several central metabolic
reactions including glyceraldehyde-3-phosphate dehydrogenase,
pyruvate dehydrogenase, pyruvate formate lyase and NAD(P)-dependent
formate dehydrogenase, isocitrate dehydrogenase,
alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and
malate dehydrogenase. Additionally, reducing equivalents can be
generated from glucose 6-phosphate-1-dehydrogenase and
6-phosphogluconate dehydrogenase of the pentose phosphate pathway.
Overall, at most twelve reducing equivalents can be obtained from a
C6 glycolysis intermediate (e.g., glucose-6-phosphate,
fructose-6-phosphate, fructose-1,6-diphosphate) and at most six
reducing equivalents can be generated from a C3 glycolysis
intermediate (e.g., dihydroxyacetone phosphate,
glyceraldehyde-3-phosphate).
[0119] In some embodiments, the at least one exogenous nucleic acid
included in the non-naturally occurring microbial organism of the
invention is a heterologous nucleic acid. Accordingly, in some
embodiments, the at least one exogenous nucleic acid encoding a
formaldehyde fixation pathway enzyme described herein is a
heterologous nucleic acid. In some embodiments, the at least one
exogenous nucleic acid encoding a formate assimilation pathway
enzyme described herein is a heterologous nucleic acid. In some
embodiments, the at least one exogenous nucleic acid encoding a
methanol metabolic pathway enzyme described herein is a
heterologous nucleic acid. In some embodiments, the at least one
exogenous nucleic acid encoding a methanol oxidation pathway enzyme
described herein is a heterologous nucleic acid. In some
embodiments, the at least one exogenous nucleic acid encoding a
hydrogenase or a carbon monoxide dehydrogenase is a heterologous
nucleic acid.
[0120] In some embodiments, the non-naturally occurring microbial
organism of the invention is in a substantially anaerobic culture
medium.
[0121] In some embodiments, a non-naturally occurring microbial
organism described herein further includes a pathway capable of
producing succinyl-CoA, malonyl-CoA, and/or acetoacetyl-CoA,
wherein the pathway converts acetyl-CoA to succinyl-CoA,
malonyl-CoA, and/or acetoacetyl-CoA by one or more enzymes.
Accordingly, in some embodiments the microbial organism includes a
succinyl-CoA pathway, wherein the pathway converts acetyl-CoA to
the succinyl-CoA by one or more enzymes. In some embodiments, the
microbial organism includes a malonyl-CoA pathway, wherein the
pathway converts acetyl-CoA to malonyl-CoA by one or more enzymes.
In some embodiments, the microbial organism includes an
acetoacetyl-CoA pathway, wherein the pathway converts acetyl-CoA to
acetoacetyl-CoA by one or more enzymes.
[0122] In some embodiments, the invention provides that a
non-naturally occurring microbial organism as described herein
further includes a pathway capable of producing a bioderived
compound as described herein. In some aspects, the bioderived
compound is an alcohol, a glycol, an organic acid, an alkene, a
diene, an organic amine, an organic aldehyde, a vitamin, a
nutraceutical or a pharmaceutical.
[0123] In some embodiments, the non-naturally occurring microbial
organism of the invention includes a pathway for production of an
alcohol as described herein. Accordingly, in some embodiments, the
alcohol is selected from: (i) a biofuel alcohol, wherein said
biofuel is a primary alcohol, a secondary alcohol, a diol or triol
comprising C3 to C10 carbon atoms; (ii) n-propanol or isopropanol;
and (iii) a fatty alcohol, wherein said fatty alcohol comprises C4
to C27 carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon
atoms, or C12 to C14 carbon atoms. In some aspects, the biofuel
alcohol is selected from 1-propanol, isopropanol, 1-butanol,
isobutanol, 1-pentanol, isopentenol, 2-methyl-1-butanol,
3-methyl-1-butanol, 1-hexanol, 3-methyl-1-pentanol, 1-heptanol,
4-methyl-1-hexanol, and 5-methyl-1-hexanol.
[0124] In some embodiments, the non-naturally occurring microbial
organism of the invention includes a pathway for production of an
diol. Accordingly, in some embodiments, the diol is a propanediol
or a butanediol. In some aspects, the butanediol is 1,4 butanediol,
1,3-butanediol or 2,3-butanediol.
[0125] In some embodiments, the non-naturally occurring microbial
organism of the invention includes a pathway for production of a
bioderived compound selected from: (i) 1,4-butanediol or an
intermediate thereto, wherein said intermediate is optionally
4-hydroxybutanoic acid (4-HB); (ii) butadiene (1,3-butadiene) or an
intermediate thereto, wherein said intermediate is optionally
1,4-butanediol, 1,3-butanediol, 2,3-butanediol, crotyl alcohol,
3-buten-2-ol (methyl vinyl carbinol) or 3-buten-1-ol;
1,3-butanediol or an intermediate thereto, wherein said
intermediate is optionally 3-hydroxybutyrate (3-HB),
2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol; (iv) adipate,
6-aminocaproic acid, caprolactam, hexamethylenediamine, levulinic
acid or an intermediate thereto, wherein said intermediate is
optionally adipyl-CoA or 4-aminobutyryl-CoA; (v) methacrylic acid
or an ester thereof, 3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or
an intermediate thereto, wherein said ester is optionally methyl
methacrylate or poly(methyl methacrylate); (vi) 1,2-propanediol
(propylene glycol), 1,3-propanediol, glycerol, ethylene glycol,
diethylene triethylene glycol, dipropylene glycol, tripropylene
glycol, neopentyl glycol, bisphenol A or an intermediate thereto;
(vii) succinic acid or an intermediate thereto; and (viii) a fatty
alcohol, a fatty aldehyde or a fatty acid comprising C4 to C27
carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms, or
C12 to C14 carbon atoms, wherein said fatty alcohol is optionally
dodecanol (C12; lauryl alcohol), tridecyl alcohol (C13;
1-tridecanol, tridecanol, isotridecanol), myristyl alcohol (C14;
1-tetradecanol), pentadecyl alcohol (C15; 1-pentadecanol,
pentadecanol), cetyl alcohol (C16; 1-hexadecanol), heptadecyl
alcohol (C17; 1-n-heptadecanol, heptadecanol) and stearyl alcohol
(C18; 1-octadecanol) or palmitoleyl alcohol (C16 unsaturated;
cis-9-hexadecen-1-ol). Accordingly, in some embodiments, the
non-naturally occurring microbial organism of the invention
includes a pathway for production of 1,4-butanediol or an
intermediate thereto, wherein said intermediate is optionally
4-hydroxybutanoic acid (4-HB). In some embodiments, the
non-naturally occurring microbial organism of the invention
includes a pathway for production of butadiene (1,3-butadiene) or
an intermediate thereto, wherein said intermediate is optionally
1,4-butanediol, 1,3-butanediol, 2,3-butanediol, crotyl alcohol,
3-buten-2-ol (methyl vinyl carbinol) or 3-buten-1-ol. In some
embodiments, the non-naturally occurring microbial organism of the
invention includes a pathway for production of 1,3-butanediol or an
intermediate thereto, wherein said intermediate is optionally
3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotyl alcohol or
3-buten-1-ol. In some embodiments, the non-naturally occurring
microbial organism of the invention includes a pathway for
production of adipate, 6-aminocaproic acid, caprolactam,
hexamethylenediamine, levulinic acid or an intermediate thereto,
wherein said intermediate is optionally adipyl-CoA or
4-aminobutyryl-CoA. In some embodiments, the non-naturally
occurring microbial organism of the invention includes a pathway
for production of methacrylic acid or an ester thereof,
3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate
thereto, wherein said ester is optionally methyl methacrylate or
poly(methyl methacrylate). In some embodiments, the non-naturally
occurring microbial organism of the invention includes a pathway
for production of 1,2-propanediol (propylene glycol),
1,3-propanediol, glycerol, ethylene glycol, diethylene glycol,
triethylene glycol, dipropylene glycol, tripropylene glycol,
neopentyl bisphenol A or an intermediate thereto. In some
embodiments, the non-naturally occurring microbial organism of the
invention includes a pathway for production of succinic acid or an
intermediate thereto. In some embodiments, the non-naturally
occurring microbial organism of the invention includes a pathway
for production of a fatty alcohol, a fatty aldehyde or a fatty acid
comprising C4 to C27 carbon atoms, C8 to C18 carbon atoms, C12 to
C18 carbon atoms, or C12 to C14 carbon atoms, wherein said fatty
alcohol is optionally dodecanol (C12; lauryl alcohol), tridecyl
alcohol (C13; 1-tridecanol, tridecanol, isotridecanol), myristyl
alcohol (C14; 1-tetradecanol), pentadecyl alcohol (C15;
1-pentadecanol, pentadecanol), cetyl alcohol (C16; 1-hexadecanol),
heptadecyl alcohol (C17; 1-n-heptadecanol, heptadecanol) and
stearyl alcohol (C18; 1-octadecanol) or palmitoleyl alcohol (C16
unsaturated; cis-9-hexadecen-1-ol).
[0126] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a 1,3-butanediol
pathway and an exogenous nucleic acid encoding a 1,3-butanediol
pathway enzyme expressed in a sufficient amount to produce
1,3-butanediol as depicted in FIG. 5. Accordingly, in some
embodiments, the 1,3-butanediol pathway comprises a pathway
selected from: (1) 5A, 5B, 5D, 5E, and 5H; (2) 5A, 5B, 5D, 5F, 5G,
and 5H; (3) 5C, 5D, 5E, and 5H; (4) 5C, 5D, 5F, 5G, and 5H; (5) 5A,
5B, 5D and 5V; and (6) 5C, 5D and 5V wherein 5A is an acetyl-CoA
carboxylase, wherein 5B is an acetoacetyl-CoA synthase, wherein 5C
is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is an
acetoacetyl-CoA reductase (ketone reducing), wherein 5E is a
3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 5F is a
3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, wherein
5G is a 3-hydroxybutyrate reductase, wherein 5H is a
3-hydroxybutyraldehyde reductase, wherein 5V is a
3-hydroxybutyryl-CoA reductase (alcohol forming).
[0127] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a crotyl alcohol
pathway and an exogenous nucleic acid encoding a crotyl alcohol
pathway enzyme expressed in a sufficient amount to produce crotyl
alcohol as depicted in FIG. 5. Accordingly, in some embodiments,
the crotyl alcohol pathway comprises a pathway selected from: (1)
5A, 5B, 5D, 5J, 5K, and 5N; (2) 5A, 5B, 5D, 5J, 5L, 5M, and 5N; (3)
5C, 5D, 5J, 5K, and 5N; (4) 5C, 5D, 5J, 5L, 5M, and 5N; (5) 5A, 5B,
5D, 5J and 5U; and (6) 5C, 5D, 5J and 5U, wherein 5A is an
acetyl-CoA carboxylase, wherein 5B is an acetoacetyl-CoA synthase,
wherein 5C is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 5D
is an acetoacetyl-CoA reductase (ketone reducing), wherein 5J is a
3-hydroxybutyryl-CoA dehydratase, wherein 5K is a crotonyl-CoA
reductase (aldehyde forming), wherein 5L is a crotonyl-CoA
hydrolase, crotonyl-CoA transferase or crotonyl-CoA synthetase,
wherein 5M is a crotonate reductase, wherein 5N is a crotonaldehyde
reductase, wherein 5U is a crotonyl-CoA reductase (alcohol
forming).
[0128] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a butadiene pathway and
an exogenous nucleic acid encoding a butadiene pathway enzyme
expressed in a sufficient amount to produce butadiene as depicted
in FIGS. 5 and 6. Accordingly, in some embodiments, the butadiene
pathway comprises a pathway selected from: (1) 5A, 5B, 5D, 5E, 5H,
6A, 6B, 6C, and 6G; (2) 5A, 5B, 5D, 5F, 5G, 5H, 6A, 6B, 6C, and 6G;
(3) 5C, 5D, 5E, 5H, 6A, 6B, 6C, and 6G; (4) 5C, 5D, 5F, 5G, 5H, 6A,
6B, 6C, and 6G; (5) 5A, 5B, 5D, 5E, 5H, 6A, 6F, and 6G; (6) 5A, 5B,
5D, 5F, 5G, 5H, 6A, 6F, and 6G; (7) 5C, 5D, 5E, 5H, 6A, 6F, and 6G;
(8) 5C, 5D, 5F, 5G, 5H, 6A, 6F, and 6G; (9) 5A, 5B, 5D, 5E, 5H, 6E,
6C, and 6G; (10) 5A, 5B, 5D, 5F, 5G, 5H, 6E, 6C, and 6G; (11) 5C,
5D, 5E, 5H, 6E, 6C, and 6G; (12) 5C, 5D, 5F, 5G, 5H, 6E, 6C, and
6G; (13) 5A, 5B, 5D, 5E, 5H, 6D, and 6G; (14) 5A, 5B, 5D, 5F, 5G,
5H, 6D, and 6G; (15) 5C, 5D, 5E, 5H, 6D, and 6G; (16) 5C, 5D, 5F,
5G, 5H, 6D, and 6G; (17) 5A, 5B, 5D, 5J, 5K, 5N, and 5S; (18) 5A,
5B, 5D, 5J, 5L, 5M, 5N, and 5S; (19) 5C, 5D, 5J, 5K, 5N, and 5S;
(20) 5C, 5D, 5J, 5L, 5M, 5N, and 5S; (21) 5A, 5B, 5D, 5J, 5K, 5N,
5R, and 5Q; (22) 5A, 5B, 5D, 5J, 5L, 5M, 5N, 5R, and 5Q; (23) 5C,
5D, 5J, 5K, 5N, 5R, and 5Q; (24) 5C, 5D, 5J, 5L, 5M, 5N, 5R, and
5Q; (25) 5A, 5B, 5D, 5J, 5K, 5N, 5O, 5P, and 5Q; (26) 5A, 5B, 5D,
5J, 5L, 5M, 5N, 5O, 5P, and 5Q; (27) 5C, 5D, 5J, 5K, 5N, 5O, 5P,
and 5Q; (28) 5C, 5D, 5J, 5L, 5M, 5N, 5O, 5P, and 5Q; (29) 5A, 5B,
5D, 5J, 5K, 5N, 5O, and 5T; (30) 5A, 5B, 5D, 5J, 5L, 5M, 5N, 5O,
and 5T; (31) 5C, 5D, 5J, 5K, 5N, 5O, and 5T; (32) 5C, 5D, 5J, 5L,
5M, 5N, 5O, and 5T, (33) 5A, 5B, 5D, 5V, 6A, 6B, 6C, and 6G; (34)
5C, 5D, 5V, 6A, 6B, 6C, and 6G; (35) 5A, 5B, 5D, 5J, 5U, and 5S;
(36) 5C, 5D, 5J, 5U, and 5S; (37) 5A, 5B, 5D, 5J, 5U, 5R, and 5Q;
(38) 5C, 5D, 5J, 5U, 5R, and 5Q; (39) 5A, 5B, 5D, 5J, 5U, 5O, 5P,
and 5Q; (40) 5C, 5D, 5J, 5U, 5O, 5P, and 5Q; (41) 5A, 5B, 5D, 5J,
5U, 5O, and 5T; and (42) 5C, 5D, 5J, 5U, 5O, and 5T, wherein 5A is
an acetyl-CoA carboxylase, wherein 5B is an acetoacetyl-CoA
synthase, wherein 5C is an acetyl-CoA:acetyl-CoA acyltransferase,
wherein 5D is an acetoacetyl-CoA reductase (ketone reducing),
wherein 5E is a 3-hydroxybutyryl-CoA reductase (aldehyde forming),
wherein 5F is a 3-hydroxybutyryl-CoA hydrolase,
3-hydroxybutyryl-CoA transferase or 3-hydroxybutyryl-CoA
synthetase, wherein 5G is a 3-hydroxybutyrate reductase, wherein 5H
is a 3-hydroxybutyraldehyde reductase, wherein 5J is a
3-hydroxybutyryl-CoA dehydratase, wherein 5K is a crotonyl-CoA
reductase (aldehyde forming), wherein 5L is a crotonyl-CoA
hydrolase, crotonyl-CoA transferase or crotonyl-CoA synthetase,
wherein 5M is a crotonate reductase, wherein 5N is a crotonaldehyde
reductase, wherein 50 is a crotyl alcohol kinase, wherein 5P is a
2-butenyl-4-phosphate kinase, wherein 5Q is a butadiene synthase,
wherein 5R is a crotyl alcohol diphosphokinase, wherein 5S is
chemical dehydration or a crotyl alcohol dehydratase, wherein 5T is
a butadiene synthase (monophosphate), wherein 5T is a butadiene
synthase (monophosphate), wherein 5U is a crotonyl-CoA reductase
(alcohol forming), wherein 5V is a 3-hydroxybutyryl-CoA reductase
(alcohol forming), wherein 6A is a 1,3-butanediol kinase, wherein
6B is a 3-hydroxybutyrylphosphate kinase, wherein 6C is a
3-hydroxybutyryldiphosphate lyase, wherein 6D is a 1,3-butanediol
diphosphokinase, wherein 6E is a 1,3-butanediol dehydratase,
wherein 6F is a 3-hydroxybutyrylphosphate lyase, wherein 6G is a
3-buten-2-ol dehydratase or chemical dehydration.
[0129] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a 3-buten-2-ol pathway
and an exogenous nucleic acid encoding a 3-buten-2-ol pathway
enzyme expressed in a sufficient amount to produce 3-buten-2-ol as
depicted in FIGS. 5 and 6. Accordingly, in some embodiments, the
3-buten-2-ol pathway comprises a pathway selected from: (1) 5A, 5B,
5D, 5E, 5H, 6A, 6B, and 6C; (2) 5A, 5B, 5D, 5F, 5G, 5H, 6A, 6B, and
6C; (3) 5C, 5D, 5E, 5H, 6A, 6B, and 6C; (4) 5C, 5D, 5F, 5G, 5H, 6A,
6B, and 6C; (5) 5A, 5B, 5D, 5E, 5H, 6A, and 6F; (6) 5A, 5B, 5D, 5F,
5G, 5H, 6A, and 6F; (7) 5C, 5D, 5E, 5H, 6A, and 6F; (8) 5C, 5D, 5F,
5G, 5H, 6A, and 6F; (9) 5A, 5B, 5D, 5E, 5H, 6E, and 6C; (10) 5A,
5B, 5D, 5F, 5G, 5H, 6E, and 6C; (11) 5C, 5D, 5E, 5H, 6E, and 6C;
(12) 5C, 5D, 5F, 5G, 5H, 6E, and 6C; (13) 5A, 5B, 5D, 5E, 5H, and
6D; (14) 5A, 5B, 5D, 5F, 5G, 5H, and 6D; (15) 5C, 5D, 5E, 5H, and
6D; (16) 5C, 5D, 5F, 5G, 5H, and 6D; (17) 5A, 5B, 5D, 5V, 6A, 6B,
and 6C; (18) 5C, 5D, 5V, 6A, 6B, and 6C; (19) 5A, 5B, 5D, 5V, 6A,
and 6F; (20) 5C, 5D, 5V, 6A, and 6F; (21) 5A, 5B, 5D, 5V, 6E, and
6C; (22) 5C, 5D, 5V, 6E, and 6C; (23) 5A, 5B, 5D, 5V and 6D; and
(24) 5C, 5D, 5V and 6D, wherein 5A is an acetyl-CoA carboxylase,
wherein 5B is an acetoacetyl-CoA synthase, wherein 5C is an
acetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is an
acetoacetyl-CoA reductase (ketone reducing), wherein 5E is a
3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 5F is a
3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA transferase or
3-hydroxybutyryl-CoA synthetase, wherein 5G is a 3-hydroxybutyrate
reductase, wherein 5H is a 3-hydroxybutyraldehyde reductase,
wherein 5V is a 3-hydroxybutyryl-CoA reductase (alcohol forming),
wherein 6A is a 1,3-butanediol kinase, wherein 6B is a
3-hydroxybutyrylphosphate kinase, wherein 6C is a
3-hydroxybutyryldiphosphate lyase, wherein 6D is a 1,3-butanediol
diphosphokinase, wherein 6E is a 1,3-butanediol dehydratase,
wherein 6F is a 3-hydroxybutyrylphosphate lyase.
[0130] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a 1,4-butanediol
pathway and an exogenous nucleic acid encoding a 1,4-butanediol
pathway enzyme expressed in a sufficient amount to produce
1,4-butanediol as depicted in FIG. 7. Accordingly, in some
embodiments, the 1,4-butanediol pathway comprises a pathway
selected from: (1) 7B, 7C, 7D, 7E, 7F, and 7G; (2) 7A, 7H, 7C, 7D,
7E, 7F, and 7G; (3) 7I, 7D, 7E, 7F, and 7G; (4) 7B, 7C, 7K, and 7G;
(5) 7A, 7H, 7C, 7K, and 7G; (6) 7I, 7K, and 7G; (7) 7B, 7C, 7D, 7L,
and 7G; (8) 7A, 7H, 7C, 7D, 7L, and 7G; (9) 7I, 7D, 7L, and 7G;
(10) 7B, 7C, 7J, 7F, and 7G; (11) 7A, 7H, 7C, 7J, 7F, and 7G; (12)
7I, 7J, 7F, and 7G; (13) 7B, 7C, 7D, 7E, and 7M; (14) 7A, 7H, 7C,
7D, 7E, and 7M; and (15) 7I, 7D, 7E, and 7M, wherein 7A is a
succinyl-CoA transferase or a succinyl-CoA synthetase, wherein 7B
is a succinyl-CoA reductase (aldehyde forming), wherein 7C is a
4-HB dehydrogenase, wherein 7D is a 4-HB kinase, wherein 7E is a
phosphotrans-4-hydroxybutyrylase, wherein 7F is a
4-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 7G is a
1,4-butanediol dehydrogenase, wherein 7H is a succinate reductase,
wherein 71 is a succinyl-CoA reductase (alcohol forming), wherein
7J is a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA
synthetase, wherein 7K is a 4-HB reductase, wherein 7L is a
4-hydroxybutyryl-phosphate reductase, wherein 7M is a
4-hydroxybutyryl-CoA reductase (alcohol forming).
[0131] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises an adipate pathway and
an exogenous nucleic acid encoding an adipate pathway enzyme
expressed in a sufficient amount to produce adipate as depicted in
FIG. 8. Accordingly, in some embodiments, the adipate pathway
comprises 8A, 8B, 8C, 8D and 8L, wherein 8A is a 3-oxoadipyl-CoA
thiolase, wherein 8B is a 3-oxoadipyl-CoA reductase, wherein 8C is
a 3-hydroxyadipyl-CoA dehydratase, wherein 8D is a
5-carboxy-2-pentenoyl-CoA reductase, wherein 8L is an adipyl-CoA
hydrolase, adipyl-CoA ligase, adipyl-CoA transferase, or
phosphotransadipylase/adipate kinase.
[0132] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a 6-aminocaproate
pathway and an exogenous nucleic acid encoding a 6-aminocaproate
pathway enzyme expressed in a sufficient amount to produce
6-aminocaproate as depicted in FIG. 8. Accordingly, in some
embodiments, the 6-aminocaproate pathway comprises 8A, 8B, 8C, 8D,
8E, and 8F, wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is
a 3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoA
dehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,
wherein 8E is an adipyl-CoA reductase (aldehyde forming), wherein
8F is a 6-aminocaproate transaminase or 6-aminocaproate
dehydrogenase.
[0133] In some embodiments, a non-naturally occurring microbial
organism of the invention further includes a caprolactam pathway
and an exogenous nucleic acid encoding a caprolactam pathway enzyme
expressed in a sufficient amount to produce caprolactam as depicted
in FIG. 8. Accordingly, in some embodiments, the caprolactam
pathway comprises: (1) 8A, 8B, 8C, 8D, 8E, 8F, and 8H; or (2) 8A,
8B, 8C, 8D, 8E, 8F, 8G, and 8I, wherein 8A is a 3-oxoadipyl-CoA
thiolase, wherein 8B is a 3-oxoadipyl-CoA reductase, wherein 8C is
a 3-hydroxyadipyl-CoA dehydratase, wherein 8D is a
5-carboxy-2-pentenoyl-CoA reductase, wherein 8E is an adipyl-CoA
reductase (aldehyde forming), wherein 8F is a 6-aminocaproate
transaminase or 6-aminocaproate dehydrogenase, wherein 8G is a
6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA
synthase, wherein 8H is an amidohydrolase, wherein 81 is
spontaneous cyclization.
[0134] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a hexamethylenediamine
pathway and an exogenous nucleic acid encoding a
hexamethylenediamine pathway enzyme expressed in a sufficient
amount to produce hexamethylenediamine as depicted in FIG. 8.
Accordingly, in some embodiments, the hexamethylenediamine pathway
comprises 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8J, 8K, wherein 8A is a
3-oxoadipyl-CoA thiolase, wherein 8B is a 3-oxoadipyl-CoA
reductase, wherein 8C is a 3-hydroxyadipyl-CoA dehydratase, wherein
8D is a 5-carboxy-2-pentenoyl-CoA reductase, wherein 8E is an
adipyl-CoA reductase (aldehyde forming), wherein 8F is a
6-aminocaproate transaminase or 6-aminocaproate dehydrogenase,
wherein 8G is a 6-aminocaproyl-CoA/acyl-CoA transferase or
6-aminocaproyl-CoA synthase, wherein 8J is a 6-aminocaproyl-CoA
reductase (aldehyde forming), wherein 8K is a hexamethylenediamine
transaminase or hexamethylenediamine dehydrogenase.
[0135] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a methacrylic acid
pathway and an exogenous nucleic acid encoding a methacrylic acid
pathway enzyme expressed in a sufficient amount to produce
methacrylic acid as depicted in FIGS. 9 and 10. Accordingly, in
some embodiments, the methacrylic acid pathway comprises a pathway
selected from: (1) 9A, 9B, 9C, 9D, and 9E; (2) 9A, 9F, and 9E; (3)
9A, 9B, 9F, and 9E; (4) 9A, 9C, 9D, and 9E; and (5) 10A, 10B, 10C,
10D, and 10E, wherein 9A is a methylmalonyl-CoA mutase, wherein 9B
is a methylmalonyl-CoA epimerase, wherein 9C is a methylmalonyl-CoA
reductase (aldehyde forming), wherein 9D is a methylmalonate
semialdehyde reductase, wherein 9E is a 3-hydroxyisobutyrate
dehydratase, wherein 9F is a methylmalonyl-CoA reductase (alcohol
forming), wherein 10A is an acetyl-CoA:acetyl-CoA acyltransferase,
wherein 10B is an acetoacetyl-CoA reductase (ketone reducing),
wherein 10C is a 3-hydroxybutyrl-CoA mutase, wherein 10D is a
2-hydroxyisobutyryl-CoA dehydratase, wherein 10E is a
methacrylyl-CoA synthetase, methacrylyl-CoA hydrolase, or
methacrylyl-CoA transferase.
[0136] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a 2-hydroxyisobutyric
acid pathway and an exogenous nucleic acid encoding a
2-hydroxyisobutyric acid pathway enzyme expressed in a sufficient
amount to produce 2-hydroxyisobutyric acid as depicted in FIG. 10.
Accordingly, in some embodiments, the 2-hydroxyisobutyric acid
pathway comprises 10A, 10B, 10C, and 10F, wherein 10A is an
acetyl-CoA:acetyl-CoA acyltransferase, wherein 10B is an
acetoacetyl-CoA reductase (ketone reducing), wherein 10C is a
3-hydroxybutyrl-CoA mutase, wherein 10F is a
2-hydroxyisobutyryl-CoA synthetase, 2-hydroxyisobutyryl-CoA
hydrolase, or 2-hydroxyisobutyryl-CoA transferase.
[0137] In some embodiments, a non-naturally occurring microbial
organism of the invention further comprises a succinyl-CoA pathway
and an exogenous nucleic acid encoding a succinyl-CoA pathway
enzyme expressed in a sufficient amount to produce succinyl-CoA as
depicted in FIG. 4. Accordingly, in some embodiments, the
succinyl-CoA pathway comprises a pathway selected from: (1) 4H, 4I,
4J, and 4K; (2) 4H, 4I, 4N, and 4E; (3) 4H, 4I, 4N, 4C, 4D, and 4E;
(4) 4L, 4H, 4I, 4J, and 4K; (5) 4L, 4H, 4I, 4N, and 4E; (6) 4L, 4H,
4I, 4N, 4C, 4D, and 4E; (7) 4A, 4H, 4I, 4J, and 4K; (8) 4A, 4H, 4I,
4N, and 4E; (9) 4A, 4H, 4I, 4N, 4C, 4D, and 4E; (10) 4M, 4C, 4D,
and 4E; (11) 4F, 4L, 4H, 4I, 4J, and 4K; (12) 4F, 4L, 4H, 4I, 4N,
and 4E; (13) 4F, 4L, 4H, 4I, 4N, 4C, 4D, and 4E; (14) 4F, 4M, 4C,
4D, and 4E; (15) 4F, 4G, 4H, 4I, 4J, and 4K; (16) 4F, 4G, 4H, 4I,
4N, and 4E; and (17) 4F, 4G, 4H, 4I, 4N, 4C, 4D, and 4E, wherein 4A
is a PEP carboxylase or PEP carboxykinase, wherein 4B is a malate
dehydrogenase, wherein 4C is a fumarase, wherein 4D is a fumarate
reductase, wherein 4E is a succinyl-CoA synthetase or succinyl-CoA
transferase, wherein 4F is a pyruvate kinase or PTS-dependent
substrate import, wherein 4G is a pyruvate dehydrogenase, pyruvate
formate lyase, or pyruvate:ferredoxin oxidoreductase, wherein 4H is
a citrate synthase, wherein 41 is an aconitase, wherein 4J is an
isocitrate dehydrogenase, wherein 4K is an alpha-ketoglutarate
dehydrogenase, wherein 4L is a pyruvate carboxylase, wherein 4M is
a malic enzyme, wherein 4N is an isocitrate lyase and malate
synthase.
[0138] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an acetyl-CoA or
bioderived compound pathway, wherein the non-naturally occurring
microbial organism comprises at least one exogenous nucleic acid
encoding an enzyme or protein that converts a substrate to a
product selected from the group consisting of MeOH to Fald, Fald to
H6P, H6P to F6P, Fald to DHA and G3P, DHA and G3P to F6P, F6P to
ACTP and E4P, ACTP to ACCOA, ACTP to acetate, acetate to ACCOA,
Xu5P to ACTP and G3P, G3P to PYR, PYR to formate and ACCOA, PYR to
CO.sub.2 and ACCOA, CO.sub.2 to formate, formate to Fald, formate
to Formyl-CoA, Formyl-CoA to Fald, Formate to FTHF, FTHF to
methenyl-THF, methenyl-THF to methylene-THF, methylene-THF to Fald,
methylene-THF to glycine, glycine to serine, serine to PYR,
methylene-THF to methyl-THF, methyl-THF to ACCOA, G3P to PEP, PEP
to PYR, PYR to ACCOA, PEP to OAA, OAA to MAL, MAL to FUM, FUM to
SUCC, SUCC to SUCCOA, ACCOA and OAA to CIT, CIT to ICIT, ICIT to
AKG, AKG to SUCCOA, PYR to OAA, PYR to MAL, ICIT to MAL and SUCC,
ACCOA to MALCOA, MALCOA and ACCOA to AACOA, ACCOA to AACOA, ACCOA
to 3HBCOA, 3HBCOA to 3HBALD, 3HBALD to 13BDO; 13BDO to Butadiene,
3HBCOA to CROTCOA, CROTCOA to CROTALD, CROTCOA to CROT, CROT to
CROTALD, CROTALD to CROTALC, CROTALD to CROT-Pi, CROT-Pi to
CROT-PPi, CROT-Ppi to butadiene, CROTALD to CROT-PPi, CROT-Pi to
butadiene, 1,3-butanediol to 3-hydroxybutyryl phosphate,
3-hydroxybutyryl phosphate to 3-hydroxybutyryl diphosphate;
3-hydroxybutyryl diphosphate to 3-buten-2-ol, 3-buten-2-ol to
butadiene, 1,3-butandediol to 3-buten-2-ol, 1,3-butanediol to
3-hydroxybutyryl diphosphate, 3-hydroxybutyryl phosphate to
3-buten-2-ol, succinyl-CoA to succinate, succinyl-CoA to succinate
semialdehyde, succinate semialdehyde to 4-hydroxybutymte,
4-hydroxybutyrate to 4-hydroxybutyryl-phosphate,
4-hydroxybutyryl-phosphate to 4-hydroxybutyryl-CoA,
4-hydroxybutyryl-CoA to 4-hydroxybutanal, 4-hydroxybutanal to
1,4-butanediol, succinate to succinate semialdehyde, succinyl-CoA
to 4-hydroxybutyrate, 4-hydroxybutyrate to 4-hydroxybutyryl-CoA,
4-hydroxybutyrate to 4-hydroxybutanal, 4-hydroxybutyryl-phosphate
to 4-hydroxybutanal, 4-hydroxybutyryl-CoA to 1,4-butanediol,
succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, 3-oxoadipyl-CoA to
3-hydroxyadiply-CoA, 3-hydroxyadiply-CoA to
5-carboxy-2-pentenoyl-CoA, 5-carboxy-2-pentenoyl-CoA to adipyl-CoA,
adipyl-CoA to adipate, adipyl-CoA to adipate semialdehyde, adipate
semialdehyde to 6-aminocaproate, 6-aminocaproate to caprolactam,
6-aminocaproate to 6-aminocaproyl-CoA, 6-aminocaproyl-CoA to
6-aminocaproate semialdehyde, 6-aminocaproate semialdehyde to
hexamethylenediamine, succinyl-CoA to (R)-methylmalonyl-CoA,
(R)-methylmalonyl-CoA to methylmalonate semialdehyde,
(R)-methylmalonyl-CoA to (S)-methylmalonyl-CoA,
(S)-methylmalonyl-CoA to methylmalonate semialdehyde,
(R)-methylmalonyl-CoA to 3-hydroxyisobutyrate,
(S)-methylmalonyl-CoA to 3-hydroxyisobutyrate, 3-hydroxyisobutyrate
to methacrylic acid, 3-hydroxybutyryl-CoA to
2-hydroxyisobutyryl-CoA, 2-hydroxyisobutyryl-CoA to
2-hydroxyisobutyric acid, 2-hydroxyisobutyryl-CoA to
methacrylyl-CoA, methacrylyl-CoA to methacrylic acid. One skilled
in the art will understand that these are merely exemplary and that
any of the substrate-product pairs disclosed herein suitable to
produce a desired product and for which an appropriate activity is
available for the conversion of the substrate to the product can be
readily determined by one skilled in the art based on the teachings
herein. Thus, the invention provides a non-naturally occurring
microbial organism containing at least one exogenous nucleic acid
encoding an enzyme or protein, where the enzyme or protein converts
the substrates and products of an acetyl-CoA or a bioderived
compound pathway, such as that shown in FIG. 1-10.
[0139] While generally described herein as a microbial organism
that contains an acetyl-CoA or a bioderived compound pathway, it is
understood that the invention additionally provides a non-naturally
occurring microbial organism comprising at least one exogenous
nucleic acid encoding an acetyl-CoA or a bioderived compound
pathway enzyme expressed in a sufficient amount to produce an
intermediate of an acetyl-CoA or a bioderived compound pathway. For
example, as disclosed herein, an acetyl-CoA or a bioderived
compound pathway is exemplified in FIGS. 1-10. Therefore, in
addition to a microbial organism containing an acetyl-CoA or a
bioderived compound pathway that produces acetyl-CoA or a
bioderived compound, the invention additionally provides a
non-naturally occurring microbial organism comprising at least one
exogenous nucleic acid encoding an acetyl-CoA or a bioderived
compound pathway enzyme, where the microbial organism produces an
acetyl-CoA or a bioderived compound pathway intermediate, for
example, acetate, ACTP, G3P, PYR, Formate, Fald, formyl-CoA, FTHF,
Methenyl-THF, Methylene-THF, Glycine, Serine, Methyl-THF, H6P, F6P,
DHA, S-hydroxymethyglutathione, S-formylglutathione, OAA, CIT,
ICIT, MAL, FUM, AKG, SUCC, SUCCOA, MALCOA, AACOA, 3HBCOA, 3HBALD,
3HB, CROTCOA, CROT, CROALD, CROT-Pi, CROT-PPi, 3-hydroxybutyryl
phosphate, 3-hydroxybutyryl diphosphate, succinate semialdehyde,
4-hydroxybutyrate, 4-hydroxybutyryl-phosphate,
4-hydroxybutyryl-CoA, 4-hydroxybutanal, 3-oxoadipyl-CoA,
3-hydroxyadipyl-CoA, 5-hydroxy-2-pentenoyl-CoA, adipate
semialdehyde, 6-aminocaproyl-CoA, 6-aminocaproate semialdehyde,
(R)-methylmalonyl-CoA, (S)-methylmalonyl-CoA, methylmalonate
semialdehyde, 3-hydrdoxyisobutyrate, 2-hydroxyisobutyryl-CoA, and
methacrylyl-CoA.
[0140] It is understood that any of the pathways disclosed herein,
as described in the Examples and exemplified in the Figures,
including the pathways of FIGS. 1-10, can be utilized to generate a
non-naturally occurring microbial organism that produces any
pathway intermediate or product, as desired. As disclosed herein,
such a microbial organism that produces an intermediate can be used
in combination with another microbial organism expressing
downstream pathway enzymes to produce a desired product. However,
it is understood that a non-naturally occurring microbial organism
that produces an acetyl-CoA or a bioderived compound pathway
intermediate can be utilized to produce the intermediate as a
desired product.
[0141] In one embodiment, the invention provides a non-naturally
occurring microbial organism having an acetyl-CoA pathway, wherein
said acetyl-CoA pathway comprises a pathway selected from: (1) 1T
and 1V; (2) 1T, 1W, and 1X; (3) 1U and 1V; (4) 1U, 1W, and 1X;
wherein 1T is a fructose-6-phosphate phosphoketolase, wherein 1U is
a xylulose-5-phosphate phosphoketolase, wherein 1V is a
phosphotransacetylase, wherein 1W is an acetate kinase, wherein 1X
is an acetyl-CoA transferase, an acetyl-CoA synthetase, or an
acetyl-CoA ligase; wherein said non-naturally occurring microbial
organism further comprises a pathway capable of producing a
bioderived compound, wherein said bioderived compounds is selected
from the group consisting of (i) 1,4-butanediol or an intermediate
thereto, wherein said intermediate is optionally 4-hydroxybutanoic
acid (4-HB); (ii) butadiene (1,3-butadiene) or an intermediate
thereto, wherein said intermediate is optionally 1,4-butanediol,
1,3-butanediol, 2,3-butanediol, crotyl alcohol, 3-buten-2-ol
(methyl vinyl carbinol) or 3-buten-1-ol; (iii) 1,3-butanediol or an
intermediate thereto, wherein said intermediate is optionally
3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotyl alcohol or
3-buten-1-ol; (iv) adipate, 6-aminocaproic acid, caprolactam,
hexamethylenediamine, levulinic acid or an intermediate thereto,
wherein said intermediate is optionally adipyl-CoA or
4-aminobutyryl-CoA; (v) methacrylic acid or an ester thereof,
3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate
thereto, wherein said ester is optionally methyl methacrylate or
poly(methyl methacrylate); (vi) 1,2-propanediol (propylene glycol),
1,3-propanediol, glycerol, ethylene glycol, diethylene glycol,
triethylene glycol, dipropylene glycol, tripropylene glycol,
neopentyl glycol, bisphenol A or an intermediate thereto; (vii)
succinic acid or an intermediate thereto; and (viii) a fatty
alcohol, a fatty aldehyde or a fatty acid comprising C4 to C27
carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms, or
C12 to C14 carbon atoms, wherein said fatty alcohol is optionally
dodecanol (C12; lauryl alcohol), tridecyl alcohol (C13;
1-tridecanol, tridecanol, isotridecanol), myristyl alcohol (C14;
1-tetradecanol), pentadecyl alcohol (C15; 1-pentadecanol,
pentadecanol), cetyl alcohol (C16; 1-hexadecanol), heptadecyl
alcohol (C17; 1-n-heptadecanol, heptadecanol) and stearyl alcohol
(C18; 1-octadecanol) or palmitoleyl alcohol (C16 unsaturated;
cis-9-hexadecen-1-ol). In some aspects, the non-naturally occurring
microbial organism having an acetyl-CoA pathway can further
comprise a 1,3-butanediol pathway and an exogenous nucleic acid
encoding a 1,3-butanediol pathway enzyme expressed in a sufficient
amount to produce 1,3-butanediol, wherein said 1,3-butanediol
pathway comprises a pathway selected from: (1) 5A, 5B, 5D, 5E, and
5H; (2) 5A, 5B, 5D, 5F, 5G, and 5H; (3) 5C, 5D, 5E, and 5H; (4) 5C,
5D, 5F, 5G, and 5H; (5) 5A, 5B, 5D and 5V; and (6) 5C, 5D and 5V,
wherein 5A is an acetyl-CoA carboxylase, wherein 5B is an
acetoacetyl-CoA synthase, wherein 5C is an acetyl-CoA:acetyl-CoA
acyltransferase, wherein 5D is an acetoacetyl-CoA reductase (ketone
reducing), wherein 5E is a 3-hydroxybutyryl-CoA reductase (aldehyde
forming), wherein 5F is a 3-hydroxybutyryl-CoA hydrolase,
transferase or synthetase, wherein 5G is a 3-hydroxybutyrate
reductase, wherein 5H is a 3-hydroxybutyraldehyde reductase,
wherein 5V is a 3-hydroxybutyryl-CoA reductase (alcohol
forming).
[0142] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a crotyl
alcohol pathway and an exogenous nucleic acid encoding a crotyl
alcohol pathway enzyme expressed in a sufficient amount to produce
crotyl alcohol, wherein said crotyl alcohol pathway comprises a
pathway selected from: (1) 5A, 5B, 5D, 5J, 5K, and 5N; (2) 5A, 5B,
5D, 5J, 5L, 5M, and 5N; (3) 5C, 5D, 5J, 5K, and 5N; (4) 5C, 5D, 5J,
5L, 5M, and 5N; (5) 5A, 5B, 5D, 5J and 5U; and (6) 5C, 5D, 5J and
5U, wherein 5A is an acetyl-CoA carboxylase, wherein 5B is an
acetoacetyl-CoA synthase, wherein 5C is an acetyl-CoA:acetyl-CoA
acyltransferase, wherein 5D is an acetoacetyl-CoA reductase (ketone
reducing), wherein 5J is a 3-hydroxybutyryl-CoA dehydratase,
wherein 5K is a crotonyl-CoA reductase (aldehyde forming), wherein
5L is a crotonyl-CoA hydrolase, crotonyl-CoA transferase or
crotonyl-CoA synthetase, wherein 5M is a crotonate reductase,
wherein 5N is a crotonaldehyde reductase, wherein 5U is a
crotonyl-CoA reductase (alcohol forming).
[0143] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
butadiene pathway and an exogenous nucleic acid encoding a
butadiene pathway enzyme expressed in a sufficient amount to
produce butadiene, wherein said butadiene pathway comprises a
pathway selected from: (1) 5A, 5B, 5D, 5E, 5H, 6A, 6B, 6C, and 6G;
(2) 5A, 5B, 5D, 5F, 5G, 5H, 6A, 6B, 6C, and 6G; (3) 5C, 5D, 5E, 5H,
6A, 6B, 6C, and 6G; (4) 5C, 5D, 5F, 5G, 5H, 6A, 6B, 6C, and 6G; (5)
5A, 5B, 5D, 5E, 5H, 6A, 6F, and 6G; (6) 5A, 5B, 5D, 5F, 5G, 5H, 6A,
6F, and 6G; (7) 5C, 5D, 5E, 5H, 6A, 6F, and 6G; (8) 5C, 5D, 5F, 5G,
5H, 6A, 6F, and 6G; (9) 5A, 5B, 5D, 5E, 5H, 6E, 6C, and 6G; (10)
5A, 5B, 5D, 5F, 5G, 5H, 6E, 6C, and 6G; (11) 5C, 5D, 5E, 5H, 6E,
6C, and 6G; (12) 5C, 5D, 5F, 5G, 5H, 6E, 6C, and 6G; (13) 5A, 5B,
5D, 5E, 5H, 6D, and 6G; (14) 5A, 5B, 5D, 5F, 5G, 5H, 6D, and 6G;
(15) 5C, 5D, 5E, 5H, 6D, and 6G; (16) 5C, 5D, 5F, 5G, 5H, 6D, and
6G; (17) 5A, 5B, 5D, 5J, 5K, 5N, and 5S; (18) 5A, 5B, 5D, 5J, 5L,
5M, 5N, and 5S; (19) 5C, 5D, 5J, 5K, 5N, and 5S; (20) 5C, 5D, 5J,
5L, 5M, 5N, and 5S; (21) 5A, 5B, 5D, 5J, 5K, 5N, 5R, and 5Q; (22)
5A, 5B, 5D, 5J, 5L, 5M, 5N, 5R, and 5Q; (23) 5C, 5D, 5J, 5K, 5N,
5R, and 5Q; (24) 5C, 5D, 5J, 5L, 5M, 5N, 5R, and 5Q; (25) 5A, 5B,
5D, 5J, 5K, 5N, 5O, 5P, and 5Q; (26) 5A, 5B, 5D, 5J, 5L, 5M, 5N,
5O, 5P, and 5Q; (27) 5C, 5D, 5J, 5K, 5N, 5O, 5P, and 5Q; (28) 5C,
5D, 5J, 5L, 5M, 5N, 5O, 5P, and 5Q; (29) 5A, 5B, 5D, 5J, 5K, 5N,
5O, and 5T; (30) 5A, 5B, 5D, 5J, 5L, 5M, 5N, 5O, and 5T; (31) 5C,
5D, 5J, 5K, 5N, 5O, and 5T; (32) 5C, 5D, 5J, 5L, 5M, 5N, 5O, and
5T; (33) 5A, 5B, 5D, 5V, 6A, 6B, 6C, and 6G; (34) 5C, 5D, 5V, 6A,
6B, 6C, and 6G; (35) 5A, 5B, 5D, 5J, 5U, and 5S; (36) 5C, 5D, 5J,
5U, and 5S; (37) 5A, 5B, 5D, 5J, 5U, 5R, and 5Q; (38) 5C, 5D, 5J,
5U, 5R, and 5Q; (39) 5A, 5B, 5D, 5J, 5U, 5O, 5P, and 5Q; (40) 5C,
5D, 5J, 5U, 5O, 5P, and 5Q; (41) 5A, 5B, 5D, 5J, 5U, 50, and 5T;
and (42) 5C, 5D, 5J, 5U, 5O, and 5T, wherein 5A is an acetyl-CoA
carboxylase, wherein 5B is an acetoacetyl-CoA synthase, wherein 5C
is an acetyl-CoA:acetyl-CoA acyltransferase, wherein 5D is an
acetoacetyl-CoA reductase (ketone reducing), wherein 5E is a
3-hydroxybutyryl-CoA reductase (aldehyde forming), wherein 5F is a
3-hydroxybutyryl-CoA hydrolase, 3-hydroxybutyryl-CoA transferase or
3-hydroxybutyryl-CoA synthetase, wherein 5G is a 3-hydroxybutyrate
reductase, wherein 5H is a 3-hydroxybutyraldehyde reductase,
wherein 5J is a 3-hydroxybutyryl-CoA dehydratase, wherein 5K is a
crotonyl-CoA reductase (aldehyde forming), wherein 5L is a
crotonyl-CoA hydrolase, crotonyl-CoA transferase or crotonyl-CoA
synthetase, wherein 5M is a crotonate reductase, wherein 5N is a
crotonaldehyde reductase, wherein 50 is a crotyl alcohol kinase,
wherein 5P is a 2-butenyl-4-phosphate kinase, wherein 5Q is a
butadiene synthase, wherein 5R is a crotyl alcohol diphosphokinase,
wherein 5S is chemical dehydration or a crotyl alcohol dehydratase,
wherein 5T is a butadiene synthase (monophosphate), wherein 5U is a
crotonyl-CoA reductase (alcohol forming), wherein 5V is a
3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 6A is a
1,3-butanediol kinase, wherein 6B is a 3-hydroxybutyrylphosphate
kinase, wherein 6C is a 3-hydroxybutyryldiphosphate lyase, wherein
6D is a 1,3-butanediol diphosphokinase, wherein 6E is a
1,3-butanediol dehydratase, wherein 6F is a
3-hydroxybutyrylphosphate lyase, wherein 6G is a 3-buten-2-ol
dehydratase or chemical dehydration.
[0144] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
3-buten-2-ol pathway and an exogenous nucleic acid encoding a
3-buten-2-ol pathway enzyme expressed in a sufficient amount to
produce 3-buten-2-ol, wherein said 3-buten-2-ol pathway comprises a
pathway selected from: (1) 5A, 5B, 5D, 5E, 5H, 6A, 6B, and 6C; (2)
5A, 5B, 5D, 5F, 5G, 5H, 6A, 6B, and 6C; (3) 5C, 5D, 5E, 5H, 6A, 6B,
and 6C; (4) 5C, 5D, 5F, 5G, 5H, 6A, 6B, and 6C; (5) 5A, 5B, 5D, 5E,
5H, 6A, and 6F; (6) 5A, 5B, 5D, 5F, 5G, 5H, 6A, and 6F; (7) 5C, 5D,
5E, 5H, 6A, and 6F; (8) 5C, 5D, 5F, 5G, 5H, 6A, and 6F; (9) 5A, 5B,
5D, 5E, 5H, 6E, and 6C; (10) 5A, 5B, 5D, 5F, 5G, 5H, 6E, and 6C;
(11) 5C, 5D, 5E, 5H, 6E, and 6C; (12) 5C, 5D, 5F, 5G, 5H, 6E, and
6C; (13) 5A, 5B, 5D, 5E, 5H, and 6D; (14) 5A, 5B, 5D, 5F, 5G, 5H,
and 6D; (15) 5C, 5D, 5E, 5H, and 6D; (16) 5C, 5D, 5F, 5G, 5H, and
6D; (17) 5A, 5B, 5D, 5V, 6A, 6B, and 6C; (18) 5C, 5D, 5V, 6A, 6B,
and 6C; (19) 5A, 5B, 5D, 5V, 6A, and 6F; (20) 5C, 5D, 5V, 6A, and
6F; (21) 5A, 5B, 5D, 5V, 6E, and 6C; (22) 5C, 5D, 5V, 6E, and 6C;
(23) 5A, 5B, 5D, 5V and 6D; and (24) 5C, 5D, 5V and 6D, wherein 5A
is an acetyl-CoA carboxylase, wherein 5B is an acetoacetyl-CoA
synthase, wherein 5C is an acetyl-CoA:acetyl-CoA acyltransferase,
wherein 5D is an acetoacetyl-CoA reductase (ketone reducing),
wherein 5E is a 3-hydroxybutyryl-CoA reductase (aldehyde forming),
wherein 5F is a 3-hydroxybutyryl-CoA hydrolase,
3-hydroxybutyryl-CoA transferase or 3-hydroxybutyryl-CoA
synthetase, wherein 5G is a 3-hydroxybutyrate reductase, wherein 5H
is a 3-hydroxybutyraldehyde reductase, wherein 5V is a
3-hydroxybutyryl-CoA reductase (alcohol forming), wherein 6A is a
1,3-butanediol kinase, wherein 6B is a 3-hydroxybutyrylphosphate
kinase, wherein 6C is a 3-hydroxybutyryldiphosphate lyase, wherein
6D is a 1,3-butanediol diphosphokinase, wherein 6E is a
1,3-butanediol dehydratase, wherein 6F is a
3-hydroxybutyrylphosphate lyase.
[0145] In some aspects the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
1,4-butanediol pathway and an exogenous nucleic acid encoding a
1,4-butanediol pathway enzyme expressed in a sufficient amount to
produce 1,4-butanediol, wherein said 1,4-butanediol pathway
comprises a pathway selected from: (1) 7B, 7C, 7D, 7E, 7F, and 7G;
(2) 7A, 7H, 7C, 7D, 7E, 7F, and 7G; (3) 7I, 7D, 7E, 7F, and 7G; (4)
7B, 7C, 7K, and 7G; (5) 7A, 7H, 7C, 7K, and 7G; (6) 7I, 7K, and 7G;
(7) 7B, 7C, 7D, 7L, and 7G; (8) 7A, 7H, 7C, 7D, 7L, and 7G; (9) 7I,
7D, 7L, and 7G; (10) 7B, 7C, 7J, 7F, and 7G; (11) 7A, 7H, 7C, 7J,
7F, and 7G; (12) 7I, 7J, 7F, and 7G; (13) 7B, 7C, 7D, 7E, and 7M;
(14) 7A, 7H, 7C, 7D, 7E, and 7M; and (15) 7I, 7D, 7E, and 7M,
wherein 7A is a succinyl-CoA transferase or a succinyl-CoA
synthetase, wherein 7B is a succinyl-CoA reductase (aldehyde
forming), wherein 7C is a 4-HB dehydrogenase, wherein 7D is a 4-HB
kinase, wherein 7E is a phosphotrans-4-hydroxybutyrylase, wherein
7F is a 4-hydroxybutyryl-CoA reductase (aldehyde forming), wherein
7G is a 1,4-butanediol dehydrogenase, wherein 7H is a succinate
reductase, wherein 71 is a succinyl-CoA reductase (alcohol
forming), wherein 7J is a 4-hydroxybutyryl-CoA transferase or
4-hydroxybutyryl-CoA synthetase, wherein 7K is a 4-HB reductase,
wherein 7L is a 4-hydroxybutyryl-phosphate reductase, wherein 7M is
a 4-hydroxybutyryl-CoA reductase (alcohol forming).
[0146] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise an
adipate pathway and an exogenous nucleic acid encoding an adipate
pathway enzyme expressed in a sufficient amount to produce adipate,
wherein said adipate pathway comprises 8A, 8B, 8C, 8D and 8L,
wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is a
3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoA
dehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,
wherein 8L is an adipyl-CoA hydrolase, adipyl-CoA ligase,
adipyl-CoA transferase, or phosphotransadipylase/adipate
kinase.
[0147] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
6-aminocaproate pathway and an exogenous nucleic acid encoding a
6-aminocaproate pathway enzyme expressed in a sufficient amount to
produce 6-aminocaproate, wherein said 6-aminocaproate pathway
comprises 8A, 8B, 8C, 8D, 8E, and 8F, wherein 8A is a
3-oxoadipyl-CoA thiolase, wherein 8B is a 3-oxoadipyl-CoA
reductase, wherein 8C is a 3-hydroxyadipyl-CoA dehydratase, wherein
8D is a 5-carboxy-2-pentenoyl-CoA reductase, wherein 8E is an
adipyl-CoA reductase (aldehyde forming), wherein 8F is a
6-aminocaproate transaminase or 6-aminocaproate dehydrogenase.
[0148] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
caprolactam pathway and an exogenous nucleic acid encoding a
caprolactam pathway enzyme expressed in a sufficient amount to
produce caprolactam, wherein said caprolactam pathway comprises:
(1) 8A, 8B, 8C, 8D, 8E, 8F, and 8H; or (2) 8A, 8B, 8C, 8D, 8E, 8F,
8G, and 8I, wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is
a 3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoA
dehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,
wherein 8E is an adipyl-CoA reductase (aldehyde forming), wherein
8F is a 6-aminocaproate transaminase or 6-aminocaproate
dehydrogenase, wherein 8G is a 6-aminocaproyl-CoA/acyl-CoA
transferase or 6-aminocaproyl-CoA synthase, wherein 8H is an
amidohydrolase, wherein 81 is spontaneous cyclization.
[0149] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
hexamethylenediamine pathway and an exogenous nucleic acid encoding
a hexamethylenediamine pathway enzyme expressed in a sufficient
amount to produce hexamethylenediamine, wherein said
hexamethylenediamine pathway comprises 8A, 8B, 8C, 8D, 8E, 8F, 8G,
8J, 8K, wherein 8A is a 3-oxoadipyl-CoA thiolase, wherein 8B is a
3-oxoadipyl-CoA reductase, wherein 8C is a 3-hydroxyadipyl-CoA
dehydratase, wherein 8D is a 5-carboxy-2-pentenoyl-CoA reductase,
wherein 8E is an adipyl-CoA reductase (aldehyde forming), wherein
8F is a 6-aminocaproate transaminase or 6-aminocaproate
dehydrogenase, wherein 8G is a 6-aminocaproyl-CoA/acyl-CoA
transferase or 6-aminocaproyl-CoA synthase, wherein 8J is a
6-aminocaproyl-CoA reductase (aldehyde forming), wherein 8K is a
hexamethylenediamine transaminase or hexamethylenediamine
dehydrogenase.
[0150] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
methacrylic acid pathway and an exogenous nucleic acid encoding a
methacrylic acid pathway enzyme expressed in a sufficient amount to
produce methacrylic acid, wherein said methacrylic acid pathway
comprises a pathway selected from: (1) 9A, 9B, 9C, 9D, and 9E; (2)
9A, 9F, and 9E; (3) 9A, 9B, 9F, and 9E; (4) 9A, 9C, 9D, and 9E; and
(5) 10A, 10B, 10C, 10D, and 10E, wherein 9A is a methylmalonyl-CoA
mutase, wherein 9B is a methylmalonyl-CoA epimerase, wherein 9C is
a methylmalonyl-CoA reductase (aldehyde forming), wherein 9D is a
methylmalonate semialdehyde reductase, wherein 9E is a
3-hydroxyisobutyrate dehydratase, wherein 9F is a methylmalonyl-CoA
reductase (alcohol forming), wherein 10A is an
acetyl-CoA:acetyl-CoA acyltransferase, wherein 10B is an
acetoacetyl-CoA reductase (ketone reducing), wherein 10C is a
3-hydroxybutyrl-CoA mutase, wherein 10D is a
2-hydroxyisobutyryl-CoA dehydratase, wherein 10E is a
methacrylyl-CoA synthetase, methacrylyl-CoA hydrolase, or
methacrylyl-CoA transferase.
[0151] In some aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can further comprise a
2-hydroxyisobutyric acid pathway and an exogenous nucleic acid
encoding a 2-hydroxyisobutyric acid pathway enzyme expressed in a
sufficient amount to produce 2-hydroxyisobutyric acid, wherein said
2-hydroxyisobutyric acid pathway comprises 10A, 10B, 10C, and 10F,
wherein 10A is an acetyl-CoA:acetyl-CoA acyltransferase, wherein
10B is an acetoacetyl-CoA reductase (ketone reducing), wherein 10C
is a 3-hydroxybutyrl-CoA mutase, wherein 10F is a
2-hydroxyisobutyryl-CoA synthetase, 2-hydroxyisobutyryl-CoA
hydrolase, or 2-hydroxyisobutyryl-CoA transferase.
[0152] In certain embodiments, such as the above non-naturally
occurring microbial organisms having an acetyl-CoA pathway and
1,4-butanediol pathway, an adipate pathway, a 6-aminocaproate
pathway, a caprolactam pathway, a hexamethylenediamine pathway or a
methacrylic acid pathway, the non-naturally occurring microbial
organism can further comprise a succinyl-CoA pathway and an
exogenous nucleic acid encoding a succinyl-CoA pathway enzyme
expressed in a sufficient amount to produce succinyl-CoA, wherein
said succinyl-CoA pathway comprises a pathway selected from: (1)
4H, 4I, 4J, and 4K; (2) 4H, 4I, 4N, and 4E; (3) 4H, 4I, 4N, 4C, 4D,
and 4E; (4) 4L, 4H, 4I, 4J, and 4K; (5) 4L, 4H, 4I, 4N, and 4E; (6)
4L, 4H, 4I, 4N, 4C, 4D, and 4E; (7) 4A, 4H, 4I, 4J, and 4K; (8) 4A,
4H, 4I, 4N, and 4E; (9) 4A, 4H, 4I, 4N, 4C, 4D, and 4E; (10) 4M,
4C, 4D, and 4E; (11) 4F, 4L, 4H, 4I, 4J, and 4K; (12) 4F, 4L, 4H,
4I, 4N, and 4E; (13) 4F, 4L, 4H, 4I, 4N, 4C, 4D, and 4E; (14) 4F,
4M, 4C, 4D, and 4E; (15) 4F, 4G, 4H, 4I, 4J, and 4K; (16) 4F, 4G,
4H, 4I, 4N, and 4E; and (17) 4F, 4G, 4H, 4I, 4N, 4C, 4D, and 4E,
wherein 4A is a PEP carboxylase or PEP carboxykinase, wherein 4B is
a malate dehydrogenase, wherein 4C is a fumarase, wherein 4D is a
fumarate reductase, wherein 4E is a succinyl-CoA synthetase or
succinyl-CoA transferase, wherein 4F is a pyruvate kinase or
PTS-dependent substrate import, wherein 4G is a pyruvate
dehydrogenase, pyruvate formate lyase, or pyruvate:ferredoxin
oxidoreductase, wherein 4H is a citrate synthase, wherein 41 is an
aconitase, wherein 4J is an isocitrate dehydrogenase, wherein 4K is
an alpha-ketoglutarate dehydrogenase, wherein 4L is a pyruvate
carboxylase, wherein 4M is a malic enzyme, wherein 4N is an
isocitrate lyase and malate synthase.
[0153] In certain aspects, the non-naturally occurring microbial
organism having an acetyl-CoA pathway can be a microbial organism
species selected from a bacteria, yeast, or fungus.
[0154] The invention further provides non-naturally occurring
microbial organisms that have elevated or enhanced synthesis or
yields of acetyl-CoA or a bioderived compound and methods of using
those non-naturally occurring organisms to produce such
biosynthetic products, the bioderived compound including alcohols,
diols, fatty acids, glycols, organic acids, alkenes, dienes,
organic amines, organic aldehydes, vitamins, nutraceuticals and
pharmaceuticals. The enhanced synthesis of intracellular acetyl-CoA
enables enhanced production of bioderived compounds for which
acetyl-CoA is an intermediate and further, may have been
rate-limiting.
[0155] The non-naturally occurring microbial organisms having
enhanced yields of a biosynthetic product include one or more of
the various pathway configurations employing a methanol
dehydrogenase for methanol oxidation, a formaldehyde fixation
pathway and/or an acetyl-CoA enhancing pathway, e.g.
phosphoketolase, for directing the carbon from methanol into
acetyl-CoA and other desired products via formaldehyde fixation.
The various different methanol oxidation and formaldehyde fixation
configurations exemplified below can be engineered in conjunction
with any or each of the various methanol oxidation, formaldehyde
fixation, formate reutilization, acetyl-CoA or bioderived compound
pathways exemplified previously and herein. The metabolic
modifications exemplified below increase biosynthetic product
yields over, for example, endogenous methanol utilization pathways
because they further focus methanol derived carbon into the
assimilation pathways described herein, decrease inefficient use of
methanol carbon through competing methanol utilization and/or
formaldehyde fixation pathways and/or increase the production of
reducing equivalents.
[0156] In this regard, methylotrophs microbial organisms utilize
methanol as the sole source of carbon and energy. In such
methylotrophic organisms, the oxidation of methanol to formaldehyde
is catalyzed by one of three different enzymes: NADH dependent
methanol dehydrogenase (MeDH), PQQ-dependent methanol dehydrogenase
(MeDH-PQQ) and alcohol oxidase (AOX). Methanol oxidase is a
specific type of AOX with activity on methanol. Gram positive
bacterial methylotrophs such as Bacillus methanolicus utilize a
cytosolic MeDH which generates reducing equivalents in the form
ofNADH. Gram negative bacterial methylotrophs utilize periplasmic
PQQ-containing methanol dehydrogenase enzymes which transfer
electrons from methanol to specialized cytochromes CL, and
subsequently to a cytochrome oxidase (Afolabi et al, Biochem
40:9799-9809 (2001)). Eukaryotic methylotrophs employ a peroxisomal
oxygen-consuming and hydrogen-peroxide producing alcohol
oxidase.
[0157] Bacterial methylotrophs are found in in the genera Bacillus,
Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis
and Hyphomicrobium. These organisms utilize either the serine cycle
(type or the RuMP cycle (type I) to further assimilate formaldehyde
into central metabolism (Hanson and Hanson, Microbiol Rev
60:439-471 (1996)). As described previously, the RuMP pathway
combines formaldehyde with ribulose monophosphate to form
hexulose-6-phosphate, which is further converted to
fructose-6-phosphate (see FIG. 1, step C). In the serine cycle
formaldehyde is initially converted to 5,10-methylene-THF, which is
combined with glycine to form serine. Overall, the reactions of the
serine cycle produce one equivalent of acetyl-CoA from three
equivalents of methanol (Anthony, Science Prog 94:109-37 (2011)).
The RuMP cycle also yields one equivalent of acetyl-CoA from three
equivalents methanol in the absence of phosphoketolase activity or
a formate assimilation pathway. Genetic tools are available for
numerous prokaryotic methylotrophs and methanotrophs.
[0158] Eukaryotic methylotrophs are found in the genera Candida,
Pichia, Ogataea, Kuraishia and Komagataella. Particularly useful
methylotrophic host organisms are those with well-characterized
genetic tools and gene expression systems such as Hansenula
polymorpha, Pichia pastor's, Candida boidinii and Pichia
methanolica (for review see Yurimoto et al, Int J Microbiol
(2011)). The initial step of methanol assimilation in eukaryotic
methylotrophs occurs in the peroxisomes, where methanol and oxygen
are oxidized to formaldehyde and hydrogen peroxide by alcohol
oxidase (AOX). Formaldehyde assimilation with xylulose-5-phosphate
via DHA synthase also occurs in the peroxisomes. During growth on
methanol, the two enzymes DHA synthase and AOX together comprise
80% of the total cell protein (Horiguchi et al, J Bacteriol
183:6372-83 (2001)). DHA synthase products, DHA and
glyceraldehyde-3-phosphate, are secreted into the cytosol where
they undergo a series of rearrangements catalyzed by pentose
phosphate pathway enzymes, and are ultimately converted to cellular
constituents and xylulose-5-phosphate, which is transported back
into the peroxisomes. The initial step of formaldehyde
dissimilation, catalyzed by S-(hydroxymethyl)-glutathione synthase,
also occurs in the peroxisomes. Like the bacterial methylotrophic
pathways described above, eukaryotic methylotrophic pathways
convert three equivalents of methanol to at most one equivalent of
acetyl-CoA because they lack phosphoketolase activity or a formate
assimilation pathway.
[0159] As exemplified further below, the various configurations of
metabolic modifications disclosed herein for enhancing product
yields via methanol derived carbon include enhancing methanol
oxidation and production of reducing equivalents using either and
an endogenous NADH dependent methanol dehydrogenase, an exogenous
NADH dependent methanol dehydrogenase, both an endogenous NADH
dependent methanol dehydrogenase and exogenous NADH dependent
methanol dehydrogenase alone or in combination with one or more
metabolic modifications that attenuate, for example, DHA synthase
and/or AOX. In addition, other metabolic modifications as
exemplified below that reduce carbon flux away from methanol
oxidation and formaldehyde fixation also can be included, alone or
in combination, with the methanol oxidation and formaldehyde
fixation pathway configurations disclosed herein that enhance
carbon flux into product precursors such as acetyl-CoA and,
therefore, enhance product yields.
[0160] Accordingly, the microbial organism of the invention having
one or more of any of the above and/or below metabolic
modifications to a methanol utilization pathway and/or formaldehyde
assimilation pathway configurations for enhancing product yields
can be combined with any one or more, including all of the
previously described methanol oxidation, formaldehyde fixation,
formate reutilization, and/or acetyl-CoA pathways to enhance the
yield and/or production of a product such as any of the bioderived
compounds described herein.
[0161] Given the teachings and guidance provided herein, the
methanol oxidation and formaldehyde fixation pathway configurations
can be equally engineered into both prokaryotic and eukaryotic
organisms. In prokaryotic microbial organisms, for example, one
skilled in the art will understand that utilization of an
endogenous methanol oxidation pathway enzyme or expression of an
exogenous nucleic acid encoding a methanol oxidation pathway enzyme
will naturally occur cytosolically because prokaryotic organisms
lack peroxisomes. In eukaryotic microbial organisms one skilled in
the art will understand that certain methanol oxidation pathways
occur in the peroxisome as described above and that cytosolic
expression of the methanol oxidation pathway or pathways described
herein to enhance product yields can be beneficial. The peroxisome
located pathways and competing pathways remain or, alternatively,
attenuated as described below to further enhance methanol oxidation
and formaldehyde fixation.
[0162] With respect to eukaryotic microbial host organisms, those
skilled in the art will know that yeasts and other eukaryotic
microorganisms exhibit certain characteristics distinct from
prokaryotic microbial organisms. When such characteristics are
desirable, one skilled in the art can choose to use such eukaryotic
microbial organisms as a host for engineering the various different
methanol oxidation and formaldehyde fixation configurations
exemplified herein for enhancing product yields. For example, yeast
are robust organisms, able to grow over a wide pH range and able to
tolerate more impurities in the feedstock. Yeast also ferment under
low growth conditions and are not susceptible to infection by
phage. Less stringent aseptic design requirements can also reduce
production costs. Cell removal, disposal and propagation are also
cheaper, with the added potential for by-product value for animal
feed applications. The potential for cell recycle and
semi-continuous fermentation offers benefits in increased overall
yields and rates. Other benefits include: potential for extended
fermentation times under low growth conditions, lower viscosity
broth (vs E. coli) with insoluble hydrophobic products, the ability
to employ large fermenters with external loop heat exchangers.
[0163] Eukaryotic host microbial organisms suitable for engineering
carbon efficient methanol utilization capability can be selected
from, and the non-naturally occurring microbial organisms generated
in, for example, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. As described
previously, exemplary yeasts or fungi include species selected from
the genera Saccharomyces, Schizosaccharomyces, Schizochytrium,
Rhodotorula, Thraustochytrium, Aspergillus, Kluyveromyces,
Issatchenkia, Yarrowia, Candida, Pichia, Ogataea, Kuraishia,
Hansenula and Komagataella. Useful host organisms include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula
polymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces
lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus
niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae,
Yarrowia lipolytica, Issatchenkia orientalis and the like.
[0164] The methanol oxidation and/or formaldehyde assimilation
pathway configurations described herein for enhancing product
yields include, for example, a NADH-dependent methanol
dehydrogenase (MeDH), one or more formaldehyde assimilation
pathways and/or one or more phosphoketolases. Such engineered
pathways provide a yield advantage over endogenous pathways found
in methylotrophic organisms. For example, methanol assimilation via
methanol dehydrogenase provides reducing equivalents in the useful
form of NADH, whereas alcohol oxidase and PQQ-dependent methanol
dehydrogenase do not. Several product pathways described herein
have several NADH-dependent enzymatic steps. In addition, deletion
of redox-inefficient methanol oxidation enzymes as described
further below, combined with increased cytosolic or peroxisomal
expression of an NADH-dependent methanol dehydrogenase, improves
the ability of the organism to extract useful reducing equivalents
from methanol. In some aspects, if NADH-dependent methanol
dehydrogenase is engineered into the peroxisome, an efficient means
of shuttling redox in the form of NADH out of the peroxisome and
into the cytosol can be included. Further employment of a
formaldehyde assimilation pathway in combination with a
phosphoketolase or formate assimilation pathway enables high yield
conversion of methanol to acetyl-CoA, and subsequently to
acetyl-CoA derived products.
[0165] For example, in a eukaryotic organism such as Pichia
pastoris, deleting the endogenous alcohol oxidase and peroxisomal
formaldehyde assimilation and dissimilation pathways, and
expressing redox and carbon-efficient cytosolic methanol
utilization pathways significantly improves the yield of dodecanol,
an acetyl-CoA derived product. The maximum docidecanol yield of
Pichia pastoris from methanol using endogenous methanol oxidase and
formaldehyde assimilation enzymes is 0.256 g dodecanol/g methanol.
Adding one or more heterologous cytosolic phosphoketolase enzymes,
in combination with a formaldehyde assimilation pathway such as the
DHA pathway or the RUMP pathway, boosts the dodecanol yield to
0.306 g dodecanol/g methanol. Deletion of peroxisomal methanol
oxidase and formaldehyde assimilation pathway enzymes (alcohol
oxidase, DHA synthase), and replacement with cytosolic methanol
dehydrogenase (NADH dependent) and formaldehyde assimilation
pathways, together with a phosphoketolase, provides a significant
boost of yield to 0.422 g/g.
TABLE-US-00008 Strain design (assumes DHA pathway) Max FA yield (g
dodecanol/g MeOH) Pichia + AOX + fatty acid pathway 0.256 Pichia +
AOX + PK 0.306 Pichia + MeDH + PK 0.422
[0166] The combination of NADH-dependent methanol dehydrogenase and
phosphoketolase together results in a significant boost in yield
for other acetyl-CoA derived products. For 13-BDO production as
shown via the pathway in FIG. 5, methanol dehydrogenase in
combination with phosphoketolase improves the yield from 0.469 to
0.703 g 13-BDO/g methanol.
TABLE-US-00009 Strain design (assumes RuMP pathway) MeOH per
1,3-BDO (mol/mol) 13-BDO per MeOH (g/g) 13-BDO + AOX 6 .469 13-BDO
+ AOX + PK 5.778 .487 13-BDO + MeDH + PK 4 .703
[0167] Metabolic modifications for enabling redox- and
carbon-efficient cytosolic methanol utilization in a eukaryotic or
prokaryotic organism are exemplified in further detail below.
[0168] In one embodiment, the invention provides cytosolic
expression of one or more methanol oxidation and/or formaldehyde
assimilation pathways Engineering into a host microbial organism
carbon- and redox-efficient cytosolic formaldehyde assimilation can
be achieved by expression of one or more endogenous or exogenous
methanol oxidation pathways and/or one or more endogenous or
exogenous formaldehyde assimilation pathway enzymes in the cytosol.
An exemplary pathway for methanol oxidation includes NADH dependent
methanol dehydrogenase as shown in FIG. 1. Exemplary pathways for
converting cytosolic formaldehyde into glycolytic intermediates
also are shown in FIG. 1. Such pathways include methanol oxidation
via expression of an cytosolic NADH dependent methanol
dehydrogenase, formaldehyde fixation via expression of cytosolic
DHA synthase, both methanol oxidation via expression of an
cytosolic NADH dependent methanol dehydrogenase and formaldehyde
fixation via expression of cytosolic DHA synthase alone or together
with the metabolic modifications exemplified below that attenuate
less beneficial methanol oxidation and/or formaldehyde fixation
pathways. Such attenuating metabolic modifications include, for
example, attenuation of alcohol oxidase, attenuation of DHA kinase
and/or when utilization of ribulose-5-phosphate (Ru5P) pathway for
formaldehyde fixation attenuation of DHA synthase.
[0169] For example, in the carbon-efficient DHA pathway of
formaldehyde assimilation shown in FIG. 1, step D, formaldehyde is
converted to dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate
(GAP) by DHA synthase (FIG. 1D). DHA and G3P are then converted to
fructose-6-phosphate in one step by F6P aldolase (FIG. 1C) or in
three steps by DHA kinase, FBP aldolase and
fructose-1,6-bisphosphatase (not shown). Formation of F6P from DHA
and G3P by F6P aldolase is more ATP efficient than using DHA
kinase, FBP aldolase, and fructose-1,6-bisphosphatase.
Rearrangement of F6P and E4P by enzymes of the pentose phosphate
pathway (transaldolase, transketolase, R5P epimerase and Ru5P
epimerase) regenerates xylulose-5-phosphate, the DHA synthase
substrate. Conversion of F6P to acetyl-phosphate and E4P (FIG. 1T),
or Xu5P to G3P and acetyl-phosphate (FIGS. 1T and 1U) by one or
more phosphoketolase enzymes results in the carbon-efficient
generation of cytosolic acetyl-CoA. Exemplary enzymes catalyzing
each step of the carbon efficient DHA pathway are described
elsewhere herein.
[0170] An alternate carbon efficient pathway for formaldehyde
assimilation proceeding through ribulose-5-phosphate (Ru5P) is
shown in FIG. 1, step B. The formaldehyde assimilation enzyme of
this pathway is 3-hexulose-6-phosphate synthase, which combines
ru5p and formaldehyde to form hexulose-6-phosphate (FIG. 1B).
6-Phospho-3-hexuloisomerase converts H6P to F6P (FIG. 1C).
Regeneration of Ru5P from F6P proceeds by pentose phosphate pathway
enzymes. Carbon-efficient phosphoketolase enzymes catalyze the
conversion of F6P and/or Xu5P to acetyl-phosphate and pentose
phosphate intermediates. Exemplary enzymes catalyzing each step of
the carbon efficient RuMP pathway are described elsewhere herein.
Yet another approach is to combine the RuMP and DHA pathways
together (FIG. 1).
[0171] Thus, in this embodiment, conversion of cytosolic
formaldehyde into glycolytic intermediates can occur via expression
of a cytosolic 3-hexulose-6-phosphate (3-Hu6P) synthase and
6-phospho-3-hexuloisomerase. Thus, exemplary pathways that can be
engineered into a microbial organism of the invention can include
methanol oxidation via expression of a cytosolic NADH dependent
methanol dehydrogenase, formaldehyde fixation via expression of
cytosolic 3-Hu6P synthase and 6-phospho-3-hexuloisomerase, both
methanol oxidation via expression of an cytosolic NADH dependent
methanol dehydrogenase and formaldehyde fixation via expression of
cytosolic 3-Hu6P synthase and 6-phospho-3-hexuloisomerase alone or
together with the metabolic modifications exemplified below that
attenuate less beneficial methanol oxidation and/or formaldehyde
fixation pathways. Such attenuating metabolic modifications
include, for example, attenuation of alcohol oxidase, attenuation
of DHA kinase and/or attenuation of DHA kinase and/or attenuation
of DHA synthase (e.g. when ribulose-5-phosphate (Ru5P) pathway for
formaldehyde fixation is utilized).
[0172] In yet another embodiment increased product yields can be
accomplished by engineering into the host microbial organism of the
invention both the RuMP and DHA pathways as shown in FIG. 1. In
this embodiment, the microbial organisms can have cytosolic
expression of one or more methanol oxidation and/or formaldehyde
assimilation pathways. The formaldehyde assimilation pathways can
include both assimilation through cytosolic DHA synthase and 3-Hu6P
synthase. Such pathways include methanol oxidation via expression
of a cytosolic NADH dependent methanol dehydrogenase, formaldehyde
fixation via expression of cytosolic DHA synthase and 3-Hu6P
synthase, both methanol oxidation via expression of an cytosolic
NADH dependent methanol dehydrogenase and formaldehyde fixation via
expression of cytosolic DHA synthase and 3-Hu6P synthase alone or
together with the metabolic modifications exemplified previously
and also below that attenuate less beneficial methanol oxidation
and/or formaldehyde fixation pathways. Such attenuating metabolic
modifications include, for example, attenuation of alcohol oxidase,
attenuation of DHA kinase and/or attenuation of DHA kinase and/or
attenuation of DHA synthase (e.g. when ribulose-5-phosphate (Ru5P)
pathway for formaldehyde fixation is utilized).
[0173] Increasing the expression and/or activity of one or more
formaldehyde assimilation pathway enzymes in the cytosol can be
utilized to assimilate formaldehyde at a high rate. Increased
activity can be achieved by increased expression, altering the
ribosome binding site, altering the enzyme activity, or altering
the sequence of the gene to ensure, for example, that codon usage
is balanced with the needs of the host organism, or that the enzyme
is targeted to the cytosol as disclosed herein.
[0174] In some embodiments, the invention provides a non-naturally
occurring microbial organism as described herein, wherein the
microbial organism further includes attenuation of one or more
endogenous enzymes, which enhances carbon flux through acetyl-CoA.
For example, in some aspects, the endogenous enzyme can be selected
from DHA kinase, methanol oxidase, PQQ-dependent methanol
dehydrogenase, DHA synthase or any combination thereof.
Accordingly, in some aspects, the attenuation is of the endogenous
enzyme DHA kinase. In some aspects, the attenuation is of the
endogenous enzyme methanol oxidase. In some aspects, the
attenuation is of the endogenous enzyme PQQ-dependent methanol
dehydrogenase. In some aspects, the attenuation is of the
endogenous enzyme DHA synthase. The invention also provides a
microbial organism wherein attenuation is of any combination of two
or three endogenous enzymes described herein. For example, a
microbial organism of the invention can include attenuation of DHA
kinase and DHA synthase, or alternatively methanol oxidase and
PQQ-dependent methanol dehydrogenase, or alternatively DHA kinase,
methanol oxidase, and PQQ-dependent methanol dehydrogenase, or
alternatively DHA kinase, methanol oxidase, and DHA synthase. The
invention also provides a microbial organism wherein attenuation is
of all endogenous enzymes described herein. For example, in some
aspects, a microbial organism described herein includes attenuation
of DHA kinase, methanol oxidase, PQQ-dependent methanol
dehydrogenase and DHA synthase.
[0175] In some embodiments, the invention provides a non-naturally
occurring microbial organism as described herein, wherein the
microbial organism further includes attenuation of one or more
endogenous enzymes of a competing formaldehyde assimilation or
dissimilation pathway. Examples of these endogenous enzymes are
disclosed in FIG. 1 and described in Example XIII. It is understood
that a person skilled in the art would be able to readily identify
enzymes of such competing pathways. Competing pathways can be
dependent upon the host microbial organism and/or the exogenous
nucleic acid introduced into the microbial organism as described
herein. Accordingly, in some aspects of the invention, the
microbial organism includes attenuation of one, two, three, four,
five, six, seven, eight, nine, ten or more endogenous enzymes of a
competing formaldehyde assimilation or dissimilation pathway.
[0176] In some embodiments, the invention provides a non-naturally
occurring microbial organism as described herein, wherein the
microbial organism further includes a gene disruption of one or
more endogenous nucleic acids encoding enzymes, which enhances
carbon flux through acetyl-CoA. For example, in some aspects, the
endogenous enzyme can be selected from DHA kinase, methanol
oxidase, PQQ-dependent methanol dehydrogenase, DHA synthase or any
combination thereof. According, in some aspects, the gene
disruptiondisruption is of an endogenous nucleic acid encoding the
enzyme DHA kinase. In some aspects, the gene disruptiondisruption
is of an endogenous nucleic acid encoding the enzyme methanol
oxidase. In some aspects, the gene disruptiondisruption is of an
endogenous nucleic acid encoding the enzyme PQQ-dependent methanol
dehydrogenase. In some aspects, the gene disruption is of an
endogenous nucleic acid encoding the enzyme DHA synthase. The
invention also provides a microbial organism wherein the gene
disruption is of any combination of two or three nucleic acids
encoding endogenous enzymes described herein. For example, a
microbial organism of the invention can include a gene disruption
of DHA kinase and DHA synthase, or alternatively methanol oxidase
and PQQ-dependent methanol dehydrogenase, or alternatively DHA
kinase, methanol oxidase, and PQQ-dependent methanol dehydrogenase,
or alternatively DHA kinase, methanol oxidase, and DHA synthase.
The invention also provides a microbial organism wherein all
endogenous nucleic acids encoding enzymes described herein are
disrupted. For example, in some aspects, a microbial organism
described herein includes disruption of DHA kinase, methanol
oxidase, PQQ-dependent methanol dehydrogenase and DHA synthase.
[0177] In some embodiments, the invention provides a non-naturally
occurring microbial organism as described herein, wherein the
microbial organism further includes a gene disruption of one or
more endogenous enzymes of a competing formaldehyde assimilation or
dissimilation pathway. Examples of these endogenous enzymes are
disclosed in FIG. 1 and described in Example XIII. It is understood
that a person skilled in the art would be able to readily identify
enzymes of such competing pathways. Competing pathways can be
dependent upon the host microbial organism and/or the exogenous
nucleic acid introduced into the microbial organism as described
herein. Accordingly, in some aspects of the invention, the
microbial organism includes a gene disruption of one, two, three,
four, five, six, seven, eight, nine, ten or more endogenous nucleic
acids encoding enzymes of a competing formaldehyde assimilation or
dissimilation pathway.
[0178] In the case of gene disruptions, a particularly useful
stable genetic alteration is a gene deletion. The use of a gene
deletion to introduce a stable genetic alteration is particularly
useful to reduce the likelihood of a reversion to a phenotype prior
to the genetic alteration. For example, stable growth-coupled
production of a biochemical can be achieved, for example, by
deletion of a gene encoding an enzyme catalyzing one or more
reactions within a set of metabolic modifications. The stability of
growth-coupled production of a biochemical can be further enhanced
through multiple deletions, significantly reducing the likelihood
of multiple compensatory reversions occurring for each disrupted
activity.
[0179] Also provided is a method of producing a non-naturally
occurring microbial organisms having stable growth-coupled
production of acetyl-CoA or a bioderived compound. The method can
include identifying in silico a set of metabolic modifications that
increase production of acetyl-CoA or a bioderived compound, for
example, increase production during exponential growth; genetically
modifying an organism to contain the set of metabolic modifications
that increase production of acetyl-CoA or a bioderived compound,
and culturing the genetically modified organism. If desired,
culturing can include adaptively evolving the genetically modified
organism under conditions requiring production of acetyl-CoA or a
bioderived compound. The methods of the invention are applicable to
bacterium, yeast and fungus as well as a variety of other cells and
microorganism, as disclosed herein.
[0180] Thus, the invention provides a non-naturally occurring
microbial organism comprising one or more gene disruptions that
confer increased synthesis or production of acetyl-CoA or a
bioderived compound. In one embodiment, the one or more gene
disruptions confer growth-coupled production of acetyl-CoA or a
bioderived compound, and can, for example, confer stable
growth-coupled production of acetyl-CoA or a bioderived compound.
In another embodiment, the one or more gene disruptions can confer
obligatory coupling of acetyl-CoA or a bioderived compound
production to growth of the microbial organism. Such one or more
gene disruptions reduce the activity of the respective one or more
encoded enzymes.
[0181] The non-naturally occurring microbial organism can have one
or more gene disruptions included in a gene encoding a enzyme or
protein disclosed herein. As disclosed herein, the one or more gene
disruptions can be a deletion. Such non-naturally occurring
microbial organisms of the invention include bacteria, yeast,
fungus, or any of a variety of other microorganisms applicable to
fermentation processes, as disclosed herein.
[0182] Thus, the invention provides a non-naturally occurring
microbial organism, comprising one or more gene disruptions, where
the one or more gene disruptions occur in genes encoding proteins
or enzymes where the one or more gene disruptions confer increased
production of acetyl-CoA or a bioderived compound. The production
of acetyl-CoA or a bioderived compound can be growth-coupled or not
growth-coupled. In a particular embodiment, the production of
acetyl-CoA or a bioderived compound can be obligatorily coupled to
growth of the organism, as disclosed herein.
[0183] The invention provides non naturally occurring microbial
organisms having genetic alterations such as gene disruptions that
increase production of acetyl-CoA or a bioderived compound, for
example, growth-coupled production of acetyl-CoA or a bioderived
compound. Product production can be, for example, obligatorily
linked to the exponential growth phase of the microorganism by
genetically altering the metabolic pathways of the cell, as
disclosed herein. The genetic alterations can increase the
production of the desired product or even make the desired product
an obligatory product during the growth phase. Metabolic
alterations or transformations that result in increased production
and elevated levels of acetyl-CoA or a bioderived compound
biosynthesis are exemplified herein. Each alteration corresponds to
the requisite metabolic reaction that should be functionally
disrupted. Functional disruption of all reactions within one or
more of the pathways can result in the increased production of
acetyl-CoA or a bioderived compound by the engineered strain during
the growth phase.
[0184] Each of these non-naturally occurring alterations result in
increased production and an enhanced level of acetyl-CoA or a
bioderived compound, for example, during the exponential growth
phase of the microbial organism, compared to a strain that does not
contain such metabolic alterations, under appropriate culture
conditions. Appropriate conditions include, for example, those
disclosed herein, including conditions such as particular carbon
sources or reactant availabilities and/or adaptive evolution.
[0185] Given the teachings and guidance provided herein, those
skilled in the art will understand that to introduce a metabolic
alteration such as attenuation of an enzyme, it can be necessary to
disrupt the catalytic activity of the one or more enzymes involved
in the reaction. Alternatively, a metabolic alteration can include
disrupting expression of a regulatory protein or cofactor necessary
for enzyme activity or maximal activity. Furthermore, genetic loss
of a cofactor necessary for an enzymatic reaction can also have the
same effect as a disruption of the gene encoding the enzyme.
Disruption can occur by a variety of methods including, for
example, deletion of an encoding gene or incorporation of a genetic
alteration in one or more of the encoding gene sequences. The
encoding genes targeted for disruption can be one, some, or all of
the genes encoding enzymes involved in the catalytic activity. For
example, where a single enzyme is involved in a targeted catalytic
activity, disruption can occur by a genetic alteration that reduces
or eliminates the catalytic activity of the encoded gene product.
Similarly, where the single enzyme is multimeric, including
heteromeric, disruption can occur by a genetic alteration that
reduces or destroys the function of one or all subunits of the
encoded gene products. Destruction of activity can be accomplished
by loss of the binding activity of one or more subunits required to
form an active complex, by destruction of the catalytic subunit of
the multimeric complex or by both. Other functions of multimeric
protein association and activity also can be targeted in order to
disrupt a metabolic reaction of the invention. Such other functions
are well known to those skilled in the art. Similarly, a target
enzyme activity can be reduced or eliminated by disrupting
expression of a protein or enzyme that modifies and/or activates
the target enzyme, for example, a molecule required to convert an
apoenzyme to a holoenzyme. Further, some or all of the functions of
a single polypeptide or multimeric complex can be disrupted
according to the invention in order to reduce or abolish the
catalytic activity of one or more enzymes involved in a reaction or
metabolic modification of the invention. Similarly, some or all of
enzymes involved in a reaction or metabolic modification of the
invention can be disrupted so long as the targeted reaction is
reduced or eliminated.
[0186] Given the teachings and guidance provided herein, those
skilled in the art also will understand that an enzymatic reaction
can be disrupted by reducing or eliminating reactions encoded by a
common gene and/or by one or more orthologs of that gene exhibiting
similar or substantially the same activity. Reduction of both the
common gene and all orthologs can lead to complete abolishment of
any catalytic activity of a targeted reaction. However, disruption
of either the common gene or one or more orthologs can lead to a
reduction in the catalytic activity of the targeted reaction
sufficient to promote coupling of growth to product biosynthesis.
Exemplified herein are both the common genes encoding catalytic
activities for a variety of metabolic modifications as well as
their orthologs. Those skilled in the art will understand that
disruption of some or all of the genes encoding a enzyme of a
targeted metabolic reaction can be practiced in the methods of the
invention and incorporated into the non-naturally occurring
microbial organisms of the invention in order to achieve the
increased production of acetyl-CoA or a bioderived compound or
growth-coupled product production.
[0187] Given the teachings and guidance provided herein, those
skilled in the art also will understand that enzymatic activity or
expression can be attenuated using well known methods. Reduction of
the activity or amount of an enzyme can mimic complete disruption
of a gene if the reduction causes activity of the enzyme to fall
below a critical level that is normally required for a pathway to
function. Reduction of enzymatic activity by various techniques
rather than use of a gene disruption can be important for an
organism's viability. Methods of reducing enzymatic activity that
result in similar or identical effects of a gene disruption
include, but are not limited to: reducing gene transcription or
translation; destabilizing mRNA, protein or catalytic RNA; and
mutating a gene that affects enzyme activity or kinetics (See,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed.,
Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md. (1999). Natural or imposed regulatory controls can
also accomplish enzyme attenuation including: promoter replacement
(See, Wang et al., Mol. Biotechnol. 52(2):300-308 (2012)); loss or
alteration of transcription factors (Dietrick et al., Annu. Rev.
Biochem. 79:563-590 (2010); and Simicevic et al., Mol. Biosyst.
6(3):462-468 (2010)); introduction of inhibitory RNAs or peptides
such as siRNA, antisense RNA, RNA or peptide/small-molecule binding
aptamers, ribozymes, aptazymes and riboswitches (Wieland et al.,
Methods 56(3):351-357 (2012); O'Sullivan, Anal. Bioanal. Chem.
372(1):44-48 (2002); and Lee et al., Curr. Opin. Biotechnol.
14(5):505-511(2003)); and addition of drugs or other chemicals that
reduce or disrupt enzymatic activity such as an enzyme inhibitor,
an antibiotic or a target-specific drug.
[0188] One skilled in the art will also understand and recognize
that attenuation of an enzyme can be done at various levels. For
example, at the gene level, a mutation causing a partial or
complete null phenotype, such as a gene disruption, or a mutation
causing epistatic genetic effects that mask the activity of a gene
product (Miko, Nature Education 1(1) (2008)), can be used to
attenuate an enzyme. At the gene expression level, methods for
attenuation include: coupling transcription to an endogenous or
exogenous inducer, such as isopropylthio-.beta.-galactoside (IPTG),
then adding low amounts of inducer or no inducer during the
production phase (Donovan et al., J. Ind. Microbiol. 16(3):145-154
(1996); and Hansen et al., Curr. Microbiol. 36(6):341-347 (1998));
introducing or modifying a positive or a negative regulator of a
gene; modify histone acetylation/deacetylation in a eukaryotic
chromosomal region where a gene is integrated (Yang et al., Curr.
Opin. Genet. Dev. 13(2):143-153 (2003) and Kurdistani et al., Nat.
Rev. Mol. Cell Biol. 4(4):276-284 (2003)); introducing a
transposition to disrupt a promoter or a regulatory gene
(Bleykasten-Brosshans et al., C. R Biol. 33(8-9):679-686 (2011);
and McCue et al., PLoS Genet. 8(2):e1002474 (2012)); flipping the
orientation of a transposable element or promoter region so as to
modulate gene expression of an adjacent gene (Wang et al., Genetics
120(4):875-885 (1988); Hayes, Annu. Rev. Genet. 37:3-29 (2003); in
a diploid organism, deleting one allele resulting in loss of
heterozygosity (Daigaku et al., Mutation Research/Fundamental and
Molecular Mechanisms of Mutagenesis 600(1-2)177-183 (2006));
introducing nucleic acids that increase RNA degradation (Houseley
et al., Cell, 136(4):763-776 (2009); or in bacteria, for example,
introduction of a transfer-messenger RNA (tmRNA) tag, which can
lead to RNA degradation and ribosomal stalling (Sunoham et al., RNA
10(3):378-386 (2004); and Sunoham et al., J. Biol. Chem.
279:15368-15375 (2004)). At the translational level, attenuation
can include: introducing rare codons to limit translation (Angov,
Biotechnol. J. 6(6):650-659 (2011)); introducing RNA interference
molecules that block translation (Castel et al., Nat. Rev. Genet.
14(2):100-112 (2013); and Kawasaki et al., Curr. Opin. Mol. Ther.
7(2):125-131(2005); modifying regions outside the coding sequence,
such as introducing secondary structure into an untranslated region
(UTR) to block translation or reduce efficiency of translation
(Ringner et al., PLoS Comput. Biol. 1(7):e72 (2005)); adding RNAase
sites for rapid transcript degradation (Pasquinelli, Nat. Rev.
Genet. 13(4):271-282 (2012); and Anaiano et al., FEMS Microbiol.
Rev. 34(5):883-932 (2010); introducing antisense RNA oligomers or
antisense transcripts (Nashizawa et al., Front. Biosci. 17:938-958
(2012)); introducing RNA or peptide aptamers, ribozymes, aptazymes,
riboswitches (Wieland et al., Methods 56(3):351-357 (2012);
O'Sullivan, Anal. Bioanal. Chem. 372(1):44-48 (2002); and Lee et
al., Curr. Opin. Biotechnol. 14(5):505-511(2003)); or introducing
translational regulatory elements involving RNA structure that can
prevent or reduce translation that can be controlled by the
presence or absence of small molecules (Araujo et al., Comparative
and Functional Genomics, Article ID 475731, 8 pages (2012)). At the
level of enzyme localization and/or longevity, enzyme attenuation
can include: adding a degradation tag for faster protein turnover
(Hochstrasser, Annual Rev. Genet. 30:405-439 (1996); and Yuan et
al., PLoS One 8(4):e62529 (2013)); or adding a localization tag
that results in the enzyme being secreted or localized to a
subcellular compartment in a eukaryotic cell, where the enzyme
would not be able to react with its normal substrate (Nakai et al.
Genomics 14(4):897-911 (1992); and Russell et al., J. Bact.
189(21)7581-7585 (2007)). At the level of post-translational
regulation, enzyme attenuation can include: increasing
intracellular concentration of known inhibitors; or modifying
post-translational modified sites (Mann et al., Nature Biotech.
21:255-261 (2003)). At the level of enzyme activity, enzyme
attenuation can include: adding an endogenous or an exogenous
inhibitor, such as an enzyme inhibitor, an antibiotic or a
target-specific drug, to reduce enzyme activity; limiting
availability of essential cofactors, such as vitamin B12, for an
enzyme that requires the cofactor; chelating a metal ion that is
required for enzyme activity; or introducing a dominant negative
mutation. The applicability of a technique for attenuation
described above can depend upon whether a given host microbial
organism is prokaryotic or eukaryotic, and it is understand that a
determination of what is the appropriate technique for a given host
can be readily made by one skilled in the art.
[0189] In some embodiments, microaerobic designs can be used based
on the growth-coupled formation of the desired product. To examine
this, production cones can be constructed for each strategy by
first maximizing and, subsequently minimizing the product yields at
different rates of biomass formation feasible in the network. If
the rightmost boundary of all possible phenotypes of the mutant
network is a single point, it implies that there is a unique
optimum yield of the product at the maximum biomass formation rate
possible in the network. In other cases, the rightmost boundary of
the feasible phenotypes is a vertical line, indicating that at the
point of maximum biomass the network can make any amount of the
product in the calculated range, including the lowest amount at the
bottommost point of the vertical line. Such designs are given a low
priority.
[0190] The acetyl-CoA or a bioderived compound production
strategies identified herein can be disrupted to increase
production of acetyl-CoA or a bioderived compound. Accordingly, the
invention also provides a non-naturally occurring microbial
organism having metabolic modifications coupling acetyl-CoA or a
bioderived compound production to growth of the organism, where the
metabolic modifications includes disruption of one or more genes
selected from the genes encoding proteins and/or enzymes shown in
the various tables disclosed herein.
[0191] Each of the strains can be supplemented with additional
deletions if it is determined that the strain designs do not
sufficiently increase the production of acetyl-CoA or a bioderived
compound and/or couple the formation of the product with biomass
formation. Alternatively, some other enzymes not known to possess
significant activity under the growth conditions can become active
due to adaptive evolution or random mutagenesis. Such activities
can also be knocked out. However, the genes disclosed herein allows
the construction of strains exhibiting high-yield synthesis or
production of acetyl-CoA or a bioderived compound, including
growth-coupled production of acetyl-CoA or a bioderived
compound.
[0192] The invention is described herein with general reference to
the metabolic reaction, reactant or product thereof, or with
specific reference to one or more nucleic acids or genes encoding
an enzyme associated with or catalyzing, or a protein associated
with, the referenced metabolic reaction, reactant or product.
Unless otherwise expressly stated herein, those skilled in the art
will understand that reference to a reaction also constitutes
reference to the reactants and products of the reaction. Similarly,
unless otherwise expressly stated herein, reference to a reactant
or product also references the reaction, and reference to any of
these metabolic constituents also references the gene or genes
encoding the enzymes that catalyze or proteins involved in the
referenced reaction, reactant or product. Likewise, given the well
known fields of metabolic biochemistry, enzymology and genomics,
reference herein to a gene or encoding nucleic acid also
constitutes a reference to the corresponding encoded enzyme and the
reaction it catalyzes or a protein associated with the reaction as
well as the reactants and products of the reaction.
[0193] As disclosed herein, the bioderived compounds adipate,
6-aminocaproate, methacrylic acid, 2-hydroxyisobutyric acid, as
well as other intermediates, are carboxylic acids, which can occur
in various ionized forms, including fully protonated, partially
protonated, and fully deprotonated forms. Accordingly, the suffix
"-ate," or the acid form, can be used interchangeably to describe
both the free acid form as well as any deprotonated form, in
particular since the ionized form is known to depend on the pH in
which the compound is found. It is understood that carboxylate
products or intermediates includes ester forms of carboxylate
products or pathway intermediates, such as O-carboxylate and
S-carboxylate esters. O- and S-carboxylates can include lower
alkyl, that is C1 to C6, branched or straight chain carboxylates.
Some such O- or S-carboxylates include, without limitation, methyl,
ethyl, n-propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl,
pentyl, hexyl O- or S-carboxylates, any of which can further
possess an unsaturation, providing for example, propenyl, butenyl,
pentyl, and hexenyl O- or S-carboxylates. O-carboxylates can be the
product of a biosynthetic pathway. Exemplary O-carboxylates
accessed via biosynthetic pathways can include, without limitation,
methyl adipate, ethyl adipate, and n-propyl adipate. Other
biosynthetically accessible O-carboxylates can include medium to
long chain groups, that is C7-C22, O-carboxylate esters derived
from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl,
lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl,
heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl
alcohols, any one of which can be optionally branched and/or
contain unsaturations. O-carboxylate esters can also be accessed
via a biochemical or chemical process, such as esterification of a
free carboxylic acid product or transesterification of an O- or
S-carboxylate. S-carboxylates are exemplified by CoA S-esters,
cysteinyl S-esters, alkylthioesters, and various aryl and
heteroaryl thioesters.
[0194] The non-naturally occurring microbial organisms of the
invention can be produced by introducing expressible nucleic acids
encoding one or more of the enzymes or proteins participating in
one or more acetyl-CoA or a bioderived compound biosynthetic
pathways. Depending on the host microbial organism chosen for
biosynthesis, nucleic acids for some or all of a particular
acetyl-CoA or a bioderived compound biosynthetic pathway can be
expressed. For example, if a chosen host is deficient in one or
more enzymes or proteins for a desired biosynthetic pathway, then
expressible nucleic acids for the deficient enzyme(s) or protein(s)
are introduced into the host for subsequent exogenous expression.
Alternatively, if the chosen host exhibits endogenous expression of
some pathway genes, but is deficient in others, then an encoding
nucleic acid is needed for the deficient enzyme(s) or protein(s) to
achieve acetyl-CoA or a bioderived compound biosynthesis. Thus, a
non-naturally occurring microbial organism of the invention can be
produced by introducing exogenous enzyme or protein activities to
obtain a desired biosynthetic pathway or a desired biosynthetic
pathway can be obtained by introducing one or more exogenous enzyme
or protein activities that, together with one or more endogenous
enzymes or proteins, produces a desired product such as acetyl-CoA
or a bioderived compound.
[0195] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable or suitable to fermentation processes.
Exemplary bacteria include any species selected from the order
Enterobacteriales, family Enterobacteriaceae, including the genera
Escherichia and Klebsiella, the order Aeromonadales, family
Succinivibrionaceae, including the genus Anaerobiospirillum; the
order Pasteurellales, family Pasteurellaceae, including the genera
Actinobacillus and Mannheimia; the order Rhizobiales, family
Bradyrhizobiaceae, including the genus Rhizobium; the order
Bacillales, family Bacillaceae, including the genus Bacillus; the
order Actinomycetales, families Corynebacteriaceae and
Streptomycetaceae, including the genus Corynebacterium and the
genus Streptomyces, respectively; order Rhodospirillales, family
Acetobacteraceae, including the genus Gluconobacter; the order
Sphingomonadales, family Sphingomonadaceae, including the genus
Zymomonas; the order Lactobacillales, families Lactobacillaceae and
Streptococcaceae, including the genus Lactobacillus and the genus
Lactococcus, respectively; the order Clostridiales, family
Clostridiaceae, genus Clostridium; and the order Pseudomonadales,
family Pseudomonadaceae, including the genus Pseudomonas.
Non-limiting species of host bacteria include Escherichia coli,
Klebsiella oxytoca, Anaerobiospirillum succiniciproducens,
Actinobacillus succinogenes, Mannheimia succiniciproducens,
Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum,
Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis,
Lactobacillus plantarum, Streptomyces coelicolor, Clostridium
acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.
Exemplary bacterial methylotrophs include, for example, Bacillus,
Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis
and Hyphomicrobium.
[0196] Similarly, exemplary species of yeast or fungi species
include any species selected from the order Saccharomycetales,
family Saccaromycetaceae, including the genera Saccharomyces,
Kluyveromyces and Pichia; the order Saccharomycetales, family
Dipodascaceae, including the genus Yarrowia; the order
Schizosaccharomycetales, family Schizosaccaromycetaceae, including
the genus Schizosaccharomyces; the order Eurotiales, family
Trichocomaceae, including the genus Aspergillus; and the order
Mucorales, family Mucoraceae, including the genus Rhizopus.
Non-limiting species of host yeast or fungi include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger,
Pichia pastor's, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia
lipolytica, and the like. E. coli is a particularly useful host
organism since it is a well characterized microbial organism
suitable for genetic engineering. Other particularly useful host
organisms include yeast such as Saccharomyces cerevisiae and yeasts
or fungi selected from the genera Saccharomyces,
Schizosaccharomyces, Schizochytrium, Rhodotorula, Thraustochytrium,
Aspergillus, Kluyveromyces, Issatchenkia, Yarrowia, Candida,
Pichia, Ogataea, Kuraishia, Hansenula and Komagataella. Useful host
organisms include Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii,
Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,
Aspergillus niger, Pichia pastor's, Rhizopus arrhizus, Rhizopus
oryzae, Yarrowia lipolytica, Issatchenkia orientalis and the like.
It is understood that any suitable microbial host organism can be
used to introduce metabolic and/or genetic modifications to produce
a desired product.
[0197] Depending on the acetyl-CoA or the bioderived compound
biosynthetic pathway constituents of a selected host microbial
organism, the non-naturally occurring microbial organisms of the
invention will include at least one exogenously expressed
acetyl-CoA or a bioderived compound pathway-encoding nucleic acid
and up to all encoding nucleic acids for one or more acetyl-CoA or
a bioderived compound biosynthetic pathways. For example,
acetyl-CoA or a bioderived compound biosynthesis can be established
in a host deficient in a pathway enzyme or protein through
exogenous expression of the corresponding encoding nucleic acid. In
a host deficient in all enzymes or proteins of an acetyl-CoA or a
bioderived compound pathway, exogenous expression of all enzyme or
proteins in the pathway can be included, although it is understood
that all enzymes or proteins of a pathway can be expressed even if
the host contains at least one of the pathway enzymes or proteins.
For example, exogenous expression of all enzymes or proteins in a
pathway for production of acetyl-CoA or a bioderived compound can
be included, such as a fructose-6-phosphate phosphoketolase and a
phosphotransacetylase (see, e.g. FIG. 1), or a xylulose-5-phosphate
phosphoketolase and a phosphotransacetylase (see, e.g. FIG. 1), or
a methanol dehydrogenase, a 3-hexulose-6-phosphate synthase, a
6-phospho-3-hexuloisomerase, a fructose-6-phosphate phosphoketolase
and a phosphotransacetylase (see, e.g. FIG. 1), or an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase
(ketone reducing), a 3-hydroxybutyryl-CoA reductase (aldehyde
forming), and a 3-hydroxybutyraldehyde reductase (see, e.g. FIG.
5), or a succinyl-CoA reductase (aldehyde forming), a 4-HB
dehydrogenase, a 4-HB kinase, a phosphotrans-4-hydroxybutyrylase, a
4-hydroxybutyryl-CoA reductase (aldehyde forming), and a
1,4-butanediol dehydrogenase (see, e.g. FIG. 7), or a
3-oxoadipyl-CoA thiolase, a 3-oxoadipyl-CoA reductase, a
3-hydroxyadipyl-CoA dehydratase, a 5-carboxy-2-pentenoyl-CoA
reductase, an adipyl-CoA reductase (aldehyde forming), and
6-aminocaproate transaminase (see, e.g. FIG. 8), or an
acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase
(ketone reducing), a 3-hydroxybutyrl-CoA mutase, a
2-hydroxyisobutyryl-CoA dehydratase, and a methacrylyl-CoA
synthetase (see, e.g. FIG. 10).
[0198] Given the teachings and guidance provided herein, those
skilled in the art will understand that the number of encoding
nucleic acids to introduce in an expressible form will, at least,
parallel the acetyl-CoA or the bioderived compound pathway
deficiencies of the selected host microbial organism. Therefore, a
non-naturally occurring microbial organism of the invention can
have one, two, three, four, five, six, seven, eight, nine, ten,
eleven, or twelve up to all nucleic acids encoding the enzymes or
proteins constituting an acetyl-CoA or a bioderived compound
biosynthetic pathway disclosed herein. In some embodiments, the
non-naturally occurring microbial organisms also can include other
genetic modifications that facilitate or optimize acetyl-CoA or a
bioderived compound biosynthesis or that confer other useful
functions onto the host microbial organism. One such other
functionality can include, for example, augmentation of the
synthesis of one or more of the acetyl-CoA or the bioderived
compound pathway precursors such as Fald, H6P, DHA, G3P,
malonyl-CoA, acetoacetyl-CoA, PEP, PYR and Succinyl-CoA.
[0199] Generally, a host microbial organism is selected such that
it produces the precursor of an acetyl-CoA or a bioderived compound
pathway, either as a naturally produced molecule or as an
engineered product that either provides de novo production of a
desired precursor or increased production of a precursor naturally
produced by the host microbial organism. For example, malonyl-CoA,
acetoacetyl-CoA and pyruvate are produced naturally in a host
organism such as E. coli. A host organism can be engineered to
increase production of a precursor, as disclosed herein. In
addition, a microbial organism that has been engineered to produce
a desired precursor can be used as a host organism and further
engineered to express enzymes or proteins of an acetyl-CoA or a
bioderived compound pathway.
[0200] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize acetyl-CoA or a bioderived
compound. In this specific embodiment it can be useful to increase
the synthesis or accumulation of an acetyl-CoA or a bioderived
compound pathway product to, for example, drive acetyl-CoA or a
bioderived compound pathway reactions toward acetyl-CoA or a
bioderived compound production. Increased synthesis or accumulation
can be accomplished by, for example, overexpression of nucleic
acids encoding one or more of the above-described acetyl-CoA or a
bioderived compound pathway enzymes or proteins. Overexpression of
the enzyme or enzymes and/or protein or proteins of the acetyl-CoA
or the bioderived compound pathway can occur, for example, through
exogenous expression of the endogenous gene or genes, or through
exogenous expression of the heterologous gene or genes. Therefore,
naturally occurring organisms can be readily generated to be
non-naturally occurring microbial organisms of the invention, for
example, producing acetyl-CoA or a bioderived compound, through
overexpression of one, two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve, that is, up to all nucleic acids
encoding acetyl-CoA or a bioderived compound biosynthetic pathway
enzymes or proteins. In addition, a non-naturally occurring
organism can be generated by mutagenesis of an endogenous gene that
results in an increase in activity of an enzyme in the acetyl-CoA
or the bioderived compound biosynthetic pathway.
[0201] In particularly useful embodiments, exogenous expression of
the encoding nucleic acids is employed. Exogenous expression
confers the ability to custom tailor the expression and/or
regulatory elements to the host and application to achieve a
desired expression level that is controlled by the user. However,
endogenous expression also can be utilized in other embodiments
such as by removing a negative regulatory effector or induction of
the gene's promoter when linked to an inducible promoter or other
regulatory element. Thus, an endogenous gene having a naturally
occurring inducible promoter can be up-regulated by providing the
appropriate inducing agent, or the regulatory region of an
endogenous gene can be engineered to incorporate an inducible
regulatory element, thereby allowing the regulation of increased
expression of an endogenous gene at a desired time. Similarly, an
inducible promoter can be included as a regulatory element for an
exogenous gene introduced into a non-naturally occurring microbial
organism.
[0202] It is understood that, in methods of the invention, any of
the one or more exogenous nucleic acids can be introduced into a
microbial organism to produce a non-naturally occurring microbial
organism of the invention. The nucleic acids can be introduced so
as to confer, for example, an acetyl-CoA or a bioderived compound
biosynthetic pathway onto the microbial organism. Alternatively,
encoding nucleic acids can be introduced to produce an intermediate
microbial organism having the biosynthetic capability to catalyze
some of the required reactions to confer acetyl-CoA or a bioderived
compound biosynthetic capability. For example, a non-naturally
occurring microbial organism having an acetyl-CoA or a bioderived
compound biosynthetic pathway can comprise at least two exogenous
nucleic acids encoding desired enzymes or proteins, such as the
combination of a 3-hexulose-6-phosphate synthase and a
fructose-6-phosphate phosphoketolase, or alternatively a
xylulose-5-phosphate phosphoketolase and an acetyl-CoA transferase,
or alternatively a fructose-6-phosphate phosphoketolase and a
formate reductase, or alternatively a xylulose-5-phosphate
phosphoketolase and a methanol dehydrogenase, and the like. Thus,
it is understood that any combination of two or more enzymes or
proteins of a biosynthetic pathway can be included in a
non-naturally occurring microbial organism of the invention.
Similarly, it is understood that any combination of three or more
enzymes or proteins of a biosynthetic pathway can be included in a
non-naturally occurring microbial organism of the invention, for
example, a methanol dehydrogenase, a fructose-6-phosphate aldolase,
and a fructose-6-phosphate phosphoketolase, or alternatively a
methanol methyltransferase, fructose-6-phosphate phosphoketolase
and a 3-hydroxybutyraldehyde reductase, or alternatively a
xylulose-5-phosphate phosphoketolase, a pyruvate formate lyase and
a 4-hydroxybutyryl-CoA reductase (alcohol forming), or
alternatively a fructose-6-phosphate aldolase, a
phosphotransacetylase, and a 3-hydroxyisobutyrate dehydratase, and
so forth, as desired, so long as the combination of enzymes and/or
proteins of the desired biosynthetic pathway results in production
of the corresponding desired product. Similarly, any combination of
four, five, six, seven, eight, nine, ten, eleven, twelve or more
enzymes or proteins of a biosynthetic pathway as disclosed herein
can be included in a non-naturally occurring microbial organism of
the invention, as desired, so long as the combination of enzymes
and/or proteins of the desired biosynthetic pathway results in
production of the corresponding desired product.
[0203] In addition to the biosynthesis of acetyl-CoA or a
bioderived compound as described herein, the non-naturally
occurring microbial organisms and methods of the invention also can
be utilized in various combinations with each other and/or with
other microbial organisms and methods well known in the art to
achieve product biosynthesis by other routes. For example, one
alternative to produce acetyl-CoA or a bioderived compound other
than use of the acetyl-CoA or the bioderived compound producers is
through addition of another microbial organism capable of
converting an acetyl-CoA or a bioderived compound pathway
intermediate to acetyl-CoA or a bioderived compound. One such
procedure includes, for example, the fermentation of a microbial
organism that produces an acetyl-CoA or a bioderived compound
pathway intermediate. The acetyl-CoA or the bioderived compound
pathway intermediate can then be used as a substrate for a second
microbial organism that converts the acetyl-CoA or the bioderived
compound pathway intermediate to acetyl-CoA or a bioderived
compound. The acetyl-CoA or the bioderived compound pathway
intermediate can be added directly to another culture of the second
organism or the original culture of the acetyl-CoA or the
bioderived compound pathway intermediate producers can be depleted
of these microbial organisms by, for example, cell separation, and
then subsequent addition of the second organism to the fermentation
broth can be utilized to produce the final product without
intermediate purification steps.
[0204] In other embodiments, the non-naturally occurring microbial
organisms and methods of the invention can be assembled in a wide
variety of subpathways to achieve biosynthesis of, for example,
acetyl-CoA or a bioderived compound. In these embodiments,
biosynthetic pathways for a desired product of the invention can be
segregated into different microbial organisms, and the different
microbial organisms can be co-cultured to produce the final
product. In such a biosynthetic scheme, the product of one
microbial organism is the substrate for a second microbial organism
until the final product is synthesized. For example, the
biosynthesis of acetyl-CoA or a bioderived compound can be
accomplished by constructing a microbial organism that contains
biosynthetic pathways for conversion of one pathway intermediate to
another pathway intermediate or the product. Alternatively,
acetyl-CoA or a bioderived compound also can be biosynthetically
produced from microbial organisms through co-culture or
co-fermentation using two organisms in the same vessel, where the
first microbial organism produces an acetyl-CoA or a bioderived
compound intermediate and the second microbial organism converts
the intermediate to acetyl-CoA or a bioderived compound.
[0205] Given the teachings and guidance provided herein, those
skilled in the art will understand that a wide variety of
combinations and permutations exist for the non-naturally occurring
microbial organisms and methods of the invention together with
other microbial organisms, with the co-culture of other
non-naturally occurring microbial organisms having subpathways and
with combinations of other chemical and/or biochemical procedures
well known in the art to produce acetyl-CoA or a bioderived
compound.
[0206] Similarly, it is understood by those skilled in the art that
a host organism can be selected based on desired characteristics
for introduction of one or more gene disruptions to increase
synthesis or production of acetyl-CoA or a bioderived compound.
Thus, it is understood that, if a genetic modification is to be
introduced into a host organism to disrupt a gene, any homologs,
orthologs or paralogs that catalyze similar, yet non-identical
metabolic reactions can similarly be disrupted to ensure that a
desired metabolic reaction is sufficiently disrupted. Because
certain differences exist among metabolic networks between
different organisms, those skilled in the art will understand that
the actual genes disrupted in a given organism may differ between
organisms. However, given the teachings and guidance provided
herein, those skilled in the art also will understand that the
methods of the invention can be applied to any suitable host
microorganism to identify the cognate metabolic alterations needed
to construct an organism in a species of interest that will
increase acetyl-CoA or a bioderived compound biosynthesis. In a
particular embodiment, the increased production couples
biosynthesis of acetyl-CoA or a bioderived compound to growth of
the organism, and can obligatorily couple production of acetyl-CoA
or a bioderived compound to growth of the organism if desired and
as disclosed herein.
[0207] Sources of encoding nucleic acids for an acetyl-CoA or a
bioderived compound pathway enzyme or protein can include, for
example, any species where the encoded gene product is capable of
catalyzing the referenced reaction. Such species include both
prokaryotic and eukaryotic organisms including, but not limited to,
bacteria, including archaea and eubacteria, and eukaryotes,
including yeast, plant, insect, animal, and mammal, including
human. Exemplary species for such sources include, for example,
Escherichia coli, Abies grandis, Acetobacter aceti, Acetobacter
pasteurians, Achromobacter denitnficans, Acidaminococcus
fermentans, Acinetobacter baumannii Naval-82, Acinetobacter baylyi,
Acinetobacter calcoaceticus, Acinetobacter sp. ADP1, Acinetobacter
sp. Strain M-1, Actinobacillus succinogenes, Actinobacillus
succinogenes 130Z, Aeropyrum pernix, Agrobacterium tumefaciens,
Alkaliphilus metalliredigenes QYF, Allochromatium vinosum DSM 180,
Aminomonas aminovorus, Amycolicicoccus subflavus DQS3-9A1,
Anaerobiospirillum succiniciproducens, Anaerotruncus colihominis,
Aquifex aeolicus VF5, Arabidopsis thaliana, Arabidopsis thaliana
col, Archaeglubus fulgidus, Archaeoglobus fulgidus, Archaeoglobus
fulgidus DSM 4304, Arthrobacter globiformis, Ascaris suum,
Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger,
Aspergillus niger CBS 513.88, Aspergillus terreus MH2624, Atopobium
parvulum DSM 20469, Azotobacter vinelandii DJ, Bacillus
alcalophilus ATCC 27647, Bacillus azotoformans LMG 9581, Bacillus
cereus, Bacillus cereus ATCC 14579, Bacillus coagulans 36D1,
Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus
methanolicus PB1, Bacillus methanolicus PB-1, Bacillus
selenitireducens MLS10, Bacillus smithii, Bacillus sphaericus,
Bacillus subtilis, Bacteroides capillosus, Bifidobacterium animalis
lactis, Bifidobacterium breve, Bifidobacterium dentium ATCC 27678,
Bifidobacterium pseudolongum subsp. globosum, Bos taurus,
Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia
multivorans, Burkholderia pyrrocinia, Burkholderia stabilis,
Burkholderia thailandensis E264, Burkholderia xenovorans, butyrate
producing bacterium L2-50, Caenorhabditis elegans, Campylobacter
curvus 525.92, Campylobacter jejuni, Candida albicans, Candida
boidinii, Candida methylica, Candida parapsilosis, Candida
tropicalis, Candida tropicalis MYA-3404, Carboxydothermus
hydrogenoformans, Carboxydothermus hydrogenoformans Z-2901,
Castellaniella defragrans, Caulobacter sp. AP07, Chlamydomonas
reinhardiii, Chlorobium phaeobacteroides DSM 266, Chlorobium
limicola, Chlorobium tepidum, Chlorojlexus aggregans DSM 9485,
Chlorojlexus aurantiacus, Chlorojlexus aurantiacus J-10-fl,
Citrobacter koseri ATCC BAA-895, Citrobacter youngae, Citrobacter
youngae ATCC 29220, Clostridium acetobutylicum, Clostridium
acetobutylicum ATCC 824, Clostridium acidurici, Clostridium
aminobutyricum, Clostridium asparagiforme DSM 15981, Clostridium
beijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridium
beijerinckii NRRL B593, Clostridium beijerinckii, Clostridium
bolteae ATCC BAA-613, Clostridium botulinum C str. Eklund,
Clostridium carboxidivorans P7, Clostridium cellulolyticum H10,
Clostridium cellulovorans 743B, Clostridium difficile, Clostridium
difficile 630, Clostridium hiranonis DSM 13275, Clostridium
hylemonae DSM 15053, Clostridium kluyveri, Clostridium kluyveri DSM
555, Clostridium ljungdahli, Clostridium ljungdahlii DSM,
Clostridium ljungdahlii DSM 13528, Clostridium methylpentosum DSM
5476, Clostridium novyi NT, Clostridium pasteurianum, Clostridium
pasteurianum DSM 525, Clostridium perfringens, Clostridium
perfringens ATCC 13124, Clostridium perfringens str. 13,
Clostridium phytofermentans ISDg, Clostridium propionicum,
Clostridium saccharobutylicum, Clostridium
saccharoperbutylacetonicum, Clostridium saccharoperbutylacetonicum
N1-4, Clostridium tetani, Comamonas sp. CNB-1, Comamonas sp. CNB-1,
Corynebacterium glutamicum, Corynebacterium glutamicum ATCC 13032,
Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum
R, Corynebacterium sp., Corynebacterium sp. U-96, Corynebacterium
variabile, Cryptosporidiumparvum Iowa II Cucumis sativus,
Cupriavidus necatorN-1, Cyanobium PCC7001, Deinococcus radiodurans
R1, Desulfatibacillum alkenivorans AK-01, Desulfotobacterium
hafniense, Desulfotobacterium metallireducens DSM15288,
Desulfotomaculum reducens MI-1, Desulfovibrio africanus,
Desulfovibrio africanus str. Walvis Bay, DesulfoVibrio
desulfuricans G20, Desulfovibrio desulfuricans subsp.
desulfiuricans sfr. ATCC 27774, Desulfovibrio fructosovorans JJ,
Desulfovibrio vulgaris str. Hildenborough, Dictyostelium discoideum
AX4, Elizabethkingia meningoseptica, Enterococcus faecalis,
Erythrobacter sp. NAP1, Escherichia coli C, Escherichia coli K12,
Escherichia coli K-12MG1655, Escherichia coli W, Eubacterium
barkeri, Eubacterium hallii DSM3353, Eubacterium rectale ATCC
33656, Euglena gracilis, Flavobacterium frigoris, Fusobacterium
nucleatum, Fusobacterium nucleatum subsp. polymorphum ATCC 10953,
Geobacillus sp. GHH01, Geobacillus sp. M10EXG, Geobacillus sp.
Y4.1MC1, Geobacillus stearothermophilus, Geobacillus
themodenitrijfcans NG80-2, Geobacillus thermoglucosidasius,
Geobacter bemidjiensis Bem, Geobacter metallireducens GS-15,
Geobacter sulfurreducens, Geobacter suflfrreducens PCA, Haemophilus
influenza, Haemophilus influenzae, Haloarcula marismortui,
Haloarcula marismortui ATCC 43049, Halobacterium salinarum,
Hansenula polymorpha DL-1, Helicobacter pylori, Helicobacter pylori
26695, Helicobacter pylori, Homo sapiens, human gut metagenome,
Hydrogenobacter thermophilus, Hydrogenobacter thermophilus TK-6,
Hyphomicrobium denitrifcans ATCC 51888, Hyphomicrobium zavarzinii,
Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae MGH
78578, Kluyveromyces lactis, Kluyveromyces lactis NRRL Y-1140,
Lactobacillus acidophilus, Lactobacillus brevis ATCC 367,
Lactobacillus paraplantarum, Lactococcus lactis, Leuconostoc
mesenteroides, Lysinibacillus fusiformis, Lysinibacillus
sphaericus, Malus x domestica, Mannheimia succiniciproducens,
marine gamma proteobacterium HTCC2080, Marine metagenome JCVI SCAF
1096627185304, Mesorhizobium loti MAFF303099, Metallosphaera
sedula, Metarhizium acridum CQMa 102, Methanocaldococcus janaschii,
Methanocaldococcus annaschii, Methanosarcina acetivorans,
Methanosarcina acetivorans C2A, Methanosarcina barkeri,
Methanosarcina mazei, Methanosarcina mazei Tuc01, Methanosarcina
thermophila, Methanothermobacter thermautotrophicus, Methylibium
petroleiphilum PM1, Methylobacillus flagellatus, Methylobacillus
fagellatus KT Methylobacter marinus, Methylobacterium extorquens,
Methylobacterium extorquens AM1, Methylococcus capsulatas,
Methylomicrobium album BG8, Methylomonas aminofaciens, Methylovorus
glucosetrophus SIP3-4, Methylovorus sp. MP688, Moorella
thermoacetica, Mus musculus, Mycobacter sp. strain JC1 DSM3803,
Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium
bovis BCG, Mycobacterium gastri, Mycobacterium marinum M,
Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155,
Mycobacterium tuberculosis, Mycoplasma pneumoniae M129,
Natranaerobius thermophilus, Nectria haematococca mpVI 77-13-4,
Neurospora crassa, Nitrososphaera gargensis Ga9.2, Nocardia
brasiliensis, Nocardia farcinica IFM10152, Nocardia iowensis,
Nocardia iowensis (sp. NRRL 5646), Nostoc sp. PCC 7120, Ogataea
parapolymorpha DL-1 (Hansenula polymorpha DL-1), Organism,
Oryctolagus cuniculus, Oxalobacter formigenes, Paenibacillus
peoriae KCTC 3763, Paracoccus denitrificans, Pelobacter
carbinolicus DSM2380, Pelotomaculum thermopropionicum, Penicillium
chrysogenum, Perkinsus marinus ATCC 50983, Photobacterium profundum
3TCK, Picea abies, Pichia pastoris, Picrophilus torridus DSM9790,
Pinus sabiniana, Plasmodium falciparum, Populus alba, Populus
tremula x Populus alba, Porphyromonas gingivalis, Porphyromonas
gingivalis W83, Propionibacterium acnes, Propionibacterium
fredenreichii sp. shermanii, Pseudomonas aeruginosa, Pseudomonas
aemginosa PA01, Pseudomonas chlororaphis, Pseudomonas fluorescens,
Pseudomonas knackmussii, Pseudomonas knackmussii (B13), Pseudomonas
mendocina, Pseudomonas putida, Pseudomonas sp, Pseudomonas syringae
pv. syringae B728a, Psychroflexus torquis ATCC 700755, Pueraria
montana, Pyrobaculum aerophilum str. IM2, Pyrobaculum islandicum
DSM4184, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus
horikoshii OT3, Ralstonia eutropha, Ralstonia eutropha H16, Rattus
norvegicus, Rhizobium leguminosarum, Rhodobacter capsulatus,
Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025,
Rhodococcus opacus B4, Rhodococcus ruber, Rhodopseudomonas
palustris, Rhodopseudomonas palustris CGA009, Rhodospirillum
rubrum, Rhodospirillum rubrum ATCC 11170, Roseburia intestinalis
L1-82, Roseburia inulinivorans, Roseburia sp. A2-183, Roseiflexus
castenholzii, Rubrivivax gelatinosus, Ruminococcus obeum ATCC
29174, Saccharomyces cerevisiae, Saccharomyces cerevisiae s288c,
Saccharomyces kluyveri, Saccharomyces serevisiae, Sachharomyces
cerevisiae, Salmonella enteric, Salmonella enterica, Salmonella
enterica subsp. arizonae serovar, Salmonella enterica subsp.
enterica serovar Typhimurium str. LT2, Salmonella enterica
Typhimurium, Salmonella typhimurium, Salmonella typhimurium LT2,
Schizosaccharomyces pombe, Sebaldella termitidis ATCC 33386,
Serratia proteamaculans, Shewanella oneidensis MR-1, Shigella
flexneri, Sinorhizobium meliloti 1021, Solanum lycopersicum,
Staphylococcus aureus, Stereum hirsutum FP-91666 SS1, Streptococcus
mutans, Streptococcus pneumonia, Streptococcus pneumoniae,
Streptococcus pyogenes ATCC 10782, Streptomyces anulatus,
Streptomyces avermitilis, Streptomyces cinnamonensis, Streptomyces
clavuligerus, Streptomyces coelicolor, Streptomyces coelicolor
A3(2), Streptomyces griseus, Streptomyces griseus subsp. griseus
NBRC 13350, Streptomyces sp CL190, Streptomyces sp. 2065,
Streptomyces sp. ACT-1, Streptomyces sp. KO-3988, Sulfolobus
acidocalarius, Sulfolobus acidocaldarius, Sulfolobus solfataricus,
Sulfolobus solfataricus P-2, Sulfolobus sp. strain 7, Sulfolobus
tokodan, Sulfurimonas denitrifcans, Sus scrofa, Synechococcus
elongatus PCC 7942, Synechococcus sp. PCC 7002, Synechocystis str.
PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica,
Thermoanaerobacter brockii HTD4, Thermoanaerobacter sp. X514,
Thermoanaerobacter tengcongensis MB4, Thermococcus kodakaraensis,
Thermococcus litoralis, Thermoplasma acidophilum, Thermoproteus
neutrophilus, Thermotoga maritima, Thermotoga maritime, Thermotoga
maritime MSB8, Thermus thermophilus, Thiocapsa roseopersicina,
Tolumonas auensis DSM9187, Treponema denticola, Trichomonas
vaginalis G3, Triticum aestivum, Trypanosoma brucei, Tsukamurella
paurometabola DSM 20162, Uncultured bacterium, uncultured organism,
Vibrio cholera, Vibrio harveyi ATCC BAA-1116, Xanthobacter
autotrophicus Py2, Yarrowia lipolytica, Yersinia frederiksenii,
Yersinia intermedia, Yersinia intermedia ATCC 29909, Yersinia
pestis, Zea mays, Zoogloea ramigera, Zymomonas mobilus, as well as
other exemplary species disclosed herein or available as source
organisms for corresponding genes. However, with the complete
genome sequence available for now more than 550 species (with more
than half of these available on public databases such as the NCBI),
including 395 microorganism genomes and a variety of yeast, fungi,
plant, and mammalian genomes, the identification of genes encoding
the requisite acetyl-CoA or a bioderived compound biosynthetic
activity for one or more genes in related or distant species,
including for example, homologues, orthologs, paralogs and
nonorthologous gene displacements of known genes, and the
interchange of genetic alterations between organisms is routine and
well known in the art. Accordingly, the metabolic alterations
allowing biosynthesis of acetyl-CoA or a bioderived compound
described herein with reference to a particular organism such as E.
coli can be readily applied to other microorganisms, including
prokaryotic and eukaryotic organisms alike. Given the teachings and
guidance provided herein, those skilled in the art will know that a
metabolic alteration exemplified in one organism can be applied
equally to other organisms.
[0208] In some instances, such as when an alternative acetyl-CoA or
a bioderived compound biosynthetic pathway exists in an unrelated
species, acetyl-CoA or a bioderived compound biosynthesis can be
conferred onto the host species by, for example, exogenous
expression of a paralog or paralogs from the unrelated species that
catalyzes a similar, yet non-identical metabolic reaction to
replace the referenced reaction. Because certain differences among
metabolic networks exist between different organisms, those skilled
in the art will understand that the actual gene usage between
different organisms may differ. However, given the teachings and
guidance provided herein, those skilled in the art also will
understand that the teachings and methods of the invention can be
applied to all microbial organisms using the cognate metabolic
alterations to those exemplified herein to construct a microbial
organism in a species of interest that will synthesize acetyl-CoA
or a bioderived compound.
[0209] A nucleic acid molecule encoding an acetyl-CoA or a
bioderived compound pathway enzyme or protein of the invention can
also include a nucleic acid molecule that hybridizes to a nucleic
acid disclosed herein by SEQ ID NO, GenBank and/or GI number or a
nucleic acid molecule that hybridizes to a nucleic acid molecule
that encodes an amino acid sequence disclosed herein by SEQ ID NO,
GenBank and/or GI number. Hybridization conditions can include
highly stringent, moderately stringent, or low stringency
hybridization conditions that are well known to one of skill in the
art such as those described herein. Similarly, a nucleic acid
molecule that can be used in the invention can be described as
having a certain percent sequence identity to a nucleic acid
disclosed herein by SEQ ID NO, GenBank and/or GI number or a
nucleic acid molecule that hybridizes to a nucleic acid molecule
that encodes an amino acid sequence disclosed herein by SEQ ID NO,
GenBank and/or GI number. For example, the nucleic acid molecule
can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% sequence identity to a nucleic acid
described herein.
[0210] Stringent hybridization refers to conditions under which
hybridized polynucleotides are stable. As known to those of skill
in the art, the stability of hybridized polynucleotides is
reflected in the melting temperature (T.sub.m) of the hybrids. In
general, the stability of hybridized polynucleotides is a function
of the salt concentration, for example, the sodium ion
concentration and temperature. A hybridization reaction can be
performed under conditions of lower stringency, followed by washes
of varying, but higher, stringency. Reference to hybridization
stringency relates to such washing conditions. Highly stringent
hybridization includes conditions that permit hybridization of only
those nucleic acid sequences that form stable hybridized
polynucleotides in 0.018M NaCl at 65.degree. C., for example, if a
hybrid is not stable in 0.018M NaCl at 65.degree. C., it will not
be stable under high stringency conditions, as contemplated herein.
High stringency conditions can be provided, for example, by
hybridization in 50% formamide, 5.times.Denhart's solution,
5.times.SSPE, 0.2% SDS at 42.degree. C., followed by washing in
0.1.times.SSPE, and 0.1% SDS at 65.degree. C. Hybridization
conditions other than highly stringent hybridization conditions can
also be used to describe the nucleic acid sequences disclosed
herein. For example, the phrase moderately stringent hybridization
refers to conditions equivalent to hybridization in 50% formamide,
5.times.Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 0.2.times.SSPE, 0.2% SDS, at 42.degree.
C. The phrase low stringency hybridization refers to conditions
equivalent to hybridization in 10% formamide, 5.times.Denhart's
solution, 6.times.SSPE, 0.2% SDS at 22.degree. C., followed by
washing in 1.times.SSPE, 0.2% SDS, at 37.degree. C. Denhart's
solution contains 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine
serum albumin (BSA). 20.times.SSPE (sodium chloride, sodium
phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M
sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other
suitable low, moderate and high stringency hybridization buffers
and conditions are well known to those of skill in the art and are
described, for example, in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New
York (2001); and Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (1999).
[0211] A nucleic acid molecule encoding an acetyl-CoA or a
bioderived compound pathway enzyme or protein of the invention can
have at least a certain sequence identity to a nucleotide sequence
disclosed herein. According, in some aspects of the invention, a
nucleic acid molecule encoding an acetyl-CoA or a bioderived
compound pathway enzyme or protein has a nucleotide sequence of at
least 65% identity, at least 70% identity, at least 75% identity,
at least 80% identity, at least 85% identity, at least 90%
identity, at least 91% identity, at least 92% identity, at least
93% identity, at least 94% identity, at least 95% identity, at
least 96% identity, at least 97% identity, at least 98% identity,
or at least 99% identity to a nucleic acid disclosed herein by SEQ
ID NO, GenBank and/or GI number or a nucleic acid molecule that
hybridizes to a nucleic acid molecule that encodes an amino acid
sequence disclosed herein by SEQ ID NO, GenBank and/or GI
number.
[0212] Sequence identity (also known as homology or similarity)
refers to sequence similarity between two nucleic acid molecules or
between two polypeptides. Identity can be determined by comparing a
position in each sequence, which may be aligned for purposes of
comparison. When a position in the compared sequence is occupied by
the same base or amino acid, then the molecules are identical at
that position. A degree of identity between sequences is a function
of the number of matching or homologous positions shared by the
sequences. The alignment of two sequences to determine their
percent sequence identity can be done using software programs known
in the art, such as, for example, those described in Ausubel et
al., Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md. (1999). Preferably, default parameters are used for
the alignment. One alignment program well known in the art that can
be used is BLAST set to default parameters. In particular, programs
are BLASTN and BLASTP, using the following default parameters:
Genetic code=standard; filter=none; strand=both; cutoff=60;
expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH
SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR Details of these programs
can be found at the National Center for Biotechnology
Information.
[0213] Methods for constructing and testing the expression levels
of a non-naturally occurring acetyl-CoA or a bioderived
compound-producing host can be performed, for example, by
recombinant and detection methods well known in the art. Such
methods can be found described in, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring
Harbor Laboratory, New York (2001); and Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
(1999).
[0214] Exogenous nucleic acid sequences involved in a pathway for
production of acetyl-CoA or a bioderived compound can be introduced
stably or transiently into a host cell using techniques well known
in the art including, but not limited to, conjugation,
electroporation, chemical transformation, transduction,
transfection, and ultrasound transformation. For exogenous
expression in E. coli or other prokaryotic cells, some nucleic acid
sequences in the genes or cDNAs of eukaryotic nucleic acids can
encode targeting signals such as an N-terminal mitochondrial or
other targeting signal, which can be removed before transformation
into prokaryotic host cells, if desired. For example, removal of a
mitochondrial leader sequence led to increased expression in E.
coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For
exogenous expression in yeast or other eukaryotic cells, genes can
be expressed in the cytosol without the addition of leader
sequence, or can be targeted to mitochondrion or other organelles,
or targeted for secretion, by the addition of a suitable targeting
sequence such as a mitochondrial targeting or secretion signal
suitable for the host cells. Thus, it is understood that
appropriate modifications to a nucleic acid sequence to remove or
include a targeting sequence can be incorporated into an exogenous
nucleic acid sequence to impart desirable properties. Furthermore,
genes can be subjected to codon optimization with techniques well
known in the art to achieve optimized expression of the
proteins.
[0215] An expression vector or vectors can be constructed to
include one or more acetyl-CoA or a bioderived compound
biosynthetic pathway encoding nucleic acids as exemplified herein
operably linked to expression control sequences functional in the
host organism. Expression vectors applicable for use in the
microbial host organisms of the invention include, for example,
plasmids, phage vectors, viral vectors, episomes and artificial
chromosomes, including vectors and selection sequences or markers
operable for stable integration into a host chromosome.
Additionally, the expression vectors can include one or more
selectable marker genes and appropriate expression control
sequences. Selectable marker genes also can be included that, for
example, provide resistance to antibiotics or toxins, complement
auxotrophic deficiencies, or supply critical nutrients not in the
culture media. Expression control sequences can include
constitutive and inducible promoters, transcription enhancers,
transcription terminators, and the like which are well known in the
art. When two or more exogenous encoding nucleic acids are to be
co-expressed, both nucleic acids can be inserted, for example, into
a single expression vector or in separate expression vectors. For
single vector expression, the encoding nucleic acids can be
operationally linked to one common expression control sequence or
linked to different expression control sequences, such as one
inducible promoter and one constitutive promoter. The
transformation of exogenous nucleic acid sequences involved in a
metabolic or synthetic pathway can be confirmed using methods well
known in the art. Such methods include, for example, nucleic acid
analysis such as Northern blots or polymerase chain reaction (PCR)
amplification of mRNA, or immunoblotting for expression of gene
products, or other suitable analytical methods to test the
expression of an introduced nucleic acid sequence or its
corresponding gene product. It is understood by those skilled in
the art that the exogenous nucleic acid is expressed in a
sufficient amount to produce the desired product, and it is further
understood that expression levels can be optimized to obtain
sufficient expression using methods well known in the art and as
disclosed herein.
[0216] In some embodiments, the invention also provides a method
for producing a bioderived compound described herein. Such a method
can comprise culturing the non-naturally occurring microbial
organism as described herein under conditions and for a sufficient
period of time to produce the bioderived compound. In another
embodiment, method further includes separating the bioderived
compound from other components in the culture. In this aspect,
separating can include extraction, continuous liquid-liquid
extraction, pervaporation, membrane filtration, membrane
separation, reverse osmosis, electrodialysis, distillation,
crystallization, centrifugation, extractive filtration, ion
exchange chromatography, absorption chromatography, or
ultrafiltration.
[0217] In some embodiments, depending on the bioderived compound,
the method of the invention may further include chemically
converting a bioderived compound to the directed final compound.
For example, in some embodiments, wherein the bioderived compound
is butadiene, the method of the invention can further include
chemically dehydrating 1,3-butanediol, crotyl alcohol, or
3-buten-2-ol to produce the butadiene.
[0218] Suitable purification and/or assays to test for the
production of acetyl-CoA or a bioderived compound can be performed
using well known methods. Suitable replicates such as triplicate
cultures can be grown for each engineered strain to be tested. For
example, product and byproduct formation in the engineered
production host can be monitored. The final product and
intermediates, and other organic compounds, can be analyzed by
methods such as HPLC (High Performance Liquid Chromatography),
GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid
Chromatography-Mass Spectroscopy) or other suitable analytical
methods using routine procedures well known in the art. The release
of product in the fermentation broth can also be tested with the
culture supernatant. Byproducts and residual glucose can be
quantified by HPLC using, for example, a refractive index detector
for glucose and alcohols, and a UV detector for organic acids (Lin
et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable
assay and detection methods well known in the art. The individual
enzyme or protein activities from the exogenous DNA sequences can
also be assayed using methods well known in the art.
[0219] The bioderived compound can be separated from other
components in the culture using a variety of methods well known in
the art. Such separation methods include, for example, extraction
procedures as well as methods that include continuous liquid-liquid
extraction, pervaporation, membrane filtration, membrane
separation, reverse osmosis, electrodialysis, distillation,
crystallization, centrifugation, extractive filtration, ion
exchange chromatography, size exclusion chromatography, adsorption
chromatography, and ultrafiltration. All of the above methods are
well known in the art.
[0220] Any of the non-naturally occurring microbial organisms
described herein can be cultured to produce and/or secrete the
biosynthetic products of the invention. For example, the bioderived
compound producers can be cultured for the biosynthetic production
of a bioderived compound disclosed herein. Accordingly, in some
embodiments, the invention provides culture medium having the
bioderived compound or bioderived compound pathway intermediate
described herein. In some aspects, the culture medium can also be
separated from the non-naturally occurring microbial organisms of
the invention that produced the bioderived compound or bioderived
compound pathway intermediate. Methods for separating a microbial
organism from culture medium are well known in the art. Exemplary
methods include filtration, flocculation, precipitation,
centrifugation, sedimentation, and the like.
[0221] For the production of acetyl-CoA or a bioderived compound,
the recombinant strains are cultured in a medium with carbon source
and other essential nutrients. It is sometimes desirable and can be
highly desirable to maintain anaerobic conditions in the fermenter
to reduce the cost of the overall process. Such conditions can be
obtained, for example, by first sparging the medium with nitrogen
and then sealing the flasks with a septum and crimp-cap. For
strains where growth is not observed anaerobically, microaerobic or
substantially anaerobic conditions can be applied by perforating
the septum with a small hole for limited aeration. Exemplary
anaerobic conditions have been described previously and are
well-known in the art. Exemplary aerobic and anaerobic conditions
are described, for example, in United State publication
2009/0047719, filed Aug. 10, 2007. Fermentations can be performed
in a batch, fed-batch or continuous manner, as disclosed herein.
Fermentations can also be conducted in two phases, if desired. The
first phase can be aerobic to allow for high growth and therefore
high productivity, followed by an anaerobic phase of high
acetyl-CoA or a bioderived compound yields.
[0222] If desired, the pH of the medium can be maintained at a
desired pH, in particular neutral pH, such as a pH of around 7 by
addition of a base, such as NaOH or other bases, or acid, as needed
to maintain the culture medium at a desirable pH. The growth rate
can be determined by measuring optical density using a
spectrophotometer (600 nm), and the glucose uptake rate by
monitoring carbon source depletion over time.
[0223] The growth medium, can include, for example, any
carbohydrate source which can supply a source of carbon to the
non-naturally occurring microbial organism of the invention. Such
sources include, for example, sugars such as glucose, xylose,
arabinose, galactose, mannose, fructose, sucrose and starch; or
glycerol, alone as the sole source of carbon or in combination with
other carbon sources described herein or known in the art. In one
embodiment, the carbon source is a sugar. In one embodiment, the
carbon source is a sugar-containing biomass. In some embodiments,
the sugar is glucose. In one embodiment, the sugar is xylose. In
another embodiment, the sugar is arabinose. In one embodiment, the
sugar is galactose. In another embodiment, the sugar is fructose.
In other embodiments, the sugar is sucrose. In one embodiment, the
sugar is starch. In certain embodiments, the carbon source is
glycerol. In some embodiments, the carbon source is crude glycerol.
In one embodiment, the carbon source is crude glycerol without
treatment. In other embodiments, the carbon source is glycerol and
glucose. In another embodiment, the carbon source is methanol and
glycerol. In one embodiment, the carbon source is carbon dioxide.
In one embodiment, the carbon source is formate. In one embodiment,
the carbon source is methane. In one embodiment, the carbon source
is methanol. In certain embodiments, methanol is used alone as the
sole source of carbon or in combination with other carbon sources
described herein or known in the art. In a specific embodiment, the
methanol is the only (sole) carbon source. In one embodiment, the
carbon source is chemoelectro-generated carbon (see, e.g., Liao et
al. (2012) Science 335:1596). In one embodiment, the
chemoelectro-generated carbon is methanol. In one embodiment, the
chemoelectro-generated carbon is formate. In one embodiment, the
chemoelectro-generated carbon is formate and methanol. In one
embodiment, the carbon source is a carbohydrate and methanol. In
one embodiment, the carbon source is a sugar and methanol. In
another embodiment, the carbon source is a sugar and glycerol. In
other embodiments, the carbon source is a sugar and crude glycerol.
In yet other embodiments, the carbon source is a sugar and crude
glycerol without treatment. In one embodiment, the carbon source is
a sugar-containing biomass and methanol. In another embodiment, the
carbon source is a sugar-containing biomass and glycerol. In other
embodiments, the carbon source is a sugar-containing biomass and
crude glycerol. In yet other embodiments, the carbon source is a
sugar-containing biomass and crude glycerol without treatment. In
some embodiments, the carbon source is a sugar-containing biomass,
methanol and a carbohydrate. Other sources of carbohydrate include,
for example, renewable feedstocks and biomass. Exemplary types of
biomasses that can be used as feedstocks in the methods provided
herein include cellulosic biomass, hemicellulosic biomass and
lignin feedstocks or portions of feedstocks. Such biomass
feedstocks contain, for example, carbohydrate substrates useful as
carbon sources such as glucose, xylose, arabinose, galactose,
mannose, fructose and starch. Given the teachings and guidance
provided herein, those skilled in the art will understand that
renewable feedstocks and biomass other than those exemplified above
also can be used for culturing the microbial organisms provided
herein for the production of succinate and other pathway
intermediates.
[0224] In one embodiment, the carbon source is glycerol. In certain
embodiments, the glycerol carbon source is crude glycerol or crude
glycerol without further treatment. In a further embodiment, the
carbon source comprises glycerol or crude glycerol, and also sugar
or a sugar-containing biomass, such as glucose. In a specific
embodiment, the concentration of glycerol in the fermentation broth
is maintained by feeding crude glycerol, or a mixture of crude
glycerol and sugar (e.g., glucose). In certain embodiments, sugar
is provided for sufficient strain growth. In some embodiments, the
sugar (e.g., glucose) is provided at a molar concentration ratio of
glycerol to sugar of from 200:1 to 1:200. In some embodiments, the
sugar (e.g., glucose) is provided at a molar concentration ratio of
glycerol to sugar of from 100:1 to 1:100. In some embodiments, the
sugar (e.g., glucose) is provided at a molar concentration ratio of
glycerol to sugar of from 100:1 to 5:1. In some embodiments, the
sugar (e.g., glucose) is provided at a molar concentration ratio of
glycerol to sugar of from 50:1 to 5:1. In certain embodiments, the
sugar (e.g., glucose) is provided at a molar concentration ratio of
glycerol to sugar of 100:1. In one embodiment, the sugar (e.g,
glucose) is provided at a molar concentration ratio of glycerol to
sugar of 90:1. In one embodiment, the sugar (e.g, glucose) is
provided at a molar concentration ratio of glycerol to sugar of
80:1. In one embodiment, the sugar (e.g, glucose) is provided at a
molar concentration ratio of glycerol to sugar of 70:1. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 60:1. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 50:1. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 40:1. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 30:1. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 20:1. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 10:1. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 5:1. In one embodiment,
the sugar (e.g., glucose) is provided at a molar concentration
ratio of glycerol to sugar of 2:1. In one embodiment, the sugar
(e.g., glucose) is provided at a molar concentration ratio of
glycerol to sugar of 1:1. In certain embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of glycerol to
sugar of 1:100. In one embodiment, the sugar (e.g, glucose) is
provided at a molar concentration ratio of glycerol to sugar of
1:90. In one embodiment, the sugar (e.g, glucose) is provided at a
molar concentration ratio of glycerol to sugar of 1:80. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:70. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:60. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:50. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:40. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:30. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:20. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:10. In one
embodiment, the sugar (e.g, glucose) is provided at a molar
concentration ratio of glycerol to sugar of 1:5. In one embodiment,
the sugar (e.g, glucose) is provided at a molar concentration ratio
of glycerol to sugar of 1:2. In certain embodiments of the ratios
provided above, the sugar is a sugar-containing biomass. In certain
other embodiments of the ratios provided above, the glycerol is a
crude glycerol or a crude glycerol without further treatment. In
other embodiments of the ratios provided above, the sugar is a
sugar-containing biomass, and the glycerol is a crude glycerol or a
crude glycerol without further treatment.
[0225] Crude glycerol can be a by-product produced in the
production of biodiesel, and can be used for fermentation without
any further treatment. Biodiesel production methods include (1) a
chemical method wherein the glycerol-group of vegetable oils or
animal oils is substituted by low-carbon alcohols such as methanol
or ethanol to produce a corresponding fatty acid methyl esters or
fatty acid ethyl esters by transesterification in the presence of
acidic or basic catalysts; (2) a biological method where biological
enzymes or cells are used to catalyze transesterification reaction
and the corresponding fatty acid methyl esters or fatty acid ethyl
esters are produced; and (3) a supercritical method, wherein
transesterification reaction is carried out in a supercritical
solvent system without any catalysts. The chemical composition of
crude glycerol can vary with the process used to produce biodiesel,
the transesterification efficiency, recovery efficiency of the
biodiesel, other impurities in the feedstock, and whether methanol
and catalysts were recovered. For example, the chemical
compositions of eleven crude glycerol collected from seven
Australian biodiesel producers reported that glycerol content
ranged between 38% and 96%, with some samples including more than
14% methanol and 29% ash. In certain embodiments, the crude
glycerol comprises from 5% to 99% glycerol. In some embodiments,
the crude glycerol comprises from 10% to 90% glycerol. In some
embodiments, the crude glycerol comprises from 10% to 80% glycerol.
In some embodiments, the crude glycerol comprises from 10% to 70%
glycerol. In some embodiments, the crude glycerol comprises from
10% to 60% glycerol. In some embodiments, the crude glycerol
comprises from 10% to 50% glycerol. In some embodiments, the crude
glycerol comprises from 10% to 40% glycerol. In some embodiments,
the crude glycerol comprises from 10% to 30% glycerol. In some
embodiments, the crude glycerol comprises from 10% to 20% glycerol.
In some embodiments, the crude glycerol comprises from 80% to 90%
glycerol. In some embodiments, the crude glycerol comprises from
70% to 90% glycerol. In some embodiments, the crude glycerol
comprises from 60% to 90% glycerol. In some embodiments, the crude
glycerol comprises from 50% to 90% glycerol. In some embodiments,
the crude glycerol comprises from 40% to 90% glycerol. In some
embodiments, the crude glycerol comprises from 30% to 90% glycerol.
In some embodiments, the crude glycerol comprises from 20% to 90%
glycerol. In some embodiments, the crude glycerol comprises from
20% to 40% glycerol. In some embodiments, the crude glycerol
comprises from 40% to 60% glycerol. In some embodiments, the crude
glycerol comprises from 60% to 80% glycerol. In some embodiments,
the crude glycerol comprises from 50% to 70% glycerol. In one
embodiment, the glycerol comprises 5% glycerol. In one embodiment,
the glycerol comprises 10% glycerol. In one embodiment, the
glycerol comprises 15% glycerol. In one embodiment, the glycerol
comprises 20% glycerol. In one embodiment, the glycerol comprises
25% glycerol. In one embodiment, the glycerol comprises 30%
glycerol. In one embodiment, the glycerol comprises 35% glycerol.
In one embodiment, the glycerol comprises 40% glycerol. In one
embodiment, the glycerol comprises 45% glycerol. In one embodiment,
the glycerol comprises 50% glycerol. In one embodiment, the
glycerol comprises 55% glycerol. In one embodiment, the glycerol
comprises 60% glycerol. In one embodiment, the glycerol comprises
65% glycerol. In one embodiment, the glycerol comprises 70%
glycerol. In one embodiment, the glycerol comprises 75% glycerol.
In one embodiment, the glycerol comprises 80% glycerol. In one
embodiment, the glycerol comprises 85% glycerol. In one embodiment,
the glycerol comprises 90% glycerol. In one embodiment, the
glycerol comprises 95% glycerol. In one embodiment, the glycerol
comprises 99% glycerol.
[0226] In one embodiment, the carbon source is methanol or formate.
In certain embodiments, methanol is used as a carbon source in a
formaldehyde fixation pathway provided herein. In one embodiment,
the carbon source is methanol or formate. In other embodiments,
formate is used as a carbon source in a formaldehyde fixation
pathway provided herein. In specific embodiments, methanol is used
as a carbon source in a methanol oxidation pathway provided herein,
either alone or in combination with the fatty alcohol, fatty
aldehyde, fatty acid or isopropanol pathways provided herein. In
one embodiment, the carbon source is methanol. In another
embodiment, the carbon source is formate.
[0227] In one embodiment, the carbon source comprises methanol, and
sugar (e.g, glucose) or a sugar-containing biomass. In another
embodiment, the carbon source comprises formate, and sugar (e.g.,
glucose) or a sugar-containing biomass. In one embodiment, the
carbon source comprises methanol, formate, and sugar (e.g, glucose)
or a sugar-containing biomass. In specific embodiments, the
methanol or formate, or both, in the fermentation feed is provided
as a mixture with sugar (e.g, glucose) or sugar-comprising biomass.
In certain embodiments, sugar is provided for sufficient strain
growth.
[0228] In certain embodiments, the carbon source comprises methanol
and a sugar (e.g, glucose). In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of methanol to
sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of methanol to
sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of methanol to
sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of methanol to
sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g,
glucose) is provided at a molar concentration ratio of methanol to
sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol to sugar of
90:1. In one embodiment, the sugar (e.g., glucose) is provided at a
molar concentration ratio of methanol to sugar of 80:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 70:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 60:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 50:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 40:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 30:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 20:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 10:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 5:1. In one embodiment,
the sugar (e.g., glucose) is provided at a molar concentration
ratio of methanol to sugar of 2:1. In one embodiment, the sugar
(e.g., glucose) is provided at a molar concentration ratio of
methanol to sugar of 1:1. In certain embodiments, the sugar (e.g,
glucose) is provided at a molar concentration ratio of methanol to
sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol to sugar of
1:90. In one embodiment, the sugar (e.g., glucose) is provided at a
molar concentration ratio of methanol to sugar of 1:80. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:70. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:60. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:50. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:40. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:30. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:20. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:10. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol to sugar of 1:5. In one embodiment,
the sugar (e.g., glucose) is provided at a molar concentration
ratio of methanol to sugar of 1:2. In certain embodiments of the
ratios provided above, the sugar is a sugar-containing biomass.
[0229] In certain embodiments, the carbon source comprises formate
and a sugar (e.g., glucose). In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of from 200:1 to 1:200. In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of from 100:1 to 1:100. In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of from 100:1 to 5:1. In some embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of from 50:1 to 5:1. In certain embodiments, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of formate to sugar of
90:1. In one embodiment, the sugar (e.g., glucose) is provided at a
molar concentration ratio of formate to sugar of 80:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of formate to sugar of 70:1. In one embodiment,
the sugar (e.g., glucose) is provided at a molar concentration
ratio of formate to sugar of 60:1. In one embodiment, the sugar
(e.g., glucose) is provided at a molar concentration ratio of
formate to sugar of 50:1. In one embodiment, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of formate to sugar of
30:1. In one embodiment, the sugar (e.g., glucose) is provided at a
molar concentration ratio of formate to sugar of 20:1. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of formate to sugar of 10:1. In one embodiment,
the sugar (e.g., glucose) is provided at a molar concentration
ratio of formate to sugar of 5:1. In one embodiment, the sugar
(e.g., glucose) is provided at a molar concentration ratio of
formate to sugar of 2:1. In one embodiment, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of 1:1. In certain embodiments, the sugar (e.g., glucose) is
provided at a molar concentration ratio of formate to sugar of
1:100. In one embodiment, the sugar (e.g, glucose) is provided at a
molar concentration ratio of formate to sugar of 1:90. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of formate to sugar of 1:80. In one embodiment,
the sugar (e.g., glucose) is provided at a molar concentration
ratio of formate to sugar of 1:70. In one embodiment, the sugar
(e.g., glucose) is provided at a molar concentration ratio of
formate to sugar of 1:60. In one embodiment, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of formate to sugar of
1:40. In one embodiment, the sugar (e.g., glucose) is provided at a
molar concentration ratio of formate to sugar of 1:30. In one
embodiment, the sugar (e.g., glucose) is provided at a molar
concentration ratio of formate to sugar of 1:20. In one embodiment,
the sugar (e.g., glucose) is provided at a molar concentration
ratio of formate to sugar of 1:10. In one embodiment, the sugar
(e.g., glucose) is provided at a molar concentration ratio of
formate to sugar of 1:5. In one embodiment, the sugar (e.g.,
glucose) is provided at a molar concentration ratio of formate to
sugar of 1:2. In certain embodiments of the ratios provided above,
the sugar is a sugar-containing biomass.
[0230] In certain embodiments, the carbon source comprises a
mixture of methanol and formate, and a sugar (e.g., glucose). In
certain embodiments, sugar is provided for sufficient strain
growth. In some embodiments, the sugar (e.g., glucose) is provided
at a molar concentration ratio of methanol and formate to sugar of
from 200:1 to 1:200. In some embodiments, the sugar (e.g., glucose)
is provided at a molar concentration ratio of methanol and formate
to sugar of from 100:1 to 1:100. In some embodiments, the sugar
(e.g., glucose) is provided at a molar concentration ratio of
methanol and formate to sugar of from 100:1 to 5:1. In some
embodiments, the sugar (e.g., glucose) is provided at a molar
concentration ratio of methanol and formate to sugar of from 50:1
to 5:1. In certain embodiments, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 100:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 90:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 80:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 70:1. In one embodiment, the sugar (e.g, glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 60:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 50:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 40:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 30:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 20:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 10:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 5:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 2:1. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:1. In certain embodiments, the sugar (e.g, glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:100. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:90. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:80. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:70. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:60. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:50. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:40. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:30. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:20. In one embodiment, the sugar (e.g, glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:10. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:5. In one embodiment, the sugar (e.g., glucose) is
provided at a molar concentration ratio of methanol and formate to
sugar of 1:2. In certain embodiments of the ratios provided above,
the sugar is a sugar-containing biomass.
[0231] In addition to renewable feedstocks such as those
exemplified above, the acetyl-CoA or the bioderived compound
producing microbial organisms of the invention also can be modified
for growth on syngas as its source of carbon. In this specific
embodiment, one or more proteins or enzymes are expressed in the
acetyl-CoA or the bioderived compound producing organisms to
provide a metabolic pathway for utilization of syngas or other
gaseous carbon source.
[0232] Synthesis gas, also known as syngas or producer gas, is the
major product of gasification of coal and of carbonaceous materials
such as biomass materials, including agricultural crops and
residues. Syngas is a mixture primarily of H2 and CO and can be
obtained from the gasification of any organic feedstock, including
but not limited to coal, coal oil, natural gas, biomass, and waste
organic matter. Gasification is generally carried out under a high
fuel to oxygen ratio. Although largely H2 and CO, syngas can also
include CO.sub.2 and other gases in smaller quantities. Thus,
synthesis gas provides a cost effective source of gaseous carbon
such as CO and, additionally, CO.sub.2.
[0233] Accordingly, given the teachings and guidance provided
herein, those skilled in the art will understand that a
non-naturally occurring microbial organism can be produced that
secretes the biosynthesized compounds of the invention when grown
on a carbon source such as a carbohydrate. Such compounds include,
for example, acetyl-CoA or a bioderived compound and any of the
intermediate metabolites in the acetyl-CoA or the bioderived
compound pathway. All that is required is to engineer in one or
more of the required enzyme or protein activities to achieve
biosynthesis of the desired compound or intermediate including, for
example, inclusion of some or all of the acetyl-CoA or the
bioderived compound biosynthetic pathways. Accordingly, the
invention provides a non-naturally occurring microbial organism
that produces and/or secretes acetyl-CoA or a bioderived compound
when grown on a carbohydrate or other carbon source and produces
and/or secretes any of the intermediate metabolites shown in the
acetyl-CoA or the bioderived compound pathway when grown on a
carbohydrate or other carbon source. The acetyl-CoA or the
bioderived compound producing microbial organisms of the invention
can initiate synthesis from an intermediate, for example, F6P, E4P,
formate, formyl-CoA, G3P, PYR, DHA, H6P, 3HBCOA, 3HB,
3-hydroxybutyryl phosphate, 4-hydroxybutyrate,
4-hydroxybutyryl-CoA, adipyl-CoA, adipate semialdehyde,
3-hydroxyisobutyrate, or 2-hydroxyisobutyryl-CoA.
[0234] The non-naturally occurring microbial organisms of the
invention are constructed using methods well known in the art as
exemplified herein to exogenously express at least one nucleic acid
encoding an acetyl-CoA or a bioderived compound pathway enzyme or
protein in sufficient amounts to produce acetyl-CoA or a bioderived
compound. It is understood that the microbial organisms of the
invention are cultured under conditions sufficient to produce
acetyl-CoA or a bioderived compound. Following the teachings and
guidance provided herein, the non-naturally occurring microbial
organisms of the invention can achieve biosynthesis of a bioderived
compound resulting in intracellular concentrations between about
0.1-200 mM or more. Generally, the intracellular concentration of a
bioderived compound is between about 3-150 mM, particularly between
about 5-125 mM and more particularly between about 8-100 mM,
including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular
concentrations between and above each of these exemplary ranges
also can be achieved from the non-naturally occurring microbial
organisms of the invention.
[0235] In some embodiments, culture conditions include anaerobic or
substantially anaerobic growth or maintenance conditions. Exemplary
anaerobic conditions have been described previously and are well
known in the art. Exemplary anaerobic conditions for fermentation
processes are described herein and are described, for example, in
U.S. publication 2009/0047719, filed Aug. 10, 2007. Any of these
conditions can be employed with the non-naturally occurring
microbial organisms as well as other anaerobic conditions well
known in the art. Under such anaerobic or substantially anaerobic
conditions, the acetyl-CoA or the bioderived compound producers can
synthesize a bioderived compound at intracellular concentrations of
5-10 mM or more as well as all other concentrations exemplified
herein. It is understood that, even though the above description
refers to intracellular concentrations, acetyl-CoA or a bioderived
compound producing microbial organisms can produce a bioderived
compound intracellularly and/or secrete the product into the
culture medium.
[0236] Exemplary fermentation processes include, but are not
limited to, fed-batch fermentation and batch separation; fed-batch
fermentation and continuous separation; and continuous fermentation
and continuous separation. In an exemplary batch fermentation
protocol, the production organism is grown in a suitably sized
bioreactor sparged with an appropriate gas. Under anaerobic
conditions, the culture is sparged with an inert gas or combination
of gases, for example, nitrogen, N2/CO.sub.2 mixture, argon,
helium, and the like. As the cells grow and utilize the carbon
source, additional carbon source(s) and/or other nutrients are fed
into the bioreactor at a rate approximately balancing consumption
of the carbon source and/or nutrients. The temperature of the
bioreactor is maintained at a desired temperature, generally in the
range of 22-37 degrees C., but the temperature can be maintained at
a higher or lower temperature depending on the growth
characteristics of the production organism and/or desired
conditions for the fermentation process. Growth continues for a
desired period of time to achieve desired characteristics of the
culture in the fermenter, for example, cell density, product
concentration, and the like. In a batch fermentation process, the
time period for the fermentation is generally in the range of
several hours to several days, for example, 8 to 24 hours, or 1, 2,
3, 4 or 5 days, or up to a week, depending on the desired culture
conditions. The pH can be controlled or not, as desired, in which
case a culture in which pH is not controlled will typically
decrease to pH 3-6 by the end of the run. Upon completion of the
cultivation period, the fermenter contents can be passed through a
cell separation unit, for example, a centrifuge, filtration unit,
and the like, to remove cells and cell debris. In the case where
the desired product is expressed intracellularly, the cells can be
lysed or disrupted enzymatically or chemically prior to or after
separation of cells from the fermentation broth, as desired, in
order to release additional product. The fermentation broth can be
transferred to a product separations unit. Isolation of product
occurs by standard separations procedures employed in the art to
separate a desired product from dilute aqueous solutions. Such
methods include, but are not limited to, liquid-liquid extraction
using a water immiscible organic solvent (e.g, toluene or other
suitable solvents, including but not limited to diethyl ether,
ethyl acetate, tetrahydrofuran (THF), methylene chloride,
chloroform, benzene, pentane, hexane, heptane, petroleum ether,
methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), and the like) to provide an
organic solution of the product, if appropriate, standard
distillation methods, and the like, depending on the chemical
characteristics of the product of the fermentation process.
[0237] In an exemplary fully continuous fermentation protocol, the
production organism is generally first grown up in batch mode in
order to achieve a desired cell density. When the carbon source
and/or other nutrients are exhausted, feed medium of the same
composition is supplied continuously at a desired rate, and
fermentation liquid is withdrawn at the same rate. Under such
conditions, the product concentration in the bioreactor generally
remains constant, as well as the cell density. The temperature of
the fermenter is maintained at a desired temperature, as discussed
above. During the continuous fermentation phase, it is generally
desirable to maintain a suitable pH range for optimized production.
The pH can be monitored and maintained using routine methods,
including the addition of suitable acids or bases to maintain a
desired pH range. The bioreactor is operated continuously for
extended periods of time, generally at least one week to several
weeks and up to one month, or longer, as appropriate and desired.
The fermentation liquid and/or culture is monitored periodically,
including sampling up to every day, as desired, to assure
consistency of product concentration and/or cell density. In
continuous mode, fermenter contents are constantly removed as new
feed medium is supplied. The exit stream, containing cells, medium,
and product, are generally subjected to a continuous product
separations procedure, with or without removing cells and cell
debris, as desired. Continuous separations methods employed in the
art can be used to separate the product from dilute aqueous
solutions, including but not limited to continuous liquid-liquid
extraction using a water immiscible organic solvent (e.g., toluene
or other suitable solvents, including but not limited to diethyl
ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride,
chloroform, benzene, pentane, hexane, heptane, petroleum ether,
methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), and the like), standard
continuous distillation methods, and the like, or other methods
well known in the art.
[0238] In addition to the culturing and fermentation conditions
disclosed herein, growth condition for achieving biosynthesis of
acetyl-CoA or a bioderived compound can include the addition of an
osmoprotectant to the culturing conditions. In certain embodiments,
the non-naturally occurring microbial organisms of the invention
can be sustained, cultured or fermented as described herein in the
presence of an osmoprotectant. Briefly, an osmoprotectant refers to
a compound that acts as an osmolyte and helps a microbial organism
as described herein survive osmotic stress. Osmoprotectants
include, but are not limited to, betaines, amino acids, and the
sugar trehalose. Non-limiting examples of such are glycine betaine,
praline betaine, dimethylthetin, dimethylslfonioproprionate,
3-dimethylsulfonio-2-methylproprionate, pipecolic acid,
dimethylsulfonioacetate, choline, L-camitine and ectoine. In one
aspect, the osmoprotectant is glycine betaine. It is understood to
one of ordinary skill in the art that the amount and type of
osmoprotectant suitable for protecting a microbial organism
described herein from osmotic stress will depend on the microbial
organism used. The amount of osmoprotectant in the culturing
conditions can be, for example, no more than about 0.1 mM, no more
than about 0.5 mM, no more than about 1.0 mM, no more than about
1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no
more than about 3.0 mM, no more than about 5.0 mM, no more than
about 7.0 mM, no more than about 10 mM, no more than about 50 mM,
no more than about 100 mM or no more than about 500 mM.
[0239] In some embodiments, the carbon feedstock and other cellular
uptake sources such as phosphate, ammonia, sulfate, chloride and
other halogens can be chosen to alter the isotopic distribution of
the atoms present in acetyl-CoA or a bioderived compound or any
acetyl-CoA or a bioderived compound pathway intermediate. The
various carbon feedstock and other uptake sources enumerated above
will be referred to herein, collectively, as "uptake sources."
Uptake sources can provide isotopic enrichment for any atom present
in the acetyl-CoA, bioderived compound or pathway intermediate, or
for side products generated in reactions diverging away from an
acetyl-CoA or a bioderived compound pathway. Isotopic enrichment
can be achieved for any target atom including, for example, carbon,
hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other
halogens.
[0240] In some embodiments, the uptake sources can be selected to
alter the carbon-12, carbon-13, and carbon-14 ratios. In some
embodiments, the uptake sources can be selected to alter the
oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments,
the uptake sources can be selected to alter the hydrogen,
deuterium, and tritium ratios. In some embodiments, the uptake
sources can be selected to alter the nitrogen-14 and nitrogen-15
ratios. In some embodiments, the uptake sources can be selected to
alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In
some embodiments, the uptake sources can be selected to alter the
phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some
embodiments, the uptake sources can be selected to alter the
chlorine-35, chlorine-36, and chlorine-37 ratios.
[0241] In some embodiments, the isotopic ratio of a target atom can
be varied to a desired ratio by selecting one or more uptake
sources. An uptake source can be derived from a natural source, as
found in nature, or from a man-made source, and one skilled in the
art can select a natural source, a man-made source, or a
combination thereof, to achieve a desired isotopic ratio of a
target atom. An example of a man-made uptake source includes, for
example, an uptake source that is at least partially derived from a
chemical synthetic reaction. Such isotopically enriched uptake
sources can be purchased commercially or prepared in the laboratory
and/or optionally mixed with a natural source of the uptake source
to achieve a desired isotopic ratio. In some embodiments, a target
atom isotopic ratio of an uptake source can be achieved by
selecting a desired origin of the uptake source as found in nature.
For example, as discussed herein, a natural source can be a
biobased derived from or synthesized by a biological organism or a
source such as petroleum-based products or the atmosphere. In some
such embodiments, a source of carbon, for example, can be selected
from a fossil fuel-derived carbon source, which can be relatively
depleted of carbon-14, or an environmental or atmospheric carbon
source, such as CO.sub.2, which can possess a larger amount of
carbon-14 than its petroleum-derived counterpart.
[0242] The unstable carbon isotope carbon-14 or radiocarbon makes
up for roughly 1 in 10.sup.12 carbon atoms in the earth's
atmosphere and has a half-life of about 5700 years. The stock of
carbon is replenished in the upper atmosphere by a nuclear reaction
involving cosmic rays and ordinary nitrogen (.sup.14N). Fossil
fuels contain no carbon-14, as it decayed long ago. Burning of
fossil fuels lowers the atmospheric carbon-14 fraction, the
so-called "Suess effect".
[0243] Methods of determining the isotopic ratios of atoms in a
compound are well known to those skilled in the art. Isotopic
enrichment is readily assessed by mass spectrometry using
techniques known in the art such as accelerated mass spectrometry
(AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and
Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic
Resonance (SNIF-NMR). Such mass spectral techniques can be
integrated with separation techniques such as liquid chromatography
(LC), high performance liquid chromatography (HPLC) and/or gas
chromatography, and the like.
[0244] In the case of carbon, ASTM D6866 was developed in the
United States as a standardized analytical method for determining
the biobased content of solid, liquid, and gaseous samples using
radiocarbon dating by the American Society for Testing and
Materials (ASTM) International. The standard is based on the use of
radiocarbon dating for the determination of a product's biobased
content. ASTM D6866 was first published in 2004, and the current
active version of the standard is ASTM D6866-11 (effective Apr. 1,
2011). Radiocarbon dating techniques are well known to those
skilled in the art, including those described herein.
[0245] The biobased content of a compound is estimated by the ratio
of carbon-14 (.sup.14C) to carbon-12 (.sup.12C). Specifically, the
Fraction Modem (Fm) is computed from the expression:
Fm=(S-B)/(M-B), where B, S and M represent the .sup.14C/.sup.12C
ratios of the blank, the sample and the modem reference,
respectively. Fraction Modem is a measurement of the deviation of
the .sup.14C/.sup.12C ratio of a sample from "Modem." Modem is
defined as 95% of the radiocarbon concentration (in AD 1950) of
National Bureau of Standards (NBS) Oxalic Acid I (i.e., standard
reference materials (SRM) 4990b) normalized to
.delta..sup.13C.sub.VPDB=-19 per mil (Olsson, The use of Oxalic
acid as a Standard. in, Radiocarbon Variations and Absolute
Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, New
York (1970)). Mass spectrometry results, for example, measured by
ASM, are calculated using the internationally agreed upon
definition of 0.95 times the specific activity of NBS Oxalic Acid I
(SRM 4990b) normalized to .delta..sup.13C.sub.VPDB=-19 per mil.
This is equivalent to an absolute (AD 1950).sup.14C/.sup.12C ratio
of 1.176.+-.0.010.times.10.sup.-12 (Karlen et al., Arkiv Geoftsik,
4:465-471 (1968)). The standard calculations take into account the
differential uptake of one isotope with respect to another, for
example, the preferential uptake in biological systems of C.sup.12
over C.sup.13 over C'', and these corrections are reflected as a Fm
corrected for .delta..sup.13.
[0246] An oxalic acid standard (SRM 4990b or HOx 1) was made from a
crop of 1955 sugar beet. Although there were 1000 lbs made, this
oxalic acid standard is no longer commercially available. The
Oxalic Acid II standard (HOx 2; N.I.S.T designation SRM 4990 C) was
made from a crop of 1977 French beet molasses. In the early 1980's,
a group of 12 laboratories measured the ratios of the two
standards. The ratio of the activity of Oxalic acid II to 1 is
1.2933.+-.0.001 (the weighted mean). The isotopic ratio of HOx II
is -17.8 per mil. ASTM D6866-11 suggests use of the available
Oxalic Acid II standard SRM 4990 C (Hox2) for the modem standard
(see discussion of original vs. currently available oxalic acid
standards in Mann, Radiocarbon, 25(2):519-527 (1983)). A Fm=0%
represents the entire lack of carbon-14 atoms in a material, thus
indicating a fossil (for example, petroleum based) carbon source. A
Fm=100%, after correction for the post-1950 injection of carbon-14
into the atmosphere from nuclear bomb testing, indicates an
entirely modem carbon source. As described herein, such a "modem"
source includes biobased sources.
[0247] As described in ASTM D6866, the percent modem carbon (pMC)
can be greater than 100% because of the continuing but diminishing
effects of the 1950s nuclear testing programs, which resulted in a
considerable enrichment of carbon-14 in the atmosphere as described
in ASTM D6866-11. Because all sample carbon-14 activities are
referenced to a "pre-bomb" standard, and because nearly all new
biobased products are produced in a post-bomb environment, all pMC
values (after correction for isotopic fraction) must be multiplied
by 0.95 (as of 2010) to better reflect the true biobased content of
the sample. A biobased content that is greater than 103% suggests
that either an analytical error has occurred, or that the source of
biobased carbon is more than several years old.
[0248] ASTM D6866 quantifies the biobased content relative to the
material's total organic content and does not consider the
inorganic carbon and other non-carbon containing substances
present. For example, a product that is 50% starch-based material
and 50% water would be considered to have a Biobased Content=100%
(50% organic content that is 100% biobased) based on ASTM D6866. In
another example, a product that is 50% starch-based material, 25%
petroleum-based, and 25% water would have a Biobased Content=66.7%
(75% organic content but only 50% of the product is biobased). In
another example, a product that is 50% organic carbon and is a
petroleum-based product would be considered to have a Biobased
Content=0% (50% organic carbon but from fossil sources). Thus,
based on the well known methods and known standards for determining
the biobased content of a compound or material, one skilled in the
art can readily determine the biobased content and/or prepared
downstream products that utilize of the invention having a desired
biobased content.
[0249] Applications of carbon-14 dating techniques to quantify
bio-based content of materials are known in the art (Currie et al.,
Nuclear Instruments and Methods in Physics Research B, 172:281-287
(2000)). For example, carbon-14 dating has been used to quantify
bio-based content in terephthalate-containing materials (Colonna et
al., Green Chemistry, 13:2543-2548 (2011)). Notably, polypropylene
terephthalate (PPT) polymers derived from renewable 1,3-propanediol
and petroleum-derived terephthalic acid resulted in Fm values near
30% (i.e., since 3/11 of the polymeric carbon derives from
renewable 1,3-propanediol and 8/11 from the fossil end member
terephthalic acid) (Currie et al., supra, 2000). In contrast,
polybutylene terephthalate polymer derived from both renewable
1,4-butanediol and renewable terephthalic acid resulted in
bio-based content exceeding 90% (Colonna et al., supra, 2011).
[0250] Accordingly, in some embodiments, the present invention
provides acetyl-CoA or a bioderived compound or an acetyl-CoA or a
bioderived compound pathway intermediate that has a carbon-12,
carbon-13, and carbon-14 ratio that reflects an atmospheric carbon,
also referred to as environmental carbon, uptake source. For
example, in some aspects the acetyl-CoA or the bioderived compound
or an acetyl-CoA or a bioderived compound pathway intermediate can
have an Fm value of at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98% or as much as 100%. In some such
embodiments, the uptake source is CO.sub.2. In some embodiments,
the present invention provides acetyl-CoA or a bioderived compound
or an acetyl-CoA or a bioderived compound pathway intermediate that
has a carbon-12, carbon-13, and carbon-14 ratio that reflects
petroleum-based carbon uptake source. In this aspect, the
acetyl-CoA or the bioderived compound or an acetyl-CoA or a
bioderived compound pathway intermediate can have an Fm value of
less than 95%, less than 90%, less than 85%, less than 80%, less
than 75%, less than 70%, less than 65%, less than 60%, less than
55%, less than 50%, less than 45%, less than 40%, less than 35%,
less than 30%, less than 25%, less than 20%, less than 15%, less
than 10%, less than 5%, less than 2% or less than 1%. In some
embodiments, the present invention provides acetyl-CoA or a
bioderived compound or an acetyl-CoA or a bioderived compound
pathway intermediate that has a carbon-12, carbon-13, and carbon-14
ratio that is obtained by a combination of an atmospheric carbon
uptake source with a petroleum-based uptake source. Using such a
combination of uptake sources is one way by which the carbon-12,
carbon-13, and carbon-14 ratio can be varied, and the respective
ratios would reflect the proportions of the uptake sources.
[0251] Further, the present invention relates to the biologically
produced acetyl-CoA, a bioderived compound or pathway intermediate
as disclosed herein, and to the products derived therefrom, wherein
the acetyl-CoA or the bioderived compound or an acetyl-CoA or a
bioderived compound pathway intermediate has a carbon-12,
carbon-13, and carbon-14 isotope ratio of about the same value as
the CO.sub.2 that occurs in the environment. For example, in some
aspects the invention provides bioderived acetyl-CoA or a
bioderived compound or a bioderived acetyl-CoA or a bioderived
compound intermediate having a carbon-12 versus carbon-13 versus
carbon-14 isotope ratio of about the same value as the CO.sub.2
that occurs in the environment, or any of the other ratios
disclosed herein. It is understood, as disclosed herein, that a
product can have a carbon-12 versus carbon-13 versus carbon-14
isotope ratio of about the same value as the CO.sub.2 that occurs
in the environment, or any of the ratios disclosed herein, wherein
the product is generated from a bioderived compound or a bioderived
compound pathway intermediate as disclosed herein, wherein the
bioderived product is chemically modified to generate a final
product. Methods of chemically modifying a bioderived product of a
bioderived compound, or an intermediate thereof, to generate a
desired product are well known to those skilled in the art, as
described herein. The invention further provides biobased products
having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio
of about the same value as the CO.sub.2 that occurs in the
environment, wherein the biobased products are generated directly
from or in combination with bioderived compound or a bioderived
compound pathway intermediate as disclosed herein.
[0252] Fatty alcohol, fatty aldehyde or fatty acid is a chemical
used in commercial and industrial applications. Non-limiting
examples of such applications include production of biofuels,
chemicals, polymers, surfactants, soaps, detergents, shampoos,
lubricating oil additives, fragrances, flavor materials and
acrylates. Accordingly, in some embodiments, the invention provides
biobased biofuels, chemicals, polymers, surfactants, soaps,
detergents, shampoos, lubricating oil additives, fragrances, flavor
materials and acrylates comprising one or more bioderived fatty
alcohol, fatty aldehyde or fatty acid or bioderived fatty alcohol,
fatty aldehyde or fatty acid pathway intermediate produced by a
non-naturally occurring microorganism of the invention or produced
using a method disclosed herein.
[0253] In some embodiments, the invention provides a biofuel,
chemical, polymer, surfactant, soap, detergent, shampoo,
lubricating oil additive, fragrance, flavor material or acrylate
comprising bioderived fatty alcohol, fatty aldehyde or fatty acid
or bioderived fatty alcohol, fatty aldehyde or fatty acid pathway
intermediate, wherein the bioderived fatty alcohol, fatty aldehyde
or fatty acid or bioderived fatty alcohol, fatty aldehyde or fatty
acid pathway intermediate includes all or part of the fatty
alcohol, fatty aldehyde or fatty acid or fatty alcohol, fatty
aldehyde or fatty acid pathway intermediate used in the production
of a biofuel, chemical, polymer, surfactant, soap, detergent,
shampoo, lubricating oil additive, fragrance, flavor material or
acrylate. For example, the final biofuel, chemical, polymer,
surfactant, soap, detergent, shampoo, lubricating oil additive,
fragrance, flavor material or acrylate can contain the bioderived
fatty alcohol, fatty aldehyde or fatty acid, fatty alcohol, fatty
aldehyde or fatty acid pathway intermediate, or a portion thereof
that is the result of the manufacturing of the biofuel, chemical,
polymer, surfactant, soap, detergent, shampoo, lubricating oil
additive, fragrance, flavor material or acrylate. Such
manufacturing can include chemically reacting the bioderived fatty
alcohol, fatty aldehyde or fatty acid, or bioderived fatty alcohol,
fatty aldehyde or fatty acid pathway intermediate (e.g. chemical
conversion, chemical functionalization, chemical coupling,
oxidation, reduction, polymerization, copolymerization and the
like) with itself or another compound in a reaction that produces
the final biofuel, chemical, polymer, surfactant, soap, detergent,
shampoo, lubricating oil additive, fragrance, flavor material or
acrylate. Thus, in some aspects, the invention provides a biobased
biofuel, chemical, polymer, surfactant, soap, detergent, shampoo,
lubricating oil additive, fragrance, flavor material or acrylate
comprising at least 2%, at least 3%, at least 5%, at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 95%, at least 98% or 100% bioderived
fatty alcohol, fatty aldehyde or fatty acid or bioderived fatty
alcohol, fatty aldehyde or fatty acid pathway intermediate as
disclosed herein. In some aspects, when the product is a biobased
polymer that includes or is obtained from a bioderived fatty
alcohol, fatty aldehyde or fatty acid, or or fatty alcohol, fatty
aldehyde or fatty acid pathway intermediate described herein, the
biobased polymer can be molded using methods well known in the art.
Accordingly, in some embodiments, provided herein is a molded
product comprising the biobased polymer described herein.
[0254] Butadiene is a chemical commonly used in many commercial and
industrial applications. Provided herein are a bioderived butadiene
and biobased products comprising one or more bioderived butadiene
or bioderived butadiene intermediate produced by a non-naturally
occurring microorganism of the invention or produced using a method
disclosed herein. Also provided herein are uses for bioderived
butadiene and the biobased products. Non-limiting examples are
described herein and include the following. Biobased products
comprising all or a portion of bioderived butadiene include
polymers, including synthetic rubbers and ABS resins, and
chemicals, including hexamethylenediamine (HMDA), 1,4-butanediol,
tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam,
chloroprene, sulfalone, n-octanol and octene-1. The biobased
polymers, including co-polymers, and resins include those where
butadiene is reacted with one or more other chemicals, such as
other alkenes, e.g. styrene, to manufacture numerous copolymers,
including acrylonitrile 1,3-butadiene styrene (ABS),
styrene-1,3-butadiene rubber (styrene butadiene rubber; SBR),
styrene-1,3-butadiene latex. Products comprising biobased butadiene
in the form of polymer synthetic rubber (SBR) include synthetic
rubber articles, including tires, adhesives, seals, sealants,
coatings, hose and shoe soles, and in the form of synthetic rubber
polybutadiene (polybutadiene rubber, PBR or BR) which is used in
synthetic rubber articles including tires, seals, gaskets and
adhesives and as an intermediate in production of thermoplastic
resin including acrylonitrile-butadiene-styrene (ABS) and in
production of high impact modifier of polymers such as high impact
polystyrene (HIPS). ABS is used in molded articles, including pipe,
telephone, computer casings, mobile phones, radios, and appliances.
Other biobased BD polymers include a latex, including
styrene-butadiene latex (SB), used for example in paper coatings,
carpet backing, adhesives, and foam mattresses; nitrile rubber,
used in for example hoses, fuel lines, gasket seals, gloves and
footwear; and styrene-butadiene block copolymers, used for example
in asphalt modifiers (for road and roofing construction
applications), adhesives, footwear and toys. Chemical intermediates
made from butadiene include adiponitrile, HMDA, lauryl lactam, and
caprolactam, used for example in production of nylon, including
nylon-6,6 and other nylon-6,X, and chloroprene used for example in
production of polychloroprene (neoprene). Butanediol produced from
butadiene is used for example in production of speciality polymer
resins including thermoplastic including polybutylene terephthalate
(PBT), used in molded articles including parts for automotive,
electrical, water systems and small appliances. Butadiene is also a
co-monomer for polyurethane and polyurethane-polyurea copolymers.
Butadiene is a co-monomer for biodegradable polymers, including
PBAT (poly(butylene adipate-co-terephthalate)) and PBS
(poly(butylene succinate)). Tetrahydrofuran produced from butadiene
finds use as a solvent and in production of elastic fibers.
Conversion of butadiene to THF, and subsequently to
polytetramethylene ether glycol (PTMEG) (also referred to as PTMO,
polytetramethylene oxide and PTHF, poly(tetrahydrofuran)), provides
an intermediate used to manufacture elastic fibers, e.g. spandex
fiber, used in products such as LYCRA.RTM. fibers or elastane, for
example when combined with polyurethane-polyurea copolymers. THF
also finds use as an industrial solvent and in pharmaceutical
production. PTMEG is also combined with in the production of
specialty thermoplastic elastomers (TPE), including thermoplastic
elastomer polyester (TPE-E or TPEE) and copolyester ethers (COPE).
COPES are high modulus elastomers with excellent mechanical
properties and oil/environmental resistance, allowing them to
operate at high and low temperature extremes. PTMEG and butadiene
also make thermoplastic polyurethanes (e.g. TPE-U or TPEU)
processed on standard thermoplastic extrusion, calendaring, and
molding equipment, and are characterized by their outstanding
toughness and abrasion resistance. Other biobased products of
bioderived BD include styrene block copolymers used for example in
bitumen modification, footwear, packaging, and molded extruded
products; methylmethacrylate butadiene styrene and methacrylate
butadiene styrene (MBS) resins--clear resins--used as impact
modifier for transparent thermoplastics including polycarbonate
(PC), polyvinyl carbonate (PVC) and poly)methyl methacrylate
(PMMA); sulfalone used as a solvent or chemical; n-octanol and
octene-1. Accordingly, in some embodiments, the invention provides
a biobased product comprising one or more bioderived butadiene or
bioderived butadiene intermediate produced by a non-naturally
occurring microorganism of the invention or produced using a method
disclosed herein.
[0255] Crotyl alcohol, also referred to as 2-buten-1-ol, is a
valuable chemical intermediate. Crotyl alcohol is a chemical
commonly used in many commercial and industrial applications.
Non-limiting examples of such applications include production of
crotyl halides, esters, and ethers, which in turn are chemical are
chemical intermediates in the production of monomers, fine
chemicals, such as sorbic acid, trimethylhydroquinone, crotonic
acid and 3-methoxybutanol, agricultural chemicals, and
pharmaceuticals. Exemplary fine chemical products include sorbic
acid, trimethylhydroquinone, crotonic acid and 3-methoxybutanol.
Crotyl alcohol is also a precursor to 1,3-butadiene. Crotyl alcohol
is currently produced exclusively from petroleum feedstocks. For
example Japanese Patent 47-013009 and U.S. Pat. Nos. 3,090,815,
3,090,816, and 3,542,883 describe a method of producing crotyl
alcohol by isomerization of 1,2-epoxybutane. The ability to
manufacture crotyl alcohol from alternative and/or renewable
feedstocks would represent a major advance in the quest for more
sustainable chemical production processes. Accordingly, in some
embodiments, the invention provides a biobased monomer, fine
chemical, agricultural chemical, or pharmaceutical comprising one
or more bioderived crotyl alcohol or bioderived crotyl alcohol
intermediate produced by a non-naturally occurring microorganism of
the invention or produced using a method disclosed herein.
[0256] 1,3-Butanediol is a chemical commonly used in many
commercial and industrial applications. Non-limiting examples of
such applications include its use as an organic solvent for food
flavoring agents or as a hypoglycaemic agent and its use in the
production of polyurethane and polyester resins. Moreover,
optically active 1,3-butanediol is also used in the synthesis of
biologically active compounds and liquid crystals. Still further,
1,3-butanediol can be used in commercial production of
1,3-butadiene, a compound used in the manufacture of synthetic
rubbers (e.g., tires), latex, and resins. 1,3-butanediol can also
be sued to synthesize (R)-3-hydroxybutyryl-(R)-1,3-butanediol
monoester or (R)-3-ketobutyryl-(R)-1,3-butanediol. Accordingly, in
some embodiments, the invention provides a biobased organic
solvent, hypoglycaemic agent, polyurethane, polyester resin,
synthetic rubber, latex, or resin comprising one or more bioderived
1,3-butanediol or bioderived 1,3-butanediol intermediate produced
by a non-naturally occurring microorganism of the invention or
produced using a method disclosed herein.
[0257] 3-Buten-2-ol is a chemical commonly used in many commercial
and industrial applications. Non-limiting examples of such
applications include it use as a solvent, e.g. as a viscosity
adjustor, a monomer for polymer production, or a precursor to a
fine chemical such as in production of contrast agents for imaging
(see US20110091374) or production of glycerol (see
US20120302800A1). 3-Buten-2-ol can also be used as a precursor in
the production of 1,3-butadiene. Accordingly, in some embodiments,
the invention provides a biobased solvent, polymer (or plastic or
resin made from that polymer), or fine chemical comprising one or
more bioderived 3-buten-2-ol or bioderived 3-buten-2-ol
intermediate produced by a non-naturally occurring microorganism of
the invention or produced using a method disclosed herein.
[0258] In some embodiments, the invention provides polymer,
synthetic rubber, resin, or chemical comprising bioderived
butadiene or bioderived butadiene pathway intermediate, wherein the
bioderived butadiene or bioderived butadiene pathway intermediate
includes all or part of the butadiene or butadiene pathway
intermediate used in the production of polymer, synthetic rubber,
resin, or chemical, or other biobased products described herein
(for example hexamethylenediamine (HMDA), 1,4-butanediol,
tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam,
chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR, PTMEG,
COPE). Thus, in some aspects, the invention provides a biobased
polymer, synthetic rubber, resin, or chemical or other biobased
product described herein comprising at least 2%, at least 3%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, at
least 98% or 100% bioderived butadiene or bioderived butadiene
pathway intermediate as disclosed herein. Additionally, in some
aspects, the invention provides a biobased polymer, synthetic
rubber, resin, or chemical or other biobased product described
herein (for example hexamethylenediamine (HMDA), 1,4-butanediol,
tetrahydrofuran (THF), adiponitrile, lauryl lactam, caprolactam,
chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR, PBR, PTMEG,
COPE), wherein the butadiene or butadiene pathway intermediate used
in its production is a combination of bioderived and petroleum
derived butadiene or butadiene pathway intermediate. For example, a
biobased polymer, synthetic rubber, resin, or chemical or other
biobased product described herein (for example hexamethylenediamine
(HMDA), 1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl
lactam, caprolactam, chloroprene, sulfalone, n-octanol, octene-1,
ABS, SBR, PBR, PTMEG, COPE) can be produced using 50% bioderived
butadiene and 50% petroleum derived butadiene or other desired
ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%,
40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived
precursors, so long as at least a portion of the product comprises
a bioderived product produced by the microbial organisms disclosed
herein. It is understood that methods for producing polymer,
synthetic rubber, resin, or chemical or other biobased product
described herein (for example hexamethylenediamine (HMDA),
1,4-butanediol, tetrahydrofuran (THF), adiponitrile, lauryl lactam,
caprolactam, chloroprene, sulfalone, n-octanol, octene-1, ABS, SBR,
PBR, PTMEG, COPE) using the bioderived butadiene or bioderived
butadiene pathway intermediate of the invention are well known in
the art.
[0259] In some embodiments, the invention provides organic solvent,
hypoglycaemic agent, polyurethane, polyester resin, synthetic
rubber, latex, or resin comprising bioderived 1,3-butanediol or
bioderived 1,3-butanediol pathway intermediate, wherein the
bioderived 1,3-butanediol or bioderived 1,3-butanediol pathway
intermediate includes all or part of the 1,3-butanediol or
1,3-butanediol pathway intermediate used in the production of
organic solvent, hypoglycaemic agent, polyurethane, polyester
resin, synthetic rubber, latex, or resin. Thus, in some aspects,
the invention provides a biobased organic solvent, hypoglycaemic
agent, polyurethane, polyester resin, synthetic rubber, latex, or
resin comprising at least 2%, at least 3%, at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 98% or 100%
bioderived 1,3-butanediol or bioderived 1,3-butanediol pathway
intermediate as disclosed herein. Additionally, in some aspects,
the invention provides a biobased organic solvent, hypoglycaemic
agent, polyurethane, polyester resin, synthetic rubber, latex, or
resin wherein the 1,3-butanediol or 1,3-butanediol pathway
intermediate used in its production is a combination of bioderived
and petroleum derived 1,3-butanediol or 1,3-butanediol pathway
intermediate. For example, a biobased organic solvent,
hypoglycaemic agent, polyurethane, polyester resin, synthetic
rubber, latex, or resin can be produced using 50% bioderived
1,3-butanediol and 50% petroleum derived 1,3-butanediol or other
desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%,
100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum
derived precursors, so long as at least a portion of the product
comprises a bioderived product produced by the microbial organisms
disclosed herein. It is understood that methods for producing
organic solvent, hypoglycaemic agent, polyurethane, polyester
resin, synthetic rubber, latex, or resin using the bioderived
1,3-butanediol or bioderived 1,3-butanediol pathway intermediate of
the invention are well known in the art.
[0260] In some embodiments, the invention provides monomer, fine
chemical, agricultural chemical, or pharmaceutical comprising
bioderived crotyl alcohol or bioderived crotyl alcohol pathway
intermediate, wherein the bioderived crotyl alcohol or bioderived
crotyl alcohol pathway intermediate includes all or part of the
crotyl alcohol or crotyl alcohol pathway intermediate used in the
production of monomer, fine chemical, agricultural chemical, or
pharmaceutical. Thus, in some aspects, the invention provides a
biobased monomer, fine chemical, agricultural chemical, or
pharmaceutical comprising at least 2%, at least 3%, at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, at least 98% or 100%
bioderived crotyl alcohol or bioderived crotyl alcohol pathway
intermediate as disclosed herein. Additionally, in some aspects,
the invention provides a biobased monomer, fine chemical,
agricultural chemical, or pharmaceutical wherein the crotyl alcohol
or crotyl alcohol pathway intermediate used in its production is a
combination of bioderived and petroleum derived crotyl alcohol or
crotyl alcohol pathway intermediate. For example, a biobased
monomer, fine chemical, agricultural chemical, or pharmaceutical
can be produced using 50% bioderived crotyl alcohol and 50%
petroleum derived crotyl alcohol or other desired ratios such as
60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%,
30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived
precursors, so long as at least a portion of the product comprises
a bioderived product produced by the microbial organisms disclosed
herein. It is understood that methods for producing monomer, fine
chemical, agricultural chemical, or pharmaceutical using the
bioderived crotyl alcohol or bioderived crotyl alcohol pathway
intermediate of the invention are well known in the art.
[0261] In some embodiments, the invention provides solvent (or
solvent-containing composition), polymer (or plastic or resin made
from that polymer), or a fine chemical, comprising bioderived
3-buten-2-ol or bioderived 3-buten-2-ol pathway intermediate,
wherein the bioderived 3-buten-2-ol or bioderived 3-buten-2-ol
pathway intermediate includes all or part of the 3-buten-2-ol or
3-buten-2-ol pathway intermediate used in the production of the
solvent (or composition containing the solvent), polymer (or
plastic or resin made from that polymer) or fine chemical. Thus, in
some aspects, the invention provides a biobased solvent (or
composition containing the solvent), polymer (or plastic or resin
made from that polymer) or fine chemical comprising at least 2%, at
least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30% at least 35%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98% or 100% bioderived 3-buten-2-ol or bioderived
3-buten-2-ol pathway intermediate as disclosed herein.
Additionally, in some aspects, the invention provides the biobased
solvent (or composition containing the solvent), polymer (or
plastic or resin made from that polymer) or fine chemical wherein
the 3-buten-2-ol or 3-buten-2-ol pathway intermediate used in its
production is a combination of bioderived and petroleum derived
3-buten-2-ol or 3-buten-2-ol pathway intermediate. For example, the
biobased the solvent (or composition containing the solvent),
polymer (or plastic or resin made from that polymer) or fine
chemical can be produced using 50% bioderived 3-buten-2-ol and 50%
petroleum derived 3-buten-2-ol or other desired ratios such as
60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%,
30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived
precursors, so long as at least a portion of the product comprises
a bioderived product produced by the microbial organisms disclosed
herein. It is understood that methods for producing the solvent (or
composition containing the solvent), polymer (or plastic or resin
made from that polymer) or fine chemical using the bioderived
3-buten-2-ol or bioderived 3-buten-2-ol pathway intermediate of the
invention are well known in the art.
[0262] 1,4-Butanediol and/or 4-HB are chemicals used in commercial
and industrial applications. Non-limiting examples of such
applications include production of plastics, elastic fibers,
polyurethanes, polyesters, including polyhydroxyalkanoates such as
P4HB or co-polymers thereof, PTMEG and polyurethane-polyurea
copolymers, referred to as spandex, elastane or Lycra.TM., nylons,
and the like. Moreover, 1,4-butanediol and/or 4-HB are also used as
a raw material in the production of a wide range of products
including plastics, elastic fibers, polyurethanes, polyesters,
including polyhydroxyalkanoates such as P4HB or co-polymers
thereof, PTMEG and polyurethane-polyurea copolymers, referred to as
spandex, elastane or Lycra.TM., nylons, and the like. Accordingly,
in some embodiments, provided are biobased plastics, elastic
fibers, polyurethanes, polyesters, including polyhydroxyalkanoates
such as P4HB or co-polymers thereof, PTMEG and
polyurethane-polyurea copolymers, referred to as spandex, elastane
or Lycra.TM., nylons, and the like, comprising one or more
bioderived 1,4-butanediol and/or 4-HB or bioderived 1,4-butanediol
and/or 4-HB intermediate thereof produced by a non-naturally
occurring microbial organism provided herein or produced using a
method disclosed herein.
[0263] In some embodiments, the invention provides plastics,
elastic fibers, polyurethanes, polyesters, including
polyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG
and polyurethane-polyurea copolymers, referred to as spandex,
elastane or Lycra.TM., nylons, and the like, comprising bioderived
1,4-butanediol and/or 4-HB or bioderived 1,4-butanediol and/or 4-HB
intermediate thereof, wherein the bioderived 1,4-butanediol and/or
4-HB or bioderived 1,4-butanediol and/or 4-HB intermediate thereof
includes all or part of the 1,4-butanediol and/or 4-HB or
1,4-butanediol and/or 4-HB intermediate thereof used in the
production of plastics, elastic fibers, polyurethanes, polyesters,
including polyhydroxyalkanoates such as P4HB or co-polymers
thereof, PTMEG and polyurethane-polyurea copolymers, referred to as
spandex, elastane or Lycra.TM., nylons, and the like. Thus, in some
aspects, the invention provides a biobased plastics, elastic
fibers, polyurethanes, polyesters, including polyhydroxyalkanoates
such as P4HB or co-polymers thereof, PTMEG and
polyurethane-polyurea copolymers, referred to as spandex, elastane
or Lycra.TM., nylons, and the like, comprising at least 2%, at
least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80% at least 90%, at least
95%, at least 98% or 100% bioderived 1,4-butanediol and/or 4-HB or
bioderived 1,4-butanediol and/or 4-HB intermediate thereof as
disclosed herein.
[0264] In one embodiment, the product is a plastic. In one
embodiment, the product is an elastic fiber. In one embodiment, the
product is a polyurethane. In one embodiment, the product is a
polyester. In one embodiment, the product is a
polyhydroxyalkanoate. In one embodiment, the product is a
poly-4-HB. In one embodiment, the product is a co-polymer of
poly-4-HB. In one embodiment, the product is a poly(tetramethylene
ether) glycol. In one embodiment, the product is a
polyurethane-polyurea copolymer. In one embodiment, the product is
a spandex. In one embodiment, the product is an elastane. In one
embodiment, the product is a Lycra.TM.. In one embodiment, the
product is a nylon.
[0265] Adipate, 6-aminocaproate, hexamethylenediamine and
caprolactam, as well as intermediates thereof, are chemicals used
in commercial and industrial applications. Non-limiting examples of
such applications include production of polymers, plastics, epoxy
resins, nylons (e.g., nylon-6 or nylon 6-6), textiles,
polyurethanes, plasticizers, unsaturated polyesters, fibers,
polyester polyols, polyurethane, lubricant components, PVC, food
additives, food ingredients, flavorants, gelling aids, food and
oral medicinal coatings/products, and the like. Moreover, adipate,
6-aminocaproate, hexamethylenediamine and caprolactam are also used
as a raw material in the production of a wide range of products
including polymers, plastics, epoxy resins, nylons (e.g., nylon-6
or nylon 6-6), textiles, polyurethanes, plasticizers, unsaturated
polyesters, fibers, polyester polyols, polyurethane, lubricant
components, PVC, food additives, food ingredients, flavorants,
gelling aids, food and oral medicinal coatings/products, and the
like. Accordingly, in some embodiments, provided is biobased
polymers, plastics, epoxy resins, nylons (e.g., nylon-6 or nylon
6-6), textiles, polyurethanes, plasticizers, unsaturated
polyesters, fibers, polyester polyols, polyurethane, lubricant
components, PVC, food additives, food ingredients, flavorants,
gelling aids, food and oral medicinal coatings/products, and the
like, comprising one or more of bioderived adipate,
6-aminocaproate, hexamethylenediamine or caprolactam, or a
bioderived intermediate thereof, produced by a non-naturally
occurring microbial organism provided herein or produced using a
method disclosed herein.
[0266] In one embodiment, the product is a polymer. In one
embodiment, the product is a plastic. In one embodiment, the
product is an epoxy resin. In one embodiment, the product is a
nylons (e.g., nylon-6 or nylon 6-6). In one embodiment, the product
is a textile. In one embodiment, the product is a polyurethane. In
one embodiment, the product is a plasticizer. In one embodiment,
the product is an unsaturated polyester. In one embodiment, the
product is a fiber. In one embodiment, the product is a polyester
polyol. In one embodiment, the product is a polyurethane. In one
embodiment, the product is a lubricant component. In one
embodiment, the product is a PVC. In one embodiment, the product is
a food additive. In one embodiment, the product is a food
ingredient. In one embodiment, the product is a flavorant. In one
embodiment, the product is a gelling aid. In one embodiment, the
product is a food coating. In one embodiment, the product is a food
product. In one embodiment, the product is an oral medicinal
coatings. In one embodiment, the product is an oral product
[0267] In some embodiments, provided is polymers, plastics, epoxy
resins, nylons (e.g., nylon-6 or nylon 6-6), textiles,
polyurethanes, plasticizers, unsaturated polyesters, fibers,
polyester polyols, polyurethane, lubricant components, PVC, food
additives, food ingredients, flavorants, gelling aids, food and
oral medicinal coatings/products, and the like, comprising
bioderived adipate, 6-aminocaproate, hexamethylenediamine or
caprolactam, or a bioderived intermediate thereof, wherein the
bioderived adipate, 6-aminocaproate, hexamethylenediamine or
caprolactam, or bioderived intermediate thereof, includes all or
part of an adipate, 6-aminocaproate, hexamethylenediamine or
caprolactam, or an intermediate thereof, used in the production of
polymers, plastics, epoxy resins, nylons (e.g., nylon-6 or nylon
6-6), textiles, polyurethanes, plasticizers, unsaturated
polyesters, fibers, polyester polyols, polyurethane, lubricant
components, PVC, food additives, food ingredients, flavorants,
gelling aids, food and oral medicinal coatings/products, and the
like. Thus, in some aspects, provided is a biobased polymers,
plastics, epoxy resins, nylons (e.g., nylon-6 or nylon 6-6),
textiles, polyurethanes, plasticizers, unsaturated polyesters,
fibers, polyester polyols, polyurethane, lubricant components, PVC,
food additives, food ingredients, flavorants, gelling aids, food
and oral medicinal coatings/products, and the like, comprising at
least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, at least 98% or 100% bioderived adipate,
6-aminocaproate, hexamethylenediamine or caprolactam, or a
bioderived adipate, 6-aminocaproate, hexamethylenediamine or
caprolactam intermediate, as disclosed herein.
[0268] Methacylic acid, as well as intermediates thereof such as
3-hydroxyisobutyrate, and 2-hydroxyisobutyric acid is a chemical
used in commercial and industrial applications. Non-limiting
examples of such applications include production of polymers,
co-polymers, plastics, methacrylates (e.g., a methyl methacrylate
or a butyl methacrylate), glacial methacylic acid, and the like.
2-Hydroxyisobutyric acid can be dehydrated to form methacrylic acid
as described, for example, in U.S. Pat. No. 7,186,856. Moreover,
3-hydroxyisobutyrate and methacylic acid are also used as a raw
material in the production of a wide range of products including
polymers, co-polymers, plastics, methacrylates (e.g., a methyl
methacrylate or a butyl methacrylate), glacial methacylic acid, and
the like. Accordingly, in some embodiments, the invention provides
biobased polymers, co-polymers, plastics, methacrylates (e.g., a
methyl methacrylate or a butyl methacrylate), glacial methacylic
acid, and the like, comprising one or more of bioderived methacylic
acid, 3-hydroxyisobutyrate or 2-hydroxyisobutyric acid, or a
bioderived intermediate thereof, produced by a non-naturally
occurring microorganism of the invention or produced using a method
disclosed herein.
[0269] In some embodiments, the invention provides polymers,
co-polymers, plastics, methacrylates (e.g., a methyl methacrylate
or a butyl methacrylate), glacial methacylic acid, and the like,
comprising bioderived methacylic acid, 3-hydroxyisobutyrate or
2-hydroxyisobutyric acid, or a bioderived intermediate thereof,
wherein the bioderived methacylic acid, 3-hydroxyisobutyrate or
2-hydroxyisobutyric acid, or bioderived intermediate thereof,
includes all or part of the a methacylic acid, 3-hydroxyisobutyrate
or 2-hydroxyisobutyric acid, or an intermediate thereof, used in
the production of polymers, co-polymers, plastics, methacrylates
(e.g., a methyl methacrylate or a butyl methacrylate), glacial
methacylic acid, and the like. Thus, in some aspects, the invention
provides a biobased polymers, co-polymers, plastics, methacrylates
(e.g., a methyl methacrylate or a butyl methacrylate), glacial
methacylic acid, and the like, comprising at least 2%, at least 3%,
at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 98% or 100% bioderived methacylic acid,
3-hydroxyisobutyrate or 2-hydroxyisobutyric acid, or a bioderived
methacylic acid, 3-hydroxyisobutyrate or 2-hydroxyisobutyric acid
intermediate, as disclosed herein.
[0270] Additionally, in some embodiments, the invention provides a
composition having a bioderived compound or pathway intermediate
disclosed herein and a compound other than the bioderived compound
or pathway intermediate. For example, in some aspects, the
invention provides a biobased product as described herein wherein
the bioderived compound or bioderived compound pathway intermediate
used in its production is a combination of bioderived and petroleum
derived compound or compound pathway intermediate. For example, a
biobased product described herein can be produced using 50%
bioderived compound and 50% petroleum derived compound or other
desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%,
100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum
derived precursors, so long as at least a portion of the product
comprises a bioderived product produced by the microbial organisms
disclosed herein. It is understood that methods for producing a
biobased product as described herein using the bioderived compound
or bioderived compound pathway intermediate of the invention are
well known in the art.
[0271] The invention further provides a composition comprising
bioderived compound described herein and a compound other than the
bioderived bioderived. The compound other than the bioderived
product can be a cellular portion, for example, a trace amount of a
cellular portion of, or can be fermentation broth or culture
medium, or a purified or partially purified fraction thereof
produced in the presence of, a non-naturally occurring microbial
organism of the invention. The composition can comprise, for
example, a reduced level of a byproduct when produced by an
organism having reduced byproduct formation, as disclosed herein.
The composition can comprise, for example, bioderived compound, or
a cell lysate or culture supernatant of a microbial organism of the
invention.
[0272] In certain embodiments, provided herein is a composition
comprising a bioderived compound provided herein produced by
culturing a non-naturally occurring microbial organism described
herein. In some embodiments, the composition further comprises a
compound other than said bioderived compound. In certain
embodiments, the compound other than said bioderived compound is a
trace amount of a cellular portion of a non-naturally occurring
microbial organism describe herein.
[0273] The culture conditions can include, for example, liquid
culture procedures as well as fermentation and other large scale
culture procedures. As described herein, particularly useful yields
of the biosynthetic products of the invention can be obtained under
anaerobic or substantially anaerobic culture conditions.
[0274] As described herein, one exemplary growth condition for
achieving biosynthesis of acetyl-CoA or a bioderived compound
includes anaerobic culture or fermentation conditions. In certain
embodiments, the non-naturally occurring microbial organisms of the
invention can be sustained, cultured or fermented under anaerobic
or substantially anaerobic conditions. Briefly, an anaerobic
condition refers to an environment devoid of oxygen. Substantially
anaerobic conditions include, for example, a culture, batch
fermentation or continuous fermentation such that the dissolved
oxygen concentration in the medium remains between 0 and 10% of
saturation. Substantially anaerobic conditions also includes
growing or resting cells in liquid medium or on solid agar inside a
sealed chamber maintained with an atmosphere of less than 1%
oxygen. The percent of oxygen can be maintained by, for example,
sparging the culture with an N.sub.2/CO.sub.2 mixture or other
suitable non-oxygen gas or gases.
[0275] The culture conditions described herein can be scaled up and
grown continuously for manufacturing of acetyl-CoA or a bioderived
compound. Exemplary growth procedures include, for example,
fed-batch fermentation and batch separation; fed-batch fermentation
and continuous separation, or continuous fermentation and
continuous separation. All of these processes are well known in the
art. Fermentation procedures are particularly useful for the
biosynthetic production of commercial quantities of acetyl-CoA or a
bioderived compound. Generally, and as with non-continuous culture
procedures, the continuous and/or near-continuous production of
acetyl-CoA or a bioderived compound will include culturing a
non-naturally occurring acetyl-CoA or a bioderived compound
producing organism of the invention in sufficient nutrients and
medium to sustain and/or nearly sustain growth in an exponential
phase. Continuous culture under such conditions can include, for
example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or
more. Additionally, continuous culture can include longer time
periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several
months. Alternatively, organisms of the invention can be cultured
for hours, if suitable for a particular application. It is to be
understood that the continuous and/or near-continuous culture
conditions also can include all time intervals in between these
exemplary periods. It is further understood that the time of
culturing the microbial organism of the invention is for a
sufficient period of time to produce a sufficient amount of product
for a desired purpose.
[0276] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of acetyl-CoA or a
bioderived compound can be utilized in, for example, fed-batch
fermentation and batch separation; fed-batch fermentation and
continuous separation, or continuous fermentation and continuous
separation. Examples of batch and continuous fermentation
procedures are well known in the art.
[0277] In addition to the above fermentation procedures using the
acetyl-CoA or the bioderived compound producers of the invention
for continuous production of substantial quantities of acetyl-CoA
or a bioderived compound, the acetyl-CoA or the bioderived compound
producers also can be, for example, simultaneously subjected to
chemical synthesis and/or enzymatic procedures to convert the
product to other compounds or the product can be separated from the
fermentation culture and sequentially subjected to chemical and/or
enzymatic conversion to convert the product to other compounds, if
desired.
[0278] To generate better producers, metabolic modeling can be
utilized to optimize growth conditions. Modeling can also be used
to design gene knockouts that additionally optimize utilization of
the pathway (see, for example, U.S. patent publications US
2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US
2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Pat.
No. 7,127,379). Modeling analysis allows reliable predictions of
the effects on cell growth of shifting the metabolism towards more
efficient production of acetyl-CoA or a bioderived compound.
[0279] One computational method for identifying and designing
metabolic alterations favoring biosynthesis of a desired product is
the OptKnock computational framework (Biirgard et al., Biotechnol.
Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and
simulation program that suggests gene deletion or disruption
strategies that result in genetically stable microorganisms which
overproduce the target product. Specifically, the framework
examines the complete metabolic and/or biochemical network of a
microorganism in order to suggest genetic manipulations that force
the desired biochemical to become an obligatory byproduct of cell
growth. By coupling biochemical production with cell growth through
strategically placed gene deletions or other functional gene
disruption, the growth selection pressures imposed on the
engineered strains after long periods of time in a bioreactor lead
to improvements in performance as a result of the compulsory
growth-coupled biochemical production. Lastly, when gene deletions
are constructed there is a negligible possibility of the designed
strains reverting to their wild-type states because the genes
selected by OptKnock are to be completely removed from the genome.
Therefore, this computational methodology can be used to either
identify alternative pathways that lead to biosynthesis of a
desired product or used in connection with the non-naturally
occurring microbial organisms for further optimization of
biosynthesis of a desired product.
[0280] Briefly, OptKnock is a term used herein to refer to a
computational method and system for modeling cellular metabolism.
The OptKnock program relates to a framework of models and methods
that incorporate particular constraints into flux balance analysis
(FBA) models. These constraints include, for example, qualitative
kinetic information, qualitative regulatory information, and/or DNA
microarray experimental data. OptKnock also computes solutions to
various metabolic problems by, for example, tightening the flux
boundaries derived through flux balance models and subsequently
probing the performance limits of metabolic networks in the
presence of gene additions or deletions. OptKnock computational
framework allows the construction of model formulations that allow
an effective query of the performance limits of metabolic networks
and provides methods for solving the resulting mixed-integer linear
programming problems. The metabolic modeling and simulation methods
referred to herein as OptKnock are described in, for example, U.S.
publication 2002/0168654, filed Jan. 10, 2002, in International
Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S.
publication 2009/0047719, filed Aug. 10, 2007.
[0281] Another computational method for identifying and designing
metabolic alterations favoring biosynthetic production of a product
is a metabolic modeling and simulation system termed SimPheny.RTM..
This computational method and system is described in, for example,
U.S. publication 2003/0233218, filed Jun. 14, 2002, and in
International Patent Application No. PCT/US03/18838, filed Jun. 13,
2003. SimPheny.RTM. is a computational system that can be used to
produce a network model in silico and to simulate the flux of mass,
energy or charge through the chemical reactions of a biological
system to define a solution space that contains any and all
possible functionalities of the chemical reactions in the system,
thereby determining a range of allowed activities for the
biological system. This approach is referred to as
constraints-based modeling because the solution space is defined by
constraints such as the known stoichiometry of the included
reactions as well as reaction thermodynamic and capacity
constraints associated with maximum fluxes through reactions. The
space defined by these constraints can be interrogated to determine
the phenotypic capabilities and behavior of the biological system
or of its biochemical components.
[0282] These computational approaches are consistent with
biological realities because biological systems are flexible and
can reach the same result in many different ways. Biological
systems are designed through evolutionary mechanisms that have been
restricted by fundamental constraints that all living systems must
face. Therefore, constraints-based modeling strategy embraces these
general realities. Further, the ability to continuously impose
further restrictions on a network model via the tightening of
constraints results in a reduction in the size of the solution
space, thereby enhancing the precision with which physiological
performance or phenotype can be predicted.
[0283] Given the teachings and guidance provided herein, those
skilled in the art will be able to apply various computational
frameworks for metabolic modeling and simulation to design and
implement biosynthesis of a desired compound in host microbial
organisms. Such metabolic modeling and simulation methods include,
for example, the computational systems exemplified above as
SimPheny.RTM. and OptKnock. For illustration of the invention, some
methods are described herein with reference to the OptKnock
computation framework for modeling and simulation. Those skilled in
the art will know how to apply the identification, design and
implementation of the metabolic alterations using OptKnock to any
of such other metabolic modeling and simulation computational
frameworks and methods well known in the art.
[0284] The methods described above will provide one set of
metabolic reactions to disrupt. Elimination of each reaction within
the set or metabolic modification can result in a desired product
as an obligatory product during the growth phase of the organism.
Because the reactions are known, a solution to the bilevel OptKnock
problem also will provide the associated gene or genes encoding one
or more enzymes that catalyze each reaction within the set of
reactions. Identification of a set of reactions and their
corresponding genes encoding the enzymes participating in each
reaction is generally an automated process, accomplished through
correlation of the reactions with a reaction database having a
relationship between enzymes and encoding genes.
[0285] Once identified, the set of reactions that are to be
disrupted in order to achieve production of a desired product are
implemented in the target cell or organism by functional disruption
of at least one gene encoding each metabolic reaction within the
set. One particularly useful means to achieve functional disruption
of the reaction set is by deletion of each encoding gene. However,
in some instances, it can be beneficial to disrupt the reaction by
other genetic aberrations including, for example, mutation,
deletion of regulatory regions such as promoters or cis binding
sites for regulatory factors, or by truncation of the coding
sequence at any of a number of locations. These latter aberrations,
resulting in less than total deletion of the gene set can be
useful, for example, when rapid assessments of the coupling of a
product are desired or when genetic reversion is less likely to
occur.
[0286] To identify additional productive solutions to the above
described bilevel OptKnock problem which lead to further sets of
reactions to disrupt or metabolic modifications that can result in
the biosynthesis, including growth-coupled biosynthesis of a
desired product, an optimization method, termed integer cuts, can
be implemented. This method proceeds by iteratively solving the
OptKnock problem exemplified above with the incorporation of an
additional constraint referred to as an integer cut at each
iteration. Integer cut constraints effectively prevent the solution
procedure from choosing the exact same set of reactions identified
in any previous iteration that obligatorily couples product
biosynthesis to growth. For example, if a previously identified
growth-coupled metabolic modification specifies reactions 1, 2, and
3 for disruption, then the following constraint prevents the same
reactions from being simultaneously considered in subsequent
solutions. The integer cut method is well known in the art and can
be found described in, for example, Burgard et al., Biotechnol.
Prog. 17:791-797 (2001). As with all methods described herein with
reference to their use in combination with the OptKnock
computational framework for metabolic modeling and simulation, the
integer cut method of reducing redundancy in iterative
computational analysis also can be applied with other computational
frameworks well known in the art including, for example,
SimPheny.RTM..
[0287] The methods exemplified herein allow the construction of
cells and organisms that biosynthetically produce a desired
product, including the obligatory coupling of production of a
target biochemical product to growth of the cell or organism
engineered to harbor the identified genetic alterations. Therefore,
the computational methods described herein allow the identification
and implementation of metabolic modifications that are identified
by an in silico method selected from OptKnock or SimPheny.RTM.. The
set of metabolic modifications can include, for example, addition
of one or more biosynthetic pathway enzymes and/or functional
disruption of one or more metabolic reactions including, for
example, disruption by gene deletion.
[0288] As discussed above, the OptKnock methodology was developed
on the premise that mutant microbial networks can be evolved
towards their computationally predicted maximum-growth phenotypes
when subjected to long periods of growth selection. In other words,
the approach leverages an organism's ability to self-optimize under
selective pressures. The OptKnock framework allows for the
exhaustive enumeration of gene deletion combinations that force a
coupling between biochemical production and cell growth based on
network stoichiometry. The identification of optimal gene/reaction
knockouts requires the solution of a bilevel optimization problem
that chooses the set of active reactions such that an optimal
growth solution for the resulting network overproduces the
biochemical of interest (Burgard et al., Biotechnol. Bioeng.
84:647-657 (2003)).
[0289] An in silico stoichiometric model of E. coli metabolism can
be employed to identify essential genes for metabolic pathways as
exemplified previously and described in, for example, U.S. patent
publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US
2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,
and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock
mathematical framework can be applied to pinpoint gene deletions
leading to the growth-coupled production of a desired product.
Further, the solution of the bilevel OptKnock problem provides only
one set of deletions. To enumerate all meaningful solutions, that
is, all sets of knockouts leading to growth-coupled production
formation, an optimization technique, termed integer cuts, can be
implemented. This entails iteratively solving the OptKnock problem
with the incorporation of an additional constraint referred to as
an integer cut at each iteration, as discussed above.
[0290] As disclosed herein, a nucleic acid encoding a desired
activity of an acetyl-CoA or a bioderived compound pathway can be
introduced into a host organism. In some cases, it can be desirable
to modify an activity of an acetyl-CoA or a bioderived compound
pathway enzyme or protein to increase production of acetyl-CoA or a
bioderived compound. For example, known mutations that increase the
activity of a protein or enzyme can be introduced into an encoding
nucleic acid molecule. Additionally, optimization methods can be
applied to increase the activity of an enzyme or protein and/or
decrease an inhibitory activity, for example, decrease the activity
of a negative regulator.
[0291] One such optimization method is directed evolution. Directed
evolution is a powerful approach that involves the introduction of
mutations targeted to a specific gene in order to improve and/or
alter the properties of an enzyme. Improved and/or altered enzymes
can be identified through the development and implementation of
sensitive high-throughput screening assays that allow the automated
screening of many enzyme variants (for example, >10.sup.4).
Iterative rounds of mutagenesis and screening typically are
performed to afford an enzyme with optimized properties.
Computational algorithms that can help to identify areas of the
gene for mutagenesis also have been developed and can significantly
reduce the number of enzyme variants that need to be generated and
screened. Numerous directed evolution technologies have been
developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19
(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical
and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC
Press; Otten and Quax. Biomol. Eng 22:1-9 (2005); and Sen et al.,
Appl Biochem. Biotechnol 143:212-223 (2007)) to be effective at
creating diverse variant libraries, and these methods have been
successfully applied to the improvement of a wide range of
properties across many enzyme classes. Enzyme characteristics that
have been improved and/or altered by directed evolution
technologies include, for example: selectivity/specificity, for
conversion of non-natural substrates, temperature stability, for
robust high temperature processing; pH stability, for bioprocessing
under lower or higher pH conditions; substrate or product
tolerance, so that high product titers can be achieved; binding
(K.sub.m), including broadening substrate binding to include
non-natural substrates; inhibition (K.sub.i), to remove inhibition
by products, substrates, or key intermediates; activity (kcat), to
increases enzymatic reaction rates to achieve desired flux;
expression levels, to increase protein yields and overall pathway
flux; oxygen stability, for operation of air sensitive enzymes
under aerobic conditions; and anaerobic activity, for operation of
an aerobic enzyme in the absence of oxygen.
[0292] A number of exemplary methods have been developed for the
mutagenesis and diversification of genes to target desired
properties of specific enzymes. Such methods are well known to
those skilled in the art. Any of these can be used to alter and/or
optimize the activity of an acetyl-CoA or a bioderived compound
pathway enzyme or protein. Such methods include, but are not
limited to EpPCR, which introduces random point mutations by
reducing the fidelity of DNA polymerase in PCR reactions (Pritchard
et al., J. Theor. Biol. 234:497-509 (2005)); Error-prone Rolling
Circle Amplification (epRCA), which is similar to epPCR except a
whole circular plasmid is used as the template and random 6-mers
with exonuclease resistant thiophosphate linkages on the last 2
nucleotides are used to amplify the plasmid followed by
transformation into cells in which the plasmid is re-circularized
at tandem repeats (Fujii et al., Nucleic Acids Res. 32:e145 (2004);
and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or Family
Shuffling, which typically involves digestion of two or more
variant genes with nucleases such as Dnase I or EndoV to generate a
pool of random fragments that are reassembled by cycles of
annealing and extension in the presence of DNA polymerase to create
a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA
91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994));
Staggered Extension (StEP), which entails template priming followed
by repeated cycles of 2 step PCR with denaturation and very short
duration of annealing/extension (as short as 5 sec) (Zhao et al.,
Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination
(RPR), in which random sequence primers are used to generate many
short DNA fragments complementary to different segments of the
template (Shao et al., Nucleic Acids Res 26:681-683 (1998)).
[0293] Additional methods include Heteroduplex Recombination, in
which linearized plasmid DNA is used to form heteroduplexes that
are repaired by mismatch repair (Volkov et al, Nucleic Acids Res.
27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463
(2000)); Random Chimeragenesis on Transient Templates (RACHITT),
which employs Dnase I fragmentation and size fractionation of
single stranded DNA (ssDNA) (Cow et al., Nat. Biotechnol.
19:354-359 (2001)); Recombined Extension on Truncated templates
(RETT), which entails template switching of unidirectionally
growing strands from primers in the presence of unidirectional
ssDNA fragments used as a pool of templates (Lee et al., J. Molec.
Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene
Shuffling (DOGS), in which degenerate primers are used to control
recombination between molecules; (Bergquist and Gibbs, Methods Mol.
Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72
(2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental
Truncation for the Creation of Hybrid Enzymes (ITCHY), which
creates a combinatorial library with 1 base pair deletions of a
gene or gene fragment of interest (Ostermeier et al., Proc. Natl.
Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat.
Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for
the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to
ITCHY except that phosphothioate dNTPs are used to generate
truncations (Lutz et al., Nucleic Acids Res 29:H16 (2001));
SCRATCHY, which combines two methods for recombining genes, ITCHY
and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA
98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which
mutations made via epPCR are followed by screening/selection for
those retaining usable activity (Bergquist et al., Biomol. Eng.
22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random
mutagenesis method that generates a pool of random length fragments
using random incorporation of a phosphothioate nucleotide and
cleavage, which is used as a template to extend in the presence of
"universal" bases such as inosine, and replication of an
inosine-containing complement gives random base incorporation and,
consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82
(2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et
al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which
uses overlapping oligonucleotides designed to encode "all genetic
diversity in targets" and allows a very high diversity for the
shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255
(2002)); Nucleotide Exchange and Excision Technology NexT, which
exploits a combination of dUTP incorporation followed by treatment
with uracil DNA glycosylase and then piperidine to perform endpoint
DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117
(2005)).
[0294] Further methods include Sequence Homology-Independent
Protein Recombination (SHIPREC), in which a linker is used to
facilitate fusion between two distantly related or unrelated genes,
and a range of chimeras is generated between the two genes,
resulting in libraries of single-crossover hybrids (Sieber et al.,
Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.), in which the starting materials include
a supercoiled double stranded DNA (dsDNA) plasmid containing an
insert and two primers which are degenerate at the desired site of
mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004));
Combinatorial Cassette Mutagenesis (CCM), which involves the use of
short oligonucleotide cassettes to replace limited regions with a
large number of possible amino acid sequence alterations
(Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and
Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial
Multiple Cassette Mutagenesis (CMCM), which is essentially similar
to CCM and uses epPCR at high mutation rate to identify hot spots
and hot regions and then extension by CMCM to cover a defined
region of protein sequence space (Reetz et al., Angew. Chem. Int.
Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in
which conditional is mutator plasmids, utilizing the mutD5 gene,
which encodes a mutant subunit of DNA polymerase III, to allow
increases of 20 to 4000-X in random and natural mutation frequency
during selection and block accumulation of deleterious mutations
when selection is not required (Selifonova et al., Appl. Environ.
Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol.
260:359-3680 (1996)).
[0295] Additional exemplary methods include Look-Through
Mutagenesis (LTM), which is a multidimensional mutagenesis method
that assesses and optimizes combinatorial mutations of selected
amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA
102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling
method that can be applied to multiple genes at one time or to
create a large library of chimeras (multiple mutations) of a single
gene (Tunable GeneReassembly.TM. (TGR.TM.) Technology supplied by
Verenium Corporation), in Silico Protein Design Automation (PDA),
which is an optimization algorithm that anchors the structurally
defined protein backbone possessing a particular fold, and searches
sequence space for amino acid substitutions that can stabilize the
fold and overall protein energetics, and generally works most
effectively on proteins with known three-dimensional structures
(Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002));
and Iterative Saturation Mutagenesis (ISM), which involves using
knowledge of structure/function to choose a likely site for enzyme
improvement, performing saturation mutagenesis at chosen site using
a mutagenesis method such as Stratagene QuikChange (Stratagene; San
Diego Calif.), screening/selecting for desired properties, and,
using improved clone(s), starting over at another site and continue
repeating until a desired activity is achieved (Reetz et al., Nat.
Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed
Engl. 45:7745-7751 (2006)).
[0296] Any of the aforementioned methods for mutagenesis can be
used alone or in any combination. Additionally, any one or
combination of the directed evolution methods can be used in
conjunction with adaptive evolution techniques, as described
herein.
[0297] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also provided within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
Example I
Formate Assimilation Pathways
[0298] This example describes enzymatic pathways for converting
pyruvate to formaldehyde, and optionally in combination with
producing acetyl-CoA and/or reproducing pyruvate.
Step E, FIG. 1: Formate Reductase
[0299] The conversion of formate to formaldehyde can be carried out
by a formate reductase (step E, FIG. 1). A suitable enzyme for
these transformations is the aryl-aldehyde dehydrogenase, or
equivalently a carboxylic acid reductase, from Nocardia iowensis.
Expression of the npt gene product improved activity of the enzyme
via post-transcriptional modification. The npt gene encodes a
specific phosphopantetheine transferase (PPTase) that converts the
inactive apo-enzyme to the active holo-enzyme. Information related
to these proteins and genes is shown below.
TABLE-US-00010 Protein GenBank ID GI number Organism Car AAR91681.1
40796035 Nocardia iowensis (sp. NRRL 5646) Npt ABI83656.1 114848891
Nocardia iowensis (sp. NRRL 5646)
[0300] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00011 Protein GenBank ID GI number Organism fadD9
YP_978699.1 121638475 Mycobacterium bovis BCG BCG_2812c YP_978898.1
121638674 Mycobacterium bovis BCG nfa20150 YP_118225.1 54023983
Nocardia farcinica IFM 10152 nfa40540 YP_120266.1 54026024 Nocardia
farcinica IFM 10152 SGR_6790 YP_001828302.1 182440583 Streptomyces
griseus subsp. griseus NBRC 13350 SGR_665 YP_001822177.1 182434458
Streptomyces griseus subsp. griseus NBRC 13350 MSMEG_2956
YP_887275.1 118473501 Mycobacterium smegmatis MC2 155 MSMEG 5739
YP_889972.1 118469671 Mycobacterium smegmatis MC2 155 MSMEG_2648
YP_886985.1 118471293 Mycobacterium smegmatis MC2 155 MAP1040c
NP_959974.1 41407138 Mycobacterium avium subsp. paratuberculosis
K-10 MAP2899c NP_961833.1 41408997 Mycobacterium avium subsp.
paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131
Mycobacterium marinumM MMAR 2936 YP_001851230.1 183982939
Mycobacterium marinumM MMAR_1916 YP_001850220.1 183981929
Mycobacterium marinumM TpauDRAFT_33060 ZP_04027864.1 227980601
Tsukamurella paurometabola DSM 20162 TpauDRAFT_20920 ZP_04026660.1
227979396 Tsukamurella paurometabola DSM 20162 CPCC70011320
ZP_05045132.1 254431429 Cyanobium PCC7001 DDBDRAFT_0187729
XP_636931.1 66806417 Dictyostelium discoideum AX4
[0301] An additional enzyme candidate found in Streptomyces griseus
is encoded by the griC and griD genes. This enzyme is believed to
convert 3-amino-4-hydroxybenzoic acid to
3-amino-4-hydroxybenzaldehyde. Co-expression of griC and griD with
SGR 665, an enzyme similar in sequence to the Nocardia iowensis
npt, can be beneficial. Information related to these proteins and
genes is shown below.
TABLE-US-00012 Protein GenBank ID GI number Organism griC
YP_001825755.1 182438036 Streptomycesgriseus subsp. griseus NBRC
13350 griD YP_001825756.1 182438037 Streptomycesgriseus subsp.
griseus NBRC 13350
[0302] An enzyme with similar characteristics, alpha-aminoadipate
reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis
pathways in some fungal species. This enzyme naturally reduces
alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl
group is first activated through the ATP-dependent formation of an
adenylate that is then reduced by NAD(P)H to yield the aldehyde and
AMP. Like CAR, this enzyme utilizes magnesium and requires
activation by a PPTase. Enzyme candidates for AAR and its
corresponding PPTase are shown below.
TABLE-US-00013 Protein Gen Bank ID GI number Organism LYS2
AAA34747.1 171867 Saccharomyces cerevisiae LYSS P50113.1 1708896
Saccharomyces cerevisiae LYS2 AAC02241.1 2853226 Candida albicans
LYSS AAO26020.1 28136195 Candida albicans Lys1p P40976.3 13124791
Schizosaccharomyces pombe Lys7p Q10474.1 1723561
Schizosaccharomyces pombe Lys2 CAA74300.1 3282044 Penicillium
chrysogenum
[0303] Tani et al (Agric Biol Chem, 1978, 42: 63-68; Agric Biol
Chem, 1974, 38: 2057-2058) showed that purified enzymes from
Escherichia coli strain B could reduce the sodium salts of
different organic acids (e.g. formate, glycolate, acetate, etc.) to
their respective aldehydes (e.g. formaldehyde, glycoaldehyde,
acetaldehyde, etc.). Of three purified enzymes examined by Tani et
al (1978), only the "A" isozyme was shown to reduce formate to
formaldehyde. Collectively, this group of enzymes was originally
termed glycoaldehyde dehydrogenase; however, their novel reductase
activity led the authors to propose the name glycolate reductase as
being more appropriate (Morita et al, Agric Biol Chem, 1979, 43:
185-186). Morita et al (Agric Biol Chem, 1979, 43: 185-186)
subsequently showed that glycolate reductase activity is relatively
widespread among microorganisms, being found for example in:
Pseudomonas, Agrobacterium, Escherichia, Flavobacterium,
Micrococcus, Staphylococcus, Bacillus, and others. Without wishing
to be bound by any particular theory, it is believed that some of
these glycolate reductase enzymes are able to reduce formate to
formaldehyde.
[0304] Any of these CAR or CAR-like enzymes can exhibit formate
reductase activity or can be engineered to do so.
Step F, FIG. 1 Formate Ligase, Formate Transferase, Formate
Synthetase
[0305] The acylation of formate to formyl-CoA is catalyzed by
enzymes with formate transferase, synthetase, or ligase activity
(Step F, FIG. 1). Formate transferase enzymes have been identified
in several organisms including Escherichia coli, Oxalobacter
formigenes, and Lactobacillus acidophilus. Homologs exist in
several other organisms. Enzymes acting on the CoA-donor for
formate transferase may also be expressed to ensure efficient
regeneration of the CoA-donor. For example, if oxalyl-CoA is the
CoA donor substrate for formate transferase, an additional
transferase, synthetase, or ligase may be required to enable
efficient regeneration of oxalyl-CoA from oxalate. Similarly, if
succinyl-CoA or acetyl-CoA is the CoA donor substrate for formate
transferase, an additional transferase, synthetase, or ligase may
be required to enable efficient regeneration of succinyl-CoA from
succinate or acetyl-CoA from acetate, respectively.
TABLE-US-00014 Protein Gen Bank ID GI number Organism YfdW
NP_416875.1 16130306 Escherichia coli frc O06644.3 21542067
Oxalobacter formiyenes frc ZP_04021099.1 227903294 Lactobacillus
acidophilus
[0306] Suitable CoA-donor regeneration or formate transferase
enzymes are encoded by the gene products of cat1, cat2, and cat3 of
Clostridium kluyveri. These enzymes have been shown to exhibit
succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA
acetyltransferase activity, respectively. Similar CoA transferase
activities are also present in Trichomonas vaginalis and
Trypanosoma brucei. Yet another transferase capable of the desired
conversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplary
enzymes are shown below. Genes FN0272 and FN0273 have been
annotated as a butyrate-acetoacetate CoA-transferase
TABLE-US-00015 Protein GenBank ID GI number Organism Cat1 P38946.1
729048 Clostridium kluyveri Cat2 P38942.2 1705614 Clostridium
kluyveri Cat3 EDK35586.1 146349050 Clostridium kluyveri TVAG_395550
XP_001330176 123975034 Trichomonas vaginalis G3 Tb11.02.0290
XP_828352 71754875 Trypanosoma brucei FN0272 NP_603179.1 19703617
Fusobacterium nucleatum FN0273 NP_603180.1 19703618 Fusobacterium
nucleatum FN1857 NP_602657.1 19705162 Fusobacterium nucleatum
FN1856 NP_602656.1 19705161 Fusobacterium nucleatum PG1066
NP_905281.1 34540802 Porphyromonas gingivalis W83 PG1075
NP_905290.1 34540811 Porphyromonas gingivalis W83 TTE0720
NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4 TTE0721
NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4
[0307] Additional transferase enzymes of interest include proteins
and genes shown below.
TABLE-US-00016 Protein GenBank ID GI number Organism AtoA P76459.1
2492994 Escherichia coli AtoD P76458.1 2492990 Escherichia coli
CtfA NP_149326.1 15004866 Clostridium acetobutylicum CtfB
NP_149327.1 15004867 Clostridium acetobutylicum CtfA AAP42564.1
31075384 Clostridium saccharoperbutylacetonicum CtfB AAP42565.1
31075385 Clostridium saccharoperbutylacetonicum
[0308] Succinyl-CoA:3-ketoacid-CoA transferase naturally converts
succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a
3-ketoacid. Exemplary succinyl-CoA:3:ketoacid-CoA transferases
proteins and genes are shown below.
TABLE-US-00017 Protein GenBank ID GI number Organism HPAG1 0676
YP_627417 108563101 Helicobacter pylori HPAG1_0677 YP_627418
108563102 Helicobacter pylori ScoA NP_391778 16080950 Bacillus
subtilis ScoB NP_391777 16080949 Bacillus subtilis OXCT1 NP_000427
4557817 Homo sapiens OXCT2 NP_071403 11545841 Homo sapiens
[0309] Two additional enzymes that catalyze the activation of
formate to formyl-CoA reaction are AMP-forming formyl-CoA
synthetase and ADP-forming formyl-CoA synthetase. Exemplary
enzymes, known to function on acetate, are shown below. Such
enzymes may also acylate formate naturally or can be engineered to
do so.
TABLE-US-00018 Protein GenBank ID GI Number Organism acs AAC77039.1
1790505 Escherichia coli acoE AAA21945.1 141890 Ralstonia eutropha
acs1 ABC87079.1 86169671 Methanothermobacter thermautotrophicus
acs1 AAL23099.1 16422835 Salmonella enterica ACS1 Q01574.2
257050994 Saccharomyces cerevisiae
[0310] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is
another candidate enzyme that couples the conversion of acyl-CoA
esters to their corresponding acids with the concurrent synthesis
of ATP. Several enzymes with broad substrate specificities have
been described in the literature. Such enzymes may also acylate
formate naturally or can be engineered to do so. Information
related to these proteins and genes is shown below.
TABLE-US-00019 Protein GenBank ID GI number Organism AF1211
NP_070039.1 11498810 Archaeoylobiis fulyidiis DSM 4304 AF1983
NP_070807.1 11499565 Archaeoylobiis fulyidiis DSM 4304 scs
YP_135572.1 55377722 Haloarcula marismortui ATCC 43049 PAE3250
NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC
NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949
Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida
[0311] An alternative method for adding the CoA moiety to formate
is to apply a pair of enzymes such as a phosphate-transferring
acyltransferase and a kinase. These activities enable the net
formation of formyl-CoA with the simultaneous consumption of ATP.
An exemplary phosphate transferring acyltransferase is
phosphotransacetylase, encoded by pta. Exemplary enzymes are shown
below. Such enzymes may also phosphorylate formate naturally or can
be engineered to do so.
TABLE-US-00020 Protein GenBank ID GI number Organism Pta
NP_416800.1 16130232 Escherichia coli Pta NP_461280.1 16765665
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2
PAT2 XP_001694504.1 159472743 Chlamydomonas reinhardtii PAT1
XP_001691787.1 159467202 Chlamydomonas reinhardtii
[0312] An exemplary acetate kinase is the E. coli acetate kinase,
encoded by ackA (Skarstedt and Silverstein J. Biol. Chem.
251:6775-6783 (1976)). Homologs exist in several other organisms
including Salmonella enterica and Chlamydomonas reinhardtii. It is
likely that such enzymes naturally possess formate kinase activity
or can be engineered to have this activity. Information related to
these proteins and genes is shown below:
TABLE-US-00021 Protein GenBank ID GI number Organism AckA
NP_416799.1 16130231 Escherichia coli AckA NP_461279.1 16765664
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2
ACK1 XP_001694505.1 159472745 Chlamydomonas reinhardtii ACK2
XP_001691682.1 159466992 Chlamydomonas reinhardtii
[0313] The acylation of formate to formyl-CoA can also be carried
out by a formate ligase. Such enzymes may also acylate formate
naturally or can be engineered to do so. Information related to
these proteins and genes is shown below.
TABLE-US-00022 Protein GenBank ID GI number Organism SucC
NP_415256.1 16128703 Escherichia coli SucD AAC73823.1 1786949
Escherichia coli LSC1 NP_014785 6324716 Saccharomyces cerevisiae
LSC2 NP_011760 6321683 Saccharomyces cerevisiae
[0314] Additional exemplary CoA-ligases include the rat
dicarboxylate-CoA ligase for which the sequence is yet
uncharacterized (Vamecq et al., Biochemical J. 230:683-693 (1985)),
and exemplary enzymes shown below. Such enzymes may also acylate
formate naturally or can be engineered to do so. Information
related to these proteins and genes is shown below.
TABLE-US-00023 Protein GenBank ID GI number Organism Phl CAJ15517.1
77019264 Penicillium chrysogenum PhlB ABS19624.1 152002983
Penicillium chrysogenum PaaF AAC24333.2 22711873 Pseudomonas putida
BioW NP_390902.2 50812281 Bacillus subtilis AACS NP_084486.1
21313520 Mus musculus AACS NP_076417.2 31982927 Homo sapiens
Msed_1422 YP_001191504 146304188 Metallosphaera sedula
Step G, FIG. 1: Formyl-CoA Reductase
[0315] Several acyl-CoA dehydrogenases are capable of reducing an
acyl-CoA (e.g., formyl-CoA) to its corresponding aldehyde (e.g.,
formaldehyde) (Steps F, FIG. 1). Exemplary genes that encode such
enzymes include those below. Such enzymes may be capable of
naturally converting formyl-CoA to formaldehyde or can be
engineered to do so.
TABLE-US-00024 Protein GenBank ID GI number Organism acr1
YP_047869.1 50086355 Acinetobacter calcoaceticus acr1 AAC45217
1684886 Acinetobacter baylyi acr1 BAB85476.1 18857901 Acinetobacter
sp. Strain M-1 sucD P38947.1 172046062 Clostridium kluyveri sucD
NP_904963.1 34540484 Porphyromonas gingivalis bphG BAA03892.1
425213 Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc
mesenteroides Bld AAP42563.1 31075383 Clostridium
saccharoperbutylacetonicum Ald ACL06658.1 218764192
Desulfatibacillum alkenivorans AK-01 Ald YP_001452373 157145054
Citrobacter koseri ATCC BAA-895 pduP NP_460996.1 16765381
Salmonella enterica Typhimurium pduP ABJ64680.1 116099531
Lactobacillus brevis ATCC 367 BselDRAFT_1651 ZP_02169447 163762382
Bacillus selenitireducens MLS10
[0316] An additional enzyme type that converts an acyl-CoA to its
corresponding aldehyde is malonyl-CoA reductase which transforms
malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key
enzyme in autotrophic carbon fixation via the 3-hydroxypropionate
cycle in thermoacidophilic archaeal bacteria (Berg et al., Science
318:1782-1786 (2007); Thauer, Science 318:1732-1733 (2007)). The
enzyme utilizes NADPH as a cofactor. Although the aldehyde
dehydrogenase functionality of these enzymes is similar to the
bifunctional dehydrogenase from Chloroflexus aurantiacus, there is
little sequence similarity. Both malonyl-CoA reductase enzyme
candidates have high sequence similarity to aspartate-semialdehyde
dehydrogenase, an enzyme catalyzing the reduction and concurrent
dephosphorylation of aspartyl-4-phosphate to aspartate
semialdehyde. Additional gene candidates can be found by sequence
homology to proteins in other organisms including Sulfolobus
solfataricus and Sulfolobus acidocaldarius and have been listed
below. Such enzymes may be capable of naturally converting
formyl-CoA to formaldehyde or can be engineered to do so.
TABLE-US-00025 Protein GenBank ID GI number Organism Msed_0709
YP_001190808.1 146303492 Metallosphaera sedula Mcr NP_378167.1
15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958 Sulfolobus
solfataricus Saci 2370 YP_256941.1 70608071 Sulfolobus
acidocaldarius Ald AAT66436 9473535 Clostridium beijerinckii eutE
AAA80209 687645 Salmonella typhimurium eutE P77445 2498347
Escherichia coli
Step H, FIG. 1: Formyltetrahydrofolate Synthetase
[0317] Formyltetrahydrofolate synthetase ligates formate to
tetrahydrofolate at the expense of one ATP. This enzyme is found in
several other organisms as listed below.
TABLE-US-00026 Protein GenBank ID GI number Organism Moth_0109
YP_428991.1 83588982 Moorella thermoacetica CHY_2385 YP_361182.1
78045024 Carboxydothermus hydrogenoformans FHS P13419.1 120562
Clostridium acidurici CcarbDRAFT_1913 ZP_05391913.1 255524966
Clostridium carboxidivorans P7 CcarbDRAFT_2946 ZP_05392946.1
255526022 Clostridium carboxidivorans P7 Dhaf_0555 ACL18622.1
219536883 Desulfitobacterium hafniense fhs YP_001393842.1 153953077
Clostridium kluyveri DSM 555 fhs YP_003781893.1 300856909
Clostridium ljungdahlii DSM 13528 MGA3_08300 EIJ83208.1 387590889
Bacillus methanolicus MGA3 PB1_13509 ZP_10132113.1 387929436
Bacillus methanolicus PB1
Steps I and J, FIG. 1: Formyltetrahydrofolate Synthetase and
Methylenetetrahydrofolate Dehydrogenase
[0318] In M. thermoacetica, E coli, and C. hydrogenoformans,
methenyltetrahydrofolate cyclohydrolase and
methylenetetrahydrofolate dehydrogenase are carried out by the
bi-functional gene products. Several other organisms also encode
for this bifunctional protein as tabulated below.
TABLE-US-00027 Protein GenBank ID GI number Organism Moth_1516
YP_430368.1 83590359 Moorella thermoacetica folD NP_415062.1
16128513 Escherichia coli CHY_1878 YP_360698.1 78044829
Carboxydothermus hydrogenoformans CcarbDRAFT_2948 ZP_05392948.1
255526024 Clostridium carboxidivorans P7 folD ADK16789.1 300437022
Clostridium ljungdahlii DSM 13528 folD-2 NP_951919.1 39995968
Geobacter sulfurreducens PCA folD YP_725874.1 113867385 Ralstonia
eutropha H16 folD NP_348702.1 15895353 Clostridium acetobutylicum
ATCC 824 folD YP_696506.1 110800457 Clostridium perfringens
MGA3_09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3
PB1_14689 ZP_10132349.1 387929672 Bacillus methanolicus PB1
Steps K, FIG. 1: Formaldehyde-Forming Enzyme or Spontaneous
[0319] Methylene-THF, or active formaldehyde, will spontaneously
decompose to formaldehyde and THF. To achieve higher rates, a
formaldehyde-forming enzyme can be applied. Such an activity can be
obtained by engineering an enzyme that reversibly forms
methylene-THF from THF and a formaldehyde donor, to release flee
formaldehyde. Such enzymes include glycine cleavage system enzymes
which naturally transfer a formaldehyde group from methylene-THF to
glycine (see Step L, FIG. 1 for candidate enzymes). Additional
enzymes include serine hydroxymethyltransferase (see Step M, FIG. 1
for candidate enzymes), dimethylglycine dehydrogenase (Porter, et
al., Arch Biochem Biophys. 1985, 243(2) 396-407; Brizio et al.,
2004, (37) 2, 434-442), sarcosine dehydrogenase (Porter, et al.,
Arch Biochem Biophys. 1985, 243(2) 396-407), and dimethylglycine
oxidase (Leys, et al., 2003, The EMBO Journal 22(16)
4038-4048).
TABLE-US-00028 Protein GenBank ID GI number Organism dmgo
ZP_09278452.1 359775109 Arthrobacter globiformis dmgo
YP_002778684.1 226360906 Rhodococcus opacus B4 dmgo EFY87157.1
322695347 Metarhizium acridum CQMa 102 shd AAD53398.2 5902974 Homo
sapiens shd NP_446116.1 GI: 25742657 Rattus norvegicus dmgdh
NP_037523.2 24797151 Homo sapiens dmgdh Q63342.1 2498527 Rattus
norvegicus
Step L, FIG. 1: Glycine Cleavage System
[0320] The reversible NAD(P)H-dependent conversion of
5,10-methylenetetrahydrofolate and CO.sub.2 to glycine is catalyzed
by the glycine cleavage complex, also called glycine cleavage
system, composed of four protein components; P, H, T and L. The
glycine cleavage complex is involved in glycine catabolism in
organisms such as E. coli and glycine biosynthesis in eukaryotes
(Kikuchi et al, Proc Jpn Acad Ser 84:246 (2008)). The glycine
cleavage system of E. coli is encoded by four genes: gcvPHT and
1pdA (Okamura et al, Eur J Biochem 216:539-48 (1993); Heil et al,
Microbiol 148:2203-14 (2002)). Activity of the glycine cleavage
system in the direction of glycine biosynthesis has been
demonstrated in vivo in Saccharomyces cerevisiae (Maaheimo et al,
Eur J Biochem 268:2464-79 (2001)). The yeast GCV is encoded by
GCV1, GCV2, GCV3 and LPD1.
TABLE-US-00029 Protein GenBank ID GI Number Organism gcvP
AAC75941.1 1789269 Escherichia coli gcvT AAC75943.1 1789272
Escherichia coli gcvH AAC75942.1 1789271 Escherichia coli lpdA
AAC73227.1 1786307 Escherichia coli GCV1 NP_010302.1 6320222
Saccharomyces cerevisiae GCV2 NP_013914.1 6323843 Saccharomyces
cerevisiae GCV3 NP_009355.3 269970294 Saccharomyces cerevisiae LPD1
NP_116635.1 14318501 Saccharomyces cerevisiae
Step M, FIG. 1: Serine Hydroxymethyltransferase
[0321] Conversion of glycine to serine is catalyzed by serine
hydroxymethyltransferase, also called glycine
hydroxymethyltranferase. This enzyme reversibly converts glycine
and 5,10-methylenetetrahydrofolate to serine and THF. Serine
methyltransferase has several side reactions including the
reversible cleavage of 3-hydroxyacids to glycine and an aldehyde,
and the hydrolysis of 5,10-methenyl-THF to 5-formyl-THF. Exemplary
enzymes are listed below.
TABLE-US-00030 Protein GenBank ID GI Number Organism glyA
AAC75604.1 1788902 Escherichia coli SHM1 NP_009822.2 37362622
Saccharomyces cerevisiae SHM2 NP_013159.1 6323087 Saccharomyces
cerevisiae glyA AAA64456.1 496116 Methylobacterium extorquens glyA
AAK60516.1 14334055 Corynebacterium glutamicum
Step N, FIG. 1: Serine Deaminase
[0322] Serine can be deaminated to pyruvate by serine deaminase.
Exemplary enzymes are listed below.
TABLE-US-00031 Protein GenBank ID GI Number Organism sdaA
YP_490075.1 388477887 Escherichia coli sdaB YP_491005.1 388478813
Escherichia coli tdcG YP_491301.1 388479109 Escherichia coli tdcB
YP_491307.1 388479115 Escherichia coli sdaA YP_225930.1 62390528
Corynebacterium sp.
Step O, FIG. 1: Methylenetetrahydrofolate Reductase
[0323] In M. thermoacetica, this enzyme is oxygen-sensitive and
contains an iron-sulfur cluster (Clark and Ljungdahl, J. Biol.
Chem. 259:10845-10849 (1984). This enzyme is encoded by metF in E.
coli (Sheppard et al., J. Bacteriol. 181:718-725 (1999) and
CHY_1233 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65
(2005). The M. thermoacetica genes, and its C. hydrogenoformans
counterpart, are located near the CODH/ACS gene cluster, separated
by putative hydrogenase and heterodisulfide reductase genes. Some
additional gene candidates found bioinformatically are listed
below. In Acetobacterium woodii metF is coupled to the Rnf complex
through RnfC2 (Poehlein et al, PLoS One. 7:e33439). Homologs of
RnfC are found in other organisms by blast search. The Rnf complex
is known to be a reversible complex (Fuchs (2011) Annu Rev.
Microbiol. 65:631-658).
TABLE-US-00032 Protein GenBank ID GI number Organism Moth_1191
YP_430048.1 83590039 Moorella thermoacetica Moth_1192 YP_430049.1
83590040 Moorella thermoacetica metF NP_418376.1 16131779
Escherichia coli CHY_1233 YP_360071.1 78044792 Carboxydothermus
hydrogenoformans CLJU_c37610 YP_003781889.1 300856905 Clostridium
ljungdahlii DSM 13528 DesfrDRAFT_3717 ZP_07335241.1 303248996
Desulfovibrio fructosovorans JJ CcarbDRAFT_2950 ZP_05392950.1
255526026 Clostridium carboxidivoransP7 Ccel74_010100023124
ZP_07633513.1 307691067 Clostridium cellulovorans 743B Cphy_3110
YP_001560205.1 160881237 Clostridium phytofermentans ISDg
Step P, FIG. 1: Acetyl-CoA Synthase
[0324] Acetyl-CoA synthase is the central enzyme of the carbonyl
branch of the Wood-Ljungdahl pathway. It catalyzes the synthesis of
acetyl-CoA from carbon monoxide, coenzyme A, and the methyl group
from a methylated corrinoid-iron-sulfur protein. The
corrinoid-iron-sulfur-protein is methylated by
methyltetrahydrofolate via a methyltransferase. Expression in a
foreign host entails introducing one or more of the following
proteins and their corresponding activities:
Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE),
Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly
protein (AcsF), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB and
AcsC), Carbon monoxide dehydrogenase (AcsA), and Nickel-protein
assembly protein (CooC).
[0325] The genes used for carbon-monoxide dehydrogenase/acetyl-CoA
synthase activity typically reside in a limited region of the
native genome that can be an extended operon (Ragsdale, S W., Crit.
Rev. Biochem. Mol. Biol. 39:165-195 (2004); Morton et at, J. Biol.
Chem. 266:23824-23828 (1991); Roberts et al., Proc. Natl. Acad.
Sci. USA. 86:32-36 (1989). Each of the genes in this operon from
the acetogen, M. thermoacetica, has already been cloned and
expressed actively in E. coli (Morton et al. supra; Roberts et al.
supra; Lu et al., J. Biol. Chem. 268:5605-5614 (1993). The protein
sequences of these genes can be identified by the following GenBank
accession numbers.
TABLE-US-00033 Protein GenBank ID GI number Organism AcsE YP_430054
83590045 Moorella thermoacetica AcsD YP_430055 83590046 Moorella
thermoacetica AcsF YP_430056 83590047 Moorella thermoacetica Orf7
YP_430057 83590048 Moorella thermoacetica AcsC YP_430058 83590049
Moorella thermoacetica AcsB YP_430059 83590050 Moorella
thermoacetica AcsA YP_430060 83590051 Moorella thermoacetica CooC
YP_430061 83590052 Moorella thermoacetica
[0326] The hydrogenic bacterium, Carboxydothermus hydrogenoformans,
can utilize carbon monoxide as a growth substrate by means of
acetyl-CoA synthase (Wu et al., PLoS Genet. 1:e65 (2005)). In
strain Z-2901, the acetyl-CoA synthase enzyme complex lacks carbon
monoxide dehydrogenase due to a frameshift mutation (Wu et al.
supra (2005)), whereas in strain DSM 6008, a functional
unframeshifted full-length version of this protein has been
purified (Svetlitchnyi et al., Proc. Natl. Acad. Sci. USA.
101:446-451 (2004)). The protein sequences of the C.
hydrogenoformans genes from strain Z-2901 can be identified by the
following GenBank accession numbers.
TABLE-US-00034 Protein GenBank ID GI number Organism AcsE YP_360065
78044202 Carboxydothermus hydrogenoformans AcsD YP_360064 78042962
Carboxydothermus hydrogenoformans AcsF YP_360063 78044060
Carboxydothermus hydrogenoformans Orf7 YP_360062 78044449
Carboxydothermus hydrogenoformans AcsC YP_360061 78043584
Carboxydothermus hydrogenoformans AcsB YP_360060 78042742
Carboxydothermus hydrogenoformans CooC YP_360059 78044249
Carboxydothermus hydrogenoformans
[0327] Homologous ACS/CODH genes can also be found in the draft
genome assembly of Clostridium carboxidivorans P7.
TABLE-US-00035 Protein GenBank ID GI Number Organism AcsA
ZP_05392944.1 255526020 Clostridium carboxidivorans P7 CooC
ZP_05392945.1 255526021 Clostridium carboxidivorans P7 AcsF
ZP_05392952.1 255526028 Clostridium carboxidivorans P7 AcsD
ZP_05392953.1 255526029 Clostridium carboxidivorans P7 AcsC
ZP_05392954.1 255526030 Clostridium carboxidivorans P7 AcsE
ZP_05392955.1 255526031 Clostridium carboxidivorans P7 AcsB
ZP_05392956.1 255526032 Clostridium carboxidivorans P7 Orf7
ZP_05392958.1 255526034 Clostridium carboxidivorans P7
[0328] The methanogenic archaeon, Methanosarcina acetivorans, can
also grow on carbon monoxide, exhibits acetyl-CoA synthase/carbon
monoxide dehydrogenase activity, and produces both acetate and
formate (Lessner et al., Proc. Natl. Acad. Sci. USA.
103:17921-17926 (2006)). This organism contains two sets of genes
that encode ACS/CODH activity (Rother and Metcalf, Proc. Natl. Acad
Sci. USA. 101:16929-16934 (2004)). The protein sequences of both
sets of M. acetivorans genes are identified by the following
GenBank accession numbers.
TABLE-US-00036 Protein GenBank ID GI number Organism AcsC NP_618736
20092661 Methanosarcina acetivorans AcsD NP_618735 20092660
Methanosarcina acetivorans AcsF, CooC NP_618734 20092659
Methanosarcina acetivorans AcsB NP_618733 20092658 Methanosarcina
acetivorans AcsEps NP_618732 20092657 Methanosarcina acetivorans
AcsA NP_618731 20092656 Methanosarcina acetivorans AcsC NP_615961
20089886 Methanosarcina acetivorans AcsD NP_615962 20089887
Methanosarcina acetivorans AcsF, CooC NP_615963 20089888
Methanosarcina acetivorans AcsB NP_615964 20089889 Methanosarcina
acetivorans AcsEps NP_615965 20089890 Methanosarcina acetivorans
AcsA NP_615966 20089891 Methanosarcina acetivorans
[0329] The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly
referred to as the gamma, delta, beta, epsilon, and alpha subunits
of the methanogenic CODH/ACS. Homologs to the epsilon encoding
genes are not present in acetogens such as M. thermoacetica or
hydrogenogenic bacteria such as C. hydrogenoformans. Hypotheses for
the existence of two active CODH/ACS operons in M. acetivorans
include catalytic properties (i.e., K.sub.m, V.sub.max, k.sub.cat)
that favor carboxidotrophic or aceticlastic growth or differential
gene regulation enabling various stimuli to induce CODH/ACS
expression (Rother et al., Arch. Microbiol. 188:463-472
(2007)).
Step Y, FIG. 1: Glyceraldehydes-3-Phosphate Dehydrogenase and
Enzymes of Lower Glycolysis
[0330] Enzymes comprising Step Y, G3P to PYR include:
Glyceraldehyde-3-phosphate dehydrogenase; Phosphoglycerate kinase;
Phosphoglyceromutase; Enolase; Pyruvate kinase or PTS-dependent
substrate import.
[0331] Glyceraldehyde-3-phosphate dehydrogenase enzymes include:
NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, exemplary
enzymes are:
TABLE-US-00037 Protein GenBank ID GI Number Organism gapN
AAA91091.1 642667 Streptococcus mutans NP-GAPDH AEC07555.1
330252461 Arabidopsis thaliana GAPN AAM77679.2 82469904 Triticum
aestivum gapN CAI56300.1 87298962 Clostridium acetobutylicum
NADP-GAPDH 2D2I_A 112490271 Synechococcus elongatus PCC 7942
NADP-GAPDH CAA62619.1 4741714 Synechococcus elongatus PCC 7942 GDP1
XP_455496.1 50310947 Kluyveromyces lactis NRRL Y-1140 HP1346
NP_208138.1 15645959 Helicobacter pylori 26695
and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase,
exemplary enzymes are:
TABLE-US-00038 Protein GenBank ID GI Number Organism TDH1
NP_012483.1 6322409 Saccharomyces cerevisiae s288c TDH2 NP_012542.1
6322468 Saccharomyces cerevisiae s288c TDH3 NP_011708.1 632163
Saccharomyces cerevisiae s288c KLLA0A11858g XP_451516.1 50303157
Kluyveromyces lactis NRRL Y-1140 KLLA0F20988g XP_456022.1 50311981
Kluyveromyces lactis NRRL Y-1140 ANI_1_256144 XP_001397496.1
145251966 Aspergillus niger CBS 513.88 YALI0C06369g XP_501515.1
50548091 Yarrowia lipolytica CTRG_05666 XP_002551368.1 255732890
Candida tropicalis MYA-3404 HPODL 1089 EFW97311.1 320583095
Hansenula polymorpha DL-1 gapA YP_490040.1 388477852 Escherichia
coli
[0332] Phosphoglycerate kinase enzymes include:
TABLE-US-00039 Protein GenBank ID GI Number Organism PGK1
NP_009938.2 10383781 Saccharomyces cerevisiae s288c PGK BAD83658.1
57157302 Candida boidinii PGK EFW98395.1 320584184 Hansenula
polymorpha DL-1 pgk EIJ77825.1 387585500 Bacillus methanolicus MGA3
pgk YP_491126.1 388478934 Escherichia coli
[0333] Phosphoglyceromutase (aka phosphoglycerate mutase) enzymes
include;
TABLE-US-00040 Protein GenBank ID GI Number Organism GPM1
NP_012770.1 6322697 Saccharomyces cerevisiae s288c GPM2 NP_010263.1
6320183 Saccharomyces cerevisiae s288c GPM3 NP_014585.1 6324516
Saccharomyces cerevisiae s288c HPODL 1391 EFW96681.1 320582464
Hansenula polymorpha DL-1 HPODL_0376 EFW97746.1 320583533 Hansenula
polymorpha DL-1 gpmI EIJ77827.1 387585502 Bacillus methanolicus
MGA3 gpmA YP_489028.1 388476840 Escherichia coli gpmM AAC76636.1
1790041 Escherichia coli
[0334] Enolase (also known as phosphopyruvate hydratase and
2-phosphoglycerate dehydratase) enzymes include:
TABLE-US-00041 Protein GenBank ID GI Number Organism ENO1
NP_011770.3 398366315 Saccharomyces cerevisiae s288c ENO2
AAB68019.1 458897 Saccharomyces cerevisiae s288c HPODL 2596
EFW95743.1 320581523 Hansenula polymorpha DL-1 eno EIJ77828.1
387585503 Bacillus methanolicus MGA3 eno AAC75821.1 1789141
Escherichia coli
[0335] Pyruvate kinase (also known as phosphoenolpyruvate kinase
and phosphoenolpyruvate kinase) or PTS-dependent substrate import
enzymes include those below. Pyruvate kinase, also known as
phosphoenolpyruvate synthase (EC 2.7.9.2), converts pyruvate and
ATP to PEP and AMP. This enzyme is encoded by the PYK1 (Burke et
al., J. Biol. Chem. 258:2193-2201 (1983)) and PYK2 (Boles et al.,
J. Bacteriol. 179:2987-2993 (1997)) genes in S. cerevisiae. In E.
coli, this activity is catalyzed by the gene products of pykF and
pykA. Note that pykA and pykF are genes encoding separate enzymes
potentially capable of carrying out the PYK reaction. Selected
homologs of the S. cerevisiae enzymes are also shown in the table
below.
TABLE-US-00042 Protein GenBank ID GI Number Organism PYK1 NP_009362
6319279 Saccharomyces cerevisiae PYK2 NP_014992 6324923
Saccharomyces cerevisiae pykF NP_416191.1 16129632 Escherichia coli
pykA NP_416368.1 16129807 Escherichia coli KLLA0F23397g XP_456122.1
50312181 Kluyveromyces lactis CaO19.3575 XP_714934.1 68482353
Candida albicans CaO19.11059 XP_714997.1 68482226 Candida albicans
YALI0F09185p XP_505195 210075987 Yarrowia lipolytica ANI_1_1126064
XP_001391973 145238652 Aspergillus niger MGA3_03005 EIJ84220.1
387591903 Bacillus methanolicus MGA3 HPODL_1539 EFW96829.1
320582612 Hansenula polymorpha DL-1
[0336] PTS-dependent substrate uptake systems catalyze a
phosphotransfer cascade that couples conversion of PEP to pyruvate
with the transport and phosphorylation of carbon substrates. For
example, the glucose PTS system transports glucose, releasing
glucose-6-phosphate into the cytoplasm and concomitantly converting
phosphoenolpyruvate to pyruvate. PTS systems are comprised of
substrate-specific and non-substrate-specific components. In E.
coli the two non-specific components are encoded by ptsI (Enzyme I)
and ptsH (HPr). The sugar-dependent components are encoded by crr
and ptsG. Pts systems have been extensively studied and are
reviewed, for example in Postma et al, Microbiol Rev 57: 543-94
(1993).
TABLE-US-00043 Protein GenBank ID GI Number Organism ptsG AC74185.1
1787343 Escherichia coli ptsI AAC75469.1 1788756 Escherichia coli
ptsH AAC75468.1 1788755 Escherichia coli err AAC75470.1 1788757
Escherichia coli
[0337] The IIA[Glc] component mediates the transfer of the
phosphoryl group from histidine protein Hpr (ptsH) to the IIB[Glc]
(ptsG) component. A truncated variant of the crr gene was
introduced into 1,4-butanediol producing strains.
[0338] Alternatively, Phosphoenolpyruvate phosphatase (EC 3.1.3.60)
catalyzes the hydrolysis of PEP to pyruvate and phosphate. Numerous
phosphatase enzymes catalyze this activity, including alkaline
phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2),
phosphoglycerate phosphatase (EC 3.1.3.20) and PEP phosphatase (EC
3.1.3.60). PEP phosphatase enzymes have been characterized in
plants such as Vignia radiate, Bruguiera sexangula and Brassica
nigra. Exemplary enzymes are listed below. Enzyme engineering
and/or removal of targeting sequences may be required for alkaline
phosphatase enzymes to function in the cytoplasm.
TABLE-US-00044 Protein GenBank ID GI Number Organism phyA O00092.1
41017447 Aspergillus fumigatus Acp5 P13686.3 56757583 Homo sapiens
phoA NP_414917.2 49176017 Escherichia coli phoX ZP_01072054.1
86153851 Campylobacter jejuni PHO8 AAA34871.1 172164 Saccharomyces
cerevisiae SaurJH1_2706 YP_001317815.1 150395140 Staphylococcus
aureus
Step Q, FIG. 1: Pyruvate Formate Lyase
[0339] Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded by pflB
in E. coli, can convert pyruvate into acetyl-CoA and formate. The
activity of PFL can be enhanced by an activating enzyme encoded by
pflA. Keto-acid formate-lyase (EC 2.3.1.-), also known as
2-ketobutyrate formate-lyase (KFL) and pyruvate formate-lyase 4, is
the gene product of tdcE in E. coli. This enzyme catalyzes the
conversion of 2-ketobutyrate to propionyl-CoA and formate during
anaerobic threonine degradation, and can also substitute for
pyruvate formate-lyase in anaerobic catabolism. The enzyme is
oxygen-sensitive and, like PflB, can require post-translational
modification by PFL-AE to activate a glycyl radical in the active
site (Hesslinger et al., Mol Microbiol 27:477-492 (1998)).
Exemplary enzymes are listed below.
TABLE-US-00045 Protein GenBank ID GI Number Organism pflB NP_415423
16128870 Escherichia coli pflA NP_415422.1 16128869 Escherichia
coli tdcE AAT48170.1 48994926 Escherichia coli PflD NP_070278.1
11499044 Archaeglubus fulgidus Pfl CAA03993 2407931 Lactococcus
lactis Pfl BAA09085 1129082 Streptococcus mutans PFL1
XP_001689719.1 159462978 Chlamydomonas reinhardtii pflA1
XP_001700657.1 159485246 Chlamydomonas reinhardtii Pfl Q46266.1
2500058 Clostridium pasteurianum Act CAA63749.1 1072362 Clostridium
pasteurianum
Step R, FIG. 1: Pyruvate Dehydrogenase, Pyruvate Ferredoxin
Oxidoreductase, Pyruvate:NADP+ Oxidoreductase
[0340] The pyruvate dehydrogenase (PDH) complex catalyzes the
conversion of pyruvate to acetyl-CoA (FIG. 3H). The E. coli PDH
complex is encoded by the genes aceEF and 1pdA. Enzyme engineering
efforts have improved the E. coli PDH enzyme activity under
anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858
(2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007);
Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to
the E. coli PDH, the B. subtilis complex is active and required for
growth under anaerobic conditions (Nakano et al., 179:6749-6755
(1997)). The Klebsiella pneumoniae PDH, characterized during growth
on glycerol, is also active under anaerobic conditions (Menzel et
al., 56:135-142 (1997)). Comparative kinetics of Rattus norvegicus
PDH and BCKAD indicate that BCKAD has higher activity on
2-oxobutanoate as a substrate (Paxton et al., Biochem J.
234:295-303 (1986)). The S. cerevisiae PDH complex can consist of
an E2 (LAT1) core that binds E1 (PDA1, PDB1), E3 (LPD1), and
Protein X (PDX1) components (Pronk et al., Yeast 12:1607-1633
(1996)). The PDH complex of S. cerevisiae is regulated by
phosphorylation of E1 involving PKP1 (PDH kinase I), PTCS (PDH
phosphatase I), PKP2 and PTC6. Modification of these regulators may
also enhance PDH activity. Coexpression of lipoyl ligase (LplA of
E. coli and AIM22 in S. cerevisiae) with PDH in the cytosol may be
necessary for activating the PDH enzyme complex. Increasing the
supply of cytosolic lipoate, either by modifying a metabolic
pathway or media supplementation with lipoate, may also improve PDH
activity.
TABLE-US-00046 Gene Accession No. GI Number Organism aceE
NP_414656.1 16128107 Escherichia coli aceF NP_414657.1 16128108
Escherichia coli lpd NP_414658.1 16128109 Escherichia coli lplA
NP_418803.1 16132203 Escherichia coli pdhA P21881.1 3123238
Bacillus subtilis pdhB P21882.1 129068 Bacillus subtilis pdhC
P21883.2 129054 Bacillus subtilis pdhD P21880.1 118672 Bacillus
subtilis aceE YP_001333808.1 152968699 Klebsiella pneumoniae aceF
YP_001333809.1 152968700 Klebsiella pneumoniae lpdA YP_001333810.1
152968701 Klebsiella pneumoniae Pdha1 NP_001004072.2 124430510
Rattus norvegicus Pdha2 NP_446446.1 16758900 Rattus norvegicus Dlat
NP_112287.1 78365255 Rattus norvegicus Dld NP_955417.1 40786469
Rattus norvegicus LAT1 NP_014328 6324258 Saccharomyces cerevisiae
PDA1 NP_011105 37362644 Saccharomyces cerevisiae PDB1 NP_009780
6319698 Saccharomyces cerevisiae LPD1 NP_116635 14318501
Saccharomyces cerevisiae PDX1 NP_011709 6321632 Saccharomyces
cerevisiae AIM22 NP_012489.2 83578101 Saccharomyces cerevisiae
[0341] As an alternative to the large multienzyme PDH complexes
described above, some organisms utilize enzymes in the 2-ketoacid
oxidoreductase family (OFOR) to catalyze acylating oxidative
decarboxylation of 2-keto-acids. Unlike the PDH complexes, PFOR
enzymes contain iron-sulfur clusters, utilize different cofactors
and use ferredoxin or flavodixin as electron acceptors in lieu of
NAD(P)H. Pyruvate ferredoxin oxidoreductase (PFOR) can catalyze the
oxidation of pyruvate to form acetyl-CoA (FIG. 3H). Several
additional PFOR enzymes are described in Ragsdale, Chem. Rev.
103:2333-2346 (2003). Finally, flavodoxin reductases (e.g., fqrB
from Helicobacter pylori or Campylobacter jejuni (St Maurice et
al., J. Bacteriol. 189:4764-4773 (2007))) or Rnf-type proteins
(Seedorf et al., Proc. Natl. Acad. Sci. USA. 105:2128-2133 (2008);
Hellmann et al., J Bacteriol. 190:784-791 (2008)) provide a means
to generate NADH or NADPH from the reduced ferredoxin generated by
PFOR These proteins are identified below.
TABLE-US-00047 Protein GenBank ID GI Number Organism Por CAA70873.1
1770208 Desulfovibrio africanus Por YP_428946.1 83588937 Moorella
thermoacetica ydbK NP_415896.1 16129339 Escherichia coli fqrB
NP_207955.1 15645778 Helicobacter pylori fqrB YP_001482096.1
157414840 Campylobacter jejuni RnfC EDK33306.1 146346770
Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri
RnfG EDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1
146346773 Clostridium kluyveri RnfA EDK33310.1 146346774
Clostridium kluyveri RnfB EDK33311.1 146346775 Clostridium
kluyveri
[0342] Pyruvate:NADP oxidoreductase (PNO) catalyzes the conversion
of pyruvate to acetyl-CoA. This enzyme is encoded by a single gene
and the active enzyme is a homodimer, in contrast to the
multi-subunit PDH enzyme complexes described above. The enzyme from
Euglena gracilis is stabilized by its cofactor, thiamin
pyrophosphate (Nakazawa et al, Arch Biochem Biophys 411:183-8
(2003)). The mitochondrial targeting sequence of this enzyme should
be removed for expression in the cytosol. The PNO protein of E
gracilis and other NADP-dependent pyruvate:NADP+ oxidoreductase
enzymes are listed in the table below.
TABLE-US-00048 Protein GenBank ID GI Number Organism PNO Q94IN5.1
33112418 Euglena gracilis cgd4_690 XP_625673.1 66356990
Cryptosporidium parvumIowa II TPP_PFOR_PNO XP_002765111.11
294867463 Perkinsus marinus ATCC 50983
Step S, FIG. 1: Formate Dehydrogenase
[0343] Formate dehydrogenase (FDH) catalyzes the reversible
transfer of electrons from formate to an acceptor. Enzymes with FDH
activity utilize various electron Gathers such as, for example,
NADH (EC 1.2.1.2), NADPH (EC 1.2.1.43), quinols (EC 1.1.5.6),
cytochromes (EC 1.2.2.3) and hydrogenases (EC 1.1.99.33). The loci,
Moth_2312 is responsible for encoding the alpha subunit of formate
dehydrogenase while the beta subunit is encoded by Moth_2314.
Another set of genes encoding formate dehydrogenase activity with a
propensity for CO.sub.2 reduction is encoded by Sfum_2703 through
Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur J
Biochem. 270:2476-2485 (2003)); Reda et al., PNAS 105:10654-10658
(2008)). A similar set of genes presumed to carry out the same
function are encoded by CHY_0731, CHY_0732, and CHY_0733 in C.
hydrogenoformans (Wu et al., PLoS Genet 1:e65 (2005)).
[0344] Several EM8 enzymes have been identified that have higher
specificity for NADP as the cofactor as compared to NAD. This
enzyme has been deemed as the NADP-dependent formate dehydrogenase
and has been reported from 5 species of the Burkholderia cepacia
complex. More gene candidates can be identified using sequence
homology of proteins deposited in Public databases such as NCBI,
JGI and the metagenomic databases.
TABLE-US-00049 Protein GenBank ID GI Number Organism Moth_2312
YP_431142 148283121 Moorella thermoacetica Moth_2314 YP_431144
83591135 Moorella thermoacetica Sfum 2703 YP_846816.1 116750129
Syntrophobacter fiimaroxidans Sfum 2704 YP_846817.1 116750130
Syntrophobacter fiimaroxidans Sfum_2705 YP_846818.1 116750131
Syntrophobacter fiimaroxidans Sfum_2706 YP_846819.1 116750132
Syntrophobacter fiimaroxidans CHY 0731 YP_359585.1 78044572
Carboxydothermus hydrogenoformans CHY 0732 YP_359586.1 78044500
Carboxydothermus hydrogenoformans CHY 0733 YP_359587.1 78044647
Carboxydothermus hydrogenoformans CcarbDRAFT_0901 ZP_05390901.1
255523938 Clostridium carboxidivorans P7 CcarbDRAFT_4380
ZP_05394380.1 255527512 Clostridium carboxidivorans P7 fdhA,
MGA3_06625 EIJ82879.1 387590560 Bacillus methanolicus MGA3 fdhA,
PB1_11719 ZP10131761.1 387929084 Bacillus methanolicus PB1 fdhD,
MGA3_06630 EIJ82880.1 387590561 Bacillus methanolicus MGA3 fdhD,
PB1_11724 ZP_10131762.1 387929085 Bacillus methanolicus PB1 fdh
ACF35003.1 194220249 Burkholderia stabilis fdh ACF35004.1 194220251
Burkholderia pyrrocinia fdh ACF35002.1 194220247 Burkholderia
cenocepacia fdh ACF35001.1 194220245 Burkholderia multivorans fdh
ACF35000.1 194220243 Burkholderia cepacia FDH1 AAC49766.1 2276465
Candida boidinii fdh CAA57036.1 1181204 Candida methylica FDH2
P0CF35.1 294956522 Saccharomyces cerevisiae S288c FDH1 NP_015033.1
6324964 Saccharomyces cerevisiae S288c fdsG YP_725156.1 113866667
Ralstonia eutropha fdsB YP_725157.1 113866668 Ralstonia eutropha
fdsA YP_725158.1 113866669 Ralstonia eutropha fdsC YP_725159.1
113866670 Ralstonia eutropha fdsD YP_725160.1 113866671 Ralstonia
eutropha
Example II
Production of Reducing Equivalents
[0345] This example describes methanol metabolic pathways and other
additional enzymes generating reducing equivalents as shown in FIG.
2.
FIG. 2, Step A--Methanol Methyltransferase
[0346] A complex of 3-methyltransferase proteins, denoted MtaA,
MtaB, and MtaC, perform the desired methanol methyltransferase
activity (Ragsdale, S W., Crit. Rev. Biochem. Mol. Biol. 39:165-195
(2004)).
[0347] MtaB is a zinc protein that can catalyze the transfer of a
methyl group from methanol to MtaC, a corrinoid protein. The
protein sequences of various MtaB and MtaC encoding genes in M.
barkeri, M. acetivorans, and M. thermoaceticum can be identified by
their following GenBank accession numbers.
TABLE-US-00050 Protein GenBank ID GI number Organism MtaB1
YP_304299 73668284 Methanosarcina barkeri MtaC1 YP_304298 73668283
Methanosarcina barkeri MtaB2 YP_307082 73671067 Methanosarcina
barkeri MtaC2 YP_307081 73671066 Methanosarcina barkeri MtaB3
YP_304612 73668597 Methanosarcina barkeri MtaC3 YP_304611 73668596
Methanosarcina barkeri MtaB1 NP_615421 20089346 Methanosarcina
acetivorans MtaB1 NP_615422 20089347 Methanosarcina acetivorans
MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2 NP_619253
20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474
Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcina
acetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaC
YP_430065 83590056 Moorella thermoacetica MtaA YP_430064 83590056
Moorella thermoacetica
[0348] In general, homology searches are an effective means of
identifying methanol methyltransferases because MtaB encoding genes
show little or no similarity to methyltransferases that act on
alternative substrates such as trimethylamine, dimethylamine,
monomethylamine, or dimethylsulfide. Mutant strains lacking two of
the sets were able to grow on methanol, whereas a strain lacking
all three sets of MtaB and MtaC genes sets could not grow on
methanol. This suggests that each set of genes plays a role in
methanol utilization.
[0349] MtaA is zinc protein that catalyzes the transfer of the
methyl group from MtaC to either Coenzyme M in methanogens or
methyltetrahydrofolate in acetogens. MtaA can also utilize
methylcobalamin as the methyl donor. Exemplary genes encoding MtaA
can be found in methanogenic archaea such as Methanosarcina barkeri
(Maeder et al., J. Bacteriol. 188:7922-7931 (2006) and
Methanosarcina acetivorans (Galagan et al., Genome Res. 12:532-542
(2002), as well as the acetogen, Moorella thermoacetica ((Das et
al., Proteins 67:167-176 (2007)). In general, MtaA proteins that
catalyze the transfer of the methyl group from CH.sub.3--MtaC are
difficult to identify bioinformatically as they share similarity to
other corrinoid protein methyltransferases and are not oriented
adjacent to the MtaB and MtaC genes on the chromosomes.
Nevertheless, a number of MtaA encoding genes have been
characterized. The protein sequences of these genes in M. barkeri
and M. acetivorans can be identified by the following GenBank
accession numbers.
TABLE-US-00051 Protein GenBank ID GI number Organism MtaA YP_304602
73668587 Methanosarcina barkeri MtaA1 NP_619241 20093166
Methanosarcina acetivorans MtaA2 NP_616548 20090473 Methanosarcina
acetivorans
[0350] The MtaA gene, YP_304602, from M. barkeri was cloned,
sequenced, and functionally overexpressed in E. coli (Harms and
Thauer, Eur. J. Biochem. 235:653-659 (1996)).
[0351] Putative MtaA encoding genes in M. thermoacetica were
identified by their sequence similarity to the characterized
methanogenic MtaA genes. Specifically, three M. thermoacetica genes
show high homology (>30% sequence identity) to YP_304602 from M.
barkeri. The protein sequences of putative MtaA encoding genes from
M. thermoacetica can be identified by the following GenBank
accession numbers.
TABLE-US-00052 Protein GenBank ID GI number Organism MtaA YP_430937
83590928 Moorella thermoacetica MtaA YP_431175 83591166 Moorella
thermoacetica MtaA YP_430935 83590926 Moorella thermoacetica MtaA
YP_430064 83590056 Moorella thermoacetica
FIG. 2, Step B--Methylenetetrahydrofolate Reductase
[0352] The conversion of methyl-THF to methylenetetrahydrofolate is
catalyzed by methylenetetrahydrofolate reductase. Some additional
gene candidates found bioinformatically are listed below. In
Acetobacterium woodii metF is coupled to the Rnf complex through
RnfC2 (Poehlein et al, PLoS One. 7:e33439). Homologs of RnfC are
found in other organisms by blast search. The Rnf complex is known
to be a reversible complex (Fuchs (2011) Annu. Rev. Microbiol.
65:631-658).
TABLE-US-00053 Protein GenBank ID GI number Organism Moth_1191
YP_430048.1 83590039 Moorella thermoacetica Moth_1192 YP_430049.1
83590040 Moorella thermoacetica metF NP_418376.1 16131779
Escherichia coli CHY_1233 YP_360071.1 78044792 Carboxydothermus
hydrogenoformans CLJU_c37610 YP_003781889.1 300856905 Clostridium
ljungdahlii DSM 13528 DesfrDRAFT_3717 ZP_07335241.1 303248996
Desulfovibrio fructosovorans JJ CcarbDRAFT_2950 ZP_05392950.1
255526026 Clostridium carboxidivorans P7 Ccel74_010100023124
ZP_07633513.1 307691067 Clostridium cellulovorans 743B Cphy_3110
YP_001560205.1 160881237 Clostridium phytofermentans ISDg
FIG. 2, Steps C and D--Methylenetetrahydrofolate Dehydrogenase,
Methenyltetrahydrofolate Cyclohydrolase
[0353] In M. thermoacetica, E. coli, and C. hydrogenoformans,
methenyltetrahydrofolate cyclohydrolase and
methylenetetrahydrofolate dehydrogenase are carried out by the
bi-functional gene products of Moth_1516, folD, and CHY 1878,
respectively (Pierce et al., Environ. Microbiol. 10:2550-2573
(2008); Wu et al., PLoS Genet. 1:e65 (2005); D'Ari and Rabinowitz,
J. Biol. Chem. 266:23953-23958 (1991)). A homolog exists in C.
carboxidivorans P7. Several other organisms also encode for this
bifunctional protein as tabulated below.
TABLE-US-00054 Protein GenBank ID GI number Organism Moth_1516
YP_430368.1 83590359 Moorella thermoacetica folD NP_415062.1
16128513 Escherichia coli CHY_1878 YP_360698.1 78044829
Carboxydothermus hydrogenoformans CcarbDRAFT_2948 ZP_05392948.1
255526024 Clostridium carboxidivorans P7 folD ADK16789.1 300437022
Clostridium ljungdahlii DSM 13528 folD-2 NP_951919.1 39995968
Geobacter sulfurreducens PCA folD YP_725874.1 113867385 Ralstonia
eutropha H16 folD NP_348702.1 15895353 Clostridium acetobutylicum
ATCC 824 folD YP_696506.1 110800457 Clostridium perfringens
MGA3_09460 EIJ83438.1 387591119 Bacillus methanolicus MGA3
PB1_14689 ZP_10132349.1 387929672 Bacillus methanolicus PB1
FIG. 2, Step E--Formyltetrahydrofolate Deformylase
[0354] This enzyme catalyzes the hydrolysis of
10-formyltetrahydrofolate (formyl-THF) to THF and formate. In E
coli, this enzyme is encoded by purU and has been overproduced,
purified, and characterized (Nagy, et al., J. Bacteriol.
3:1292-1298 (1995)).
TABLE-US-00055 Protein GenBank ID GI number Organism purU
AAC74314.1 1787483 Escherichia coli K-12 MG1655 purU BAD97821.1
63002616 Corynebacterium sp. U-96 purU EHE84645.1 354511740
Corynebacterium glutamicum ATCC 14067 purU NP_460715.1 16765100
Salmonella enterica subsp. enterica serovar Typhimurium str.
LT2
FIG. 2, Step F--Formyltetrahydrofolate Synthetase
[0355] Formyltetrahydrofolate synthetase ligates formate to
tetrahydrofolate at the expense of one ATP. This enzyme is found in
several organisms as listed below.
TABLE-US-00056 Protein GenBank ID GI number Organism Moth_0109
YP_428991.1 83588982 Moorella thermoacetica CHY_2385 YP_361182.1
78045024 Carboxydothermus hydrogenoformans FHS P13419.1 120562
Clostridium acidurici CcarbDRAFT_1913 ZP_05391913.1 255524966
Clostridium carboxidivorans P7 CcarbDRAFT_2946 ZP_05392946.1
255526022 Clostridium carboxidivorans P7 Dhaf_0555 ACL18622.1
219536883 Desulfitobacterium hafniense fhs YP_001393842.1 153953077
Clostridium kluyveri DSM 555 fhs YP_003781893.1 300856909
Clostridium ljungdahlii DSM 13528 MGA3_08300 EIJ83208.1 387590889
Bacillus methanolicus MGA3 PB1_13509 ZP_10132113.1 387929436
Bacillus methanolicus PB1
FIG. 2, Step G--Formate Hydrogen Lyase
[0356] A formate hydrogen lyase enzyme can be employed to convert
formate to carbon dioxide and hydrogen. An exemplary formate
hydrogen lyase enzyme can be found in Escherichia coli. The E. coli
formate hydrogen lyase consists of hydrogenase 3 and formate
dehydrogenase-H (Maeda et al., Appl Microbiol Biotechnol 77:879-890
(2007)). It is activated by the gene product of fhlA. (Maeda et
al., Appl Microbiol Biotechnol 77:879-890 (2007)). The addition of
the trace elements, selenium, nickel and molybdenum, to a
fermentation broth has been shown to enhance formate hydrogen lyase
activity (Soini et al., Microb. Cell Fact. 7:26 (2008)). Various
hydrogenase 3, formate dehydrogenase and transcriptional activator
genes are shown below.
TABLE-US-00057 Protein GenBank ID GI number Organism hycA NP_417205
16130632 Escherichia coli K-12 MG1655 hycB NP_417204 16130631
Escherichia coli K-12 MG1655 hycC NP_417203 16130630 Escherichia
coli K-12 MG1655 hycD NP_417202 16130629 Escherichia coli K-12
MG1655 hycE NP_417201 16130628 Escherichia coli K-12 MG1655 hycF
NP_417200 16130627 Escherichia coli K-12 MG1655 hycG NP_417199
16130626 Escherichia coli K-12 MG1655 hycH NP_417198 16130625
Escherichia coli K-12 MG1655 hycI NP_417197 16130624 Escherichia
coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coli K-12
MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655
[0357] A formate hydrogen lyase enzyme also exists in the
hyperthermophilic archaeon, Thermococcus litoralis (Takacs et al.,
BMC Microbiol 8:88 (2008)).
TABLE-US-00058 Protein GenBank ID GI number Organism mhyC ABW05543
157954626 Thermococcus litoralis mhyD ABW05544 157954627
Thermococcus litoralis mhyE ABW05545 157954628 Thermococcus
litoralis myhF ABW05546 157954629 Thermococcus litoralis myhG
ABW05547 157954630 Thermococcus litoralis myhH ABW05548 157954631
Thermococcus litoralis fdhA AAB94932 2746736 Thermococcus litoralis
fdhB AAB94931 157954625 Thermococcus litoralis
[0358] Additional formate hydrogen lyase systems have been found in
Salmonella typhimurium, Klebsiella pneumoniae, Rhodospirillum
rubrum, Methanobacterium formicicum (Vardar-Schara et al.,
Microbial Biotechnology 1:107-125 (2008)).
FIG. 2, Step H--Hydrogenase
[0359] Hydrogenase enzymes can convert hydrogen gas to protons and
transfer electrons to acceptors such as ferredoxins, NAD+, or
NADP+. Ralstonia eutropha H16 uses hydrogen as an energy source
with oxygen as a terminal electron acceptor. Its membrane-bound
uptake [NiFe]-hydrogenase is an "02-tolerant" hydrogenase
(Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009))
that is periplasmically-oriented and connected to the respiratory
chain via a b-type cytochrome (Schink and Schlegel, Biochim.
Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J
Biochem. 248, 179-186 (1997)). R. eutropha also contains an
02-tolerant soluble hydrogenase encoded by the Hox operon which is
cytoplasmic and directly reduces NAD+ at the expense of hydrogen
(Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976);
Burgdorf, J. Bact. 187(9) 3122-3132 (2005)). Soluble hydrogenase
enzymes are additionally present in several other organisms
including Geobacter sulfurreducens (Coppi, Microbiology 151,
1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J. Biol.
Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina
(Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The
Synechocystis enzyme is capable of generating NADPH from hydrogen.
Overexpression of both the Hox operon from Synechocystis str. PCC
6803 and the accessory genes encoded by the Hyp operon from Nostoc
sp. PCC 7120 led to increased hydrogenase activity compared to
expression of the Hox genes alone (Germer, J. Biol. Chem. 284(52),
36462-36472 (2009)).
TABLE-US-00059 Protein GenBank ID GI Number Organism HoxF
NP_942727.1 38637753 Ralstonia eutropha H16 HoxU NP_942728.1
38637754 Ralstonia eutropha H16 HoxY NP_942729.1 38637755 Ralstonia
eutropha H16 HoxH NP_942730.1 38637756 Ralstonia eutropha H16 HoxW
NP_942731.1 38637757 Ralstonia eutropha H16 HoxI NP_942732.1
38637758 Ralstonia eutropha H16 HoxE NP_953767.1 39997816 Geobacter
sulfurreducens HoxF NP_953766.1 39997815 Geobacter sulfurreducens
HoxU NP_953765.1 39997814 Geobacter sulfurreducens HoxY NP_953764.1
39997813 Geobacter sulfurreducens HoxH NP_953763.1 39997812
Geobacter sulfurreducens GSU2717 NP_953762.1 39997811 Geobacter
sulfurreducens HoxE NP_441418.1 16330690 Synechocystis str. PCC
6803 HoxF NP_441417.1 16330689 Synechocystis str. PCC 6803 Unknown
NP_441416.1 16330688 Synechocystis str. PCC 6803 function HoxU
NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxY NP_441414.1
16330686 Synechocystis str. PCC 6803 Unknown NP_441413.1 16330685
Synechocystis str. PCC 6803 function Unknown NP_441412.1 16330684
Synechocystis str. PCC 6803 function HoxH NP_441411.1 16330683
Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp.
PCC 7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD
NP_484739.1 17228191 Nostoc sp. PCC 7120 Unknown NP_484740.1
17228192 Nostoc sp. PCC 7120 function HypE NP_484741.1 17228193
Nostoc sp. PCC 7120 HypA NP_484742.1 17228194 Nostoc sp. PCC 7120
HypB NP_484743.1 17228195 Nostoc sp. PCC 7120 Hox1E AAP50519.1
37787351 Thiocapsa roseopersicina Hox1F AAP50520.1 37787352
Thiocapsa roseopersicina Hox1U AAP50521.1 37787353 Thiocapsa
roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsa roseopersicina
Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina
[0360] The genomes of E. coli and other enteric bacteria encode up
to four hydrogenase enzymes (Sawers, G., Antonie Van Leeuwenhoek
66:57-88 (1994); Sawers et al., J Bacteria 164:1324-1331 (1985);
Sawers and Boxer, Eur. J Biochem. 156:265-275 (1986); Sawers et
al., J Bacteriol. 168:398-404 (1986)). Given the multiplicity of
enzyme activities E. coli or another host organism can provide
sufficient hydrogenase activity to split incoming molecular
hydrogen and reduce the corresponding acceptor. Endogenous
hydrogen-lyase enzymes of E. coli include hydrogenase 3, a
membrane-bound enzyme complex using ferredoxin as an acceptor, and
hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3
and 4 are encoded by the hyc and hyfgene clusters, respectively.
Hydrogenase activity in E coli is also dependent upon the
expression of the hyp genes whose corresponding proteins are
involved in the assembly of the hydrogenase complexes (Jacobi et
al., Arch. Microbiol 158:444-451 (1992); Rangarajan et al., J
Bacteriol 190:1447-1458 (2008)). The M. thermoacetica and
Clostridium ljungdahli hydrogenases are suitable for a host that
lacks sufficient endogenous hydrogenase activity. M. thermoacetica
and C. ljungdahli can grow with CO.sub.2 as the exclusive carbon
source indicating that reducing equivalents are extracted from
H.sub.2 to enable acetyl-CoA synthesis via the Wood-Ljungdahl
pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and
Daniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J
Bacteriol. 160:466-469 (1984)). M. thermoacetica has homologs to
several hyp, hyc, and hyf genes from E. coli. These protein
sequences encoded for by these genes are identified by the
following GenBank accession numbers. In addition, several gene
clusters encoding hydrogenase functionality are present in M.
thermoacetica and C. ljungdahli (see for example US
2012/0003652).
TABLE-US-00060 Protein GenBank ID GI Number Organism HypA NP_417206
16130633 Escherichia coli HypB NP_417207 16130634 Escherichia coli
HypC NP_417208 16130635 Escherichia coli HypD NP_417209 16130636
Escherichia coli HypE NP_417210 226524740 Escherichia coli HypF
NP_417192 16130619 Escherichia coli HycA NP_417205 16130632
Escherichia coli HycB NP_417204 16130631 Escherichia coli HycC
NP_417203 16130630 Escherichia coli HycD NP_417202 16130629
Escherichia coli HycE NP_417201 16130628 Escherichia coli HycF
NP_417200 16130627 Escherichia coli HycG NP_417199 16130626
Escherichia coli HycH NP_417198 16130625 Escherichia coli HycI
NP_417197 16130624 Escherichia coli HyfA NP_416976 90111444
Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC
NP_416978 90111445 Escherichia coli HyfD NP_416979 16130409
Escherichia coli HyfE NP_416980 16130410 Escherichia coli HyfF
NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412
Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI
NP_416984 16130414 Escherichia coli HyfJ NP_416985 90111446
Escherichia coli HyfR NP_416986 90111447 Escherichia coli
[0361] Proteins in M. thermoacetica whose genes are homologous to
the E. coli hydrogenase genes are shown below.
TABLE-US-00061 Protein GenBank ID GI Number Organism Moth_2175
YP_431007 83590998 Moorella thermoacetica Moth_2176 YP_431008
83590999 Moorella thermoacetica Moth_2177 YP_431009 83591000
Moorella thermoacetica Moth_2178 YP_431010 83591001 Moorella
thermoacetica Moth_2179 YP_431011 83591002 Moorella thermoacetica
Moth_2180 YP_431012 83591003 Moorella thermoacetica Moth_2181
YP_431013 83591004 Moorella thermoacetica Moth_2182 YP_431014
83591005 Moorella thermoacetica Moth_2183 YP_431015 83591006
Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorella
thermoacetica Moth_2185 YP_431017 83591008 Moorella thermoacetica
Moth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187
YP_431019 83591010 Moorella thermoacetica Moth_2188 YP_431020
83591011 Moorella thermoacetica Moth_2189 YP_431021 83591012
Moorella thermoacetica Moth_2190 YP_431022 83591013 Moorella
thermoacetica Moth_2191 YP_431023 83591014 Moorella thermoacetica
Moth_2192 YP_431024 83591015 Moorella thermoacetica Moth_0439
YP_429313 83589304 Moorella thermoacetica Moth_0440 YP_429314
83589305 Moorella thermoacetica Moth_0441 YP_429315 83589306
Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorella
thermoacetica Moth_0809 YP_429670 83589661 Moorella thermoacetica
Moth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811
YP_429672 83589663 Moorella thermoacetica Moth_0812 YP_429673
83589664 Moorella thermoacetica Moth_0814 YP_429674 83589665
Moorella thermoacetica Moth_0815 YP_429675 83589666 Moorella
thermoacetica Moth_0816 YP_429676 83589667 Moorella thermoacetica
Moth_1193 YP_430050 83590041 Moorella thermoacetica Moth_1194
YP_430051 83590042 Moorella thermoacetica Moth_1195 YP_430052
83590043 Moorella thermoacetica Moth_1196 YP_430053 83590044
Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorella
thermoacetica Moth_1718 YP_430563 83590554 Moorella thermoacetica
Moth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883
YP_430726 83590717 Moorella thermoacetica Moth_1884 YP_430727
83590718 Moorella thermoacetica Moth_1885 YP_430728 83590719
Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorella
thermoacetica Moth_1887 YP_430730 83590721 Moorella thermoacetica
Moth_1888 YP_430731 83590722 Moorella thermoacetica Moth_1452
YP_430305 83590296 Moorella thermoacetica Moth_1453 YP_430306
83590297 Moorella thermoacetica Moth_1454 YP_430307 83590298
Moorella thermoacetica
[0362] Genes encoding hydrogenase enzymes from C. ljungdahli are
shown below.
TABLE-US-00062 Protein GenBank ID GI Number Organism CLJU_c20290
ADK15091.1 300435324 Clostridium ljungdahli CLJU_c07030 ADK13773.1
300434006 Clostridium ljungdahli CLJU_c07040 ADK13774.1 300434007
Clostridium ljungdahli CLJU_c07050 ADK13775.1 300434008 Clostridium
ljungdahli CLJU_c07060 ADK13776.1 300434009 Clostridium ljungdahli
CLJU_c07070 ADK13777.1 300434010 Clostridium ljungdahli CLJU_c07080
ADK13778.1 300434011 Clostridium ljungdahli CLJU_c14730 ADK14541.1
300434774 Clostridium ljungdahli CLJU_c14720 ADK14540.1 300434773
Clostridium ljungdahli CLJU_c14710 ADK14539.1 300434772 Clostridium
ljungdahli CLJU_c14700 ADK14538.1 300434771 Clostridium ljungdahli
CLJU_c28670 ADK15915.1 300436148 Clostridium ljungdahli CLJU_c28660
ADK15914.1 300436147 Clostridium ljungdahli CLJU_c28650 ADK15913.1
300436146 Clostridium ljungdahli CLJU_c28640 ADK15912.1 300436145
Clostridium ljungdahli
[0363] In some cases, hydrogenase encoding genes are located
adjacent to a CODH. In Rhodospirillum rubrum, the encoded
CODH/hydrogenase proteins form a membrane-bound enzyme complex that
has been indicated to be a site where energy, in the form of a
proton gradient, is generated from the conversion of CO and
H.sub.2O to CO.sub.2 and H2 (Fox et al., J Bacteriol 178:6200-6208
(1996)). The CODH-I of C. hydrogenoformans and its adjacent genes
have been proposed to catalyze a similar functional role based on
their similarity to the R rubrum CODH/hydrogenase gene cluster (Wu
et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-I
was also shown to exhibit intense CO oxidation and CO.sub.2
reduction activities when linked to an electrode (Parkin et al., J
Am. Chem. Soc. 129:10328-10329 (2007)).
TABLE-US-00063 Protein GenBank ID GI Number Organism CooL AAC45118
1515468 Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum
rubrum CooU AAC45120 1515470 Rhodospirillum rubrum CooH AAC45121
1498746 Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum
rubrum CODH (CooS) AAC45123 1498748 Rhodospirillum rubrum CooC
AAC45124 1498749 Rhodospirillum rubrum CooT AAC45125 1498750
Rhodospirillum rubrum CooJ AAC45126 1498751 Rhodospirillum rubrum
CODH-I (CooS-I) YP_360644 78043418 Carboxydothermus
hydrogenoformans CooF YP_360645 78044791 Carboxydothermus
hydrogenoformans HypA YP_360646 78044340 Carboxydothermus
hydrogenoformans CooH YP_360647 78043871 Carboxydothermus
hydrogenoformans CooU YP_360648 78044023 Carboxydothermus
hydrogenoformans CooX YP_360649 78043124 Carboxydothermus
hydrogenoformans CooL YP_360650 78043938 Carboxydothermus
hydrogenoformans CooK YP_360651 78044700 Carboxydothermus
hydrogenoformans CooM YP_360652 78043942 Carboxydothermus
hydrogenoformans CooC YP_360654.1 78043296 Carboxydothermus
hydrogenoformans CooA-1 YP_360655.1 78044021 Carboxydothermus
hydrogenoformans
[0364] Some hydrogenase and CODH enzymes transfer electrons to
ferredoxins Ferredoxins are small acidic proteins containing one or
more iron-sulfur clusters that function as intracellular electron
Gathers with a low reduction potential. Reduced ferredoxins donate
electrons to Fe-dependent enzymes such as ferredoxin-NADP.sup.+
oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and
2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H.
thermophilus gene fdx1 encodes a [4Fe-4S]-type ferredoxin that is
required for the reversible carboxylation of 2-oxoglutarate and
pyruvate by OFOR and PFOR, respectively (Yamamoto et al.,
Extremophiles 14:79-85 (2010)). The ferredoxin associated with the
Sulfolobus solfataricus 2-oxoacid:ferredoxin reductase is a
monomeric dicluster [3Fe-4S][4Fe-4S]type ferredoxin (Park et al.
2006). While the gene associated with this protein has not been
fully sequenced, the N-terminal domain shares 93% homology with the
zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a
soluble ferredoxin of unknown physiological function, fdx. Some
evidence indicates that this protein can function in iron-sulfur
cluster assembly (Takahashi and Nakamura, 1999). Additional
ferredoxin proteins have been characterized in Helicobacter pylori
(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et
al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has
been cloned and expressed in E. coli (Fujinaga and Meyer,
Biochemical and Biophysical Research Communications, 192(3):
(1993)). Acetogenic bacteria such as Moorella thermoacetica,
Clostridium carboxidivorans P7, Clostridium ljungdahli and
Rhodospirillum rubrum are predicted to encode several ferredoxins,
listed below.
TABLE-US-00064 Protein GenBank ID GI Number Organism fdx1
BAE02673.1 68163284 Hydrogenobacter thermophilus M11214.1
AAA83524.1 144806 Clostridium pasteurianum Zfx AAY79867.1 68566938
Sulfolobus acidocalarius Fdx AAC75578.1 1788874 Escherichia coli hp
0277 AAD07340.1 2313367 Helicobacter pylori fdxA CAL34484.1
112359698 Campylobacter jejuni Moth_0061 ABC18400.1 83571848
Moorella thermoacetica Moth_1200 ABC19514.1 83572962 Moorella
thermoacetica Moth_1888 ABC20188.1 83573636 Moorella thermoacetica
Moth_2112 ABC20404.1 83573852 Moorella thermoacetica Moth_1037
ABC19351.1 83572799 Moorella thermoacetica CcarbDRAFT_4383
ZP_05394383.1 255527515 Clostridium carboxidivorans P7
CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans
P7 CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium
carboxidivorans P7 CcarbDRAFT_5296 ZP_05395295.1 255528511
Clostridium carboxidivorans P7 CcarbDRAFT_1615 ZP_05391615.1
255524662 Clostridium carboxidivorans P7 CcarbDRAFT_1304
ZP_05391304.1 255524347 Clostridium carboxidivorans P7 cooF
AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxN
CAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1
83576513 Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165
Rhodospirillum rubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum
rubrum cooF AAC45122.1 1498747 Rhodospirillum rubrum fdxN
AAA26460.1 152605 Rhodospirillum rubrum Alvin_2884 ADC63789.1
288897953 Allochromatium vinosum DSM 180 Fdx YP_002801146.1
226946073 Azotobacter vinelandii DJ CKL_3790 YP_001397146.1
153956381 Clostridium kluyveri DSM 555 fer1 NP_949965.1 39937689
Rhodopseudomonas palustris CGA009 Fdx CAA12251.1 3724172 Thauera
aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermus
hydrogenoformans Fer YP_359966.1 78045103 Carboxydothermus
hydrogenoformans Fer AAC83945.1 1146198 Bacillus subtilis fdx1
NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP 003148.1
89109368 Escherichia coli K-12 CLJU_c00930 ADK13195.1 300433428
Clostridium ljungdahli CLJU_c00010 ADK13115.1 300433348 Clostridium
ljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahli
CLJU_c17980 ADK14861.1 300435094 Clostridium ljungdahli CLJU_c17970
ADK14860.1 300435093 Clostridium ljungdahli CLJU_c22510 ADK15311.1
300435544 Clostridium ljungdahli CLJU_c26680 ADK15726.1 300435959
Clostridium ljungdahli CLJU_c29400 ADK15988.1 300436221 Clostridium
ljungdahli
[0365] Ferredoxin oxidoreductase enzymes transfer electrons from
ferredoxins or flavodoxins to NAD(P)H. Two enzymes catalyzing the
reversible transfer of electrons from reduced ferredoxins to
NAD(P)+ are ferredoxin:NAD+ oxidoreductase (EC 1.18.1.3) and
ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2).
Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has a
noncovalently bound FAD cofactor that facilitates the reversible
transfer of electrons from NADPH to low-potential acceptors such as
ferredoxins or flavodoxins.
TABLE-US-00065 Protein GenBank ID GI Number Organism fqrB
NP_207955.1 15645778 Helicobacter pylori fqrB YP_001482096.1
157414840 Campylobacter jejuni RPA3954 CAE29395.1 39650872
Rhodopseudomonas palustris Fpr BAH29712.1 225320633 Hydrogenobacter
thermophilus yumC NP_3910912 255767736 Bacillus subtilis Fpr
P28861.4 399486 Escherichia coli hcaD AAC75595.1 1788892
Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea mays
NfnA YP_001393861.1 153953096 Clostridium kluyveri NfnB
YP_001393862.1 153953097 Clostridium kluyveri CcarbDRAFT_2639
ZP_05392639.1 255525707 Clostridium carboxidivorans P7
CcarbDRAFT_2638 ZP_05392638.1 255525706 Clostridium carboxidivorans
P7 CcarbDRAFT_2636 ZP_05392636.1 255525704 Clostridium
carboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241
Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1
255525514 Clostridium carboxidivorans P7 CcarbDRAFT_1084
ZP_05391084.1 255524124 Clostridium carboxidivorans P7 RnfC
EDK33306.1 146346770 Clostridium kluyveri RnfD EDK33307.1 146346771
Clostridium kluyveri RnfG EDK33308.1 146346772 Clostridium kluyveri
RnfE EDK33309.1 146346773 Clostridium kluyveri RnfA EDK33310.1
146346774 Clostridium kluyveri RnfB EDK33311.1 146346775
Clostridium kluyveri CLJU_c11410 (RnfB) ADK14209.1 300434442
Clostridium ljungdahlii CLJU_c11400 (RnfA) ADK14208.1 300434441
Clostridium ljungdahlii CLJU_c11390 (RnfE) ADK14207.1 300434440
Clostridium ljungdahlii CLJU_c11380 (RnfG) ADK14206.1 300434439
Clostridium ljungdahlii CLJU_c11370 (RnfD) ADK14205.1 300434438
Clostridium ljungdahlii CLJU_c11360 (RnfC) ADK14204.1 300434437
Clostridium ljungdahlii MOTH_1518 (NfhA) YP_430370.1 83590361
Moorella thermoacetica MOTH_1517(NfhB) YP_430369.1 83590360
Moorella thermoacetica CHY_1992 (NfhA) YP_360811.1 78045020
Carboxydothermus hydrogenoformans CHY_1993 (NfhB) YP_360812.1
78044266 Carboxydothermus hydrogenoformans CLJU_c37220 (NfnAB)
YP_003781850.1 300856866 Clostridium ljungdahlii
FIG. 2, Step I--Formate Dehydrogenase
[0366] Formate dehydrogenase (FDH) catalyzes the reversible
transfer of electrons from formate to an acceptor. Exemplary
enzymes include those described in the section for Step S, FIG. 1:
Formate dehydrogenase.
FIG. 2, Step J--Methanol Dehydrogenase
[0367] NAD+ dependent methanol dehydrogenase enzymes (EC 1.1.1.244)
catalyze the conversion of methanol and NAD+ to formaldehyde and
NADH. An enzyme with this activity was first characterized in
Bacillus methanolicus (Heggeset, et al., Applied and Environmental
Microbiology, 78(15):5170-5181 (2012)). This enzyme is zinc and
magnesium dependent, and activity of the enzyme is enhanced by the
activating enzyme encoded by act (Kloosterman et al, J Biol Chem
277:34785-92 (2002)). The act is a Nudix hydrolase. Several of
these candidates have been identified and shown to have activity on
methanol. Additional NAD(P)+ dependent enzymes can be identified by
sequence homology. Methanol dehydrogenase enzymes utilizing
different electron acceptors are also known in the art. Examples
include cytochrome dependent enzymes such as mxalF of the
methylotroph Methylobacterium extorquens (Nunn et al, Nucl Acid Res
16:7722 (1988)). Methanol dehydrogenase enzymes of methanotrophs
such as Methylococcus capsulatis function in a complex with methane
monooxygenase (MMO) (Myronova et al, Biochem 45:11905-14 (2006)).
Methanol can also be oxidized to formaldehyde by alcohol oxidase
enzymes such as methanol oxidase (EC 1.1.3.13) of Candida boidinii
(Sakai et al, Gene 114: 67-73 (1992)).
TABLE-US-00066 Protein GenBank ID GI Number Organism mdh,
MGA3_17392 EIJ77596.1 387585261 Bacillus methanolicus MGA3 mdh2,
MGA3_07340 EIJ83020.1 387590701 Bacillus methanolicus MGA3 mdh3,
MGA3_10725 EIJ80770.1 387588449 Bacillus methanolicus MGA3 act,
MGA3_09170 EIJ83380.1 387591061 Bacillus methanolicus MGA3 mdh,
PB1_17533 ZP_10132907.1 387930234 Bacillus methanolicus PB1 mdh1,
PB1_14569 ZP_10132325.1 387929648 Bacillus methanolicus PB1 mdh2,
PB1_12584 ZP_10131932.1 387929255 Bacillus methanolicus PB1 act,
PB1_14394 ZP_10132290.1 387929613 Bacillus methanolicus PB1
BFZC1_05383 ZP_07048751.1 299535429 Lysinibacillus fusiformis
BFZC1_20163 ZP_07051637.1 299538354 Lysinibacillus fusiformis
Bsph_4187 YP_001699778.1 169829620 Lysinibacillus sphaericus
Bsph_1706 YP_001697432.1 169827274 Lysinibacillus sphaericus mdh2
YP_004681552.1 339322658 Cupriavidus necator N-1 nudF1
YP_004684845.1 339325152 Cupriavidus necator N-1 BthaA_010200007655
ZP_05587334.1 257139072 Burkholderia thailandensis E264 BTH_I1076
YP_441629.1 83721454 Burkholderia thailandensis E264 (MutT/NUDIX
NTP pyrophosphatase) BalcAV_11743 ZP_10819291.1 402299711 Bacillus
alcalophilus ATCC 27647 BalcAV_05251 ZP_10818002.1 402298299
Bacillus alcalophilus ATCC 27647 alcohol dehydrogenase YP_001447544
156976638 Vibrio harveyi ATCC BAA-1116 P3TCK_27679 ZP_01220157.1
90412151 Photobacterium profundum 3TCK alcohol dehydrogenase
YP_694908 110799824 Clostridium perfringens ATCC 13124 adhB
NP_717107 24373064 Shewanella oneidensis MR-1 alcohol dehydrogenase
YP_237055 66047214 Pseudomonas syringae pv. syringae B728a alcohol
dehydrogenase YP_359772 78043360 Carboxydothermus hydrogenoformans
Z-2901 alcohol dehydrogenase YP_003990729 312112413 Geobacillus sp.
Y4.1MC1 PpeoK3_010100018471 ZP_10241531.1 390456003 Paenibacillus
peoriae KCTC 3763 OBE_12016 EKC54576 406526935 human gut metagenome
alcohol dehydrogenase YP_001343716 152978087 Actinobacillus
succinogenes 130Z dhaT AAC45651 2393887 Clostridium pasteurianum
DSM 525 alcohol dehydrogenase NP_561852 18309918 Clostridium
perfringens str. 13 BAZO_10081 ZP_11313277.1 410459529 Bacillus
azotoformans LMG 9581 alcohol dehydrogenase YP_007491369 452211255
Methanosarcina mazei Tuc01 alcohol dehydrogenase YP_004860127
347752562 Bacillus coagulans 36D1 alcohol dehydrogenase
YP_002138168 197117741 Geobacter bemidjiensis Bem DesmeDRAFT_1354
ZP_08977641.1 354558386 Desulfitobacterium metallireducens DSM
15288 alcohol dehydrogenase YP_001337153 152972007 Klebsiella
pneumoniae subsp. pneumoniae MGH 78578 alcohol dehydrogenase
YP_001113612 134300116 Desulfotomaculum reducens MI-1 alcohol
dehydrogenase YP_001663549 167040564 Thermoanaerobacter sp. X514
ACINNAV82_2382 ZP_16224338.1 421788018 Acinetobacter baumannii
Naval-82 alcohol dehydrogenase YP_005052855 374301216 Desulfovibrio
africanus str. Walvis Bay alcohol dehydrogenase AGF87161 451936849
uncultured organism DesfrDRAFT_3929 ZP_07335453.1 303249216
Desulfovibrio fructosovorans JJ alcohol dehydrogenase NP_617528
20091453 Methanosarcina acetivorans C2A alcohol dehydrogenase
NP_343875.1 15899270 Sulfolobus solfataricus P-2 adh4 YP_006863258
408405275 Nitrososphaera gargensis Ga9.2 Ta0841 NP_394301.1
16081897 Thermoplasma acidophilum PTO1151 YP_023929.1 48478223
Picrophilus torridus DSM9790 alcohol dehydrogenase ZP_10129817.1
387927138 Bacillus methanolicus PB-1 cgR_2695 YP_001139613.1
145296792 Corynebacterium glutamicum R alcohol dehydrogenase
YP_004758576.1 340793113 Corynebacterium variabile HMPREF1015_01790
ZP_09352758.1 365156443 Bacillus smithii ADH1 NP_014555.1 6324486
Saccharomyces cerevisiae NADH-dependent YP_001126968.1 138896515
Geobacillus themodenitrificans NG80-2 butanol dehydrogenase A
alcohol dehydrogenase WP_007139094.1 494231392 Flavobacterium
frigoris methanol dehydrogenase WP_003897664.1 489994607
Mycobacterium smegmatis ADH1B NP_000659.2 34577061 Homo sapiens
PMI01_01199 ZP_10750164.1 399072070 Caulobacter sp. AP07 YiaY
YP_026233.1 49176377 Escherichia coli MCA0299 YP_112833.1 53802410
Methylococcus capsulatis MCA0782 YP_113284.1 53804880 Methylococcus
capsulatis mxaI YP_002965443.1 240140963 Methylobacterium
extorquens mxaF YP_002965446.1 240140966 Methylobacterium
extorquens AOD1 AAA34321.1 170820 Candida boidinii hypothetical
protein EDA87976.1 142827286 Marine metagenome
JCVI_SCAF_1096627185304 GOS_1920437 alcohol dehydrogenase
CAA80989.1 580823 Geobacillus stearothermophilus
[0368] An in vivo assay was developed to determine the activity of
methanol dehydrogenases. This assay relies on the detection of
formaldehyde (HCHO), thus measuring the forward activity of the
enzyme (oxidation of methanol). To this end, a strain comprising a
BDOP and lacking frmA, frmB, frmR was created using Lambda Red
recombinase technology (Datsenko and Wanner, Proc. Natl. Acad. Sci.
USA, 6 97(12): 6640-5 (2000). Plasmids expressing methanol
dehydrogenases were transformed into the strain, then grown to
saturation in LB medium+antibiotic at 37.degree. C. with shaking.
Transformation of the strain with an empty vector served as a
negative control. Cultures were adjusted by O.D. and then diluted
1:10 into M9 medium+0.5% glucose+antibiotic and cultured at
37.degree. C. with shaking for 6-8 hours until late log phase.
Methanol was added to 2% v/v and the cultures were further
incubated for 30 min. with shaking at 37.degree. C. Cultures were
spun down and the supernatant was assayed for formaldehyde produced
using DETECTX Formaldehyde Detection kit (Arbor Assays; Ann Arbor,
Mich.) according to manufacturer's instructions. The frmA, frmB,
frmR deletions resulted in the native formaldehyde utilization
pathway to be deleted, which enables the formation of formaldehyde
that can be used to detect methanol dehydrogenase activity in the
non-naturally occurring microbial organism.
[0369] The activity of several enzymes was measured using the assay
described above. The results of four independent experiments are
provided in the below table.
[0370] Results of in vivo assays showing formaldehyde (HCHO)
production by various non-naturally occurring microbial organism
comprising a plasmid expressing a methanol dehydrogenase.
TABLE-US-00067 HCHO Accession number (.mu.M) Experiment 1
EIJ77596.1 >50 EIJ83020.1 >20 EIJ80770.1 >50 ZP_10132907.1
>20 ZP_10132325.1 >20 ZP_10131932.1 >50 ZP_07048751.1
>50 YP_001699778.1 >50 YP_004681552.1 >10 ZP_10819291.1
<1 Empty vector 2.33 Experiment 2 EIJ77596.1 >50 NP_00659.2
>50 YP_004758576.1 >20 ZP_09352758.1 >50 ZP_10129817.1
>20 YP_001139613.1 >20 NP_014555.1 >10 WP_007139094.1
>10 NP_343875.1 >1 YP_006863258 >1 NP_394301.1 >1
ZP_10750164.1 >1 YP_023929.1 >1 ZP_08977641.1 <1
ZP_10117398.1 <1 YP_004108045.1 <1 ZP_09753449.1 <1 Empty
vector 0.17 Experiment 3 EIJ77596.1 >50 NP_561852 >50
YP_002138168 >50 YP_026233.1 >50 YP_001447544 >50
Metalibrary >50 YP_359772 >50 ZP_01220157.1 >50
ZP_07335453.1 >20 YP_001337153 >20 YP_694908 >20 NP_717107
>20 AAC45651 >10 ZP_11313277.1 >10 ZP_16224338.1 >10
YP_001113612 >10 YP_004860127 >10 YP_003310546 >10
YP_001343716 >10 NP_717107 >10 YP_002434746 >10 Empty
vector 0.11 Experiment 4 EIJ77596.1 >20 ZP_11313277.1 >50
YP_001113612 >50 YP_001447544 >20 AGF87161 >50 EDA87976.1
>20 Empty vector -0.8
FIG. 2, Step K--Spontaneous or Formaldehyde Activating Enzyme
[0371] The conversion of formaldehyde and THF to
methylenetetrahydrofolate can occur spontaneously. It is also
possible that the rate of this reaction can be enhanced by a
formaldehyde activating enzyme. A formaldehyde activating enzyme
(Fae) has been identified in Methylobacterium extorquens AM1 which
catalyzes the condensation of formaldehyde and
tetrahydromethanopterin to methylene tetrahydromethanopterin
(Vorholt, et al., J. Bacteriol., 182(23), 6645-6650 (2000)). It is
possible that a similar enzyme exists or can be engineered to
catalyze the condensation of formaldehyde and tetrahydrofolate to
methylenetetrahydrofolate. Homologs exist in several organisms.
TABLE-US-00068 Protein GenBank ID GI Number Organism
MexAM1_META1p1766 Q9FA38.3 17366061 Methylobacterium extorquens AM1
Xaut_0032 YP_001414948.1 154243990 Xanthobacter autotrophicus Py2
Hden_1474 YP_003755607.1 300022996 Hyphomicrobium denitrificans
ATCC 51888
FIG. 2, Step L--Formaldehyde Dehydrogenase
[0372] Oxidation of formaldehyde to formate is catalyzed by
formaldehyde dehydrogenase. An NAD+ dependent formaldehyde
dehydrogenase enzyme is encoded by fdhA of Pseudomonas putida (Ito
et al, J Bacteriol 176: 2483-2491 (1994)). Additional formaldehyde
dehydrogenase enzymes include the NAD+ and glutathione independent
formaldehyde dehydrogenase from Hyphomicrobium zavarzinii (Jerome
et al, Appl Microbiol Biotechnol 77:779-88 (2007)), the glutathione
dependent formaldehyde dehydrogenase of Pichia pastoris (Sunga et
al, Gene 330:39-47 (2004)) and the NAD(P)+ dependent formaldehyde
dehydrogenase of Methylobacter marinus (Speer et al, FEMS Microbiol
Lett, 121(3):349-55 (1994)).
TABLE-US-00069 Protein GenBank ID GI Number Organism fdhA P46154.3
1169603 Pseudomonas putida faoA CAC85637.1 19912992 Hyphomicrobium
zavarzinii Fld1 CCA39112.1 328352714 Pichia pastoris fdh P47734.2
221222447 Methylobacter marinus
[0373] In addition to the formaldehyde dehydrogenase enzymes listed
above, alternate enzymes and pathways for converting formaldehyde
to formate are known in the art. For example, many organisms employ
glutathione-dependent formaldehyde oxidation pathways, in which
formaldehyde is converted to formate in three steps via the
intermediates S-hydroxymethylglutathione and S-formylglutathione
(Vorholt et al, J Bacteriol 182:6645-50 (2000)). The enzymes of
this pathway are S-(hydroxymethyl)glutathione synthase (EC
4.4.1.22), glutathione-dependent formaldehyde dehydrogenase (EC
1.1.1.284) and S-formylglutathione hydrolase (EC 3.1.2.12).
FIG. 2, Step M--Spontaneous or S-(Hydroxymethyl)Glutathione
Synthase
[0374] While conversion of formaldehyde to
S-hydroxymethylglutathione can occur spontaneously in the presence
of glutathione, it has been shown by Goenrich et al (Goenrich, et
al., J Biol Chem 277(5); 3069-72 (2002)) that an enzyme from
Paracoccus denitrificans can accelerate this spontaneous
condensation reaction. The enzyme catalyzing the conversion of
formaldehyde and glutathione was purified and named
glutathione-dependent formaldehyde-activating enzyme (Gfa).
Putative proteins with sequence identity to Gfa from P.
denitrificans are present also in Rhodobacter sphaeroides,
Sinorhizobium meliloti, and Mesorhizobium loti.
TABLE-US-00070 Protein GenBank ID GI Number Organism Gfa Q51669.3
38257308 Paracoccus denitrificans Gfa ABP71667.1 145557054
Rhodobacter sphaeroides ATCC 17025 Gfa Q92WX6.1 38257348
Sinorhizobium meliloti 1021 Gfa Q98LU4.2 38257349 Mesorhizobium
loti MAFF303099
FIG. 2, Step N--Glutathione-Dependent Formaldehyde
Dehydrogenase
[0375] Glutathione-dependent formaldehyde dehydrogenase (GS-FDH)
belongs to the family of class DI alcohol dehydrogenases.
Glutathione and formaldehyde combine non-enzymatically to form
hydroxymethylglutathione, the true substrate of the GS-FDH
catalyzed reaction. The product, S-formylglutathione, is further
metabolized to formic acid.
TABLE-US-00071 Protein GenBank ID GI Number Organism frmA
YP_488650.1 388476464 Escherichia coli K-12 MG1655 SFA1 NP_010113.1
6320033 Saccharomyces cerevisiae S288c flhA AAC44551.1 1002865
Paracoccus denitrificans adhI AAB09774.1 986949 Rhodobacter
sphaeroides
FIG. 2, Step O--S-Formylglutathione Hydrolase S-formylglutathione
hydrolase is a glutathione thiol esterase found in bacteria, plants
and animals. It catalyzes conversion of S-formylglutathione to
formate and glutathione. Exemplary enzymes are below. YeiG of E.
coli is a promiscuous serine hydrolase; its highest specific
activity is with the substrate S-formylglutathione.
TABLE-US-00072 Protein GenBank ID GI Number Organism frmB
NP_414889.1 16128340 Escherichia coli K-12 MG1655 yeiG AAC75215.1
1788477 Escherichia coli K-12 MG1655 fghA AAC44554.1 1002868
Paracoccus denitrificans
FIG. 2, Step P--Carbon Monoxide Dehydrogenase (CODH)
[0376] CODH is a reversible enzyme that interconverts CO and
CO.sub.2 at the expense or gain of electrons. The natural
physiological role of the CODH in ACS/CODH complexes is to convert
CO.sub.2 to CO for incorporation into acetyl-CoA by acetyl-CoA
synthase. Nevertheless, such CODH enzymes are suitable for the
extraction of reducing equivalents from CO due to the reversible
nature of such enzymes. Expressing such CODH enzymes in the absence
of ACS allows them to operate in the direction opposite to their
natural physiological role (i.e., CO oxidation).
[0377] In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans
P7, and several other organisms, additional CODH encoding genes are
located outside of the ACS/CODH operons. These enzymes provide a
means for extracting electrons (or reducing equivalents) from the
conversion of carbon monoxide to carbon dioxide. The M.
thermoacetica gene (Genbank Accession Number YP_430813) is
expressed by itself in an operon and is believed to transfer
electrons from CO to an external mediator like ferredoxin in a
"Ping-pong" reaction. The reduced mediator then couples to other
reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H)
carriers or ferredoxin-dependent cellular processes (Ragsdale,
Annals of the New York Academy of Sciences 1125: 129-136 (2008)).
Similar ACS-free CODH enzymes can be found in a diverse army of
organisms.
TABLE-US-00073 Protein GenBank ID GI Number Organism CODH
(putative) YP_430813 83590804 Moorella thermoacetica CODH-II
(CooS-II) YP_358957 78044574 Carboxydothermus hydrogenoformans CooF
YP_358958 78045112 Carboxydothermus hydrogenoformans CODH
(putative) ZP_05390164.1 255523193 Clostridium carboxidivorans P7
CcarbDRAFT_0341 ZP_05390341.1 255523371 Clostridium carboxidivorans
P7 CcarbDRAFT_1756 ZP_05391756.1 255524806 Clostridium
carboxidivorans P7 CcarbDRAFT_2944 ZP_05392944.1 255526020
Clostridium carboxidivorans P7 CODH YP_384856.1 78223109 Geobacter
metallireducens GS-15 Cpha266_0148 (cytochrome c) YP_910642.1
119355998 Chlorobium phaeobacteroides DSM 266 Cpha266_0149 (CODH)
YP_910643.1 119355999 Chlorobium phaeobacteroides DSM 266 Ccel_0438
YP_002504800.1 220927891 Clostridium cellulolyticum H10 Ddes_0382
(CODH) YP_002478973.1 220903661 Desulfovibrio desulfuricans subsp.
desulfuricans str. ATCC 27774 Ddes_0381 (CooC) YP_002478972.1
220903660 Desulfovibrio desulfuricans subsp. desulfuricans str.
ATCC 27774 Pcar_0057 (CODH) YP_355490.1 7791767 Pelobacter
carbinolicus DSM 2380 Pcar 0058 (CooC) YP_355491.1 7791766
Pelobacter carbinolicus DSM 2380 Pcar_0058 (HypA) YP_355492.1
7791765 Pelobacter carbinolicus DSM 2380 CooS (CODH) YP_001407343.1
154175407 Campylobacter curvus 525.92 CLJU_c09110 ADK13979.1
300434212 Clostridium ljungdahli CLJU_c09100 ADK13978.1 300434211
Clostridium ljungdahli CLJU c09090 ADK13977.1 300434210 Clostridium
ljungdahli
Example III
Methods for Formaldehyde Fixation
[0378] Provided herein are exemplary pathways, which utilize
formaldehyde produced from the oxidation of methanol (see, e.g.,
FIG. 1, step A, or FIG. 2, step J) or from formate assimilation
pathways described in Example I (see, e.g., FIG. 1) in the
formation of intermediates of certain central metabolic pathways
that can be used for the production of compounds disclosed
herein.
[0379] One exemplary pathway that can utilize formaldehyde produced
from the oxidation of methanol is shown in FIG. 1, which involves
condensation of formaldehyde and D-ribulose-5-phosphate to form
hexulose-6-phosphate (h6p) by hexulose-6-phosphate synthase (FIG.
1, step B). The enzyme can use Mg' or Me for maximal activity,
although other metal ions are useful, and even
non-metal-ion-dependent mechanisms are contemplated. H6p is
converted into fructose-6-phosphate by 6-phospho-3-hexuloisomerase
(FIG. 1, step C).
[0380] Another exemplary pathway that involves the detoxification
and assimilation of formaldehyde produced from the oxidation of
methanol is shown in FIG. 1 and proceeds through dihydroxyacetone.
Dihydroxyacetone synthase is a special transketolase that first
transfers a glycoaldehyde group from xylulose-5-phosphate to
formaldehyde, resulting in the formation of dihydroxyacetone (DHA)
and glyceraldehyde-3-phosphate (G3P), which is an intermediate in
glycolysis (FIG. 1). The DHA obtained from DHA synthase can be
further phosphorylated to form DHA phosphate and assimilated into
glycolysis and several other pathways (FIG. 1). Alternatively, or
in addition, a fructose-6-phosphate aldolase can be used to
catalyze the conversion of DHA and G3P to fructose-6-phosphate
(FIG. 1, step Z).
FIG. 1, Steps B and C--Hexulose-6-Phosphate Synthase (Step B) and
6-Phospho-3-Hexuloisomerase (Step C)
[0381] Both of the hexulose-6-phosphate synthase and
6-phospho-3-hexuloisomerase enzymes are found in several organisms,
including methanotrophs and methylotrophs where they have been. In
addition, these enzymes have been reported in heterotrophs such as
Bacillus subtilis also where they are reported to be involved in
formaldehyde detoxification. Genes for these two enzymes from the
methylotrophic bacterium Mycobacterium gastri MB19 have been fused
and E. coli strains harboring the hps-phi construct showed more
efficient utilization of formaldehyde (Orita et at, 2007, Appl
Microbiol Biotechnol. 76:439-445). In some organisms, these two
enzymes naturally exist as a fused version that is
bifunctional.
[0382] Exemplary candidate genes for hexulose-6-phopshate synthase
are:
TABLE-US-00074 Protein GenBank ID GI number Organism Hps AAR39392.1
40074227 Bacillus methanolicus MGA3 Hps EIJ81375.1 387589055
Bacillus methanolicus PB1 RmpA BAA83096.1 5706381 Methylomonas
aminofaciens RmpA BAA90546.1 6899861 Mycobacterium gastri YckG
BAA08980.1 1805418 Bacillus subtilis Hps YP_544362.1 91774606
Methylobacillus flagellatus Hps YP_545763.1 91776007
Methylobacillus flagellatus Hps AAG29505.1 11093955 Aminomonas
aminovorus SgbH YP_004038706.1 313200048 Methylovorus sp. MP688 Hps
YP_003050044.1 253997981 Methylovorus glucosetrophus SIP3-4 Hps
YP_003990382.1 312112066 Geobacillus sp. Y4.1MC1 Hps gb|AAR91478.1
40795504 Geobacillus sp. M10EXG Hps YP_007402409.1 448238351
Geobacillus sp. GHH01
[0383] Exemplary gene candidates for 6-phospho-3-hexuloisomerase
are:
TABLE-US-00075 Protein GenBank ID GI number Organism Phi AAR39393.1
40074228 Bacillus methanolicus MGA3 Phi EIJ81376.1 387589056
Bacillus methanolicus PB1 Phi BAA83098.1 5706383 Methylomonas
aminofaciens RmpB BAA90545.1 6899860 Mycobacterium gastri Phi
YP_545762.1 91776006 Methylobacillus flagellatus KT Phi
YP_003051269.1 253999206 Methylovorus glucosetrophus SIP3-4 Phi
YP_003990383.1 312112067 Geobacillus sp. Y4.1MC1 Phi YP_007402408.1
448238350 Geobacillus sp. GHH01
[0384] Candidates for enzymes where both of these functions have
been fused into a single open reading frame include the
following.
TABLE-US-00076 Protein GenBank ID GI number Organism PH1938
NP_143767.1 14591680 Pyrococcus horikoshii OT3 PF0220 NP_577949.1
18976592 Pyrococcus furiosus TK0475 YP_182888.1 57640410
Thermococcus kodakaraensis PAB1222 NP_127388.1 14521911 Pyrococcus
abyssi MCA2738 YP_115138.1 53803128 Methylococcus capsulatas
Metal_3152 EIC30826.1 380884949 Methylomicrobium album BG8
FIG. 1, Step D--Dihydroxyacetone Synthase
[0385] The dihydroxyacetone synthase enzyme in Candida boidinii
uses thiamine pyrophosphate and Mg.sup.2+ as cofactors and is
localized in the peroxisome. The enzyme from the methanol-growing
carboxydobacterium, Mycobacter sp. strain JC1 DSM 3803, was also
found to have DHA synthase and kinase activities. Several other
mycobacteria, excluding only Mycobacterium tuberculosis, can use
methanol as the sole source of carbon and energy and are reported
to use dihydroxyacetone synthase.
TABLE-US-00077 Protein GenBank ID GI number Organism DAS1
AAC83349.1 3978466 Candida boidinii HPODL_2613 EFW95760.1 320581540
Ogataea parapolymorpha DL-1 (Hansenula polymorpha DL-1) AAG12171.2
18497328 Mycobacter sp. strain JC1 DSM 3803
FIG. 1, Step Z--Fructose-6-Phosphate Aldolase
[0386] Fructose-6-phosphate aldolase (F6P aldolase) can catalyze
the combination of dihydroxyacetone (DHA) and
glyceraldehyde-3-phosphate (G3P) to form fructose-6-phosphate. This
activity was recently discovered in E. coli and the corresponding
gene candidate has been termed fsa. The enzyme prefers the aldol
formation over the cleavage reaction.
[0387] The selectivity of the E. coli enzyme towards DHA can be
improved by introducing point mutations. For example, the mutation
A129S improved reactivity towards DHA by over 17 fold in terms of
K.sub.cat/K.sub.m (Gutierrez et al., Chem Commun (Carob), 2011,
47(20), 5762-5764). Genes similar to fsa have been found in other
genomes by sequence homology. Some exemplary gene candidates have
been listed below.
TABLE-US-00078 Gene Protein accession no. GI number Organism fsa
AAC73912.2 87081788 Escherichia coli K12 talC AAC76928.1 1790382
Escherichia coli K12 fsa WP_017209835.1 515777235 Clostridium
beijerinckii DR_1337 AAF10909.1 6459090 Deinococcus radiodurans R1
talC NP_213080.1 15605703 Aquifex aeolicus VF5 MJ 0960 NP_247955.1
15669150 Methanocaldococcus janaschii mipB NP_993370.2 161511381
Yersinia pestis
[0388] As Described Below, there is an Energetic Advantage to Using
F6P Aldolase in the DHA Pathway.
[0389] The assimilation of formaldehyde formed by the oxidation of
methanol can proceed either via the dihydroxyacetone (DHA) pathway
(step D, FIG. 1) or the Ribulose monophosphate (RuMP) pathway
(steps B and C, FIG. 1). In the RuMP pathway, formaldehyde combines
with ribulose-5-phosphate to form F6P. F6P is then either
metabolized via glycolysis or used for regeneration of
ribulose-5-phosphate to enable further formaldehyde assimilation.
Notably, ATP hydrolysis is not required to form F6P from
formaldehyde and ribulose-5-phosphate via the RuMP pathway.
[0390] In contrast, in the DHA pathway, formaldehyde combines with
xylulose-5-phosphate (X5P) to form dihydroxyacetone (DHA) and
glyceraldehyde-3-phosphate (G3P). Some of the DHA and G3P must be
metabolized to F6P to enable regeneration of xylulose-5-phosphate.
In the standard DHA pathway, DHA and G3P are converted to F6P by
three enzymes: DHA kinase, fructose bisphosphate aldolase, and
fructose bisphosphatase. The net conversion of DHA and G3P to F6P
requires ATP hydrolysis as described below. First, DHA is
phosphorylated to form DHA phosphate (DHAP) by DHA kinase at the
expense of an ATP. DHAP and G3P are then combined by fructose
bisphosphate aldolase to form fructose-1,6-diphosphate (FDP). FDP
is converted to F6P by fructose bisphosphatase, thus wasting a high
energy phosphate bond.
[0391] A more ATP efficient sequence of reactions is enabled if DHA
synthase functions in combination with F6P aldolase as opposed to
in combination with DHA kinase, fructose bisphosphate aldolase, and
fructose bisphosphatase. F6P aldolase enables direct conversion of
DHA and G3P to F6P, bypassing the need for ATP hydrolysis. Overall,
DHA synthase when combined with F6P aldolase is identical in energy
demand to the RuMP pathway. Both of these formaldehyde assimilation
options (i.e., RuMP pathway, DHA synthase+F6P aldolase) are
superior to DHA synthase combined with DHA kinase, fructose
bisphosphate aldolase, and fructose bisphosphatase in terms of ATP
demand.
Example IV
In Vivo Labeling Assay for Conversion of Methanol to CO.sub.2
[0392] This example describes a functional methanol pathway in a
microbial organism.
[0393] Strains with functional reductive TCA branch and pyruvate
formate lyase deletion were grown aerobically in LB medium
overnight, followed by inoculation of M9 high-seed media containing
IPTG and aerobic growth for 4 hrs. These strains had methanol
dehydrogenase/ACT pairs in the presence and absence of formaldehyde
dehydrogenase or formate dehydrogenase. ACT is an activator protein
(a Nudix hydrolase). At this time, strains were pelleted,
resuspended in fresh M9 medium high-seed media containing 2%
.sup.13CH.sub.3OH, and sealed in anaerobic vials. Head space was
replaced with nitrogen and strains grown for 40 hours at 37.degree.
C. Following growth, headspace was analyzed for .sup.13CO.sub.2.
Media was examined for residual methanol as well as 1,4-butanediol
and byproducts. All constructs expressing methanol dehydrogenase
(MeDH) mutants and MeDH/ACT pairs grew to slightly lower ODs than
strains containing empty vector controls. This is likely due to the
high expression of these constructs (Data not shown). One construct
(2315/2317) displayed significant accumulation of labeled CO.sub.2
relative to controls in the presence of FalDH, FDH or no
coexpressed protein. This shows a functional MeOH pathway in E.
coli and that the endogenous glutathione-dependent formaldehyde
detoxification genes (frmAB) are sufficient to carry flux generated
by the current MeDH/ACT constructs.
[0394] 2315 is internal laboratory designation for the MeDH from
Bacillus methanolicus MGA3 (GenBank Accession number EIJ77596.1; GI
number 387585261), and 2317 is internal laboratory designation for
the activator protein from the same organism (locus tag:
MGA3_09170; GenBank Accession number EIJ83380; GI number:
387591061).
[0395] Sequence analysis of the NADH-dependent MeDH from Bacillus
methanolicus places the enzyme in the alcohol dehydrogenase family
III. It does not contain any tryptophan residues, resulting in a
low extinction coefficient (18,500 M.sup.-1, cm.sup.-1) and should
be detected on SDS gels by Coomassie staining.
[0396] The enzyme has been characterized as a multisubunit complex
built from 43 kDa subunits containing one Zn and 1-2 Mg atoms per
subunit. Electron microscopy and sedimentation studies determined
it to be a decamer, in which two rings with five-fold symmetry are
stacked on top of each other (Vonck et al., J. Biol. Chem.
266:3949-3954, 1991). It is described to contain a tightly but not
covalently bound cofactor and requires exogenous NAD.sup.+ as
e.sup.--acceptor to measure activity in vitro. A strong increase
(10-40-fold) of in vitro activity was observed in the presence of
an activator protein (ACT), which is a homodimer (21 kDa subunits)
and contains one Zn and one Mg atom per subunit.
[0397] The mechanism of the activation was investigated by
Kloosterman et al., J. Biol. Chem. 277:34785-34792, 2002, showing
that ACT is a Nudix hydrolase and Hektor et al., J. Biol. Chem.
277:46966-46973, 2002, demonstrating that mutation of residue S97
to G or Tin MeDH changes activation characteristics along with the
affinity for the cofactor. While mutation of residues G15 and D88
had no significant impact, a role of residue G13 for stability as
well as of residues G95, D100, and K103 for the activity is
suggested. Both papers together propose a hypothesis in which ACT
cleaves MeDH-bound NAD.sup.+. MeDH retains AMP bound and enters an
activated cycle with increased turnover. The stoichiometric ratio
between ACT and MeDH is not well defined in the literature.
Kloosterman et al., supra determine the ratio of dimeric Act to
decameric MeDH for full in vitro activation to be 10:1. In
contrast, Arfman et al. J. Biol. Chem. 266:3955-3960, 1991
determined a ratio of 3:1 in vitro for maximum and a 1:6 ratio for
significant activation, but observe a high sensitivity to dilution.
Based on expression of both proteins in Bacillus, the authors
estimate the ratio in vivo to be around 1:17.5.
[0398] However, our in vitro experiments with purified activator
protein (2317A) and methanol dehydrogenase (2315A) showed the ratio
of ACT to MeDH to be 10:1. This in vitro test was done with 5 M
methanol, 2 mM NAD and 10 .mu.M methanol dehydrogenase 2315A at pH
7.4.
Example V
Phosphoketolase-Dependent Acetyl-CoA Synthesis Enzymes
[0399] This Example provides genes that can be used for enhancing
carbon flux through acetyl-CoA using phosphoketolase enzymes.
FIG. 1, Step T--Fructose-6-Phosphate Phosphoketolase
[0400] Conversion of fructose-6-phosphate and phosphate to
acetyl-phosphate and erythrose-5-phosphate can be carried out by
fructose-6-phosphate phosphoketolase (EC 4.1.2.22). Conversion of
fructose-6-phosphate and phosphate to acetyl-phosphate and
erythrose-5-phosphate is one of the key reactions in the
Bifidobacterium shunt. There is evidence for the existence of two
distinct phosphoketolase enzymes in bifidobacteria (Sgorbati et al,
1976, Antonie Van Leeuwenhoek, 42(1-2) 49-57; Grill et al, 1995,
Curr Microbiol, 31(1); 49-54). The enzyme from Bifidobacterium
dentium appeared to be specific solely for fructose-6-phosphate
(EC: 4.1.2.22) while the enzyme from Bifidobacterium pseudolongum
subsp. globosum is able to utilize both fructose-6-phosphate and
D-xylulose 5-phosphate (EC: 4.1.2.9) (Sgorbati et al, 1976, Antonie
Van Leeuwenhoek, 42(1-2) 49-57). The enzyme encoded by the xfp
gene, originally discovered in Bifidobacterium animalis lactis, is
the dual-specificity enzyme (Meile et al., 2001, J Bacteriol, 183,
2929-2936; Yin et al, 2005, FEMS Microbiol Lett, 246(2); 251-257).
Additional phosphoketolase enzymes can be found in Leuconostoc
mesenteroides (Lee et al, Biotechnol Lett. 2005 June;
27(12):853-8), Clostridium acetobutylicum ATCC 824 (Servinsky et
al, Journal of Industrial Microbiology & Biotechnology, 2012,
39, 1859-1867), Aspergillus nidulans (Kocharin et al, 2013,
Biotechnol Bioeng, 110(8), 2216-2224; Papini, 2012, Appl Microbiol
Biotechnol, 95 (4), 1001-1010), Bifidobacterium breve (Suziki et
al, 2010, Acta Crystallogr Sect F Struct Biol Cryst Commun., 66(Pt
8):941-3), Lactobacillus paraplantarum (Jeong et al, 2007, J
Microbiol Biotechnol, 17(5), 822-9).
TABLE-US-00079 Protein GENBANK ID GI NUMBER ORGANISM xfp
YP_006280131.1 386867137 Bifidobacterium animalis lactis xfp
AAV66077.1 55818565 Leuconostoc mesenteroides CAC1343 NP_347971.1
15894622 Clostridium acetobutylicum ATCC 824 xpkA CBF76492.1
259482219 Aspergillus nidulans xfp WP_003840380.1 489937073
Bifidobacterium dentium ATCC 27678 xfp AAR98788.1 41056827
Bifidobacterium pseudolongum subsp. globosum xfp WP_022857642.1
551237197 Bifidobacterium pseudolongum subsp. globosum xfp
ADF97524.1 295314695 Bifidobacterium breve xfp AAQ64626.1 34333987
Lactobacillus paraplantarum
FIG. 1, Step U--Xylulose-5-Phosphate Phosphoketolase
[0401] Conversion of xylulose-5-phosphate and phosphate to
acetyl-phosphate and glyceraldehyde-3-phosphate can be carried out
by xylulose-5-phosphate phosphoketolase (EC 4.1.2.9). There is
evidence for the existence of two distinct phosphoketolase enzymes
in bifidobacteria (Sgorbati et al, 1976, Antonie Van Leeuwenhoek,
42(1-2) 49-57; Grill et al, 1995, Curr Microbiol, 31(1); 49-54).
The enzyme from Bifidobacterium dentium appeared to be specific
solely for fructose-6-phosphate (EC: 4.1.2.22) while the enzyme
from Bifidobacterium pseudolongum subsp. globosum is able to
utilize both fructose-6-phosphate and D-xylulose 5-phosphate (EC:
4.1.2.9) (Sgorbati et al, 1976, Antonie Van Leeuwenhoek, 42(1-2)
49-57). Many characterized enzymes have dual-specificity for
xylulose-5-phosphate and fructose-6-phosphate. The enzyme encoded
by the xfp gene, originally discovered in Bifidobacterium animalis
lactis, is the dual-specificity enzyme (Meile et al., 2001, J
Bacteriol, 183, 2929-2936; Yin et al, 2005, FEMS Microbiol Lett,
246(2); 251-257). Additional phosphoketolase enzymes can be found
in Leuconostoc mesenteroides (Lee et al, Biotechnol Let 2005 June;
27(12):853-8), Clostridium acetobutylicum ATCC 824 (Servinsky et
al, Journal of Industrial Microbiology & Biotechnology, 2012,
39, 1859-1867), Aspergillus nidulans (Kocharin et al, 2013,
Biotechnol Bioeng, 110(8), 2216-2224; Papini, 2012, Appl Microbiol
Biotechnol, 95 (4), 1001-1010), Bifidobacterium breve (Suziki et
al, 2010, Acta Crystallogr Sect F Struct Biol Cryst Commun., 66(Pt
8):941-3), and Lactobacillus paraplantarum (Jeong et al, 2007, J
Microbiol Biotechnol, 17(5), 822-9).
TABLE-US-00080 Protein GENBANK ID GI NUMBER ORGANISM xfp
YP_006280131.1 386867137 Bifidobacterium animalis lactis xfp
AAV66077.1 55818565 Leuconostoc mesenteroides CAC1343 NP_347971.1
15894622 Clostridium acetobutylicum ATCC 824 xpkA CBF76492.1
259482219 Aspergillus nidulans xfp AAR98788.1 41056827
Bifidobacterium pseudolongum subsp. globosum xfp WP_022857642.1
551237197 Bifidobacterium pseudolongum subsp. globosum xfp
ADF97524.1 295314695 Bifidobacterium breve xfp AAQ64626.1 34333987
Lactobacillus paraplantarum
FIG. 1, Step V--Phosphotransacetylase
[0402] The formation of acetyl-CoA from acetyl-phosphate can be
catalyzed by phosphotransacetylase (EC 2.3.1.8). The pta gene from
E. coli encodes an enzyme that reversibly converts acetyl-CoA into
acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569
(969)). Additional acetyltransferase enzymes have been
characterized in Bacillus subtilis (Rado and Hoch, Biochim.
Biophys. Acta 321:114-125 (1973), Clostridium kluyveri (Stadtman,
E., Methods Enzymol. 1:5896-599 (1955), and Thermotoga maritima
(Bock et al., J. Bacteriol. 181:1861-1867 (1999)). This reaction
can also be catalyzed by some phosphotransbutyrylase enzymes (EC
2.3.1.19), including the ptb gene products from Clostridium
acetobutylicum (Wiesenbom et al., App. Environ. Microbiol.
55:317-322 (1989); Walter et al., Gene 134:107-111 (1993)).
Additional ptb genes are found in butyrate-producing bacterium
L2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004) and
Bacillus megaterium (Vazquez et al., Curr. Microbiol. 42:345-349
(2001). Homologs to the E. coli pta gene exist in several other
organisms including Salmonella enterica and Chlamydomonas
reinhardtii.
TABLE-US-00081 Protein GenBank ID GI Number Organism Pta
NP_416800.1 71152910 Escherichia coli Pta P39646 730415 Bacillus
subtilis Pta A5N801 146346896 Clostridium kluyveri Pta Q9X0L4
6685776 Thermotoga maritime Ptb NP_349676 34540484 Clostridium
acetobutylicum Ptb AAR19757.1 38425288 butyrate-producing bacterium
L2-50 Ptb CAC07932.1 10046659 Bacillus megaterium Pta NP_461280.1
16765665 Salmonella enterica subsp. enterica serovar Typhimurium
str. LT2 PAT2 XP_001694504.1 159472743 Chlamydomonas reinhardtii
PAT1 XP_001691787.1 159467202 Chlamydomonas reinhardtii
FIG. 1, Step W--Acetate Kinase
[0403] Acetate kinase (EC 2.7.2.1) can catalyze the reversible
ATP-dependent phosphorylation of acetate to acetylphosphate.
Exemplary acetate kinase enzymes have been characterized in many
organisms including E. coli, Clostridium acetobutylicum and
Methanosarcina thermophila (Ingram-Smith et al., J. Bacteriol.
187:2386-2394 (2005); Fox and Roseman, J. Biol. Chem.
261:13487-13497 (1986); Winzer et al., Microbioloy 143 (Pt
10):3279-3286 (1997)). Acetate kinase activity has also been
demonstrated in the gene product of E. coli purT (Marolewski et
al., Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes
(EC 2.7.2.7), for example buk1 and buk2 from Clostridium
acetobutylicum, also accept acetate as a substrate (Hartmanis, M
G., J. Biol. Chem. 262:617-621 (1987)). Homologs exist in several
other organisms including Salmonella enterica and Chlamydomonas
reinhardtii.
TABLE-US-00082 Protein GenBank ID GI Number Organism ackA
NP_416799.1 16130231 Escherichia coli Ack AAB18301.1 1491790
Clostridium acetobutylicum Ack AAA72042.1 349834 Methanosarcina
thermophila purT AAC74919.1 1788155 Escherichia coli buk1 NP_349675
15896326 Clostridium acetobutylicum buk2 Q97II1 20137415
Clostridium acetobutylicum ackA NP_461279.1 16765664 Salmonella
typhimurium ACK1 XP_001694505.1 159472745 Chlamydomonas reinhardtii
ACK2 XP_001691682.1 159466992 Chlamydomonas reinhardtii
FIG. 1, Step X--Acetyl-CoA Transferase, Synthetase, or Ligase
[0404] The acylation of acetate to acetyl-CoA can be catalyzed by
enzymes with acetyl-CoA synthetase, ligase or transferase activity.
Two enzymes that can catalyze this reaction are AMP-forming
acetyl-CoA synthetase or ligase (EC 6.2.1.1) and ADP-forming
acetyl-CoA synthetase (EC 6.2.1.13). AMP-forming acetyl-CoA
synthetase (ACS) is the predominant enzyme for activation of
acetate to acetyl-CoA. ADP-forming acetyl-CoA synthetases are
reversible enzymes with a generally broad substrate range (Musfeldt
and Schonheit, J. Bacteriol. 184:636-644 (2002)). The
aforementioned proteins are shown below.
TABLE-US-00083 Protein GenBank ID GI Number Organism Acs AAC77039.1
1790505 Escherichia coli acoE AAA21945.1 141890 Ralstonia eutropha
acs1 ABC87079.1 86169671 Methanothermobacter thermautotrophicus
acs1 AAL23099.1 16422835 Salmonella enterica ACS1 Q01574.2
257050994 Saccharomyces cerevisiae AF1211 NP_070039.1 11498810
Archaeoglobus fulgidus AF1983 NP_070807.1 11499565 Archaeoglobus
fulgidus Scs YP_135572.1 55377722 Haloarcula marismortui PAE3250
NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC
NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949
Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida
[0405] An acetyl-CoA transferase that can utilize acetate as the
CoA acceptor is acetoacetyl-CoA transferase, encoded by the E. coli
atoA (alpha subunit) and atoD (beta subunit) genes. This and other
proteins are identified below.
TABLE-US-00084 Protein GenBank ID GI Number Organism atoA P76459.1
2492994 Escherichia coli K12 atoD P76458.1 2492990 Escherichia coli
K12 actA YP_226809.1 62391407 Corynebacterium glutamicum ATCC 13032
cg0592 YP_224801.1 62389399 Corynebacterium glutamicum ATCC 13032
ctfA NP_149326.1 15004866 Clostridium acetobutylicum ctfB
NP_149327.1 15004867 Clostridium acetobutylicum ctfA AAP42564.1
31075384 Clostridium saccharoperbutylacetonicum ctfB AAP42565.1
31075385 Clostridium saccharoperbutylacetonicum
[0406] Additional exemplary acetyl-CoA transferase candidates are
catalyzed by the gene products of cat1, cat2, and cat3 of
Clostridium kluyveri which have been shown to exhibit succinyl-CoA,
4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity,
respectively. Similar CoA transferase activities are also present
in Trichomonas vaginalis (van Grinsven et al., J. Biol. Chem.
283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J.
Biol. Chem. 279:45337-45346 (2004)). These and other proteins are
identified below.
TABLE-US-00085 Protein GenBank ID GI Number Organism cat1 P38946.1
729048 Clostridium kluyveri cat2 P38942.2 172046066 Clostridium
kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri TVAG_395550
XP_001330176 123975034 Trichomonas vaginalis G3 Tb11.02.0290
XP_828352 71754875 Trypanosoma brucei
Example VI
Acetyl-CoA and Succinyl-CoA Synthesis Enzymes
[0407] This Example provides genes that can be used for conversion
of glycolysis intermediate glyceraldehyde-3-phosphate (G3P) to
acetyl-CoA and/or succinyl-CoA as depicted in the pathways of FIG.
4.
[0408] A. PEP Carboxylase or PEP Carboxykinase.
[0409] Carboxylation of phosphoenolpyruvate to oxaloacetate is
catalyzed by phosphoenolpyruvate carboxylase. Exemplary PEP
carboxylase enzymes are below.
TABLE-US-00086 Protein GenBank ID GI Number Organism Ppc NP_418391
16131794 Escherichia coli ppcA AAB58883 28572162 Methylobacterium
extorquens Ppc ABB53270 80973080 Corynebacterium glutamicum
[0410] An alternative enzyme for converting phosphoenolpyruvate to
oxaloacetate is PEP carboxykinase, which simultaneously forms an
ATP while carboxylating PEP. In most organisms PEP carboxykinase
serves a gluconeogenic function and converts oxaloacetate to PEP at
the expense of one ATP. S. cerevisiae is one such organism whose
native PEP carboxykinase, PCK1, serves a gluconeogenic role
(Valdes-Hevia et al., FEBS Lett. 258:313-316 (1989). E. coli is
another such organism, as the role of PEP carboxykinase in
producing oxaloacetate is believed to be minor when compared to PEP
carboxylase, which does not form ATP, possibly due to the higher
K.sub.m for bicarbonate of PEP carboxykinase (Kim et al., Appl.
Environ. Microbiol. 70:1238-1241 (2004)). Nevertheless, activity of
the native E. coli PEP carboxykinase from PEP towards oxaloacetate
has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon
et al., J. Microbiol. Biotechnol. 16:1448-1452 (2006)). These
strains exhibited no growth defects and had increased succinate
production at high NaHCO.sub.3 concentrations. Mutant strains of E.
coli can adopt Pck as the dominant CO2-fixing enzyme following
adaptive evolution (Zhang et al. 2009). In some organisms,
particularly rumen bacteria, PEP carboxykinase is quite efficient
in producing oxaloacetate from PEP and generating ATP. Examples of
PEP carboxykinase genes are shown below.
TABLE-US-00087 Protein GenBank ID GI Number Organism PCK1 NP_013023
6322950 Saccharomyces cerevisiae pck NP_417862.1 16131280
Escherichia coli pckA YP_089485.1 52426348 Mannheimia
succiniciproducens pckA O09460.1 3122621 Anaerobiospirillum
succiniciproducens pckA Q6W6X5 75440571 Actinobacillus succinogenes
pckA P43923.1 1172573 Haemophilus influenza
[0411] B. Malate Dehydrogenase.
[0412] Oxaloacetate is converted into malate by malate
dehydrogenase (EC 1.1.1.37), an enzyme which functions in both the
forward and reverse direction. Exemplary enzymes are show
below.
TABLE-US-00088 Protein GenBank ID GI Number Organism MDH1 NP_012838
6322765 Saccharomyces cerevisiae MDH2 NP_014515 116006499
Saccharomyces cerevisiae MDH3 NP_010205 6320125 Saccharomyces
cerevisiae Mdh NP_417703.1 16131126 Escherichia coli
[0413] C. Fumarase.
[0414] Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible
hydration of fumarate to malate. The three fumarases of E. coli,
encoded by fumA, fumB and fumC, are regulated under different
conditions of oxygen availability. FumB is oxygen sensitive and is
active under anaerobic conditions. FumA is active under
microanaerobic conditions, and FumC is active under aerobic growth
conditions (Tseng et al., J. Bacteriol. 183:461-467 (2001); Woods
et al., Biochim. Biophys. Acta 954:14-26 (1988); Guest et al., J.
Gen. Microbiol. 131:2971-2984 (1985)). Additional fumarase enzymes
are shown below.
TABLE-US-00089 Protein GenBank ID GI Number Organism fumA
NP_416129.1 16129570 Escherichia coli fumB NP_418546.1 16131948
Escherichia coli fumC NP_416128.1 16129569 Escherichia coli FUM1
NP_015061 6324993 Saccharomyces cerevisiae fumC Q8NRN8.1 39931596
Corynebacterium glutamicum fumC O69294.1 9789756 Campylobacter
jejuni fumC P84127 75427690 Thermus thermophilus fumH P14408.1
120605 Rattus norvegicus MmcB YP_001211906 147677691 Pelotomaculum
thermopropionicum MmcC YP_001211907 147677692 Pelotomaculum
thermopropionicum
[0415] D. Fumarate Reductase.
[0416] Fumarate reductase catalyzes the reduction of fumarate to
succinate. The fumarate reductase of E. coli, composed of four
subunits encoded by frdABCD, is membrane-bound and active under
anaerobic conditions. The electron donor for this reaction is
menaquinone and the two protons produced in this reaction do not
contribute to the proton gradient (Iverson et al., Science
284:1961-1966 (1999)). The yeast genome encodes two soluble
fumarate reductase isozymes encoded by FRDS1 and FRDS2.
TABLE-US-00090 Protein GenBank ID GI Number Organism FRDS1 P32614
418423 Saccharomyces cerevisiae FRDS2 NP_012585 6322511
Saccharomyces cerevisiae frdA NP_418578.1 16131979 Escherichia coli
frdB NP_418577.1 16131978 Escherichia coli frdC NP_418576.1
16131977 Escherichia coli frdD NP_418475.1 16131877 Escherichia
coli
[0417] E. Succinyl-CoA Synthetase or Transferase.
[0418] The ATP-dependent acylation of succinate to succinyl-CoA is
catalyzed by succinyl-CoA synthetase (EC 6.2.1.5). The product of
the LSC1 and LSC2 genes of S. cerevisiae and the sucC and sucD
genes of E. coli naturally form a succinyl-CoA synthetase complex
that catalyzes the formation of succinyl-CoA from succinate with
the concomitant consumption of one ATP, a reaction which is
reversible in vivo. These proteins are identified below:
TABLE-US-00091 Protein GenBank ID GI Number Organism LSC1 NP_014785
6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683
Saccharomyces cerevisiae sucC NP_415256.1 16128703 Escherichia coli
sucD AAC73823.1 1786949 Escherichia coli
[0419] Succinyl-CoA transferase converts succinate and an acyl-CoA
donor to succinyl-CoA and an acid. Succinyl-CoA transferase enzymes
include ygfH of E. coli, cat1 of Clostridium kluyveri, and other
exemplary enzymes shown below. Additional CoA transferases,
described herein, are also suitable candidates.
TABLE-US-00092 Gene GI # Accession No. Organism ygfH AAC75957.1
1789287 Escherichia coli cat1 P38946.1 729048 Clostridium kluyveri
CIT292_04485 ZP_03838384.1 227334728 Citrobacter youngae SARI 04582
YP_001573497.1 161506385 Salmonella enterica yinte0001_14430
ZP_04635364.1 238791727 Yersinia intermedia pcaI 24985644
AAN69545.1 Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas
putida pcaI 50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776
AAC37147.1 Acinetobacter sp. ADP1 pcaI 21224997 NP_630776.1
Streptomyces coelicolor pcaJ 21224996 NP_630775.1 Streptomyces
coelicolor catI 75404583 Q8VPF3 Pseudomonas knackmussii catJ
75404582 Q8VPF2 Pseudomonas knackmussii HPAG1 0676 108563101
YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418
Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB
16080949 NP_391777 Bacillus subtilis OXCT1 NP_000427 4557817 Homo
sapiens OXCT2 NP_071403 11545841 Homo sapiens
[0420] F. Pyruvate Kinase or PTS-Dependent Substrate Import. See
Elsewhere Herein.
[0421] G. Pyruvate Dehydrogenase, Pyruvate Formate Lyase or
Pyruvate:Ferredoxin Oxidoreductase.
[0422] Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the
reversible oxidation of pyruvate to form acetyl-CoA. Exemplary PFOR
enzymes are found in Desulfovibrio africanus (Pieulle et al., J.
Bacteriol. 179:5684-5692 (1997)) and other Desulfovibrio species
(Vita et al., Biochemistry, 47: 957-64 (2008)). The M.
thermoacetica PFOR is also well characterized (Menon and Ragsdale,
Biochemistry 36:8484-8494 (1997)) and was shown to have high
activity in the direction of pyruvate synthesis during autotrophic
growth (Furdui and Ragsdale, J. Biol. Chem. 275:28494-28499
(2000)). Further, E. coli possesses an uncharacterized open reading
frame, ydbK, encoding a protein that is 51% identical to the M.
thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity
in E. coli has been described (Blaschkowski et al., Eur. J.
Biochem. 123:563-569 (1982)). Finally, flavodoxin reductases (e.g.,
fqrB from Helicobacter pylori or Campylobacter jejuni) (St Maurice
et al., J. Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins
(Seedorf et al., Proc. Natl. Acad. Sci. USA. 105:2128-2133 (2008);
and Hellmann, J. Bacteriol 190:784-791 (2008)) provide a means to
generate NADH or NADPH from the reduced ferredoxin generated by
PFOR
TABLE-US-00093 Protein GenBank ID GI Number Organism
DesfrDRAFT_0121 ZP_07331646.1 303245362 Desulfovibrio
fructosovorans JJ Por CAA70873.1 1770208 Desulfovibrio africanus
por YP_012236.1 46581428 Desulfovibrio vulgaris str. Hildenborough
Dde_3237 ABB40031.1 78220682 DesulfoVibrio desulfuricans G20
Ddes_0298 YP_002478891.1 220903579 Desulfovibrio desulfuricans
subsp. desulfuricans str. ATCC 27774 Por YP_428946.1 83588937
Moorella thermoacetica YdbK NP_415896.1 16129339 Escherichia
coli
[0423] The conversion of pyruvate into acetyl-CoA can be catalyzed
by several other enzymes or their combinations thereof. For
example, pyruvate dehydrogenase can transform pyruvate into
acetyl-CoA with the concomitant reduction of a molecule of NAD into
NADH. It is a multi-enzyme complex that catalyzes a series of
partial reactions which results in acylating oxidative
decarboxylation of pyruvate. The enzyme comprises of three
subunits: pyruvate decarboxylase (E1), dihydrolipoamide
acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). This
enzyme is naturally present in several organisms, including E. coli
and S. cerevisiae. Enzyme engineering efforts have improved the E.
coli PDH enzyme activity under anaerobic conditions.
TABLE-US-00094 Gene Accession No. GI # Organism aceE NP_414656.1
16128107 Escherichia coli aceF NP_414657.1 16128108 Escherichia
coli lpd NP_414658.1 16128109 Escherichia coli pdhA P21881.1
3123238 Bacillus subtilis pdhB P21882.1 129068 Bacillus subtilis
pdhC P21883.2 129054 Bacillus subtilis pdhD P21880.1 118672
Bacillus subtilis LAT1 NP_014328 6324258 Saccharomyces cerevisiae
PDA1 NP_011105 37362644 Saccharomyces cerevisiae PDB1 NP_009780
6319698 Saccharomyces cerevisiae LPD1 NP_116635 14318501
Saccharomyces cerevisiae PDX1 NP_011709 6321632 Saccharomyces
cerevisiae
[0424] Yet another enzyme that can catalyze this conversion is
pyruvate formate lyase. This enzyme catalyzes the conversion of
pyruvate and CoA into acetyl-CoA and formate. Pyruvate formate
lyase is a common enzyme in prokaryotic organisms that is used to
help modulate anaerobic redox balance. Exemplary enzymes are below.
Both pflB and tdcE from E. coli require the presence of pyruvate
formate lyase activating enzyme, encoded by pflA. Further, a short
protein encoded by yflD in E. coli can associate with and restore
activity to oxygen-cleaved pyruvate formate lyase.
TABLE-US-00095 Protein GenBank ID GI Number Organism pflB NP_415423
16128870 Escherichia coli pflA NP_415422.1 16128869 Escherichia
coli tdcE AAT48170.1 48994926 Escherichia coli yfiD AAC75632.1
1788933 Escherichia coli pfl Q46266.1 2500058 Clostridium
pasteurianum act CAA63749.1 1072362 Clostridium pasteurianum
[0425] Further, different enzymes can be used in combination to
convert pyruvate into acetyl-CoA in multiple steps. For example, in
S. cerevisiae, acetyl-CoA is obtained in the cytosol by first
decarboxylating pyruvate to form acetaldehyde; the latter is
oxidized to acetate by acetaldehyde dehydrogenase and subsequently
activated to form acetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA
synthetase is a native enzyme in several other organisms including
E. coli (Kumari et al., J. Bacteriol. 177:2878-2886 (1995)),
Salmonella enterica (Starai et al., Microbiology 151:3793-3801
(2005); Starai et al., J. Biol. Chem. 280:26200-26205 (2005)), and
Moorella thermoacetica (described already). Alternatively, acetate
can be activated to form acetyl-CoA by acetate kinase and
phosphotransacetylase. Acetate kinase first converts acetate into
acetyl-phosphate with the accompanying use of an ATP molecule.
Acetyl-phosphate and CoA are next converted into acetyl-CoA with
the release of one phosphate by phosphotransacetylase. Exemplary
enzymes encoding acetate kinase, acetyl-CoA synthetase and
phosphotransacetlyase are described above.
[0426] Yet another way of converting pyruvate to acetyl-CoA is via
pyruvate oxidase. Pyruvate oxidase converts pyruvate into acetate,
using ubiquinone as the electron acceptor. In E. coli, this
activity is encoded by poxB. PoxB has similarity to pyruvate
decarboxylase of S. cerevisiae and Zymomonas mobilis. The enzyme
has a thiamin pyrophosphate cofactor (Koland and Gennis,
Biochemistry 21:4438-4442 (1982)); O'Brien et al., Biochemistry
16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem.
255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD)
cofactor. Acetate can then be converted into acetyl-CoA by either
acetyl-CoA synthetase or by acetate kinase and
phosphotransacetylase, as described earlier. Some of these enzymes
can also catalyze the reverse reaction from acetyl-CoA to
pyruvate.
[0427] H. Citrate Synthase.
[0428] Citrate synthases are well known in the art. For exampe, the
gltA gene of E. coli encodes for a citrate synthase. It was
previously shown that this gene is inhibited allosterically by
NADH, and the amino acids involved in this inhibition have been
identified (Pereira et al., J. Biol. Chem. 269(1):412-417 (1994);
Stokell et al., J. Biol. Chem. 278(37):35435-35443 (2003)). An NADH
insensitive citrate synthase can be encoded by gltA, such as an
R163L mutant of gltA. Other citrate synthase enzymes are less
sensitive to NADH, including the aarA enzyme of Acetobacter aceti
(Francois et al, Biochem 45:13487-99 (2006)).
TABLE-US-00096 Protein GenBank ID GI number Organism gltA
NP_415248.1 16128695 Escherichia coli AarA P20901.1 116462
Acetobacter aceti CITI NP_014398.1 6324328 Saccharomyces cerevisiae
CS NP_999441.1 47523618 Sus scrofa
[0429] I. Aconitase.
[0430] Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein
catalyzing the reversible isomerization of citrate and iso-citrate
via the intermediate cis-aconitate. Two aconitase enzymes of E.
coli are encoded by acnA and acnB. AcnB is the main catabolic
enzyme, while AcnA is more stable and appears to be active under
conditions of oxidative or acid stress. Exemplary enzymes are
below.
TABLE-US-00097 Protein GenBank ID GI Number Organism acnA AAC7438.1
1787531 Escherichia coli acnB AAC73229.1 2367097 Escherichia coli
HP0779 NP_207572.1 15645398 Helicobacter pylori 26695 H16_B0568
CAJ95365.1 113529018 Ralstonia eutropha DesfrDRAFT_3783
ZP_07335307.1 303249064 Desulfovibrio fructosovorans JJ Suden_1040
(acnB) ABB44318.1 78497778 Sulfurimonas denitrificans Hydth_0755
ADO45152.1 308751669 Hydrogenobacter thermophilus CT0543 (acn)
AAM71785.1 21646475 Chlorobium tepidum Clim_2436 YP_001944436.1
189347907 Chlorobium limicola Clim_0515 ACD89607.1 189340204
Chlorobium limicola acnA NP_460671.1 16765056 Salmonella
typhimurium acnB NP_459163.1 16763548 Salmonella typhimurium ACO1
AAA34389.1 170982 Saccharomyces cerevisiae
[0431] J. Isocitrate Dehydrogenase.
[0432] Isocitrate dehydrogenase catalyzes the decarboxylation of
isocitrate to 2-oxoglutarate coupled to the reduction of
NAD(P).sup.+. Exemplary enzymes are listed below.
TABLE-US-00098 Protein GenBank ID GI Number Organism Icd ACI84720.1
209772816 Escherichia coli IDP1 AAA34703.1 171749 Saccharomyces
cerevisiae Idh BAC00856.1 21396513 Chlorobium limicola Icd
AAM71597.1 21646271 Chlorobium tepidum icd NP_952516.1 39996565
Geobacter sulfurreducens icd YP_393560. 78777245 Sulfurimonas
denitrificans
[0433] K. AKG Dehydrogenase.
[0434] Alpha-ketoglutarate dehydrogenase (AKGD) converts
alpha-ketoglutarate to succinyl-CoA and is the primary site of
control of metabolic flux through the TCA cycle (Hansford, Curr.
Top. Bioenerg. 10:217-278 (1980)). Exemplary AKGDH enzymes are
listed below.
TABLE-US-00099 Gene GI # Accession No. Organism sucA 16128701
NP_415254.1 Escherichia coli sucB 16128702 NP_415255.1 Escherichia
coli lpd 16128109 NP_414658.1 Escherichia coli odhA 51704265
P23129.2 Bacillus subtilis odhB 129041 P16263.1 Bacillus subtilis
pdhD 118672 P21880.1 Bacillus subtilis KGD1 6322066 NP_012141.1
Saccharomyces cerevisiae KGD2 6320352 NP_010432.1 Saccharomyces
cerevisiae LPD1 14318501 NP_116635.1 Saccharomyces cerevisiae
[0435] The conversion of alpha-ketoglutarate to succinyl-CoA can
also be catalyzed by alpha-ketoglutamte:ferredoxin oxidoreductase
(EC 1.2.7.3), also known as 2-oxoglutamte synthase or
2-oxoglutamte:ferredoxin oxidoreductase (OFOR). OFOR and
pyruvate:ferredoxin oxidoreductase (PFOR) are members of a diverse
family of 2-oxoacid:ferredoxin (flavodoxin) oxidoreductases which
utilize thiamine pyrophosphate, CoA and iron-sulfur clusters as
cofactors and ferredoxin, flavodoxin and FAD as electron carriers
(Adams et al., Archaea. Adv. Protein Chem. 48:101-180 (1996)).
Exemplary OFOR enzymes are found in organisms such as
Hydrogenobacter thermophilus, Desulfobacter hydrogenophilus and
Chlorobium species (Shiba et al. 1985; Evans et al., Proc. Natl.
Acad. ScI. USA. 55:92934 (1966); Buchanan, 1971). The two-subunit
enzyme from H. thermophilus, encoded by korAB, has been cloned and
expressed in E. coli. A five subunit OFOR from the same organism
with strict substrate specificity for succinyl-CoA, encoded by
forDABGE, was recently identified and expressed in E. coli.
Exemplary OFOR are below.
TABLE-US-00100 Protein GenBank ID GI Number Organism korA BAB21494
12583691 Hydrogenobacter thermophilus korB BAB21495 12583692
Hydrogenobacter thermophilus forD BAB62132.1 14970994
Hydrogenobacter thermophilus forA BAB62133.1 14970995
Hydrogenobacter thermophilus forB BAB62134.1 14970996
Hydrogenobacter thermophilus forG BAB62135.1 14970997
Hydrogenobacter thermophilus forE BAB62136.1 14970998
Hydrogenobacter thermophilus Clim 0204 ACD89303.1 189339900
Chlorobium limicola Clim 0205 ACD89302.1 189339899 Chlorobium
limicola Clim 1123 ACD90192.1 189340789 Chlorobium limicola
Clim_1124 ACD90193.1 189340790 Chlorobium limicola korA CAA12243.2
19571179 Thauera aromatica korB CAD27440.1 19571178 Thauera
aromatica
[0436] L. Pyruvate Carboxylase.
[0437] Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate
to oxaloacetate at the cost of one ATP. Pyruvate carboxylase
enzymes are below.
TABLE-US-00101 Protein GenBank ID GI Number Organism PYC1 NP_011453
6321376 Saccharomyces cerevisiae PYC2 NP_009777 6319695
Saccharomyces cerevisiae Pyc YP_890857.1 118470447 Mycobacterium
smegmatis
[0438] M. Malic Enzyme.
[0439] Malic enzyme can be applied to convert CO.sub.2 and pyruvate
to malate at the expense of one reducing equivalent. Malic enzymes
for this purpose can include, without limitation, malic enzyme
(NAD-dependent) and malic enzyme (NADP-dependent). For example, one
of the E. coli malic enzymes (Takeo, I Biochem. 66:379-387 (1969))
or a similar enzyme with higher activity can be expressed to enable
the conversion of pyruvate and CO.sub.2 to malate. By fixing carbon
to pyruvate as opposed to PEP, malic enzyme allows the high-energy
phosphate bond from PEP to be conserved by pyruvate kinase whereby
ATP is generated in the formation of pyruvate or by the
phosphotransferase system for glucose transport.
TABLE-US-00102 Protein GenBank ID GI Number Organism maeA NP_415996
90111281 Escherichia coli maeB NP_416958 16130388 Escherichia coli
NAD-ME P27443 126732 Ascaris suum
Example VII
1,3-Butanediol, Crotyl Alcohol, 3-Buten-2-ol, and Butadiene
Synthesis Enzymes
[0440] This Example provides genes that can be used for conversion
of acetyl-CoA to 1,3-butanediol, crotyl alcohol, 3-buten-2-ol,
butadiene as depicted in the pathways of FIGS. 5 and 6.
[0441] FIG. 5. Pathways for converting 1,3-butanediol to
3-buten-2-ol and/or butadiene. A) acetyl-CoA carboxylase, B) an
acetoacetyl-CoA synthase, C) an acetyl-CoA:acetyl-CoA
acyltransferase, D) an acetoacetyl-CoA reductase (ketone reducing),
E) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), F) a
3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, G) a
3-hydroxybutyrate reductase, H) a 3-hydroxybutyraldehyde reductase,
I) chemical dehydration or corresponding step in FIG. 6, J) a
3-hydroxybutyryl-CoA dehydratase, K) a crotonyl-CoA reductase
(aldehyde forming), L) a crotonyl-CoA hydrolase, transferase or
synthetase, M) a crotonate reductase, N) a crotonaldehyde
reductase, O) a crotyl alcohol kinase, P) a 2-butenyl-4-phosphate
kinase, Q) a butadiene synthase, R) a crotyl alcohol
diphosphokinase, S) chemical dehydration or a crotyl alcohol
dehydratase, T) a butadiene synthase (monophosphate), T) a
butadiene synthase (monophosphate), U) a crotonyl-CoA reductase
(alcohol forming), and V) a 3-hydroxybutyryl-CoA reductase (alcohol
forming).
[0442] A. Acetyl-CoA Carboxylase.
[0443] Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the
ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This
enzyme is biotin dependent and is the first reaction of fatty acid
biosynthesis initiation in several organisms. Exemplary enzymes are
below.
TABLE-US-00103 Protein GenBank ID GI Number Organism ACC1
CAA96294.1 1302498 Saccharomyces cerevisiae KLLA0F06072g
XP_455355.1 50310667 Kluyveromyces lactis ACC1 XP_718624.1 68474502
Candida albicans YALI0C11407p XP_501721.1 50548503 Yarrowia
lipolytica ANI_1_1724104 XP_001395476.1 145246454 Aspergillus niger
accA AAC73296.1 1786382 Escherichia coli accB AAC76287.1 1789653
Escherichia coli accC AAC76288.1 1789654 Escherichia coli accD
AAC75376.1 1788655 Escherichia coli
[0444] B. Acetoacetyl-CoA Synthase.
[0445] The conversion of malonyl-CoA and acetyl-CoA substrates to
acetoacetyl-CoA can be catalyzed by a CoA synthetase in the 2.3.1
family of enzymes. Several enzymes catalyzing the CoA synthetase
activities have been described in the literature and represent
suitable candidates.
[0446] 3-Oxoacyl-CoA products such as acetoacetyl-CoA,
3-oxopentanoyl-CoA, 3-oxo-5-hydroxypentanoyl-CoA can be synthesized
from acyl-CoA and malonyl-CoA substrates by 3-oxoacyl-CoA
synthases. As enzymes in this class catalyze an essentially
irreversible reaction, they are particularly useful for metabolic
engineering applications for overproducing metabolites, fuels or
chemicals derived from 3-oxoacyl-CoA intermediates such as
acetoacetyl-CoA. Acetoacetyl-CoA synthase, for example, has been
heterologously expressed in organisms that biosynthesize butanol
(Lan et al, PNAS USA (2012)) and poly-(3-hydroxybutyrate)
(Matsumoto et al, Biosci Biotech Biochem, 75:364-366 (2011). Other
acetoacetyl-CoA synthase genes can be identified by sequence
homology to fhsA
TABLE-US-00104 Protein GenBank ID GI Number Organism fhsA
BAJ83474.1 325302227 Streptomyces sp CL190 AB183750.1: 11991 . . .
12971 BAD86806.1 57753876 Streptomyces sp. KO-3988 epzT ADO43379.1
312190954 Strentomvces cinnamonensis ppzT CAX48662.1 238623523
Streptomyces anulatus O3I 22085 ZP 09840373.1 378817444 Nocardia
brasiliensis
[0447] C. Acetyl-CoA:acetyl-CoA Acyltransferase (Acetoacetyl-CoA
Thiolase).
[0448] Acetoacetyl-CoA thiolase (also known as acetyl-CoA
acetyltransferase) converts two molecules of acetyl-CoA into one
molecule each of acetoacetyl-CoA and CoA. Exemplary acetoacetyl-CoA
thiolase enzymes include genes/proteins identified in the Table
below.
TABLE-US-00105 Gene GenBank ID GI Number Organism AtoB NP_416728
16130161 Escherichia coli ThlA NP_349476.1 15896127 Clostridium
acetobutylicum ThlB NP_149242.1 15004782 Clostridium acetobutylicum
ERG10 NP_015297 6325229 Saccharomyces cerevisiae phbA P07097.4
135759 Zoogloea ramigera
[0449] D. Acetoacetyl-CoA Reductase.
[0450] A suitable enzyme activity is 1.1.1.a Oxidoreductase (oxo to
alcohol). See herein. In addition, Acetoacetyl-CoA reductase (EC
1.1.1.36) catalyzes the reduction of acetoacetyl-CoA to
3-hydroxybutyryl-CoA. This enzyme participates in the acetyl-CoA
fermentation pathway to butyrate in several species of Clostridia
and has been studied in detail (Jones et al., Microbiol Rev.
50:484-524 (1986)). Acetoacetyl-CoA reductase also participates in
polyhydroxybutyrate biosynthesis in many organisms, and has also
been used in metabolic engineering applications for overproducing
PHB and 3-hydroxyisobutyrate. Additional exemplary genes include
those below. The Z ramigera gene is NADPH-dependent and the gene
has been expressed in E coli (Peoples et al., Mol. Microbiol
3:349-357 (1989)). Substrate specificity studies on the gene led to
the conclusion that it could accept 3-oxopropionyl-CoA as a
substrate besides acetoacetyl-CoA (Ploux et al., Eur. J Biochem.
174:177-182 (1988)). The enzyme from Candida tropicalis is a
component of the peroxisomal fatty acid beta-oxidation
multifunctional enzyme type 2 (MFE-2). The dehydrogenase B domain
of this protein is catalytically active on acetoacetyl-CoA.
TABLE-US-00106 Protein Genbank ID GI Number Organism fadB P21177.2
119811 Escherichia coli fadJ P77399.1 3334437 Escherichia coli paaH
NP_415913.1 16129356 Escherichia coli Hbd2 EDK34807.1 146348271
Clostridium kluyveri Hbd1 EDK32512.1 146345976 Clostridium kluyveri
phaC NP_745425.1 26990000 Pseudomonas putida paaC ABF82235.1
106636095 Pseudomonas fluorescens HSD17B10 O02691.3 3183024 Bos
taurus phbB P23238.1 130017 Zoogloea ramigera phaB YP_353825.1
77464321 Rhodobacter sphaeroides phaB BAA08358 675524 Paracoccus
denitrificans Hbd NP_349314.1 15895965 Clostridium acetobutylicum
Hbd AAM14586.1 20162442 Clostridium beijerinckii Msed 1423
YP_001191505 146304189 Metallosphaera sedula Msed_0399 YP_001190500
146303184 Metallosphaera sedula Msed 0389 YP_001190490 146303174
Metallosphaera sedula Msed_1993 YP_001192057 146304741
Metallosphaera sedula Fox2 Q02207 399508 Candida tropicalis
[0451] E. 3-Hydroxybutyryl-CoA Reductase (Aldehyde Forming).
[0452] An EC 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) provides
suitable enzyme activity. Acyl-CoA reductases or acylating aldehyde
dehydrogenases reduce an acyl-CoA to its corresponding aldehyde.
Exemplary enzymes include fatty acyl-CoA reductase, succinyl-CoA
reductase (EC 1.2.1.76), acetyl-CoA reductase, butyryl-CoA
reductase, propionyl-CoA reductase (EC 1.2.1.3) and others shown in
the table below.
TABLE-US-00107 EC Number Enzyme name 1.2.1.10 Acetaldehyde
dehydrogenase (acetylating) 1.2.1.42 (Fatty) acyl-CoA reductase
1.2.1.44 Cinnamoyl-CoA reductase 1.2.1.50 Long chain fatty acyl-CoA
reductase 1.2.1.57 Butanal dehydrogenase 1.2.1.75 Malonate
semialdehyde dehydrogenase 1.2.1.76 Succinate semialdehyde
dehydrogenase 1.2.1.81 Sulfoacetaldehyde dehydrogenase 1.2.1. --
Propanal dehydrogenase 1.2.1.-- Hexanal dehydrogenase 1.2.1.--
4-Hydroxybutyraldehyde dehydrogenase
[0453] Exemplary fatty acyl-CoA reductases enzymes are encoded by
acr1 of Acinetobacter calcoaceticus (Reiser, Journal of
Bacteriology 179:2969-2975 (1997)) and Acinetobacter sp. M-1
(Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)).
Enzymes with succinyl-CoA reductase activity are encoded by sucD of
Clostridium kluyveri (Sohling, J. Bacteriol. 178:871-880 (1996))
and sucD of P. gingivalis (Takahashi, J. Bacteriol 182:4704-4710
(2000)). Additional succinyl-CoA reductase enzymes are exemplified
below.
TABLE-US-00108 Protein GenBank ID GI Number Organism acr1
YP_047869.1 50086359 Acinetobacter calcoaceticus acr1 AAC45217
1684886 Acinetobacter baylyi acr1 BAB85476.1 18857901 Acinetobacter
sp. Strain M-1 MSED 0709 YP_001190808.1 146303492 Metallosphaera
sedula Tneu 0421 ACB39369.1 170934108 Thermoproteus neutrophilus
sucD P38947.1 172046062 Clostridium kluyveri sucD NP_904963.1
34540484 Porphyromonas gingivalis bphG BAA03892.1 425213
Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc mesenteroides
bld AAP42563.1 31075383 Clostridium saccharoperbutylacetonicum pduP
NP_460996 16765381 Salmonella typhimurium LT2 eutE NP_416950
16130380 Escherichia coli
[0454] An additional enzyme that converts an acyl-CoA to its
corresponding aldehyde is malonyl-CoA reductase which transforms
malonyl-CoA to malonic semialdehyde. Although the aldehyde
dehydrogenase functionality of these enzymes is similar to the
bifunctional dehydrogenase from Chlorojlexus aurantiacus, there is
little sequence similarity. Both malonyl-CoA reductase enzyme
candidates have high sequence similarity to aspartate-semialdehyde
dehydrogenase, an enzyme catalyzing the reduction and concurrent
dephosphorylation of aspartyl-4-phosphate to aspartate
semialdehyde. Additional gene candidates can be found by sequence
homology to proteins in other organisms including Sulfolobus
solfataricus and Sulfolobus acidocaldarius and have been listed
below. Yet another candidate for CoA-acylating aldehyde
dehydrogenase is the ald gene from Clostridium beijerinckii
reported to reduce acetyl-CoA and butyryl-CoA to their
corresponding aldehydes.
TABLE-US-00109 Protein GenBank ID GI Number Organism Msed_0709
YP_001190808.1 146303492 Metallosphaera sedula Mcr NP_378167.1
15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958 Sulfolobus
solfataricus Saci_2370 YP_256941.1 70608071 Sulfolobus
acidocaldarius Ald AAT66436 49473535 Clostridium beijerinckii eutE
AAA80209 687645 Salmonella typhimurium
[0455] 4-Hydroxybutyryl-CoA reductase catalyzes the reduction of
4-hydroxybutyryl-CoA to its corresponding aldehyde. Several
acyl-CoA dehydrogenases are capable of catalyzing this activity.
The succinate semialdehyde dehydrogenases (SucD) of Clostridium
kluyveri and P. gingivalis were shown in ref (WO2008/115840) to
convert 4-hydroxybutyryl-CoA to 4-hydroxybutanal as part of a
pathway to produce 1,4-butanediol. Many butyraldehyde
dehydrogenases are also active on 4-hydroxybutyraldehyde, including
bld of Clostridium saccharoperbutylacetonicum and bphG of
Pseudomonas sp (Powlowski et al., J. Bacterial. 175:377-385
(1993)). These and additional proteins with 4-hydroxybutyryl-CoA
reductase activity are identified below.
TABLE-US-00110 Protein GenBank ID GI Number Organism bphG
BAA03892.1 425213 Pseudomonas sp ald YP_001310903.1 150018649
Clostridium beijerinckii NCIMB 8052 Ald ZP_03778292.1 225569267
Clostridium hylemonae DSM 15053 Ald ZP_03705305.1 225016072
Clostridium methylpentosum DSM 5476 Ald ZP_03715465.1 225026273
Eubacterium hallii DSM 3353 Ald ZP_01962381.1 153809713
Ruminococcus obeum ATCC 29174 Ald YP_003701164.1 297585384 Bacillus
selenitireducens MLS10 Ald AAP42563.1 31075383 Clostridium
saccharoperbutylacetonicum N1-4 Ald YP_795711.1 116334184
Lactobacillus brevis ATCC 367 Ald YP_002434126.1 218782808
Desulfatibacillum alkenivorans AK-01 Ald YP_001558295.1 160879327
Clostridium phytofermentans ISDg Ald ZP_02089671.1 160942363
Clostridium bolteae ATCC BAA-613 Ald ZP_01222600.1 90414628
Photobacterium profundum 3TCK Ald YP_001452373.1 157145054
Citrobacter koseri ATCC BAA-895 Ald NP_460996.1 16765381 Salmonella
enterica typhimurium Ald YP_003307836.1 269119659 Sebaldella
termitidis ATCC 33386 Ald ZP_04969437.1 254302079 Fusobacterium
nucleatum subsp. polymorphum ATCC 10953 Ald YP_002892893.1
237808453 Tolumonas auensis DSM 9187 Ald YP_426002.1 83592250
Rhodospirillum rubrum ATCC 11170
[0456] F. 3-Hydroxybutyryl-CoA Hydrolase, Transferase or
Synthetase.
[0457] An EC 3.1.2.a CoA hydrolase, EC 2.8.3.a CoA transferase,
and/or an EC 6.2.1.a CoA synthetase provide suitable enzyme
activity. See herein.
[0458] G. 3-Hydroxybutyrate Reductase.
[0459] An EC 1.2.1.e Oxidoreductase (acid to aldehyde) provides
suitable activity. See herein.
[0460] H. 3-Hydroxybutyraldehyde Reductase.
[0461] An EC 1.1.1.a Oxidoreductase (oxo to alcohol) provides
suitable activity. See herein.
[0462] I. Chemical Dehydration or Alternatively See Corresponding
Enzymatic Pathway in FIG. 6.
[0463] J. 3-Hydroxybutyryl-CoA Dehydratase.
[0464] An EC 4.2.1. Hydro-lyase provides suitable enzyme activity,
and are described below and herein. The enoyl-CoA hydratase of
Pseudomonas putida, encoded by ech, catalyzes the conversion of
3-hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al., Arch.
Microbiol 117:99-108 (1978)). This transformation is also catalyzed
by the crt gene product of Clostridium acetobutylicum, the crt1
gene product of C. kluyveri, and other clostridial organisms Atsumi
et al., Metab Eng 10:305-311 (2008); Boynton et al., J Bacterial.
178:3015-3024 (1996); Hillmer et al., FEBS Lett. 21:351-354
(1972)). Additional enoyl-CoA hydratase candidates are described
below.
TABLE-US-00111 Protein GenBank No. GI No. Organism ech NP_745498.1
26990073 Pseudomonas putida crt NP_349318.1 15895969 Clostridium
acetobutylicum crt1 YP_001393856 153953091 Clostridium kluyveri
phaA ABF82233.1 26990002 Pseudomonas putida phaB ABF82234.1
26990001 Pseudomonas putida paaA NP_745427.1 106636093 Pseudomonas
fluorescens paaB NP_745426.1 106636094 Pseudomonas fluorescens maoC
NP_415905.1 16129348 Escherichia coli paaF NP_415911.1 16129354
Escherichia coli paaG NP_415912.1 16129355 Escherichia coli
[0465] K. Crotonyl-CoA Reductase (Aldehyde Forming).
[0466] An EC 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) provides
suitable enzyme activity. Acyl-CoA reductases in the 1.2.1 family
reduce an acyl-CoA to its corresponding aldehyde. Several acyl-CoA
reductase enzymes have been described in the open literature and
represent suitable candidates for this step. These are described
above.
[0467] L. Crotonyl-CoA Hydrolase, Transferase or Synthetase.
[0468] An EC 3.1.2.a CoA hydrolase, EC 2.8.3.a CoA transferase,
and/or an EC 6.2.1.a CoA synthetase provide suitable enzyme
activity, and are described in the following sections.
[0469] EC 3.1.2.a CoA Hydrolase.
[0470] Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to
their corresponding acids. Several such enzymes have been described
in the literature and represent suitable candidates for these
steps.
[0471] For example, the enzyme encoded by acot12 from Rattus
norvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun.
71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and
malonyl-CoA. Exemplary enzymes are shown below.
TABLE-US-00112 Protein GenBank Accession No. GI Number Organism
acot12 NP_570103.1 18543355 Rattus norvegicus tesB NP_414986
16128437 Escherichia coli acot8 CAA15502 3191970 Homo sapiens acot8
NP_570112 51036669 Rattus norvegicus tesA NP_415027 16128478
Escherichia coli ybgC NP_415264 16128711 Escherichia coli paaI
NP_415914 16129357 Escherichia coli ybdB NP_415129 16128580
Escherichia coli ACH1 NP_009538 6319456 Saccharomyces
cerevisiae
[0472] Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA
hydrolase which has been described to efficiently catalyze the
conversion of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate
during valine degradation (Shimomura et al., J Biol Chem.
269:14248-14253 (1994)). Genes encoding this enzyme include those
below.
TABLE-US-00113 Protein GenBank No. GI Number Organism hibch
Q5XIE6.2 146324906 Rattus norvegicus hibch Q6NVY1.2 146324905 Homo
sapiens hibch P28817.2 2506374 Saccharomyces cerevisiae BC_2292
AP09256 29895975 Bacillus cereus
[0473] EC 2.8.3.a CoA Transferase.
[0474] Enzymes in the 2.8.3 family catalyze the reversible transfer
of a CoA moiety from one molecule to another. Several CoA
transferase enzymes have been described in the open literature and
represent suitable candidates for these steps. These are described
below.
[0475] Many transferases have broad specificity and thus can
utilize CoA acceptors as diverse as acetate, succinate, propionate,
butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate,
valerate, crotonate, 3-mercaptopropionate, propionate,
vinylacetate, butyrate, among others.
TABLE-US-00114 Protein GenBank ID GI Number Organism Ach1
AAX19660.1 60396828 Roseburia sp. A2-183 ROSINTL182_07121
ZP_04743841.2 257413684 Roseburia intestinalis L1-82
ROSEINA2194_03642 ZP_03755203.1 225377982 Roseburia inulinivorans
EUBREC_3075 YP_002938937.1 238925420 Eubacterium rectale ATCC 33656
Pct CAB77207.1 7242549 Clostridium propionicum NT01CX_2372
YP_878445.1 118444712 Clostridium novyi NT Cbei_4543 YP_001311608.1
150019354 Clostridium beijerinckii CBC_A0889 ZP_02621218.1
168186583 Clostridium botulinum C str. Eklund ygfH NP_417395.1
16130821 Escherichia coli CIT292_04485 ZP_03838384.1 227334728
Citrobacter youngae ATCC 29220 SARI_04582 YP_001573497.1 161506385
Salmonella enterica subsp. arizonae serovar yinte0001_14430
ZP_04635364.1 238791727 Yersinia intermedia ATCC 29909
[0476] An additional candidate enzyme are identified below.
TABLE-US-00115 Protein GenBank ID GI Number Organism pcaI
AAN69545.1 24985644 Pseudomonas putida pcaJ NP_746082.1 26990657
Pseudomonas putida pcaI YP_046368.1 50084858 Acinetobacter sp. ADP1
pcaJ AAC37147.1 141776 Acinetobacter sp. ADP1 pcaI NP_630776.1
21224997 Streptomyces coelicolor pcaJ NP_630775.1 21224996
Streptomyces coelicolor HPAG1_0676 YP_627417 108563101 Helicobacter
pylori HPAG1_0677 YP_627418 108563102 Helicobacter pylori ScoA
NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949
Bacillus subtilis
[0477] A CoA transferase that can utilize acetate as the CoA
acceptor is acetoacetyl-CoA transferase, encoded by the E. coli
atoA (alpha subunit) and atoD (beta subunit) genes. Similar enzymes
are identified below.
TABLE-US-00116 Protein GenBank ID GI Number Organism atoA P76459.1
2492994 Escherichia coli K12 atoD P76458.1 2492990 Escherichia coli
K12 actA YP_226809.1 62391407 Corynebacterium glutamicum ATCC 13032
cg0592 YP_224801.1 62389399 Corynebacterium glutamicum ATCC 13032
ctfA NP_149326.1 15004866 Clostridium acetobutylicum ctfB
NP_149327.1 15004867 Clostridium acetobutylicum ctfA AAP42564.1
31075384 Clostridium saccharoperbutylacetonicum ctfB AAP42565.1
31075385 Clostridium saccharoperbutylacetonicum
[0478] Additional exemplary transferase candidates are catalyzed by
the gene products of cat1, cat2, and cat3 of Clostridium kluyveri
which have been shown to exhibit succinyl-CoA,
4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity,
respectively. Similar CoA transferase activities are identified
below.
TABLE-US-00117 Protein GenBank ID GI Number Organism cat1 P38946.1
729048 Clostridium kluyveri cat2 P38942.2 172046066 Clostridium
kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri TVAG_395550
XP_001330176 123975034 Trichomonas vaginalis G3 Tb11.02.0290
XP_828352 71754875 Trypanosoma brucei
[0479] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from
anaerobic bacterium Acidaminococcus fermentans reacts with diacid
glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett.
405:209-212 (1997)). The genes encoding this enzyme are gctA and
gctB. These proteins are identified below.
TABLE-US-00118 Protein GenBank ID GI Number Organism gctA
CAA57199.1 559392 Acidaminococcus fermentans gctB CAA57200.1 559393
Acidaminococcus fermentans
[0480] EC 6.2.1.a CoA Synthase (Acid-Thiol Ligase).
[0481] The conversion of acyl-CoA substrates to their acid products
can be catalyzed by a CoA acid-thiol ligase or CoA synthetase in
the 6.2.1 family of enzymes, several of which are reversible.
Several enzymes catalyzing CoA acid-thiol ligase or CoA synthetase
activities have been described in the literature and represent
suitable candidates for these steps.
[0482] For example, ADP-forming acetyl-CoA synthetase (ACD, EC
6.2.1.13) is an enzyme that couples the conversion of acyl-CoA
esters to their corresponding acids with the concomitant synthesis
of ATP. The enzymes from A. fulgidus, H marismortui and P.
aerophilum have all been cloned, functionally expressed, and
characterized in E. coli (Brasen and Schonheit, supra; Musfeldt and
Schonheit, J Bacteriol. 184:636-644 (2002)). An additional
candidate is succinyl-CoA synthetase, encoded by sucCD of E. coli
and LSC1 and LSC2 genes of Saccharomyces cerevisiae. These enzymes
catalyze the formation of succinyl-CoA from succinate with the
concomitant consumption of one ATP in a reaction which is
reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)).
Exemplary enzyme are below.
TABLE-US-00119 Protein GenBank ID GI Number Organism AF1211
NP_070039.1 11498810 Archaeoglobus fulgidus AF1983 NP_070807.1
11499565 Archaeoglobus fulgidus Scs YP_135572.1 55377722 Haloarcula
marismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum
str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1
1786949 Escherichia coli LSC1 NP_014785 6324716 Saccharomyces
cerevisiae LSC2 NP_011760 6321683 Saccharomyces cerevisiae paaF
AAC24333.2 22711873 Pseudomonas putida matB AAC83455.1 3982573
Rhizobium leguminosarum
[0483] Another candidate enzyme for these steps is
6-carboxyhexanoate-CoA ligase, also known as pimeloyl-CoA ligase
(EC 6.2.1.14), which naturally activates pimelate to pimeloyl-CoA
during biotin biosynthesis in gram-positive bacteria. Exemplary
enzymes are below.
TABLE-US-00120 Protein GenBank ID GI Number Organism bioW
NP_390902.2 50812281 Bacillus subtilis bioW CAA10043.1 3850837
Pseudomonas mendocina bioW P22822.1 115012 Bacillus sphaericus
[0484] Additional CoA-ligases are listed below.
TABLE-US-00121 Protein Accession No. GI No. Organism phl CAJ15517.1
77019264 Penicillium chrysogenum phlB ABS19624.1 152002983
Penicillium chrysogenum paaF AAC24333.2 22711873 Pseudomonas putida
bioW NP_390902.2 50812281 Bacillus subtilis AACS NP_084486.1
21313520 Mus musculus AACS NP_076417.2 31982927 Homo sapiens
[0485] Like enzymes in other classes, certain enzymes in the EC
class 6.2.1 have been determined to have broad substrate
specificity. The acyl CoA ligase from Pseudomonas putida has been
demonstrated to work on several aliphatic substrates including
acetic, propionic, butyric, valeric, hexanoic, heptanoic, and
octanoic acids and on aromatic compounds such as phenylacetic and
phenoxyacetic acids (Femandez-Valverde et al., Applied and
Environmental Microbiology 59:1149-1154 (1993)). A related enzyme,
malonyl CoA synthetase (6.3.4.9) from Rhizobium trifoli could
convert several diacids, namely, ethyl-, propyl-, allyl-,
isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-,
cyclobutyl-, and benzyl-malonate into their corresponding
monothioesters (Pohl et al., J. Am. Chem. Soc. 123:5822-5823
(2001)).
[0486] M. Crotonate Reductase.
[0487] A suitable enzyme activity is an 1.2.1.e Oxidoreductase
(acid to aldehyde), which include the following.
[0488] The conversion of an acid to an aldehyde is
thermodynamically unfavorable and typically requires energy-rich
cofactors and multiple enzymatic steps. Direct conversion of the
acid to aldehyde by a single enzyme is catalyzed by an acid
reductase enzyme in the 1.2.1 family. Exemplary acid reductase
enzymes include carboxylic acid reductase, alpha-aminoadipate
reductase and retinoic acid reductase. Carboxylic acid reductase
(CAR), found in Nocardia iowensis, catalyzes the magnesium, ATP and
NADPH-dependent reduction of carboxylic acids to their
corresponding aldehydes. CAR requires post-translational activation
by a phosphopantetheine transferase (PPTase) that converts the
inactive apo-enzyme to the active holo-enzyme (Hansen et al., Appl.
Environ. Microbiol 75:2765-2774 (2009)). Expression of the npt
gene, encoding a specific PPTase, product improved activity of the
enzyme. Exemplary enzymes include those below.
TABLE-US-00122 Gene GenBank Accession No. GI No. Organism car
AAR91681.1 40796035 Nocardia iowensis npt ABI83656.1 114848891
Nocardia iowensis griC YP_001825755.1 182438036 Streptomyces
griseus griD YP_001825756.1 182438037 Streptomyces griseus
[0489] Additional car and npt genes can be identified based on
sequence homology.
TABLE-US-00123 Gene name GI No. GenBank Accession No. Organism
fadD9 121638475 YP_978699.1 Mycobacterium bovis BCG BCG_2812c
121638674 YP_978898.1 Mycobacterium bovis BCG nfa20150 54023983
YP_118225.1 Nocardia farcinica IFM 10152 nfa40540 54026024
YP_120266.1 Nocardia farcinica IFM 10152 SGR_6790 182440583
YP_001828302.1 Streptomyces griseus subsp. griseus NBRC 13350
SGR_665 182434458 YP_001822177.1 Streptomyces griseus subsp.
griseus NBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacterium
smegmatis MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacterium
smegmatis MC2 155 MSMEG_2648 YP_886985.1 118471293 Mycobacterium
smegmatis MC2 155 MAP1040C NP_959974.1 41407138 Mycobacterium avium
subsp. paratuberculosis K-10 MAP2899C NP_961833.1 41408997
Mycobacterium avium subsp. paratuberculosis K-10 MMAR_2117
YP_001850422.1 183982131 Mycobacterium marinum M MMAR_2936
YP_001851230.1 183982939 Mycobacterium marinum M MMAR_1916
YP_001850220.1 183981929 Mycobacterium marinum M TpauDRAFT_33060
ZP_04027864.1 227980601 Tsukamurella paurometabola DSM 20162
TpauDRAFT_20920 ZP_04026660.1 ZP_04026660.1 Tsukamurella
paurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429
Cyanobium PCC7001 DDBDRAFT_0187729 XP_636931.1 66806417
Dictyostelium discoideum AX4
[0490] An enzyme with similar characteristics, alpha-aminoadipate
reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis
pathways in some fungal species. Like CAR, this enzyme utilizes
magnesium and requires activation by a PPTase. Enzyme candidates
for AAR and its corresponding PPTase are below.
TABLE-US-00124 Gene GenBank Accession No. GI No. Organism LYS2
AAA34747.1 171867 Saccharomyces cerevisiae LYS5 P50113.1 1708896
Saccharomyces cerevisiae LYS2 AAC02241.1 2853226 Candida albicans
LYS5 AAO26020.1 28136195 Candida albicans Lys1p P40976.3 13124791
Schizosaccharomyces pombe Lys7p Q10474.1 1723561
Schizosaccharomyces pombe Lys2 CAA74300.1 3282044 Penicillium
chrysogenum
[0491] N. Crotonaldehyde Reductase.
[0492] A suitable enzyme activity is provided by an EC 1.1.1.a
Oxidoreductase (oxo to alcohol). EC 1.1.1.a Oxidoreductase (oxo to
alcohol) includes the following:
[0493] The reduction of glutarate semialdehyde to 5-hydroxyvalerate
by glutarate semialdehyde reductase entails reduction of an
aldehyde to its corresponding alcohol. Enzymes with glutarate
semialdehyde reductase activity include those below.
TABLE-US-00125 PROTEIN GENBANK ID GI NUMBER ORGANISM ATEG_00539
XP_001210625.1 115491995 Aspergillus terreus NIH2624 4hbd
AAK94781.1 15375068 Arabidopsis thaliana
[0494] Additional genes encoding enzymes that catalyze the
reduction of an aldehyde to alcohol (i.e., alcohol dehydrogenase or
equivalently aldehyde reductase) include those below. The enzyme
candidates described previously for catalyzing the reduction of
methylglyoxal to acetol or lactaldehyde are also suitable
lactaldehyde reductase enzyme candidates.
TABLE-US-00126 Protein GENBANK ID GI NUMBER ORGANISM alrA
BAB12273.1 9967138 Acinetobacter sp. strain M-1 ADH2 NP_014032.1
6323961 Saccharomyces cerevisiae yqhD NP_417484.1 16130909
Escherichia coli fucO NP_417279.1 16130706 Escherichia coli bdh I
NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.1
15896542 Clostridium acetobutylicum adhA YP_162971.1 56552132
Zymomonas mobilis bdh BAF45463.1 124221917 Clostridium
saccharoperbutylacetonicum Cbei_1722 YP_001308850 150016596
Clostridium beijerinckii Cbei_2181 YP_001309304 150017050
Clostridium beijerinckii Cbei_2421 YP_001309535 150017281
Clostridium beijerinckii GRE3 P38715.1 731691 Saccharomyces
cerevisiae ALD2 CAA89806.1 825575 Saccharomyces cerevisiae ALD3
NP_013892.1 6323821 Saccharomyces cerevisiae ALD4 NP_015019.1
6324950 Saccharomyces cerevisiae ALD5 NP_010996.2 330443526
Saccharomyces cerevisiae ALD6 ABX39192.1 160415767 Saccharomyces
cerevisiae HFD1 Q04458.1 2494079 Saccharomyces cerevisiae GOR1
NP_014125.1 6324055 Saccharomyces cerevisiae YPL113C AAB68248.1
1163100 Saccharomyces cerevisiae GCY1 CAA99318.1 1420317
Saccharomyces cerevisiae
[0495] Enzymes exhibiting 4-hydroxybutymte dehydrogenase activity
(EC 1.1.1.61) also fall into this category and exemplary enzymes
are below.
TABLE-US-00127 PROTEIN GENBANK ID GI NUMBER ORGANISM 4hbd
YP_726053.1 113867564 Ralstonia eutropha H16 4hbd L21902.1
146348486 Clostridium kluyveri DSM 555 adhI AAR91477.1 40795502
Geobacillus thermoglucosidasius
[0496] Another exemplary aldehyde reductase is methylmalonate
semialdehyde reductase, also known as 3-hydroxyisobutyrate
dehydrogenase (EC 1.1.1.31). This enzyme participates in valine,
leucine and isoleucine degradation and has been identified in
bacteria, eukaryotes, and mammals. Exemplary enzymes include those
below.
TABLE-US-00128 PROTEIN GENBANK ID GI NUMBER ORGANISM P84067 P84067
75345323 Thermus thermophilus 3hidh P31937.2 12643395 Homo sapiens
3hidh P32185.1 416872 Oryctolagus cuniculus mmsB NP_746775.1
26991350 Pseudomonas putida mmsB P28811.1 127211 Pseudomonas
aeruginosa dhat Q59477.1 2842618 Pseudomonas putida
[0497] There exist several exemplary alcohol dehydrogenases that
convert a ketone to a hydroxyl functional group. Two such enzymes
from E. coli are encoded by malate dehydrogenase (mdh) and lactate
dehydrogenase (ldhA). Exemplary enzymes include those below.
TABLE-US-00129 Protein Genbank ID GI Number Organism mdh AAC76268.1
1789632 Escherichia coli ldhA NP_415898.1 16129341 Escherichia coli
ldh YP_725182.1 113866693 Ralstonia eutropha bdh AAA58352.1 177198
Homo sapiens adh AAA23199.2 60592974 Clostridium beijerinckii NRRL
B593 adh P14941.1 113443 Thermoanaerobacter brockii HTD4 sadh
CAD36475 21615553 Rhodococcus ruber adhA AAC25556 3288810
Pyrococcus furiosus
[0498] A number of organisms encode genes that catalyze the
reduction of 3-oxobutanol to 1,3-butanediol, including those
belonging to the genus Bacillus, Brevibacterium, Candida, and
Klebsiella among others, as described by Matsuyama et al. J Mol Cat
B Enz, 11:513-521 (2001). One of these enzymes, SADH from Candida
parapsilosis, was cloned and characterized in E. coli. A mutated
Rhodococcus phenylacetaldehyde reductase (Sar268) and a Leifonia
alcohol dehydrogenase have also been shown to catalyze this
transformation at high yields (Itoh et al., Appalicrobiol
Biotechnol. 75:1249-1256 (2007)).
TABLE-US-00130 Protein Genbank ID GI Number Organism sadh
BAA24528.1 2815409 Candida parapsilosis
[0499] O. Crotyl Alcohol Kinase.
[0500] Crotyl alcohol kinase enzymes catalyze the transfer of a
phosphate group to the hydroxyl group of crotyl alcohol. The
enzymes described below naturally possess such activity or can be
engineered to exhibit this activity. Kinases that catalyze transfer
of a phosphate group to an alcohol group are members of the EC
2.7.1 enzyme class. The table below lists several useful kinase
enzymes in the EC 2.7.1 enzyme class.
TABLE-US-00131 Enzyme Commission Number Enzyme Name 2.7.1.1
hexokinase 2.7.1.2 glucokinase 2.7.1.3 ketohexokinase 2.7.1.4
fructokinase 2.7.1.5 rhamnulokinase 2.7.1.6 galactokinase 2.7.1.7
mannokinase 2.7.1.8 glucosamine kinase 2.7.1.10 phosphoglucokinase
2.7.1.11 6-phosphofructokinase 2.7.1.12 gluconokinase 2.7.1.13
dehydrogluconokinase 2.7.1.14 sedoheptulokinase 2.7.1.15 ribokinase
2.7.1.16 ribulokinase 2.7.1.17 xylulokinase 2.7.1.18
phosphoribokinase 2.7.1.19 phosphoribulokinase 2.7.1.20 adenosine
kinase 2.7.1.21 thymidine kinase 2.7.1.22 ribosylnicotinamide
kinase 2.7.1.23 NAD+ kinase 2.7.1.24 dephospho-CoA kinase 2.7.1.25
adenylyl-sulfate kinase 2.7.1.26 riboflavin kinase 2.7.1.27
erythritol kinase 2.7.1.28 triokinase 2.7.1.29 glycerone kinase
2.7.1.30 glycerol kinase 2.7.1.31 glycerate kinase 2.7.1.32 choline
kinase 2.7.1.33 pantothenate kinase 2.7.1.34 pantetheine kinase
2.7.1.35 pyridoxal kinase 2.7.1.36 mevalonate kinase 2.7.1.39
homoserine kinase 2.7.1.40 pyruvate kinase 2.7.1.41
glucose-1-phosphate phosphodismutase 2.7.1.42 riboflavin
phosphotransferase 2.7.1.43 glucuronokinase 2.7.1.44
galacturonokinase 2.7.1.45 2-dehydro-3-deoxygluconokinase 2.7.1.46
L-arabinokinase 2.7.1.47 D-ribulokinase 2.7.1.48 uridine kinase
2.7.1.49 hydroxymethylpyrimidine kinase 2.7.1.50
hydroxyethylthiazole kinase 2.7.1.51 L-fuculokinase 2.7.1.52
fucokinase 2.7.1.53 L-xylulokinase 2.7.1.54 D-arabinokinase
2.7.1.55 allose kinase 2.7.1.56 1-phosphofructokinase 2.7.1.58
2-dehydro-3-deoxygalactonokinase 2.7.1.59 N-acetylglucosamine
kinase 2.7.1.60 N-acylmannosamine kinase 2.7.1.61
acyl-phosphate-hexose phosphotransferase 2.7.1.62
phosphoramidate-hexose phosphotransferase 2.7.1.63
polyphosphate-glucose phosphotransferase 2.7.1.64 inositol 3-kinase
2.7.1.65 scyllo-inosamine 4-kinase 2.7.1.66 undecaprenol kinase
2.7.1.67 1-phosphatidylinositol 4-kinase 2.7.1.68
1-phosphatidylinositol-4-phosphate 5-kinase 2.7.1.69
protein-Np-phosphohistidine-sugar phosphotransferase 2.7.1.70
identical to EC 2.7.1.37. 2.7.1.71 shikimate kinase 2.7.1.72
streptomycin 6-kinase 2.7.1.73 inosine kinase 2.7.1.74
deoxycytidine kinase 2.7.1.76 deoxyadenosine kinase 2.7.1.77
nucleoside phosphotransferase 2.7.1.78 polynucleotide
5'-hydroxyl-kinase 2.7.1.79 diphosphate-glycerol phosphotransferase
2.7.1.80 diphosphate-serine phosphotransferase 2.7.1.81
hydroxylysine kinase 2.7.1.82 ethanolamine kinase 2.7.1.83
pseudouridine kinase 2.7.1.84 alkylglycerone kinase 2.7.1.85
.beta.-glucoside kinase 2.7.1.86 NADH kinase 2.7.1.87 streptomycin
3''-kinase 2.7.1.88 dihydrostreptomycin-6-phosphate 3'a-kinase
2.7.1.89 thiamine kinase 2.7.1.90 diphosphate-fructose-6-phosphate
1-phosphotransferase 2.7.1.91 sphinganine kinase 2.7.1.92
5-dehydro-2-deoxygluconokinase 2.7.1.93 alkylglycerol kinase
2.7.1.94 acylglycerol kinase 2.7.1.95 kanamycin kinase 2.7.1.100
S-methyl-5-thioribose kinase 2.7.1.101 tagatose kinase 2.7.1.102
hamamelose kinase 2.7.1.103 viomycin kinase 2.7.1.105
6-phosphofructo-2-kinase 2.7.1.106 glucose-1,6-bisphosphate
synthase 2.7.1.107 diacylglycerol kinase 2.7.1.108 dolichol kinase
2.7.1.113 deoxyguanosine kinase 2.7.1.114 AMP-thymidine kinase
2.7.1.118 ADP-thymidine kinase 2.7.1.119 hygromycin-B 7''-O-kinase
2.7.1.121 phosphoenolpyruvate-glycerone phosphotransferase
2.7.1.122 xylitol kinase 2.7.1.127 inositol-trisphosphate 3-kinase
2.7.1.130 tetraacyldisaccharide 4'-kinase 2.7.1.134
inositol-tetrakisphosphate 1-kinase 2.7.1.136 macrolide 2'-kinase
2.7.1.137 phosphatidylinositol 3-kinase 2.7.1.138 ceramide kinase
2.7.1.140 inositol-tetrakisphosphate 5-kinase 2.7.1.142
glycerol-3-phosphate-glucosephosphotransferase 2.7.1.143
diphosphate-purine nucleoside kinase 2.7.1.144 tagatose-6-phosphate
kinase 2.7.1.145 deoxynucleoside kinase 2.7.1.146 ADP-dependent
phosphofructokinase 2.7.1.147 ADP-dependent glucokinase 2.7.1.148
4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase 2.7.1.149
1-phosphatidylinositol-5-phosphate 4-kinase 2.7.1.150
1-phosphatidylinositol-3-phosphate 5-kinase 2.7.1.151
inositol-polyphosphate multikinase 2.7.1.153
phosphatidylinositol-4,5-bisphosphate 3-kinase 2.7.1.154
phosphatidylinositol-4-phosphate 3-kinase 2.7.1.156
adenosylcobinamide kinase 2.7.1.157 N-acetylgalactosamine kinase
2.7.1.158 inositol-pentakisphosphate 2-kinase 2.7.1.159
inositol-1,3,4-trisphosphate 5/6-kinase 2.7.1.160
2'-phosphotransferase 2.7.1.161 CTP-dependent riboflavin kinase
2.7.1.162 N-acetylhexosamine 1-kinase 2.7.1.163 hygromycin B
4-O-kinase 2.7.1.164 O-phosphoseryl-tRNASec kinase
[0501] Mevalonate kinase (EC 2.7.1.36) phosphorylates the terminal
hydroxyl group of mevalonate. Gene candidates for this step include
erg12 from S. cerevisiae, mvk from Methanocaldococcus jannaschi,
MVK from Homo sapiens, and mvk from Arabidopsis thaliana col.
Additional mevalonate kinase candidates include the
feedback-resistant mevalonate kinase from the archeon
Methanosarcina mazei (Primak et al, AEM, in press (2011)) and the
Mvk protein from Streptococcus pneumoniae (Andreassi et al, Protein
Sci, 16:983-9 (2007)). Exemplary Mvk proteins are below.
TABLE-US-00132 Protein GenBank ID GI Number Organism erg12
CAA39359.1 3684 Sachharomyces cerevisiae mvk Q58487.1 2497517
Methanocaldococcus jannaschii mvk AAH16140.1 16359371 Homo sapiens
mvk NP_851084.1 30690651 Arabidopsis thaliana mvk NP_633786.1
21227864 Methanosarcina mazei mvk NP_357932.1 15902382
Streptococcus pneumoniae
[0502] Glycerol kinase also phosphorylates the terminal hydroxyl
group in glycerol to form glycerol-3-phosphate. This reaction
occurs in several species, including Escherichia coli,
Saccharomyces cerevisiae, and Thermotoga maritima. Glycerol kinases
have been shown to have a wide range of substrate specificity.
Crans and Whiteside studied glycerol kinases from four different
organisms (Escherichia coli, S. cerevisiae, Bacillus
stearothermophilus, and Candida mycoderma) (Crans et al., J. Am.
Chem. Soc. 107:7008-7018 (2010); Nelson et al., supra, (1999)).
They studied 66 different analogs of glycerol and concluded that
the enzyme could accept a range of substituents in place of one
terminal hydroxyl group and that the hydrogen atom at C2 could be
replaced by a methyl group. Interestingly, the kinetic constants of
the enzyme from all four organisms were very similar.
TABLE-US-00133 Protein GenBank ID GI Number Organism glpK
AP_003883.1 89110103 Escherichia coli K12 glpK1 NP_228760.1
15642775 Thermotoga maritime MSB8 glpK2 NP_229230.1 15642775
Thermotoga maritime MSB8 Gut1 NP_011831.1 82795252 Saccharomyces
cerevisiae
[0503] Homoserine kinase is another possible candidate. This enzyme
is also present in a number of organisms including E. coli,
Streptomyces sp, and S. cerevisiae. The gene candidates are:
TABLE-US-00134 Protein GenBank ID GI Number Organism thrB
BAB96580.2 85674277 Escherichia coli K12 SACT1DRAFT_4809
ZP_06280784.1 282871792 Streptomyces sp. ACT-1 Thr1 AAA35154.1
172978 Saccharomyces serevisiae
[0504] P. 2-Butenyl-4-Phosphate Kinase.
[0505] 2-Butenyl-4-phosphate kinase enzymes catalyze the transfer
of a phosphate group to the phosphate group of
2-butenyl-4-phosphate. The enzymes described below naturally
possess such activity or can be engineered to exhibit this
activity. Kinases that catalyze transfer of a phosphate group to
another phosphate group are members of the EC 2.7.4 enzyme class.
The table below lists several useful kinase enzymes in the EC 2.7.4
enzyme class.
TABLE-US-00135 Enzyme Commission Number Enzyme Name 2.7.4.1
polyphosphate kinase 2.7.4.2 phosphomevalonate kinase 2.7.43
adenylate kinase 2.7.4.4 nucleoside-phosphate kinase 2.7.4.6
nucleoside-diphosphate kinase 2.7.4.7 phosphomethylpyrimidine
kinase 2.7.4.8 guanylate kinase 2.7.4.9 dTMP kinase 2.7.4.10
nucleoside-triphosphate-adenylate kinase 2.7.4.11 (deoxy)adenylate
kinase 2.7.4.12 T2-induced deoxynucleotide kinase 2.7.4.13
(deoxy)nucleoside-phosphate kinase 2.7.4.14 cytidylate kinase
2.7.4.15 thiamine-diphosphate kinase 2.7.4.16 thiamine-phosphate
kinase 2.7.4.17 3-phosphoglyceroyl-phosphate- polyphosphate
phosphotransferase 2.7.4.18 farnesyl-diphosphate kinase 2.7.4.19
5-methyldeoxycytidine-5'- phosphate kinase 2.7.4.20
dolichyl-diphosphate-polyphosphate phosphotransferase 2.7.4.21
inositol-hexakisphosphate kinase 2.7.4.22 UMP kinase 2.7.4.23
ribose 1,5-bisphosphate phosphokinase 2.7.4.24
diphosphoinositol-pentakisphosphate kinase 2.7.4.-- Farnesyl
monophosphate kinase 2.7.4.-- Geranyl-geranyl monophosphate kinase
2.7.4.-- Phytyl-phosphate kinase
[0506] Phosphomevalonate kinase enzymes are of particular interest.
Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the analogous
transformation to 2-butenyl-4-phosphate kinase. Exemplary enzymes
include those below.
TABLE-US-00136 Protein GenBank ID GI Number Organism Erg8
AAA34596.1 171479 Saccharomyces cerevisiae mvaK2 AAG02426.1 9937366
Staphylococcus aureus mvaK2 AAG02457.1 9937409 Streptococcus
pneumoniae mvaK2 AAG02442.1 9937388 Enterococcus faecalis
[0507] Famesyl monophosphate kinase enzymes catalyze the CTP
dependent phosphorylation of famesyl monophosphate to famesyl
diphosphate. Similarly, geranylgeranyl phosphate kinase catalyzes
CTP dependent phosphorylation. Enzymes with these activities were
identified in the microsomal fraction of cultured Nicotiana tabacum
(Thai et al, PNAS 96:13080-5 (1999)). However, the associated genes
have not been identified to date.
[0508] Q. Butadiene Synthase.
[0509] Butadiene synthase catalyzes the conversion of
2-butenyl-4-diphosphate to 1,3-butadiene. The enzymes described
below naturally possess such activity or can be engineered to
exhibit this activity. Carbon-oxygen lyases that operate on
phosphates are found in the EC 4.2.3 enzyme class. The table below
lists several useful enzymes in EC class 4.2.3.
TABLE-US-00137 Enzyme Commission Number Enzyme Name 4.2.3.15
Myrcene synthase 4.2.3.26 Linalool synthase 4.2.3.27 Isoprene
synthase 4.2.3.36 Terpentriene sythase 4.2.3.46 (E,
E)-alpha-Farnesene synthase 4.2.3.47 Beta-Farnesene synthase
4.2.3.49 Nerolidol synthase
[0510] Particularly useful enzymes include isoprene synthase,
myrcene synthase and famesene synthase. Enzyme candidates are
described below.
[0511] Isoprene synthase naturally catalyzes the conversion of
dimethylallyl diphosphate to isoprene, but can also catalyze the
synthesis of 1,3-butadiene from 2-butenyl-4-diphosphate. Isoprene
synthases can be found in several organisms including Populus alba
(Sasaki et al., FEBS Letters, 2005, 579 (11), 2514-2518), Pueraria
montana (Lindberg et al., Metabolic Eng, 12(1):70-79 (2010);
Sharkey et al., Plant Physiol., 137(2):700-712 (2005)), and Populus
tremula x Populus alba, also called Populus canescens (Miller et
al., Planta, 2001, 213 (3), 483-487). The crystal structure of the
Populus canescens isoprene synthase was determined (Koksal et al, J
Mol Biol 402:363-373 (2010)). Additional isoprene synthase enzymes
are described in (Chotani et al., WO/2010/031079, Systems Using
Cell Culture for Production of Isoprene; Cervin et al., US Patent
Application 20100003716, Isoprene Synthase Variants for Improved
Microbial Production of Isoprene).
TABLE-US-00138 Protein GenBank ID GI Number Organism ispS
BAD98243.1 63108310 Populus alba ispS AAQ84170.1 35187004 Pueraria
montana ispS CAC35696.1 13539551 Populus tremula .times. Populus
alba
[0512] Myrcene synthase enzymes catalyze the dephosphorylation of
geranyl diphosphate to beta-myrcene (EC 4.2.3.15). Exemplary
myrcene synthases are below. These enzymes were heterologously
expressed in E. coli.
TABLE-US-00139 Protein GenBank ID GI Number Organism MST2
ACN58229.1 224579303 Solanum lycopersicum TPS-Myr AAS47690.2
77546864 Picea abies G-myr O24474.1 17367921 Abies grandis TPS10
EC07543.1 330252449 Arabidopsis thaliana
[0513] Famesyl diphosphate is converted to alpha-famesene and
beta-famesene by alpha-famesene synthase and beta-famesene
synthase, respectively. Exemplary alpha-famesene synthase enzymes
include those below.
TABLE-US-00140 Protein GenBank ID GI Number Organism TPS03 A4FVP2.1
205829248 Arabidopsis thaliana TPS02 P0CJ43.1 317411866 Arabidopsis
thaliana TPS-Far AAS47697.1 44804601 Picea abies afs AAU05951.1
51537953 Cucumis sativus eafar Q84LB2.2 75241161 Malus .times.
domestica TPS1 Q84ZW8.1 75149279 Zea mays
[0514] R. Crotyl Alcohol Diphosphokinase.
[0515] Crotyl alcohol diphosphokinase enzymes catalyze the transfer
of a diphosphate group to the hydroxyl group of crotyl alcohol. The
enzymes described below naturally possess such activity or can be
engineered to exhibit this activity. Kinases that catalyze transfer
of a diphosphate group are members of the EC 2.7.6 enzyme class.
The table below lists several useful kinase enzymes in the EC 2.7.6
enzyme class.
TABLE-US-00141 Enzyme Commission Number Enzyme Name 2.7.6.1
ribose-phosphate diphosphokinase 2.7.6.2 thiamine diphosphokinase
2.7.6.3 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine
diphosphokinase 2.7.6.4 nucleotide diphosphokinase 2.7.6.5 GTP
diphosphokinase
[0516] Of particular interest are ribose-phosphate diphosphokinase
enzymes; exemplary enzymes are below.
TABLE-US-00142 Protein GenBank ID GI Number Organism prs
NP_415725.1 16129170 Escherichia coli prsA NP_109761.1 13507812
Mycoplasma pneumoniae M129 TPK1 BAH19964.1 222424006 Arabidopsis
thaliana col TPK2 BAH57065.1 227204427 Arabidopsis thaliana col
[0517] S. Chemical Dehydration or Crotyl Alcohol Dehydratase.
[0518] Converting crotyl alcohol to butadiene using a crotyl
alcohol dehydratase can include combining the activities of the
enzymatic isomerization of crotyl alcohol to 3-buten-2-ol then
dehydration of 3-buten-2-ol to butadiene. An exemplary bifunctional
enzyme with isomerase and dehydratase activities is the linalool
dehydratase/isomemse of Castellaniella defragrans. This enzyme
catalyzes the isomerization of geraniol to linalool and the
dehydration of linalool to myrcene, reactants similar in structure
to crotyl alcohol, 3-buten-2-ol and butadiene (Brodkorb et al, J
Biol Chem 285:30436-42 (2010)). Enzyme accession numbers and
homologs are listed in the table below.
TABLE-US-00143 Protein GenBank ID GI Number Organism Ldi E1XUJ2.1
403399445 Castelleniella defragrans STEHIDRAFT_68678 EIM80109.1
389738914 Stereum hirsutum FP-91666 SS1 NECHADRAFT_82460
XP_003040778.1 302883759 Nectria haematococca mpVI 77-13-4
AS9A_2751 YP_004493998.1 333920417 Amycolicicoccus subflavus
DQS3-9A1
[0519] Alternatively, a fusion protein or protein conjugate can be
generated using well know methods in the art to generate a
bi-functional (dual-functional) enzyme having both the isomemse and
dehydratase activities. The fusion protein or protein conjugate can
include at least the active domains of the enzymes (or respective
genes) of the isomemse and dehydratase reactions. For the first
step, the conversion of crotyl alcohol to 3-buten-2-ol, enzymatic
conversion can be catalyzed by a crotyl alcohol isomemse
(classified as EC 5.4.4). A similar isomerization, the conversion
of 2-methyl-3-buten-2-ol to 3-methyl-2-buten-1-ol, is catalyzed by
cell extracts of Pseudomonas putida MB-1 (Malone et al, AEM 65 (6):
2622-30 (1999)). The extract may be used in vitro, or the protein
or gene(s) associated with the isomemse activity can be isolated
and used, even though they have not been identified to date.
[0520] Alternatively, either or both steps can be done by chemical
conversion, or by enzymatic conversion (in vivo or in vitro), or
any combination. Enzymes having the desired activity for the
conversion of 3-buten-2-ol to butadiene are provided elsewhere
herein.
[0521] T. Butadiene Synthase (Monophosphate).
[0522] Butadiene synthase (monophosphate) catalyzes the conversion
of 2-butenyl-4-phosphate to 1,3-butadiene. Butadiene synthase
enzymes are of the EC 4.2.3 enzyme class as described herein that
possess such activity or can be engineered to exhibit this
activity. Diphosphate lyase enzymes catalyze the conversion of
alkyl diphosphates to alkenes. Carbon-oxygen lyases that operate on
phosphates are found in the EC 4.2.3 enzyme class. The table below
lists several useful enzymes in EC class 4.2.3. Exemplary enzyme
candidates are also phosphate lyases.
TABLE-US-00144 Enzyme Commission No. Enzyme Name 4.2.3.5 Chorismate
synthase 4.2.3.15 Myrcene synthase 4.2.3.27 Isoprene synthase
4.2.3.36 Terpentriene sythase 4.2.3.46 (E, E)-alpha-Farnesene
synthase 4.2.3.47 Beta-Farnesene synthase
[0523] Phosphate lyase enzymes catalyze the conversion of alkyl
phosphates to alkenes. Carbon-oxygen lyases that operate on
phosphates are found in the EC 4.2.3 enzyme class. The table below
lists several relevant enzymes in EC class 4.2.3.
TABLE-US-00145 Enzyme Commission Number Enzyme Name 4.2.3.5
Chorismate synthase 4.2.3.15 Myrcene synthase 4.2.3.26 Linalool
synthase 4.2.3.27 Isoprene synthase 4.2.3.36 Terpentriene sythase
4.2.3.46 (E, E)-alpha-Farnesene synthase 4.2.3.47 Beta-Farnesene
synthase 4.2.3.49 Nerolidol synthase 4.2.3.-- Methylbutenol
synthase
[0524] Isoprene synthase enzymes catalyzes the conversion of
dimethylallyl diphosphate to isoprene. Additional isoprene synthase
enzymes are below and are described in (Chotani et al.,
WO/2010/031079, Systems Using Cell Culture for Production of
Isoprene; Cervin et al., US Patent Application 20100003716,
Isoprene Synthase Variants for Improved Microbial Production of
Isoprene).
TABLE-US-00146 Protein GenBank ID GI Number Organism ispS
BAD98243.1 63108310 Populus alba ispS AAQ84170.1 35187004 Pueraria
montana ispS CAC35696.1 13539551 Populus tremula .times. Populus
alba Tps-MBO1 AEB53064.1 328834891 Pinus sabiniana
[0525] Chorismate synthase (EC 4.2.3.5) participates in the
shikimate pathway, catalyzing the dephosphorylation of
5-enolpyruvylshikimate-3-phosphate to chorismate. The enzyme
requires reduced flavin mononucleotide (FMN) as a cofactor,
although the net reaction of the enzyme does not involve a redox
change. In contrast to the enzyme found in plants and bacteria, the
chorismate synthase in fungi is also able to reduce FMN at the
expense of NADPH (Macheroux et al., Planta 207:325-334 (1999)).
Representative monofunctional enzymes and bifunctional fungal
enzymes are below.
TABLE-US-00147 Gene GenBank Accession No. GI No. Organism aroC
NP_416832.1 16130264 Escherichia coli aroC ACH47980.1 197205483
Streptococcus pneumoniae U25818.1: 19 . . . 1317 AAC49056.1 976375
Neurospora crassa ARO2 CAA42745.1 3387 Saccharomyces cerevisiae
[0526] Myrcene synthase enzymes catalyze the dephosphorylation of
geranyl diphosphate to beta-myrcene (EC 4.2.3.15). Exemplary
myrcene synthases are below. These enzymes were heterologously
expressed in E coli.
TABLE-US-00148 Protein GenBank ID GI Number Organism MST2
ACN58229.1 224579303 Solanum lycopersicum TPS-Myr AAS47690.2
77546864 Picea abies G-myr O24474.1 17367921 Abies grandis TPS10
EC07543.1 330252449 Arabidopsis thaliana
[0527] Famesyl diphosphate is converted to alpha-famesene and
beta-famesene by alpha-famesene synthase and beta-famesene
synthase, respectively. Exemplary alpha-famesene synthase enzymes
are below.
TABLE-US-00149 Protein GenBank ID GI Number Organism TPS03 A4FVP2.1
205829248 Arabidopsis thaliana TPS02 P0CJ43.1 317411866 Arabidopsis
thaliana TPS-Far AAS47697.1 44804601 Picea abies afs AAU05951.1
51537953 Cucumis sativus eafar Q84LB2.2 75241161 Malus .times.
domestica TPS1 Q84ZW8.1 75149279 Zea mays
[0528] U. Crotonyl-CoA Reductase (Alcohol Forming) and V)
3-Hydroxybutyryl-CoA Reductase (Alcohol Forming).
[0529] The direct conversion of crotonyl-CoA and
3-hydroxybutyryl-CoA substrates to their corresponding alcohols is
catalyzed by bifunctional enzymes with acyl-CoA reductase (aldehyde
forming) activity and aldehyde reductase or alcohol dehydrogenase
activities. Exemplary bifunctional oxidoreductases that convert an
acyl-CoA to alcohol are described elsewhere herein.
[0530] FIG. 6 shows pathways for converting 1,3-butanediol to
3-buten-2-ol and/or butadiene. Enzymes in FIG. 6 are A.
1,3-butanediol kinase, B. 3-hydroxybutyrylphosphate kinase, C.
3-hydroxybutyryldiphosphate lyase, D. 1,3-butanediol
diphosphokinase, E. 1,3-butanediol dehydratase, F.
3-hydroxybutyrylphosphate lyase, G. 3-buten-2-ol dehydratase or
chemical reaction.
[0531] A. 1,3-Butanediol Kinase.
[0532] Phosphorylation of 1,3-butanediol to
3-hydroxybutyrylphosphate is catalyzed by an alcohol kinase enzyme.
Alcohol kinase enzymes catalyze the transfer of a phosphate group
to a hydroxyl group. Kinases that catalyze transfer of a phosphate
group to an alcohol group are members of the EC 2.7.1 enzyme class.
The table herein in the section on Crotyl Alcohol Kinase lists
several useful kinase enzymes in the EC 2.7.1 enzyme class.
[0533] Mevalonate kinase (EC 2.7.1.36) phosphorylates the terminal
hydroxyl group of mevalonate. Gene candidates for this step include
erg12 from S. cerevisiae, mvk from Methanocaldococcus jannaschi,
MVK from Homo sapiens, and mvk from Arabidopsis thaliana col.
Additional mevalonate kinase candidates include those described
herein in the section on Crotyl Alcohol Kinase.
[0534] Glycerol kinase also phosphorylates the terminal hydroxyl
group in glycerol to Ellin glycerol-3-phosphate. Additional
glycerol kinase candidates include those described herein in the
section on Crotyl Alcohol Kinase. Homoserine kinase is another
similar enzyme candidate. Additional homoserine kinase candidates
include those described herein in the section on Crotyl Alcohol
Kinase.
[0535] B. 3-Hydroxybutyrylphosphate Kinase.
[0536] Alkyl phosphate kinase enzymes catalyze the transfer of a
phosphate group to the phosphate group of an alkyl phosphate. The
enzymes described herein and in the section for
2-Butenyl-4-phosphate Kinase naturally possess such activity or can
be engineered to exhibit this activity, and include several useful
kinase enzymes in the EC 2.7.4 enzyme class.
[0537] Phosphomevalonate kinase enzymes are of particular interest.
Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the phosphorylation
of phosphomevalonate. Exemplary candidates include those listed
herein in the section 2-Butenyl-4-phosphate Kinase.
[0538] Famesyl monophosphate kinase enzymes catalyze the CTP
dependent phosphorylation of famesyl monophosphate to famesyl
diphosphate. Similarly, geranylgeranyl phosphate kinase catalyzes
CTP dependent phosphorylation. Enzymes with these activities were
identified in the microsomal fraction of cultured Nicotiana tabacum
(Thai et al, PNAS 96:13080-5 (1999)). However, the associated genes
have not been identified to date.
[0539] C. 3-Hydroxybutyryldiphosphate Lyase.
[0540] Diphosphate lyase enzymes catalyze the conversion of alkyl
diphosphates to alkenes. Carbon-oxygen lyases that operate on
phosphates are found in the EC 4.2.3 enzyme class. The table below
lists several useful enzymes in EC class 4.2.3. described herein.
Exemplary enzyme candidates also include phosphate lyases described
herein.
TABLE-US-00150 Enzyme Commission No. Enzyme Name 4.2 3.5 Chorismate
synthase 4.2.3.15 Myrcene synthase 4.2.3.27 Isoprene synthase
4.2.3.36 Terpentriene sythase 4.2.3.46 (E, E)-alpha-Farnesene
synthase 4.2.3.47 Beta-Farnesene synthase
[0541] D. 1,3-Butanediol Dehydratase.
[0542] Exemplary dehydratase enzymes suitable for dehydrating
1,3-butanediol to 3-buten-2-ol include oleate hydratases, acyclic
1,2-hydratases and linalool dehydratase. Exemplary enzyme
candidates are described above.
[0543] E. 1,3-Butanediol Diphosphokinase.
[0544] Diphosphokinase enzymes catalyze the transfer of a
diphosphate group to an alcohol group. The enzymes described in the
section on Crotyl Alcohol Diphosphokinase naturally possess such
activity. Kinases that catalyze transfer of a diphosphate group are
members of the EC 2.7.6 enzyme class.
[0545] Of particular interest are ribose-phosphate diphosphokinase
enzymes, also described in the section on Crotyl Alcohol
Diphosphokinase.
[0546] F. 3-Hydroxybutyrylphosphate Lyase.
[0547] Phosphate lyase enzymes catalyze the conversion of alkyl
phosphates to alkenes. Carbon-oxygen lyases that operate on
phosphates are found in the EC 4.2.3 enzyme class. The section
herein on Butadiene Synthase (monophosphate) enzymesdescribes
relevant enzymes in EC class 4.2.3.
[0548] Isoprene synthase enzymes catalyzes the conversion of
dimethylallyl diphosphate to isoprene and suitable enzymes are
described in the section on Butadiene Synthase (monophosphate).
[0549] Chorismate synthase (EC 4.2.3.5) participates in the
shikimate pathway, catalyzing the dephosphorylation of
5-enolpyruvylshikimate-3-phosphate to chorismate and suitable
enzymes are described in the section on Butadiene
[0550] Synthase (monophosphate).
[0551] Myrcene synthase enzymes catalyze the dephosphorylation of
geranyl diphosphate to beta-myrcene (EC 4.2.3.15). Exemplary
myrcene synthases are described in the section on Butadiene
Synthase (monophosphate).
[0552] Famesyl diphosphate is converted to alpha-famesene and
beta-famesene by alpha-famesene synthase and beta-famesene
synthase, respectively. Exemplary alpha-famesene synthase enzymes
include those described in the section on Butadiene Synthase
(monophosphate).
[0553] G. G. 3-Buten-2-ol Dehydratase.
[0554] Dehydration of 3-buten-2-ol to butadiene is catalyzed by a
3-buten-2-ol dehydratase enzyme or by chemical dehydration.
Exemplary dehydratase enzymes suitable for dehydrating 3-buten-2-ol
include oleate hydratase, acyclic 1,2 hydratase and linalool
dehydratase enzymes. Exemplary enzymes are described above.
Example VIII
1,4-Butanediol Synthesis Enzymes
[0555] This Example provides genes that can be used for conversion
of succinyl-CoA to 1,4-butanediol as depicted in the pathways of
FIG. 7.
[0556] A) Succinyl-CoA Transferase (designated as EB1) or
Succinyl-CoA Synthetase (designated as EB2A).
[0557] The conversion of succinate to succinyl-CoA is catalyzed by
EB1 or EB2A (synthetase or ligase). Exemplary EB1 and EB2A enzymes
are described above.
[0558] B) Succinyl-CoA Reductase (Aldehyde Forming).
[0559] Enzymes with succinyl-CoA reductase activity are encoded by
sucD of Clostridium kluyveri (Sohling, J. Bacteriol. 178:871-880
(1996)) and sucD of Porphyromonas gingivalis (Takahashi, J.
Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductase
enzymes participate in the 3-hydroxypropionate/4-HB cycle of
thermophilic archaea such as Metallosphaera sedula (Berg et al.,
Science 318:1782-1786 (2007)) and Thermoproteus neutrophilus
(Ramos-Vera et al., J Bacteriol, 191:4286-4297 (2009)). These and
other exemplary succinyl-CoA reductase enzymes are described
above.
[0560] C) 4-HB Dehydrogenase (Designated as EB4).
[0561] Enzymes exhibiting EB4 activity (EC 1.1.1.61) have been
characterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci.
49:379-387 (2004), Clostridium kluyveri (Wolff and Kenealy, Protein
Expr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz
et al., J. Biol. Chem. 278:41552-41556 (2003)). Other EB4 enzymes
are found in Porphyromonas gingivalis and gbd of an uncultured
bacterium. Accession numbers of these genes are listed in the table
below.
TABLE-US-00151 Protein GenBank ID GI Number Organism 4hbd
YP_726053.1 113867564 Ralstonia eutropha H16 4hbd L21902.1
146348486 Clostridium kluyveri DSM 555 4hbd Q94B07 75249805
Arabidopsis thaliana 4-hBd NP_904964.1 34540485 Porphyromonas
gingivalis W83 gbd AF148264.1 5916168 Uncultured bacterium
[0562] D) 4-HB Kinase (Designated as EB5).
[0563] Activation of 4-HB to 4-hydroxybutyryl-phosphate is
catalyzed by EBS. Phosphotransferase enzymes in the EC class 2.7.2
transform carboxylic acids to phosphonic acids with concurrent
hydrolysis of one ATP. Enzymes suitable for catalyzing this
reaction include butyrate kinase, acetate kinase, aspartokinase and
gamma-glutamyl kinase. Other butyrate kinase enzymes are found in
C. butyricum, C. beijerinckii and C. tetanomorphum (Twarog and
Wolfe, J. Bacteriol. 86:112-117 (1963)). Aspartokinase catalyzes
the ATP-dependent phosphorylation of aspartate and participates in
the synthesis of several amino acids. The aspartokinase III enzyme
in E. coli, encoded by lysC, has a broad substrate range, and the
catalytic residues involved in substrate specificity have been
elucidated. Exemplary enzymes are below.
TABLE-US-00152 Gene Accession No. GI No. Organism buk1 NP_349675
15896326 Clostridium acetobutylicum buk2 Q97II1 20137415
Clostridium acetobutylicum buk2 Q9X278.1 6685256 Thermotoga
maritima lysC NP_418448.1 16131850 Escherichia coli ackA
NP_416799.1 16130231 Escherichia coli proB NP_414777.1 16128228
Escherichia coli buk YP_001307350.1 150015096 Clostridium
beijerinckii buk2 YP_001311072.1 150018818 Clostridium
beijerinckii
[0564] E) Phosphotrans-4-Hydroxybutyrylase (Designated as EB6).
[0565] EB6 catalyzes the transfer of the 4-hydroxybutyryl group
from phosphate to CoA. Acyltransferases suitable for catalyzing
this reaction include phosphotransacetylase and
phosphotransbutyrylase. The pta gene from E. coli encodes an enzyme
that can convert acetyl-phosphate into acetyl-CoA (Suzuki, Biochim.
Biophys. Acta 191:559-569 (1969)). This enzyme can also utilize
propionyl-CoA instead of acetyl-CoA (Hesslinger et al., Mol.
Microbiol 27:477-492 (1998)). Similarly, the ptb gene from C.
acetobutylicum encodes an enzyme that can convert butyryl-CoA into
butyryl-phosphate (Walter et al., Gene 134:107-111 (1993)); Huang
et al., J Mol. Microbiol. Biotechno.l 2:33-38 (2000). Additional
ptb genes can be found in Clostridial organisms, butyrate producing
bacterium L2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004))
and Bacillus megaterium (Vazquez et al., Curr. Microbiol.
42:345-349 (2001)).
TABLE-US-00153 Gene Accession No. GI No. Organism pta NP_416800.1
16130232 Escherichia coli ptb NP_349676 15896327 Clostridium
acetobutylicum ptb YP_001307349.1 150015095 Clostridium
beijerinckii ptb AAR19757.1 38425288 butyrate-producing bacterium
L2-50 ptb CAC07932.1 10046659 Bacillus megaterium
[0566] F) 4-Hydroxybutyryl-CoA Reductase (Aldehyde Forming).
[0567] Enzymes with this activity are described above.
[0568] G) 1,4-Butanediol Dehydrogenase (Designated as EB8).
[0569] EB8 catalyzes the reduction of 4-hydroxybutyraldehyde to
1,4-butanediol. Enzymes with 1,4-butanediol activity are listed in
the table below.
TABLE-US-00154 Protein GenBank ID GI Number Organism alrA
BAB12273.1 9967138 Acinetobacter sp. strain M-1 ADH2 NP_014032.1
6323961 Saccharomyces cerevisiae fucO NP_417279.1 16130706
Escherichia coli yqhD NP_417484.1 16130909 Escherichia coli bdh I
NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.1
15896542 Clostridium acetobutylicum bdh BAF45463.1 124221917
Clostridium saccharoperbutylacetonicum Cbei_1722 YP_001308850
150016596 Clostridium beijerinckii Cbei_2181 YP_001309304 150017050
Clostridium beijerinckii Cbei 2421 YP_001309535 150017281
Clostridium beijerinckii 14bdh AAC76047.1 1789386 Escherichia coli
K-12 MG1655 14bdh YP_001309304.1 150017050 Clostridium beijerinckii
NCIMB 8052 14bdh P13604.1 113352 Clostridium saccharobutylicum
14bdh ZP_03760651.1 225405462 Clostridium asparagiforme DSM 15981
14bdh ZP_02083621.1 160936248 Clostridium bolteae ATCC BAA-613
14bdh YP_003845251.1 302876618 Clostridium cellulovorans 743B 14bdh
ZP_03294286.1 210624270 Clostridium hiranonis DSM 13275 14bdh
ZP_03705769.1 225016577 Clostridium methylpentosum DSM 5476 14bdh
YP_003179160.1 257783943 Atopobium parvulum DSM 20469 14bdh
YP_002893476.1 237809036 Tolumonas auensis DSM 9187 14bdh
ZP_05394983.1 255528157 Clostridium carboxidivorans P7
[0570] H) Succinate Reductase.
[0571] Direct reduction of succinate to succinate semialdehyde is
catalyzed by a carboxylic acid reductase. Exemplary enzymes for
catalyzing this transformation are also those described below and
herein for K) 4-Hydroxybutyrate reductase.
[0572] I) Succinyl-CoA Reductase (Alcohol Forming) (Designated as
EB10).
[0573] EB10 enzymes are bifunctional oxidoreductases that convert
succinyl-CoA to 4-HB. Enzyme candidates described below and herein
for M) 4-hydroxybutyryl-CoA reductase (alcohol forming) are also
suitable for catalyzing the reduction of succinyl-CoA.
[0574] J) 4-Hydroxybutyryl-CoA Transferase or 4-Hydroxybutyryl-CoA
Synthetase (Designated as EB11).
[0575] Conversion of 4-HB to 4-hydroxybutyryl-CoA is catalyzed by a
CoA transferase or synthetase. EB11 enzymes include those listed
below.
TABLE-US-00155 Protein GenBank ID GI Number Organism cat1 P38946.1
729048 Clostridium kluyveri cat2 P38942.2 172046066 Clostridium
kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri TVAG_395550
XP_001330176 123975034 Trichomonas vaginalis G3 Tb11.02.0290
XP_828352 71754875 Trypanosoma brucei cat2 CAB60036.1 6249316
Clostridium aminobutyricum cat2 NP_906037.1 34541558 Porphyromonas
gingivalis W83
[0576] 4HB-CoA synthetase catalyzes the ATP-dependent conversion of
4-HB to 4-hydroxybutyryl-CoA. AMP-forming 4-HB-CoA synthetase
enzymes are found in organisms that assimilate carbon via the
dicarboxylate/hydroxybutyrate cycle or the 3-hydroxypropionate/4-HB
cycle. Enzymes with this activity have been characterized in
Thermoproteus neutrophilus and Metallosphaera sedula. Others can be
inferred by sequence homology. ADP forming CoA synthetases, such
EB2A, are also suitable candidates.
TABLE-US-00156 Protein GenBank ID GI Number Organism Tneu 0420
ACB39368.1 170934107 Thermoproteus neutrophilus Caur_0002
YP_001633649.1 163845605 Chloroflexus aurantiacus J-10-fl Cagg 3790
YP_002465062 219850629 Chloroflexus aggregans DSM 9485 acs
YP_003431745 288817398 Hydrogenobacter thermophilus TK-6 Pisl 0250
YP_929773.1 119871766 Pyrobaculum islandicum DSM 4184 Msed 1422
ABP95580.1 145702438 Metallosphaera sedula
[0577] K) 4-HB Reductase.
[0578] Reduction of 4-HB to 4-hydroxybutanal is catalyzed by a
carboxylic acid reductase (CAR) such as the Car enzyme found in
Nocardia iowensis. This enzyme and other carboxylic acid reductases
are described above (see EC 1.2.1.e).
[0579] L) 4-Hydroxybutyryl-Phosphate Reductase (Designated as
EB14).
[0580] EB14 catalyzes the reduction of 4-hydroxybutyrylphosphate to
4-hydroxybutyraldehyde. An enzyme catalyzing this transformation
has not been identified to date. However, similar enzymes include
phosphate reductases in the EC class 1.2.1. Exemplary phosphonate
reductase enzymes include G3P dehydrogenase (EC 1.2.1.12),
aspartate-semialdehyde dehydrogenase (EC 1.2.1.11)
acetylglutamylphosphate reductase (EC 1.2.1.38) and
glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.-). Aspartate
semialdehyde dehydrogenase (ASD, EC 1.2.1.11) catalyzes the
NADPH-dependent reduction of 4-aspartyl phosphate to
aspartate-4-semialdehyde. The E. coli ASD enzyme has been shown to
accept the alternate substrate beta-3-methylaspartyl phosphate
(Shames et al., J Biol. Chem. 259:15331-15339 (1984)). A related
enzyme candidate is acetylglutamylphosphate reductase (EC
1.2.1.38), an enzyme that naturally reduces acetylglutamylphosphate
to acetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels
et al., Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis
(O'Reilly et al., Microbiology 140 (Pt 5):1023-1025 (1994)), E.
coli (Parsot et al., Gene. 68:275-283 (1988)), and other organisms.
Additional phosphate reductase enzymes of E. coli include
glyceraldehyde 3-phosphate dehydrogenase (gapA (Branlant et al.,
Eur. J. Biochem. 150:61-66 (1985))) and glutamate-5-semialdehyde
dehydrogenase (proA (Smith et al., J. Bacteriol. 157:545-551
(1984))). Genes encoding glutamate-5-semialdehyde dehydrogenase
enzymes from Salmonella typhimurium (Mahan et al., J Bacteria
156:1249-1262 (1983)) and Campylobacter jejuni (Louie et al., Mol.
Gen. Genet. 240:29-35 (1993)) were cloned and expressed in E.
coli.
TABLE-US-00157 Protein GenBank ID GI Number Organism asd
NP_417891.1 16131307 Escherichia coli asd YP_248335.1 68249223
Haemophilus influenzae asd AAB49996 1899206 Mycobacterium
tuberculosis VC2036 NP_231670 15642038 Vibrio cholera asd
YP_002301787.1 210135348 Heliobacter pylori ARG5,6 NP_010992.1
6320913 Saccharomyces cerevisiae argC NP_389001.1 16078184 Bacillus
subtilis argC NP_418393.1 16131796 Escherichia coli gapA P0A9B2.2
71159358 Escherichia coli proA NP_414778.1 16128229 Escherichia
coli proA NP_459319.1 16763704 Salmonella typhimurium proA P53000.2
9087222 Campylobacter jejuni
[0581] M) 4-Hydroxybutyryl-CoA Reductase (Alcohol Forming)
(Designated as EB15).
[0582] EB15 enzymes are bifunctional oxidoreductases that convert
an 4-hydroxybutyryl-CoA to 1,4-butanediol. Enzymes with this
activity include those below.
TABLE-US-00158 Protein GenBank ID GI Number Organism adhE
NP_415757.1 16129202 Escherichia coli adhE2 AAK09379.1 12958626
Clostridium acetobutylicum bdh I NP_349892.1 15896543 Clostridium
acetobutylicum bdh II NP_349891.1 15896542 Clostridium
acetobutylicum adhE AAV66076.1 55818563 Leuconostoc mesenteroides
adhE NP_781989.1 28211045 Clostridium tetani adhE NP_563447.1
18311513 Clostridium perfringens adhE YP_001089483.1 126700586
Clostridium difficile
Example IX
Adipate, 6-Aminocaproate, Caprolactam and Hexamethylenediamine
Synthesis Enzymes
[0583] This Example provides genes that can be used for conversion
of succinyl-CoA and acetyl-CoA to adipate, 6-aminocaproate,
caprolactam and hexamethylenediamine as depicted in the pathways of
FIG. 8.
[0584] FIG. 8. depicts enzymes: A) 3-oxoadipyl-CoA thiolase, B)
3-oxoadipyl-CoA reductase, C) 3-hydroxyadipyl-CoA dehydratase, D)
5-carboxy-2-pentenoyl-CoA reductase, E) adipyl-CoA reductase
(aldehyde forming), F) 6-aminocaproate transaminase, or
6-aminocaproate dehydrogenase, G) 6-aminocaproyl-CoA/acyl-CoA
transferase, or 6-aminocaproyl-CoA synthase, H) amidohydrolase, I)
spontaneous cyclization, J) 6-aminocaproyl-CoA reductase (aldehyde
forming), K) HMDA transaminase or HMDA dehydrogenase, L) Adipyl-CoA
hydrolase, adipyl-CoA ligase, adipyl-CoA transferase, or
phosphotransadipylase/adipate kinase.
[0585] Transformations depicted in FIG. 8 fall into at least 10
general categories of transformations shown in the Table below. The
first three digits of each label correspond to the first three
Enzyme Commission number digits which denote the general type of
transformation independent of substrate specificity. Below is
described a number of biochemically characterized candidate genes
in each category. Specifically listed are exemplary genes that can
be applied to catalyze the appropriate transformations in FIG. 8
when cloned and expressed.
TABLE-US-00159 Step Label Function FIG. 8, step B 1.1.1.a
Oxidoreductase (ketone to hydroxyl or aldehyde to alcohol) FIG. 8,
steps E 1.2.1.b Oxidoreductase (acyl-CoA to and J aldehyde) FIG. 8,
step D 1.3.1.a Oxidoreductase operating on CH--CH donors FIG. 8,
steps F 1.4.1.a Oxidoreductase operating on and K amino acids FIG.
8, step A 2.3.1.b Acyltransferase FIG. 8, steps F 2.6.1.a
Aminotransferase and K FIG. 8, steps G 2.8.3.a Coenzyme-A
transferase and L FIG. 8, steps G 6.2.1.a Acid-thiol ligase and L
FIG. 8, Step H 6.3.1.a/ Amide synthases/peptide 6.3.2.a synthases
FIG. 8, step I No enzyme Spontaneous cyclization required
[0586] FIG. 8, Step A--3-Oxoadipyl-CoA Thiolase.
[0587] EC 2.3.1.b Acyl Transferase.
[0588] The first step in the pathway combines acetyl-CoA and
succinyl-CoA to form 3-oxoadipyl-CoA. Step A can involve a
3-oxoadipyl-CoA thiolase, or equivalently, succinyl CoA:acetyl CoA
acyl transferase (.beta.-ketothiolase). Since beta-ketothiolase
enzymes catalyze reversible transformations, these enzymes can be
employed for the synthesis of 3-oxoadipyl-CoA. For example, the
ketothiolase phaA from R. eutropha combines two molecules of
acetyl-CoA to form acetoacetyl-CoA (Sato et al., J Biosci Bioeng
103:38-44 (2007)). Similarly, a .beta.-keto thiolase (bktB) has
been reported to catalyze the condensation of acetyl-CoA and
propionyl-CoA to form .beta.-ketovakryl-CoA (Slater et al., J.
Bacteriol. 180:1979-1987 (1998)) in R. eutropha. The protein
sequences for the above-mentioned gene products are well known in
the art and can be accessed in the public databases such as GenBank
using the following accession numbers.
TABLE-US-00160 Gene name GI Number GenBank ID Organism paaJ
16129358 NP_415915.1 Escherichia coli pcaF 17736947 AAL02407
Pseudomonas knackmussii (B13) phaD 3253200 AAC24332.1 Pseudomonas
putida paaE 106636097 ABF82237.1 Pseudomonas fluorescens
[0589] These exemplary sequences can be used to identify homologue
proteins in GenBank or other databases through sequence similarity
searches (for example, BLASTp). The resulting homologue proteins
and their corresponding gene sequences provide additional exogenous
DNA sequences for transformation into E. coli or other suitable
host microorganisms to generate production hosts.
[0590] For example, orthologs of paaJ from Escherichia coli K12 can
be found using the following GenBank accession numbers:
TABLE-US-00161 GI Number GenBank ID Organism 152970031
YP_001335140.1 Klebsiella pneumoniae 157371321 YP_001479310.1
Serratia proteamaculans 3253200 AAC24332.1 Pseudomonas putida
[0591] Example orthologs of pcaF from Pseudomonas knackmussii can
be found using the following GenBank accession numbers:
TABLE-US-00162 GI Number GenBank ID Organism 4530443 AAD22035.1
Streptomyces sp. 2065 24982839 AAN67000.1 Pseudomonas putida
115589162 ABJ15177.1 Pseudomonas aeruginosa
[0592] Additional native candidate genes for the ketothiolase step
include atoB, which can catalyze the reversible condensation of 2
acetyl-CoA molecules (Sato et al., J. Biosci. Bioengineer.
103:38-44 (2007)), and its homolog yqeF. Non-native gene candidates
include those in the table below. The protein sequences for each of
these exemplary gene products can be found using the following
GenBank accession numbers:
TABLE-US-00163 Gene Name GenBank ID Organism atoB NP_416728.1
Escherichia coli yqeF NP_417321.2 Escherichia coli phaA YP_725941
Ralstonia eutropha bktB AAC38322.1 Ralstonia eutropha thiA
NP_349476.1 Clostridium acetobutylicum thiB NP_149242.1 Clostridium
acetobutylicum
[0593] 2-Amino-4-oxopentanoate (AKP) thiolase or AKP thiolase
(AKPT) enzymes present additional candidates for performing step A.
AKPT is a pyridoxal phosphate-dependent enzyme participating in
omithine degradation in Clostridium sticklandii (Jeng et al.,
Biochemistry 13:2898-2903 (1974); Kenklies et al., Microbiology
145:819-826 (1999)). A gene cluster encoding the alpha and beta
subunits of AKPT (or-2 (ortA) and or-3 (ortB)) was recently
identified and the biochemical properties of the enzyme were
characterized (Fonknechten et al., J. Bacteriol. In Press (2009)).
The enzyme is capable of operating in both directions and naturally
reacts with the D-isomer of alanine. AKPT from Clostridium
sticklandii has been characterized but its protein sequence has not
yet been published. Enzymes with high sequence homology are found
in Clostridium difficile, Alkaliphilus metalliredigenes QYF,
Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis
MB4 (Fonknechten et al., supra).
TABLE-US-00164 Gene name GI Number GenBank ID Organism ortA
(.alpha.) 126698017 YP_001086914.1 Clostridium difficile 630 ortB
(.beta.) 126698018 YP_001086915.1 Clostridium difficile 630
Amet_2368 (.alpha.) 150390132 YP_001320181.1 Alkaliphilus
metalliredigenes QYF Amet_2369 (.beta.) 150390133 YP_001320182.1
Alkaliphilus metalliredigenes QYF Teth514_1478 (.alpha.) 167040116
YP_001663101.1 Thermoanaerobacter sp. X514 Teth514_1479 (.beta.)
167040117 YP_001663102.1 Thermoanaerobacter sp. X514 TTE1235
(.alpha.) 20807687 NP_622858.1 Thermoanaerobacter tengcongensis MB4
thrC (.beta.) 20807688 NP_622859.1 Thermoanaerobacter tengcongensis
MB4
[0594] Step B--3-Oxoadipyl-CoA Reductase.
[0595] EC 1.1.1.a Oxidoreductases.
[0596] Certain transformations depicted in FIG. 8 involve
oxidoreductases that convert a ketone functionality to a hydroxyl
group. For example, FIG. 8, step B involves the reduction of a
3-oxoacyl-CoA to a 3-hydroxyacyl-CoA.
[0597] Exemplary enzymes that can convert 3-oxoacyl-CoA molecules,
such as 3-oxoadipyl-CoA, into 3-hydroxyacyl-CoA molecules, such as
3-hydroxyadipyl-CoA, include enzymes whose natural physiological
roles are in fatty acid beta-oxidation or phenylacetate catabolism.
For example, subunits of two fatty acid oxidation complexes in E.
coli, encoded by fadB and fadJ, function as 3-hydroxyacyl-CoA
dehydrogenases (Binstock et al., Methods Enzymol. 71:403-411
(1981)). Furthermore, the gene products encoded by phaC in
Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA
95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens ST (Di
Gennaro et al., Arch. Microbiol. 188:117-125 (2007)) catalyze the
reverse reaction of step B in FIG. 8, that is, the oxidation of
3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism
of phenylacetate or styrene. Note that the reactions catalyzed by
such enzymes are reversible. A similar transformation is also
carried out by the gene product of hbd in Clostridium
acetobutylicum (Atsumi et al., Metab. Eng. (epub Sep. 14, 2007);
Boynton et al., J. Bacteriol. 178:3015-3024 (1996)). This enzyme
converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA. In addition,
given the proximity in E. coli of paaH to other genes in the
phenylacetate degradation operon (Nogales et al., Microbiology
153:357-365 (2007)) and the fact that paaH mutants cannot grow on
phenylacetate (Ismail et al., Eur. J Biochem. 270:3047-3054
(2003)), it is expected that the E. coli paaH gene encodes a
3-hydroxyacyl-CoA dehydrogenase.
TABLE-US-00165 Gene name GI Number GenBank ID Organism fadB 119811
P21177.2 Escherichia coli fadJ 3334437 P77399.1 Escherichia coli
paaH 16129356 NP_415913.1 Escherichia coli phaC 26990000
NP_745425.1 Pseudomonas putida paaC 106636095 ABF82235.1
Pseudomonas fluorescens
[0598] Additional exemplary oxidoreductases capable of converting
3-oxoacyl-CoA molecules to their corresponding 3-hydroxyacyl-CoA
molecules include 3-hydroxybutyryl-CoA dehydrogenases. Exemplary
enzymes are shown below.
TABLE-US-00166 Gene name GI Number GenBank ID Organism hbd 18266893
P52041.2 Clostridium acetobutylicum Hbd2 146348271 EDK34807.1
Clostridium kluyveri Hbd1 146345976 EDK32512.1 Clostridium kluyveri
HSD17B10 3183024 O02691.3 Bos taurus phbB 130017 P23238.1 Zoogloea
ramigera phaB 146278501 YP_001168660.1 Rhodobacter sphaeroides
[0599] A number of similar enzymes have been found in other species
of Clostridia and in Metallosphaera sedula (Berg et al., Science
318:1782-1786 (2007)).
TABLE-US-00167 Gene name GI Number GenBank ID Organism hbd 15895965
NP_349314.1 Clostridium acetobutylicum hbd 20162442 AAM14586.1
Clostridium beijerinckii Msed 1423 146304189 YP_001191505
Metallosphaera sedula Msed_0399 146303184 YP_001190500
Metallosphaera sedula Msed_0389 146303174 YP_001190490
Metallosphaera sedula Msed_1993 146304741 YP_001192057
Metallosphaera sedula
[0600] Step C--3-Hydroxyadipyl-CoA Dehydratase.
[0601] Step C can involve a 3-hydroxyadipyl-CoA dehydratase. The
gene product of crt from C. acetobutylicum catalyzes the
dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA (Atsumi et al.,
Metab. Eng. (epub Sep. 14, 2007); Boynton et al., J. Bacteriol.
178:3015-3024 (1996)). Homologs of this gene are strong candidates
for carrying out the third step (step C) in the synthesis pathways
exemplified in FIG. 8. In addition, genes known to catalyze the
hydroxylation of double bonds in enoyl-CoA compounds represent
additional candidates given the reversibility of such enzymatic
transformations. For example, the enoyl-CoA hydratases, phaA and
phaB, of P. putida are believed to carry out the hydroxylation of
double bonds during phenylacetate catabolism (Olivera et al., Proc.
Natl. Acad. Sci. USA 95:6419-6424 (1998)) and thus represent
additional candidates for incorporation into E. coli. Lastly, a
number of Escherichia coli genes have been shown to demonstrate
enoyl-CoA hydratase functionality including maoC (Park and Lee, J.
Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur. J.
Biochem. 270:3047-3054 (2003); Park and Lee, Biotechnol. Bioeng.
86:681-686 (2004); Park and Lee, Appl. Biochem. Biotechnol.
113-116:335-346 (2004)), and paaG (Ismail et al., supra, 2003; Park
and Lee, supra, 2003; Park and Lee, supra, 2004). The protein
sequences for each of these exemplary gene products can be found
using the following GenBank accession numbers:
TABLE-US-00168 Gene Name GenBank ID Organism maoC NP_415905.1
Escherichia coli paaF NP_415911.1 Escherichia coli paaG NP_415912.1
Escherichia coli crt NP_349318.1 Clostridium acetobutylicum paaA
NP_745427.1 Pseudomonas putida paaB NP_745426.1 Pseudomonas putida
phaA ABF82233.1 Pseudomonas fluorescens phaB ABF82234.1 Pseudomonas
fluorescens
[0602] Alternatively, beta-oxidation genes are candidates for the
first three steps in adipate synthesis. Candidate genes for the
proposed adipate synthesis pathway also include the native fatty
acid oxidation genes of E. coli and their homologs in other
organisms. The E. coli genes fadA and fadB encode a multienzyme
complex that exhibits ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA
dehydrogenase, and enoyl-CoA hydratase activities (Yang et al.,
Biochem. 30:6788-6795 (1991); Yang et al., J. Biol. Chem.
265:10424-10429 (1990); Yang et al., J. Biol. Chem. 266:16255
(1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18: 4937
(1990)). These activities are mechanistically similar to the first
three transformations shown in FIG. 8. The fadI and fadJ genes
encode similar functions and are naturally expressed only
anaerobically (Campbell et al., Mol. Microbiol. 47:793-805 (2003)).
These gene products naturally operate to degrade short, medium, and
long chain fatty-acyl-CoA compounds to acetyl-CoA, rather than to
convert succinyl-CoA and acetyl-CoA into 5-carboxy-2-pentenoyl-CoA
as proposed in FIG. 8. However, it is well known that the
ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, and
enoyl-CoA hydratase enzymes catalyze reversible transformations.
Furthermore, directed evolution and related approaches can be
applied to tailor the substrate specificities of the native
beta-oxidation machinery of E. coli. Thus these enzymes or
homologues thereof can be applied for adipate production. If the
native genes operate to degrade adipate or its precursors in vivo,
the appropriate genetic modifications are made to attenuate or
eliminate these functions. However, it may not be necessary since a
method for producing poly[(R)-3-hydroxybutyrate] in E. coli that
involves activating fadB, by knocking out a negative regulator,
fadR, and co-expressing a non-native ketothiolase, phaA from
Ralstonia eutropha, has been described (Sato et al., J. Biosci.
Bioeng. 103:38-44 (2007)). This work clearly demonstrated that a
beta-oxidation enzyme, in particular the gene product of fadB which
encodes both 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA
hydratase activities, can function as part of a pathway to produce
longer chain molecules from acetyl-CoA precursors. The protein
sequences for each of these exemplary gene products can be found
using the following GenBank accession numbers:
TABLE-US-00169 Gene Name GenBank ID Organism fadA YP_026272.1
Escherichia coli fadB NP_418288.1 Escherichia coli fadI NP_416844.1
Escherichia coli fadJ NP_416843.1 Escherichia coli fadR NP_415705.1
Escherichia coli
[0603] Step D--5-Carboxy-2-Pentenoyl-CoA Reductase. EC 1.3.1.a
Oxidoreductase Operating on CH--CH Donors.
[0604] Step D involves the conversion of 5-carboxy-2-pentenoyl-CoA
to adipyl-CoA by 5-carboxy-2-pentenoyl-CoA reductase. Enoyl-CoA
reductase enzymes are suitable enzymes for this transformation.
[0605] Whereas the ketothiolase, dehydrogenase, and enoyl-CoA
hydratase steps are generally reversible, the enoyl-CoA reductase
step is almost always oxidative and irreversible under
physiological conditions (Hollmeister et al., Biol. Chem.
280:4329-4338 (2005)). FadE catalyzes this likely irreversible
transformation in E. coli (Campbell and Cronan, J Bacteriol.
184:3759-3764 (2002)). The pathway can involve an enzyme that
reduces a 2-enoyl-CoA intermediate, not one such as FadE that will
only oxidize an acyl-CoA to a 2-enoyl-CoA compound. Furthermore,
although it has been suggested that E. coli naturally possesses
enzymes for enoyl-CoA reduction (Mizugaki et al., Biochem.
92:1649-1654 (1982); Nishimaki et al., J Biochem. 95:1315-1321
(1984)), no E. coli gene possessing this function has been
biochemically characterized.
[0606] One exemplary enoyl-CoA reductase is the gene product of bcd
from C. acetobutylicum (Boynton et al., J Bacteriol. 178:3015-3024
(1996); Atsumi et al., Metab. Eng. 2008 10(6):305-311 (2008)(Epub
Sep. 14, 2007), which naturally catalyzes the reduction of
crotonyl-CoA to butyryl-CoA. Activity of this enzyme can be
enhanced by expressing bcd in conjunction with expression of the C.
acetobutylicum etfAB genes, which encode an electron transfer
flavoprotein. An additional candidate for the enoyl-CoA reductase
step is the mitochondrial enoyl-CoA reductase from E. gracilis
(Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). A
construct derived from this sequence following the removal of its
mitochondrial targeting leader sequence was cloned in E. coli
resulting in an active enzyme (Hoffmeister et al., supra). This
approach is well known to those skilled in the art of expressing
eukaryotic genes, particularly those with leader sequences that may
target the gene product to a specific intracellular compartment, in
prokaryotic organisms. A close homolog of this gene, TDE0597, from
the prokaryote Treponema denticola represents a third enoyl-CoA
reductase which has been cloned and expressed in E. coli (Tucci et
al., FEBS Letters 581:1561-1566 (2007)).
TABLE-US-00170 Gene name GI Number GenBank ID Organism bcd 15895968
NP_349317.1 Clostridium acetobutylicum etfA 15895966 NP_349315.1
Clostridium acetobutylicum etfB 15895967 NP_349316.1 Clostridium
acetobutylicum TER 62287512 Q5EU90.1 Euglena gracilis TDE0597
42526113 NP_971211.1 Treponema denticola
[0607] Step E--Adipyl-CoA Reductase (Aldehyde Forming). EC 1.2.1.b
Oxidoreductase (Acyl-CoA to Aldehyde).
[0608] The transformation of adipyl-CoA to adipate semialdehyde in
step E can involve an acyl-CoA dehydrogenases capable of reducing
an acyl-CoA to its corresponding aldehyde. An EC 1.2.1.b
oxidoreductase (acyl-CoA to aldehyde) provides suitable enzyme
activity. Exemplary enzymes in this class are described above.
[0609] Step F--6-Aminocaproate Transaminase or 6-Aminocaproate
Dehydrogenase. EC 1.4.1.a Oxidoreductase Operating on Amino
Acids.
[0610] Step F depicts a reductive amination involving the
conversion of adipate semialdehyde to 6-aminocaproate.
[0611] Most oxidoreductases operating on amino acids catalyze the
oxidative deamination of alpha-amino acids with NAD+ or NADP+ as
acceptor, though the reactions are typically reversible. Exemplary
oxidoreductases operating on amino acids include glutamate
dehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase
(deaminating), encoded by ldh, and aspartate dehydrogenase
(deaminating), encoded by nadX. The gdhA gene product from
Escherichia coli (McPherson et al., Nucleic. Acids Res.
11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273
(1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles
1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998);
Lebbink et al., J. Mol. Biol. 289:357-369 (1999)), and gdhA1 from
Halobacterium salinarum (Ingoldsby et al., Gene. 349:237-244
(2005)) catalyze the reversible interconversion of glutamate to
2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or
both, respectively. Exemplary enzymes are described in the table
below.
TABLE-US-00171 Gene name GI Number GenBank ID Organism gdhA 118547
P00370 Escherichia coli gdh 6226595 P96110.4 Thermotoga maritima
gdhA1 15789827 NP_279651.1 Halobacterium salinarum ldh 61222614
P0A393 Bacillus cereus nadX 15644391 NP_229443.1 Thermotoga
maritima
[0612] The lysine 6-dehydrogenase (deaminating), encoded by the
lysDHgenes, catalyze the oxidative deamination of the epsilon-amino
group of L-lysine to form 2-aminoadipate-6-semialdehyde, which in
turn nonenzymatically cyclizes to form
.DELTA..sup.1-piperideine-6-carboxylate (Misono et al., J.
Bacteriol. 150:398-401 (1982)). Exemplary enzymes are shown in the
table below. Such enzymes are particularly good candidates for
converting adipate semialdehyde to 6-aminocaproate given the
structural similarity between adipate semialdehyde and
2-aminoadipate-6-semialdehyde.
TABLE-US-00172 Gene name GI Number GenBank ID Organism lysDH
13429872 BAB39707 Geobacillus stearothermophilus lysDH 15888285
NP_353966 Agrobacterium tumefaciens lysDH 74026644 AAZ94428
Achromobacter denitrificans
[0613] EC 2.6.1.a Aminotransferase.
[0614] Step F of FIG. 8 can also, in certain embodiments, involve
the transamination of a 6-aldehyde to an amine. This transformation
can be catalyzed by gamma-aminobutyrate transaminase (GABA
transaminase). Exemplary enzymes are shown in the table below.
TABLE-US-00173 Gene name GI Number GenBank ID Organism gabT
16130576 NP_417148.1 Escherichia coli puuE 16129263 NP_415818.1
Escherichia coli abat 37202121 NP_766549.2 Mus musculus gabT
70733692 YP_257332.1 Pseudomonas fluorescens abat 47523600
NP_999428.1 Sus scrofa
[0615] Additional enzyme candidates include putrescine
aminotransferases or other diamine aminotransferases. Such enzymes
are particularly well suited for carrying out the conversion of
6-aminocaproate semialdehyde to hexamethylenediamine. The E. coli
putrescine aminotransfemse is encoded by the ygjG gene and the
purified enzyme also was able to transaminate cadaverine and
spermidine (Samsonova et al., BMC Microbiol 3:2 (2003)). In
addition, activity of this enzyme on 1,7-diaminoheptane and with
amino acceptors other than 2-oxoglutarate (e.g., pyruvate,
2-oxobutanoate) has been reported (Samsonova et al., supra; Kim, K.
H., J Biol Chem 239:783-786 (1964)). A putrescine aminotransfemse
with higher activity with pyruvate as the amino acceptor than
alpha-ketoglutamte is the spuC gene of Pseudomonas aeruginosa (Lu
et al., J Bacteriol 184:3765-3773 (2002)).
TABLE-US-00174 Gene name GI Number GenBank ID Organism ygjG
145698310 NP_417544 Escherichia coli spuC 9946143 AAG03688
Pseudomonas aeruginosa
[0616] Yet additional candidate enzymes include
beta-alanine/alpha-ketoglutamte aminotransferases which produce
malonate semialdehyde from beta-alanine (WO08027742). Exemplary
enzymes are shown in the table below.
TABLE-US-00175 Gene name GI Number GenBank ID Organism SkyPYD4
98626772 ABF58893.1 Saccharomyces kluyveri SkUGA1 98626792
ABF58894.1 Saccharomyces kluyveri UGA1 6321456 NP_011533.1
Saccharomyces cerevisiae Abat 122065191 P50554.3 Rattus norvegicus
Abat 120968 P80147.2 Sus scrofa
[0617] Step G--6-Aminocaproyl-CoA/Acyl-CoA Transferase or
6-Aminocaproyl-CoA Synthase.
[0618] 2.8.3.a Coenzyme-A Transferase.
[0619] CoA transferases catalyze reversible reactions that involve
the transfer of a CoA moiety from one molecule to another. For
example, step G can be catalyzed by a 6-aminocaproyl-CoA/Acyl CoA
transferase. One candidate enzyme for these steps is the two-unit
enzyme encoded by pcaI and pcaJ in Pseudomonas, which has been
shown to have 3-oxoadipyl-CoA/succinate transferase activity
((Kaschabek and Reineke, J. Bacteriol. 177:320-325 (1995); and
Kaschabek. and Reineke, J. Bacteriol. 175:6075-6081 (1993)).
Similar enzymes based on homology exist in Acinetobacter sp. ADP1
(Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces
coelicolor. Exemplary enzymes are shown in the table below.
TABLE-US-00176 Gene name GI Number GenBank ID Organism pcaI
24985644 AAN69545.1 Pseudomonas putida pcaJ 26990657 NP_746082.1
Pseudomonas putida pcaI 50084858 YP_046368.1 Acinetobacter sp. ADP1
pcaJ 141776 AAC37147.1 Acinetobacter sp. ADP1 pcaI 21224997
NP_630776.1 Streptomyces coelicolor pcaJ 21224996 NP_630775.1
Streptomyces coelicolor HPAG1_0676 108563101 YP_627417 Helicobacter
pylori HPAG1_0677 108563102 YP_627418 Helicobacter pylori ScoA
16080950 NP_391778 Bacillus subtilis ScoB 16080949 NP_391777
Bacillus subtilis
[0620] A 3-oxoacyl-CoA transferase that can utilize acetate as the
CoA acceptor is acetoacetyl-CoA transferase, encoded by the E. coli
atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et
al., Biochem. Biophys. Res Commun. 33:902-908 (1968); Korolev et
al., Acta Crystallogr. D Biol Crystallogr. 58:2116-2121 (2002)).
This enzyme has also been shown to transfer the CoA moiety to
acetate from a variety of branched and linear acyl-CoA substrates,
including isobutyrate (Matthies et al., Appl Environ Microbiol
58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra) and
butanoate (Vanderwinkel et al., supra). Exemplary enzymes are shown
in the table below.
TABLE-US-00177 Gene name GI Number GenBank ID Organism atoA 2492994
P76459.1 Escherichia coli K12 atoD 2492990 P76458.1 Escherichia
coli K12 actA 62391407 YP_226809.1 Corynebacterium glutamicum ATCC
13032 cg0592 62389399 YP_224801.1 Corynebacterium glutamicum ATCC
13032 ctfA 15004866 NP_149326.1 Clostridium acetobutylicum ctfB
15004867 NP_149327.1 Clostridium acetobutylicum ctfA 31075384
AAP42564.1 Clostridium saccharoperbutylacetonicum ctfB 31075385
AAP42565.1 Clostridium saccharoperbutylacetonicum
[0621] The above enzymes may also exhibit the desired activities on
6-aminocaproate and 6-aminocaproyl-CoA, as in step G. Nevertheless,
additional exemplary transferase candidates are catalyzed by the
gene products of cat1, cat2, and cat3 of Clostridium kluyveri which
have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and
butyryl-CoA transferase activity, respectively (Seedorf et al.,
supra; Sohling et al., Eur. J Biochem. 212:121-127 (1993); Sohling
et al., J Bacteriol. 178:871-880 (1996)).
TABLE-US-00178 Gene name GI Number GenBank ID Organism cat1 729048
P38946.1 Clostridium kluyveri cat2 172046066 P38942.2 Clostridium
kluyveri cat3 146349050 EDK35586.1 Clostridium kluyveri
[0622] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from
anaerobic bacterium Acidaminococcus fermentans reacts with diacid
glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett.
405:209-212 (1997)). The genes encoding this enzyme are gctA and
gctB. This enzyme has reduced but detectable activity with other
CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA,
adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J. Biochem.
118:315-321 (1981)). The enzyme has been cloned and expressed in E.
coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).
TABLE-US-00179 Gene name GI Number GenBank ID Organism gctA 559392
CAA57199.1 Acidaminococcus fermentans gctB 559393 CAA57200.1
Acidaminococcus fermentans
[0623] EC 6.2.1.a Acid-Thiol Ligase.
[0624] Step G can also involve an acid-thiol ligase or synthetase
functionality (the terms ligase, synthetase, and synthase are used
herein interchangeably and refer to the same enzyme class).
Exemplary genes encoding enzymes to may out these transformations
include the sucCD genes of E. coli which naturally form a
succinyl-CoA synthetase complex. This enzyme complex naturally
catalyzes the formation of succinyl-CoA from succinate with the
contaminant consumption of one ATP, a reaction which is reversible
in vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Given the
structural similarity between succinate and adipate, that is, both
are straight chain dicarboxylic acids, it is reasonable to expect
some activity of the sucCD enzyme on adipyl-CoA.
TABLE-US-00180 Gene name GI Number GenBank ID Organism sucC
16128703 NP_415256.1 Escherichia coli sucD 1786949 AAC73823.1
Escherichia coli
[0625] Additional exemplary CoA-ligases include the rat
dicarboxylate-CoA ligase for which the sequence is yet
uncharacterized (Vamecq et al., Biochemical Journal 230:683-693
(1985)), either of the two characterized phenylacetate-CoA ligases
from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395:147-155
(2005); Wang et al., Biochem Biophy Res Commun 360(2):453-458
(2007)), the phenylacetate-CoA ligase from Pseudomonas putida
(Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and
the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Boweret
al., J. Bacteriol. 178(14):4122-4130 (1996)). Exemplary enzymes are
shown in the table below
TABLE-US-00181 Gene name GI Number GenBank ID Organism phl 77019264
CAJ15517.1 Penicillium chrysogenum phlB 152002983 ABS19624.1
Penicillium chrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida
bioW 50812281 NP_390902.2 Bacillus subtilis AACS 21313520
NP_084486.1 Mus musculus AACS 31982927 NP_076417.2 Homo sapiens
[0626] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is
another candidate enzyme that couples the conversion of acyl-CoA
esters to their corresponding acids with the concurrent synthesis
of ATP. Several enzymes with broad substrate specificities have
been described in the literature. ACD I from Archaeoglobus
fulgidus, encoded by AF1211, was shown to operate on a variety of
linear and branched-chain substrates including acetyl-CoA,
propionyl-CoA, butyryl-CoA, acetate, propionate, butyrate,
isobutyryate, isovalerate, succinate, fumarate, phenylacetate,
indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)).
The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA
synthetase) accepts propionate, butyrate, and branched-chain acids
(isovalerate and isobutyrate) as substrates, and was shown to
operate in the forward and reverse directions (Brasen et al., Arch
Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from
hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the
broadest substrate range of all characterized ACDs, reacting with
acetyl-CoA, isobutyryl-CoA (preferred substrate) and
phenylacetyl-CoA (Brasen et al., supra). The enzymes from A.
fulgidus, H marismortui and P. aerophilum have all been cloned,
functionally expressed, and characterized in E. coli (Musfeldt et
al., supra; Brasen et al., supra).
TABLE-US-00182 Gene name GI Number GenBank ID Organism AF1211
11498810 NP_070039.1 Archaeoglobus fulgidus DSM 4304 Scs 55377722
YP_135572.1 Haloarcula marismortui ATCC 43049 PAE3250 18313937
NP_560604.1 Pyrobaculum aerophilum str. IM2
[0627] Yet another option is to employ a set of enzymes with net
ligase or synthetase activity. For example, phosphotransadipylase
and adipate kinase enzymes are catalyzed by the gene products of
buk1, buk2, and ptb from C. acetobutylicum (Walter et al., Gene
134:107-111 (1993); Huang et al., J. Mol. Microbial. Biotechnol.
2:33-38 (2000)). The ptb gene encodes an enzyme that can convert
butyryl-CoA into butyryl-phosphate, which is then converted to
butyrate via either of the buk gene products with the concomitant
generation of ATP.
TABLE-US-00183 Gene name GI Number GenBank ID Organism ptb 15896327
NP_349676 Clostridium acetobutylicum buk1 15896326 NP_349675
Clostridium acetobutylicum buk2 20137415 Q97II1 Clostridium
acetobutylicum
[0628] Step H--Amidohydrolase. EC 6.3.1.a/6.3.2.a Amide
Synthases/Peptide Synthases.
[0629] The direct conversion of 6-aminocaproate to caprolactam as
in step H can involve the formation of an intramolecular peptide
bond. Ribosomes, which assemble amino acids into proteins during
translation, are nature's most abundant peptide bond-forming
catalysts. Nonribosomal peptide synthetases are peptide bond
forming catalysts that do not involve messenger mRNA (Schwarzer et
al., Nat Prod. Rep. 20:275-287 (2003)). Additional enzymes capable
of forming peptide bonds are shown in the table below.
TABLE-US-00184 Gene name GI Number GenBank ID Organism acsA
60650089 BAD90933 Pseudomonas chlororaphis puuA 87081870 AAC74379
Escherichia coli bls 41016784 Q9R8E3 Streptomyces clavuligerus
[0630] Step I--Spontaneous Cyclization.
[0631] The conversion of 6-aminocaproyl-CoA to caprolactam can
occur by spontaneous cyclization. Because 6-aminocaproyl-CoA can
cyclize spontaneously to caprolactam, it eliminates the need for a
dedicated enzyme for this step. A similar spontaneous cyclization
is observed with 4-aminobutyryl-CoA which forms pyrrolidinone
(Ohsugi et al., J Biol Chem 256:7642-7651 (1981)).
[0632] Step J--6-Aminocaproyl-CoA Reductase (Aldehyde Forming).
[0633] The transformation of 6-aminocaproyl-CoA to 6-aminocaproate
semialdehyde as in step J can involve an acyl-CoA dehydrogenases
capable of reducing an acyl-CoA to its corresponding aldehyde. An
EC 1.2.1.b oxidoreductase (acyl-CoA to aldehyde) provides suitable
enzyme activity. Exemplary enzymes in this class are described
above.
[0634] Step K--HMDA Transaminase or HMDA Dehydrogenase.
[0635] EC 1.4.1.a Oxidoreductase Operating on Amino Acids.
[0636] Step K depicts a reductive animation and entails the
conversion of 6-aminocaproate semialdehyde to
hexamethylenediamine.
[0637] Most oxidoreductases operating on amino acids catalyze the
oxidative deamination of alpha-amino acids with NAD+ or NADP+ as
acceptor, though the reactions are typically reversible. Exemplary
oxidoreductases operating on amino acids of the EC 1.4.1. class are
described elsewhere herein, for example those for Step F acting on
6-aminocaproate transaminase or 6-aminocaproate dehydrogenase. The
lysine 6-dehydrogenase (deaminating), encoded by the lysDH genes,
catalyze the oxidative deamination of the epsilon-amino group of
L-lysine to form 2-aminoadipate-6-semialdehyde, which in turn
nonenzymatically cyclizes to form A'-piperideine-6-carboxylate
(Misono et al., J. Bacteriol. 150:398-401 (1982)). Exemplary
enzymes can be found in Geobacillus stearothermophilus (Heydari et
al., Appl Environ. Microbiol 70:937-942 (2004)), Agrobacterium
tumefaciens (Hashimoto et al., J Biochem 106:76-80 (1989); Misono
et al., supra), and Achromobacter denitrificans (Ruldeekulthamrong
et al., BMB. Rep. 41:790-795 (2008)). Such enzymes are particularly
good candidates for converting adipate semialdehyde to
6-aminocaproate given the structural similarity between adipate
semialdehyde and 2-aminoadipate-6-semialdehyde.
TABLE-US-00185 Gene name GI Number GenBank ID Organism lysDH
13429872 BAB39707 Geobacillus stearothermophilus lysDH 15888285
NP_353966 Agrobacterium tumefaciens lysDH 74026644 AAZ94428
Achromobacter denitrificans
[0638] EC 2.6.1.a Aminotransferase.
[0639] Step K, in certain embodiments, can involve the
transamination of a 6-aldehyde to an amine. This transformation can
be catalyzed by gamma-aminobutyrate transaminase (GABA
transaminase). Examples are below.
TABLE-US-00186 Gene name GI Number GenBank ID Organism gabT
16130576 NP_417148.1 Escherichia coli puuE 16129263 NP_415818.1
Escherichia coli abat 37202121 NP_766549.2 Mus musculus gabT
70733692 YP_257332.1 Pseudomonas fluorescens abat 47523600
NP_999428.1 Sus scrofa
[0640] Additional enzyme candidates include putrescine
aminotransferases or other diamine aminotransferases. Such enzymes
are particularly well suited for carrying out the conversion of
6-aminocaproate semialdehyde to hexamethylenediamine. Examples are
described below and elsewhere herein.
TABLE-US-00187 Gene name GI Number GenBank ID Organism ygjG
145698310 NP_417544 Escherichia coli spuC 9946143 AAG03688
Pseudomonas aeruginosa
[0641] Yet additional candidate enzymes include
beta-alanine/alpha-ketoglutarate aminotransferases which produce
malonate semialdehyde from beta-alanine (WO08027742). Exemplary
candidates are described elsewhere herein, such as for Step F. Step
L--Adipyl-CoA Hydrolase, Adipyl-CoA Ligase, Adipyl-CoA Transferase
or Phosphotransadipylase/Adipate Kinase. Step L can involve
adipyl-CoA synthetase (also referred to as adipate-CoA ligase),
phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA
transferase, or adipyl-CoA hydrolase. From an energetic standpoint,
it is desirable for the final step in the adipate synthesis pathway
to be catalyzed by an enzyme or enzyme pair that can conserve the
ATP equivalent stored in the thioester bond of adipyl-CoA. The
product of the sucC and sucD genes of E. coli, or homologs thereof,
can potentially catalyze the final transformation shown in FIG. 8
should they exhibit activity on adipyl-CoA. The sucCD genes
naturally form a succinyl-CoA synthetase complex that catalyzes the
formation of succinyl-CoA from succinate with the concaminant
consumption of one ATP, a reaction which is reversible in vivo
(Buck et al., Biochem. 24:6245-6252 (1985)). Given the structural
similarity between succinate and adipate, that is, both are
straight chain dicarboxylic acids, it is reasonable to expect some
activity of the sucCD enzyme on adipyl-CoA. An enzyme exhibiting
adipyl-CoA ligase activity can equivalently carry out the
ATP-generating production of adipate from adipyl-CoA, here using
AMP and PPi as cofactors, when operating in the opposite
physiological. Exemplary CoA-ligases include the rat
dicarboxylate-CoA ligase for which the sequence is yet
uncharacterized (Vamecq et al., Biochem. J. 230:683-693 (1985)),
either of the two characterized phenylacetate-CoA ligases from P.
chrysogenum (Lamas-Maceiras et al., Biochem. J 395, 147-155 (2005);
Wang et al., Biochem. Biophy. Res. Commun. 360:453-458 (2007)), the
phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco
et al., J. Biol. Chem. 265:7084-7090 (1990)), and the
6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al.,
J. Bacteriol. 178:4122-4130 (1996)). The protein sequences for each
of these exemplary gene products can be found using the following
GI numbers and/or GenBank identifiers:
TABLE-US-00188 Gene name GI Number GenBank ID Organism sucC
16128703 NP_415256.1 Escherichia coli sucD 1786949 AAC73823.1
Escherichia coli
[0642] Another option, using phosphotransadipylase/adipate kinase,
is catalyzed by the gene products of buk1, buk2, and ptb from C.
acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et
al., J. Mol. Microbiol. Biotechnol. 2:33-38 (2000)), or homologs
thereof. The ptb gene encodes an enzyme that can convert
butyryl-CoA into butyryl-phosphate, which is then converted to
butyrate via either of the buk gene products with the concomitant
generation of ATP. The analogous set of transformations, that is,
conversion of adipyl-CoA to adipyl-phosphate followed by conversion
of adipyl-phosphate to adipate, can be carried out by the buk1,
buk2, and ptb gene products. The protein sequences for each of
these exemplary gene products can be found using the following GI
numbers and/or GenBank identifiers:
TABLE-US-00189 Gene name GI Number GenBank ID Organism ptb 15896327
NP_349676 Clostridium acetobutylicum buk1 15896326 NP_349675
Clostridium acetobutylicum buk2 20137415 Q97II1 Clostridium
acetobutylicum
[0643] Alternatively, an acetyltransferase capable of transferring
the CoA group from adipyl-CoA to acetate can be applied. Similar
transformations are catalyzed by the gene products of cat1, cat2,
and cat3 of Clostridium kluyveri which have been shown to exhibit
succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA
acetyltransferase activity, respectively (Sohling and Gottschalk,
J. Bacteriol. 178:871-880 (1996); Seedorf et al., Proc. Natl. Acad.
Sci. USA 105:2128-2133 (2008)). The protein sequences for each of
these exemplary gene products can be found using the following GI
numbers and/or GenBank identifiers:
TABLE-US-00190 Gene name GI Number GenBank ID Organism cat1 729048
P38946.1 Clostridium kluyveri cat2 172046066 P38942.2 Clostridium
kluyveri cat3 146349050 EDK35586.1 Clostridium kluyveri
[0644] Finally, though not as desirable from an energetic
standpoint, the conversion of adipyl-CoA to adipate can also be
carried out by an acyl-CoA hydrolase or equivalently a
thioesterase. The top E. coli gene candidate is tesB (Naggert et
al., J. Biol. Chem. 266:11044-11050 (1991)), which shows high
similarity to the human acot8, which is a dicarboxylic acid
acetyltransferase with activity on adipyl-CoA (Westin et al., J.
Biol. Chem. 280:38125-38132 (2005)). This activity has also been
characterized in the rat liver (Deana, Biochem. Int. 26:767-773
(1992)). The protein sequences for each of these exemplary gene
products can be found using the following GI numbers and/or GenBank
identifiers:
TABLE-US-00191 Gene name GI Number GenBank ID Organism tesB
16128437 NP_414986 Escherichia coli acot8 3191970 CAA15502 Homo
sapiens acot8 51036669 NP_570112 Rattus norvegicus
[0645] Other native candidate genes include those in the table
below. The protein sequences for each of these exemplary gene
products can be found using the following GI numbers and/or GenBank
identifiers:
TABLE-US-00192 Gene name GI Number GenBank ID Organism tesA
16128478 NP_415027 Escherichia coli ybgC 16128711 NP_415264
Escherichia coli paaI 16129357 NP_415914 Escherichia coli ybdB
16128580 NP_415129 Escherichia coli
[0646] EC 2.8.3.a Coenzyme-A Transferase.
[0647] CoA transferases catalyze reversible reactions that involve
the transfer of a CoA moiety from one molecule to another. For
example, step L can be catalyzed by a adipyl-CoA transferase. One
candidate enzyme for this step is the two-unit enzyme encoded by
pcaI and pcaJ in Pseudomonas, which has been shown to have
3-oxoadipyl-CoA/succinate transferase activity (Kaschabek and
Reineke, J Bacteriol. 177:320-325 (1995); and Kaschabek. and
Reineke, J. Bacteriol. 175:6075-6081 (1993)). Similar enzymes based
on homology exist in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene
146:23-30 (1994)) and Streptomyces coelicolor. Additional exemplary
succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacter
pylori (Corthesy-Theulaz et al., J. Biol. Chem. 272:25659-25667
(1997)) and Bacillus subtilis (Stols et al., Protein. Expr. Punf.
53:396-403 (2007)).
TABLE-US-00193 Gene name GI Number GenBank ID Organism pcaI
24985644 AAN69545.1 Pseudomonas putida pcaJ 26990657 NP_746082.1
Pseudomonas putida pcaI 50084858 YP_046368.1 Acinetobacter sp. ADP1
pcaJ 141776 AAC37147.1 Acinetobacter sp. ADP1 pcaI 21224997
NP_630776.1 Streptomyces coelicolor pcaJ 21224996 NP_630775.1
Streptomyces coelicolor HPAG1_0676 108563101 YP_627417 Helicobacter
pylori HPAG1_0677 108563102 YP_627418 Helicobacter pylori ScoA
16080950 NP_391778 Bacillus subtilis ScoB 16080949 NP_391777
Bacillus subtilis
[0648] A 3-oxoacyl-CoA transferase that can utilize acetate as the
CoA acceptor is acetoacetyl-CoA transferase, encoded by the E. coli
atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et
al., Biochem. Biophys. Res Commun. 33:902-908 (1968); Korolev et
al., Acta Crystallogr. D Biol Crystallogr. 58:2116-2121 (2002)).
Exemplary candidates are shown in the table below.
TABLE-US-00194 Gene name GI Number GenBank ID Organism atoA 2492994
P76459.1 Escherichia coli K12 atoD 2492990 P76458.1 Escherichia
coli K12 actA 62391407 YP_226809.1 Corynebacterium glutamicum ATCC
13032 cg0592 62389399 YP_224801.1 Corynebacterium glutamicum ATCC
13032 ctfA 15004866 NP_149326.1 Clostridium acetobutylicum ctfB
15004867 NP_149327.1 Clostridium acetobutylicum ctfA 31075384
AAP42564.1 Clostridium saccharoperbutylacetonicum ctfB 31075385
AAP42565.1 Clostridium saccharoperbutylacetonicum
[0649] The above enzymes may also exhibit the desired activities on
adipyl-CoA and adipate for step L. Nevertheless, additional
exemplary transferase candidates are catalyzed by the gene products
of cat1, cat2, and cat3 of Clostridium kluyveri which have been
shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and
butyryl-CoA transferase activity, respectively (Seedorf et al.,
Proc. Natl. Acad. Sci U S. A 105:2128-2133 (2008); Sohling et al.,
Eur. J Biochem. 212:121-127 (1993); Sohling et al., J Bacterial.
178:871-880 (1996)).
TABLE-US-00195 Gene name GI Number GenBank ID Organism cat1 729048
P38946.1 Clostridium kluyveri cat2 172046066 P38942.2 Clostridium
kluyveri cat3 146349050 EDK35586.1 Clostridium kluyveri
[0650] The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from
anaerobic bacterium Acidaminococcus fermentans reacts with diacid
glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett.
405:209-212 (1997)). The genes encoding this enzyme are gctA and
gctB. This enzyme has reduced but detectable activity with other
CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA,
adipyl-CoA and acrylyl-CoA (Buckel et al., Eur. J. Biochem.
118:315-321 (1981)). The enzyme has been cloned and expressed in E.
coli (Mack et al., Eur. J. Biochem. 226:41-51 (1994)).
TABLE-US-00196 Gene name GI Number GenBank ID Organism gctA 559392
CAA57199.1 Acidaminococcus fermentans gctB 559393 CAA57200.1
Acidaminococcus fermentans
Example X
Methacrylic Acid Synthesis Enzymes
[0651] This Example provides genes that can be used for conversion
of succinyl-CoA to methacrylic acid as depicted in the pathways of
FIG. 9.
[0652] FIG. 9. depicts 3-Hydroxyisobutymte and methacrylic acid
production are carried out by the following enzymes: A)
Methylmalonyl-CoA mutase, B) Methylmalonyl-CoA epimerase, C)
Methylmalonyl-CoA reductase (aldehyde limning), D) Methylmalonate
semialdehyde reductase, E) 3-hydroxyisobutyrate dehydratase, F)
Methylmalonyl-CoA reductase (alcohol forming).
[0653] Step A--Methylmalonyl-CoA mutase (designated as EMA2).
[0654] Methylmalonyl-CoA mutase (MCM) (EMA2) (EC 5.4.99.2) is a
cobalamin-dependent enzyme that converts succinyl-CoA to
methylmalonyl-CoA. In E coli, the reversible
adenosylcobalamin-dependent mutase participates in a three-step
pathway leading to the conversion of succinate to propionate
(Haller et al., Biochemistry 39:4622-4629 (2000)). Overexpression
of the EMA2 gene candidate along with the deletion of YgfG can be
used to prevent the decarboxylation of methylmalonyl-CoA to
propionyl-CoA and to maximize the methylmalonyl-CoA available for
MAA synthesis. EMA2 is encoded by genes scpA in Escherichia coli
(Bobik and Rasche, Anal. Bioanal. Chem. 375:344-349 (2003); Haller
et al., Biochemistry 39:4622-4629 (2000)) and mutA in Homo sapiens
(Padovani and Banenee, Biochemistry 45:9300-9306 (2006)). In
several other organisms EMA2 contains alpha and beta subunits and
is encoded by two genes. Exemplary gene candidates encoding the
two-subunit protein are Propionibacterium fredenreichii sp.
shermani mutA and mutB (Korotkova and Lidstrom, J Biol. Chem.
279:13652-13658 (2004)), Methylobacterium extorquens mcmA and mcmB
(Korotkova and Lidstrom, supra, 2004), and Ralstonia eutropha sbm1
and sbm2 (Peplinski et al., Appl. Microbiol. Biotech. 88:1145-59
(2010)). Additional enzyme candidates identified based on high
homology to the E. coli spcA gene product are also listed
below.
TABLE-US-00197 Protein GenBank ID GI Number Organism scpA
NP_417392.1 16130818 Escherichia coli K12 mutA P22033.3 67469281
Homo sapiens mutA P11652.3 127549 Propionibacterium fredenreichii
sp. shermanii mutB P11653.3 127550 Propionibacterium fredenreichii
sp. shermanii mcmA Q84FZ1 75486201 Methylobacterium extorquens mcmB
Q6TMA2 75493131 Methylobacterium extorquens Sbm1 YP_724799.1
113866310 Ralstonia eutropha H16 Sbm2 YP_726418.1 113867929
Ralstonia eutropha H16 sbm NP_838397.1 30064226 Shigella flexneri
SARI_04585 ABX24358.1 160867735 Salmonella enterica YfreA_01000861
ZP_00830776.1 77975240 Yersinia frederiksenii
[0655] These sequences can be used to identify homologue proteins
in GenBank or other databases through sequence similarity searches
(for example, BLASTp). The resulting homologue proteins and their
corresponding gene sequences provide additional exogenous DNA
sequences for transformation into E. coli or other suitable host
microorganisms to generate production hosts. Additional gene
candidates include the following, which were identified based on
high homology to the E. coli spcA gene product.
[0656] There further exists evidence that genes adjacent to the
EMA2 catalytic genes contribute to maximum activity. For example,
it has been demonstrated that the meaB gene from M. extorquens
forms a complex with EMA2, stimulates in vitro mutase activity, and
possibly protects it from irreversible inactivation (Korotkova and
Lidstrom, J. Biol. Chem. 279:13652-13658 (2004)). The M. extorquens
meaB gene product is highly similar to the product of the E coli
argKgene (BLASTp: 45% identity, e-value: 4e-67), which is adjacent
to scpA on the chromosome. No sequence for a meaB homolog in P.
freudenreichii is catalogued in GenBank. However, the
Propionibacterium acnes KPA171202 gene product, YP 055310.1, is 51%
identical to the M. extorquens meaB protein and its gene is also
adjacent to the EMA2 gene on the chromosome. A similar gene is
encoded by H16_B1839 of Ralstonia eutropha H16.
TABLE-US-00198 Gene GenBank ID GI Number Organism argK AAC75955.1
1789285 Escherichia coli K12 PPA0597 YP_055310.1 50842083
Propionibacterium acnes KPA171202 2QM8_B 158430328 Methylobacterium
extorquens H16_B1839 YP_841351.1 116695775 Ralstonia eutropha
H16
[0657] E. coli can synthesize adenosylcobalamin, a necessary
cofactor for this reaction, only when supplied with the
intermediates cobinamide or cobalamin (Lawrence and Roth. J.
Bacteriol. 177:6371-6380 (1995); Lawrence and Roth, Genetics
142:11-24 (1996)). Alternatively, the ability to synthesize
cobalamins de novo has been conferred upon E coli following the
expression of heterologous genes (Raux et al., J. Bacteriol.
178:753-767 (1996)).
[0658] Alternatively, isobutyryl-CoA mutase (ICM) (EC 5.4.99.13)
could catalyze the proposed transformation shown in FIG., step B.
ICM is a cobalamin-dependent methylmutase in the EMA2 family that
reversibly rearranges the carbon backbone of butyryl-CoA into
isobutyryl-CoA (Ratnatilleke et al., J. Biol. Chem. 274:31679-31685
(1999)). A recent study of a novel ICM in Methylibium
petroleiphilum, along with previous work, provides evidence that
changing a single amino acid near the active site alters the
substrate specificity of the enzyme (Ratnatilleke et al., J. Biol.
Chem. 274:31679-31685 (1999); Rohwerder et al., Appl. Environ.
Microbiol. 72:4128-4135. (2006)). This indicates that, if a native
enzyme is unable to catalyze or exhibits low activity for the
conversion of 4HB-CoA to 3HIB-CoA, the enzyme can be rationally
engineered to increase this activity. Exemplary genes/proteins are
identified below.
TABLE-US-00199 Gene GenBank ID GI Number Organism icmA CAB40912.1
4585853 Streptomyces coelicolor A3(2) Mpe B0541 YP_001023546.1
124263076 Methylibium petroleiphilum PM1 icm AAC08713.1 3002492
Streptomyces cinnamonensis icmB CAB59633.1 6137077 Streptomyces
cinnamonensis icmA NP_824008.1 29829374 Streptomyces avermitilis
icmB NP_824637.1 29830003 Streptomyces avermitilis
[0659] Step B--Methylmalonyl-CoA epimerase (designated as
EMA3).
[0660] Methylmalonyl-CoA epimerase (MMCE) (EMA3) is the enzyme that
interconverts (R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA. EMA3
is an essential enzyme in the breakdown of odd-numbered fatty acids
and of the amino acids valine, isoleucine, and methionine. EMA3
activity is not believed to be encoded in the E. coli genome
(Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), but is
present in other organisms; gene candidates include those shown
below. This enzymatic step may or may not be necessary depending
upon the stereospecificity of the enzyme or enzymes used for the
conversion of methylmalonyl-CoA to 3-HIB. These genes/proteins are
described below.
TABLE-US-00200 Gene GenBank ID GI Number Organism YqjC NP_390273
255767522 Bacillus subtilis MCEE Q96PE7.1 50401130 Homo sapiens
Mcee_predicted NP_001099811.1 157821869 Rattus norvegicus AF454511
AAL57846.1 18042135 Propionibacterium fredenreichii sp. shermanii
Mmce AAT92095.1 51011368 Caenorhabditis elegans AE016877 AAP08811.1
29895524 Bacillus cereus ATCC 14579
[0661] Step C--Methylmalonyl-CoA reductase (aldehyde forming)
(designated as EMA4).
[0662] The reduction of methylmalonyl-CoA to its corresponding
aldehyde, methylmalonate semialdehyde, is catalyzed by a
CoA-dependent aldehyde dehydrogenase (EC 1.2.1.-). Conversion of
methylmalonyl-CoA to methylmalonic semialdehyde, is accomplished by
a CoA-dependent aldehyde dehydrogenase. An enzyme encoded by a
malonyl-CoA reductase gene from Sulfolobus tokodaii (Alber et. al.,
J. Bacteriol 188(24):8551-8559 (2006)), has been shown to catalyze
the conversion of methylmalonyl-CoA to its corresponding aldehyde
(WO2007141208). A similar enzyme exists in Metallosphaera sedula
(Alber et. al., J. Bacteriol. 188(24):8551-8559 (2006)). Several
additional CoA dehydrogenases are capable also of reducing an
acyl-CoA to its corresponding aldehyde. The reduction of
methylmalonyl-CoA to its corresponding aldehyde, methylmalonate
semialdehyde, is catalyzed by a CoA-dependent aldehyde
dehydrogenase. Exemplary enzymes include fatty acyl-CoA reductase,
succinyl-CoA reductase (EC 1.2.1.76), acetyl-CoA reductase and
butyryl-CoA reductase. Exemplary fatty acyl-CoA reductase enzymes
are shown below. Also known is a CoA- and NADP-dependent succinate
semialdehyde dehydrogenase (also referred to as succinyl-CoA
reductase) encoded by the sucD gene in Clostridium kluyveri
(Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996); Sohling
and Gottschalk, J. Bacteriol. 178:871-880 (1996)) and sucD of P.
gingivalis (Takahashi, J. Bacteriol 182:4704-4710 (2000)).
Additional succinyl-CoA reductase enzymes participate in the
3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic
archaea. Exemplary enzymes are shown in the following table.
TABLE-US-00201 Protein GenBank ID GI Number Organism acr1
YP_047869.1 50086359 Acinetobacter calcoaceticus acr1 AAC45217
1684886 Acinetobacter baylyi acr1 BAB85476.1 18857901 Acinetobacter
sp. Strain M-1 MSED_0709 YP_001190808.1 146303492 Metallosphaera
sedula Tneu_0421 Thermoproteus neutrophilus sucD P38947.1 172046062
Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonas
gingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.1
55818563 Leuconostoc mesenteroides bld AAP42563.1 31075383
Clostridium saccharoperbutylacetonicum
[0663] An additional enzyme type that converts an acyl-CoA to its
corresponding aldehyde is malonyl-CoA reductase which transforms
malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key
enzyme in autotrophic carbon fixation via the 3-hydroxypropionate
cycle in themoacidophilic archaeal bacteria (Berg, Science
318:1782-1786 (2007); and Thauer, Science 318:1732-1733 (2007)).
The enzyme utilizes NADPH as a cofactor and has been characterized
in Metallosphaera and Sulfolobus sp. (Alber et al., J. Bacteriol.
188:8551-8559 (2006); and Hugler, J. Bacteriol. 184:2404-2410
(2002)). Exemplary enzymes include those in the following
table.
TABLE-US-00202 Gene GenBank ID GI Number Organism Msed 0709
YP_001190808.1 146303492 Metallosphaera sedula Mcr NP_378167.1
15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958 Sulfolobus
solfataricus Saci 2370 YP_256941.1 70608071 Sulfolobus
acidocaldarius Ald AAT66436 49473535 Clostridium beijerinckii eutE
AAA80209 687645 Salmonella typhimurium eutE P77445 2498347
Escherichia coli
[0664] A bifunctional enzyme with acyl-CoA reductase and alcohol
dehydrogenase activity can directly convert methylmalonyl-CoA to
3-HIB. Exemplary bifunctional oxidoreductases that convert an
acyl-CoA to alcohol include those that transform substrates such as
acetyl-CoA to ethanol (for example, aahE from E. coli (Kessler et
al., FEBS Lett. 281:59-63 (1991))) and butyryl-CoA to butanol (for
example, adhE2 from C. acetobutylicum (Fontaine et al., J.
Bacteriol. 184:821-830 (2002)). Exemplary enzymes are those in the
table below.
TABLE-US-00203 Protein GenBank ID GI Number Organism adhE
NP_415757.1 16129202 Escherichia coli adhE2 AAK09379.1 12958626
Clostridium acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides bdh I NP_349892.1 15896543 Clostridium acetobutylicum
bdh II NP_349891.1 15896542 Clostridium acetobutylicum Mcr
AAS20429.1 42561982 Chloroflexus aurantiacus Rcas 2929
YP_001433009.1 156742880 Roseiflexus castenholzii NAP1_02720
ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535
ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080
[0665] Step D--Methylmalonate Semialdehyde Reductase (Designated as
EMA5).
[0666] The reduction of methylmalonate semialdehyde to is catalyzed
by EMA5 or 3-HIB dehydrogenase. This enzyme participates in valine,
leucine and isoleucine degradation and has been identified in
bacteria, eukaryotes, and mammals. Exemplary enzymes are shown
below.
TABLE-US-00204 Protein GenBank ID GI Number Organism P84067 P84067
75345323 Thermus thermophilus 3hidh P31937.2 12643395 Homo sapiens
3hidh P32185.1 416872 Oryctolagus cuniculus mmsB NP_746775.1
26991350 Pseudomonas putida mmsB P28811.1 127211 Pseudomonas
aeruginosa dhat Q59477.1 2842618 Pseudomonas putida
[0667] Step E--3-HIB Dehydratase (designated as EMA6).
[0668] The dehydration of 3-HIB to MAA is catalyzed by an enzyme
with EMA6 activity (EC 4.2.1.-). The final step involves the
dehydration of 3-HIB to MAA The dehydration of 3-HIB to MAA is
catalyzed by an enzyme with EMA6 activity. Although no direct
evidence for this specific enzymatic transformation has been
identified, most dehydratases catalyze the alpha,beta-elimination
of water, which involves activation of the .quadrature.-hydrogen by
an electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester
group and removal of the hydroxyl group from the beta-position
(Buckel and Barker, J Bacteriol. 117:1248-1260 (1974); Martins et
al, Proc. Natl. Acad Sci. USA 101:15645-15649 (2004)). This is the
exact type of transformation proposed for the final step in the
methacrylate pathway. In addition, the proposed transformation is
highly similar to the 2-(hydroxymethyl)glutamte dehydratase of
Eubacterium barkeri, which can catalyze the conversion of
2-hydroxymethyl glutarate to 2-methylene glutarate. This enzyme has
been studied in the context of nicotinate catabolism and is encoded
by hmd (Alhapel et al., Proc. Natl. Acad. Sci. USA 103:12341-12346
(2006)). Similar enzymes with high sequence homology are found in
Bacteroides capillosus, Anaerotruncus colihominis, and
Natranaerobius thermophilius. Several enzymes are known to catalyze
the alpha, beta elimination of hydroxyl groups. Exemplary enzymes
include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-),
fumarase (EC 4.2.1.2), 2-keto-4-pentenoate dehydratase (EC
4.2.1.80), citramalate hydrolyase and dimethylmaleate
hydratase.
[0669] 2-(Hydroxymethyl)glutarate dehydratase is a
[4Fe-4S]-containing enzyme that dehydrates
2-(hydroxymethyl)glutarate to 2-methylene-glutarate, studied for
its role in nicontinate catabolism in Eubacterium barker/(formerly
Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci USA
103:12341-12346 (2006)). Similar enzymes with high sequence
homology are found in Bacteroides capillosus, Anaerotruncus
colihominis, and Natranaerobius thermophilius. These enzymes are
also homologous to the alpha- and beta-subunits of
[4Fe-4S]-containing bacterial serine dehydratases, for example, E.
coli enzymes encoded by tdcG, sdhB, and sdaA). An enzyme with
similar functionality in E. barkeri is dimethylmaleate hydratase, a
reversible Fe2+-dependent and oxygen-sensitive enzyme in the
aconitase family that hydrates dimethylmaeate to form
(2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB
(Alhapel et al., Proc Natl Acad Sci USA 103:12341-6 (2006);
Kollmann-Koch et al., Hoppe Seylers.ZPhysiol Chem. 365:847-857
(1984)).
TABLE-US-00205 Protein GenBank ID GI Number Organism Hmd ABC88407.1
86278275 Eubacterium barkeri BACCAP_02294 ZP_02036683.1 154498305
Bacteroides capillosus ANACOL_02527 ZP_02443222.1 167771169
Anaerotruncus colihominis NtherDRAFT_2368 ZP_02852366.1 169192667
Natranaerobius thermophilus dmdA ABC88408 86278276 Eubacterium
barkeri dmdB ABC88409 86278277 Eubacterium barkeri
[0670] Fumarate hydratase enzymes, which naturally catalyze the
reversible hydration of fumarate to malate. Although the ability of
fumarate hydratase to react on branched substrates with
3-oxobutanol as a substrate has not been described, a wealth of
structural information is available for this enzyme and other
researchers have successfully engineered the enzyme to alter
activity, inhibition and localization (Weaver, Acta Crystallogr. D
Biol. Crystallogr. 61:1395-1401 (2005)). E. coli has three
fumarases: FumA, FumB, and FumC that are regulated by growth
conditions. FumB is oxygen sensitive and only active under
anaerobic conditions. FumA is active under microanaerobic
conditions, and FumC is the only active enzyme in aerobic growth
(Tseng et al., J. Bacteriol. 183:461-467 (2001); Woods et al.,
Biochem. Biophys. Acta 954:14-26 (1988); Guest et al., J Gen
Microbiol 131:2971-2984 (1985)). Exemplary enzyme candidates
include those encoded by fumC from Escherichia coli and include
those in the table below.
TABLE-US-00206 Protein GenBank ID GI Number Organism fumA
NP_416129.1 16129570 Escherichia coli fumB NP_418546.1 16131948
Escherichia coli fumC NP_416128.1 16129569 Escherichia coli fumC
O69294 9789756 Campylobacter jejuni fumC P84127 75427690 Thermus
thermophilus fumH P14408 120605 Rattus norvegicus fuml P93033
39931311 Arabidopsis thaliana fumC Q8NRN8 39931596 Corynebacterium
glutamicum MmcB YP_001211906 147677691 Pelotomaculum
thermopropionicum MmcC YP_001211907 147677692 Pelotomaculum
thermopropionicum
[0671] Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate is
catalyzed by 4-hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). This
enzyme participates in aromatic degradation pathways and is
typically co-transcribed with a gene encoding an enzyme with
4-hydroxy-2-oxovalerate aldolase activity. Exemplary gene products
include those in the table below. Sequence comparison reveals
homologs in a wide range of bacteria, plants and animals. Enzymes
with highly similar sequences are contained in Klebsiella pneumonia
(91% identity, eval=2e-138) and Salmonella enterica (91% identity,
eval=4e-138), among others.
TABLE-US-00207 GenBank Gene Accession No. GI No. Organism mhpD
AAC73453.2 87081722 Escherichia coli cmtF AAB62293.1 1263188
Pseudomonas putida todG AAA61942.1 485738 Pseudomonas putida cnbE
YP_001967714.1 190572008 Comamonas sp. CNB-1 mhpD Q13VU0 123358582
Burkholderia xenovorans hpcG CAA57202.1 556840 Escherichia coli C
hpaH CAA86044.1 757830 Escherichia coli W hpaH ABR80130.1 150958100
Klebsiella pneumoniae Sari 01896 ABX21779.1 160865156 Salmonella
enterica
[0672] Another enzyme candidate is citramalate hydrolyase (EC
4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to
mesaconate. This enzyme has been studied in Methanocaldococcus
jannaschii in the context of the pyruvate pathway to
2-oxobutanoate, where it has been shown to have a broad substrate
specificity (Drevland et al., J Bacteriol. 189:4391-4400 (2007)).
This enzyme activity was also detected in Clostridium
tetanomorphum, Morganella morganii, Citrobacter amalonaticus where
it is thought to participate in glutamate degradation (Kato et al.,
Arch. Microbiol 168:457-463 (1997)). The M. jannaschii protein
sequence does not bear significant homology to genes in these
organisms.
TABLE-US-00208 Protein GenBank ID GI Number Organism leuD Q58673.1
3122345 Methanocaldococcus jannaschii
[0673] Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible
Fe.sup.2+-dependent and oxygen-sensitive enzyme in the aconitase
family that hydrates dimethylmaeate to form
(2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in
Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al.,
Hoppe Seylers.ZPhysiol Chem. 365:847-857 (1984)).
TABLE-US-00209 Protein GenBank ID GI Number Organism dmdA ABC88408
86278276 Eubacterium barkeri dmdB ABC88409.1 86278277 Eubacterium
barkeri
[0674] Step F--Methylmalonyl-CoA Reductase (Alcohol Forming)
(Designated as EMA7).
[0675] Step F can involve a combined Alcohol/Aldehyde dehydrogenase
(EC 1.2.1.-). Methylmalonyl-CoA can be reduced to 3-HIB in one step
by a multifunctional enzyme with dual acyl-CoA reductase and
alcohol dehydrogenase activity. Although the direct conversion of
methylmalonyl-CoA to 3-HIB has not been reported, this reaction is
similar to the common conversions such as acetyl-CoA to ethanol and
butyryl-CoA to butanol, which are catalyzed by CoA-dependent
enzymes with both alcohol and aldehyde dehydrogenase activities.
Gene candidates include the E. coli adhE and exemplary enzymes in
the table below.
TABLE-US-00210 Protein GenBank ID GI Number Organism Mcr
YP_001636209.1 163848165 Chloroflexus aurantiacus adhE NP_415757.1
16129202 Escherichia coli bdhI NP_349892.1 15896543 Clostridium
acetobutylicum bdhII NP_349891.1 15896542 Clostridium
acetobutylicum adhE AAV66076.1 55818563 Leuconostoc
mesenteroides
Example XI
Methacrylic Acid and 2-Hydroxyisobutyric Synthesis Enzymes
[0676] This Example provides genes that can be used for conversion
of acetyl-CoA to methacrylic acid and 2-hydroxyisobutyric as
depicted in the pathways of FIG. 10.
[0677] FIG. 10. Exemplary pathways enabling production of
2-hydroxyisobutyrate and methacrylic acid from acetyl-CoA.
2-Hydroxyisobutyrate and methacrylic acid production are carried
out by the following enzymes: A) acetyl-CoA:acetyl-CoA
acyltransferase, B) acetoacetyl-CoA reductase (ketone reducing), C)
3-hydroxybutyrl-CoA mutase, D) 2-hydroxyisobutyryl-CoA dehydratase,
E) methacrylyl-CoA synthetase, hydrolase, or transferase, F)
2-hydroxyisobutyryl-CoA synthetase, hydrolase, or transferase.
[0678] MAA biosynthesis can proceed from acetyl-CoA in a minimum of
five enzymatic steps (see FIG. 10). In this pathway, two molecules
of acetyl-CoA are combined to form acetoacetyl-CoA, which is then
reduced to 3-hydroxybutyryl-CoA. Alternatively,
4-hydroxybutyryl-CoA can be converted to 3-hydroxybutyryl-CoA by
way of 4-hydroxybutyryl-CoA dehydratase and crotonase (Martins et
al., Proc. Nat. Acad. Sci. USA 101(44) 15645-15649 (2004); Jones
and Woods, Microbiol. Rev. 50:484-524 (1986); Berg et al., Science
318(5857) 1782-1786 (2007)). A methylmutase then rearranges the
carbon backbone of 3-hydroxybutyryl-CoA to 2-hydroxyisobutyryl-CoA,
which is then dehydrated to form methacrylyl-CoA. Alternatively,
2-hydroxyisobutyryl-CoA can be converted to 2-hydroxyisobutyrate,
secreted, and recovered as product. The final step converting
methacrylyl-CoA to MAA can be performed by a single enzyme shown in
the figure or a series of enzymes.
[0679] A) Acetyl-CoA:Acetyl-CoA Acyltransferase.
[0680] Step A involves acetoacetyl-CoA thiolase (EC 2.3.1.9). The
formation of acetoacetyl-CoA from two acetyl-CoA units is catalyzed
by acetyl-CoA thiolase. This enzyme is native to E. coli, encoded
by gene atoB, and typically operates in the acetoacetate-degrading
direction during fatty acid oxidation (Duncombe and Freman, Arch.
Biochem. Biophys. 176:159-170 (1976); Freman and Duncombe, Biochim.
Biophys. Acta 580:289-297 (1979)). Additional exemplary genes
include those below.
TABLE-US-00211 Protein GenBank ID GI Number Organism atoB NP_416728
16130161 Escherichia coli thlA NP_349476.1 15896127 Clostridium
acetobutylicum thlB NP_149242.1 15004782 Clostridium acetobutylicum
thl ABA18857.1 75315385 Clostridium pasteurianum ERG10 NP_015297
6325229 Saccharomyces cerevisiae
[0681] B) Acetoacetyl-CoA Reductase (Ketone Reducing).
[0682] Step B involves acetoacetyl-CoA reductase (EC #: 1.1.1.35).
This second step entails the reduction of acetoacetyl-CoA to
3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase. This enzyme
participates in the acetyl-CoA fermentation pathway to butyrate in
several species of Clostridia and has been studied in detail (Jones
and Woods, Microbiol. Rev. 50:484-524 (1986)). The enzyme from
Clostridium acetobutylicum, encoded by hbd, has been cloned and
functionally expressed in E. coli (Youngleson et al., J. Bacteriol.
171:6800-6807 (1989)). Additionally, subunits of two fatty acid
oxidation complexes in E. coli, encoded by fadB and fadJ, function
as 3-hydroxyacyl-CoA dehydrogenases (Binstock and Schulz, Methods
Enzymol. 71 Pt C:403-411 (1981)). Exemplary enzymes are in the
following table.
TABLE-US-00212 Protein GENBANK ID GI NUMBER ORGANISM fadB P21177.2
119811 Escherichia coli fadJ P77399.1 3334437 Escherichia coli Hbd2
EDK34807.1 146348271 Clostridium kluyveri Hbd1 EDK32512.1 146345976
Clostridium kluyveri HSD17B10 O02691.3 3183024 Bos taurus phbB
P23238.1 130017 Zoogloea ramigera phaB YP_353825.1 77464321
Rhodobacter sphaeroides phaB BAA08358 675524 Paracoccus
denitrificans Hbd NP_349314.1 15895965 Clostridium acetobutylicum
Hbd AAM14586.1 20162442 Clostridium beijerinckii Msed_1423
YP_001191505 146304189 Metallosphaera sedula Msed_0399 YP_001190500
146303184 Metallosphaera sedula Msed_0389 YP_001190490 146303174
Metallosphaera sedula Msed_1993 YP_001192057 146304741
Metallosphaera sedula Fox2 Q02207 399508 Candida tropicalis
[0683] C) 3-Hydroxybutyrl-CoA Mutase.
[0684] Step C involves 3-hydroxybutyryl-CoA mutase (EC 5.4.99.-).
In this step, 3-hydroxybutyryl-CoA is rearranged to form
2-hydroxyisobutyryl-CoA (2-HIBCoA) by 3-hydroxybutyryl-CoA mutase.
This enzyme is a novel ICM-like methylmutase recently discovered
and characterized in Methylibium petroleiphilum (Ratnatilleke et
al., J. Biol. Chem. 274:31679-31685 (1999); Rohwerder et al., Appl.
Environ. Microbiol. 72:4128-4135 (2006)). This enzyme, encoded by
Mpe B0541 in Methylibium petroleiphilum PM1, has high sequence
homology to the large subunit of methylmalonyl-CoA mutase in other
organisms including Rsph17029_3657 in Rhodobacter sphaeroides and
Xaut_5021 in Xanthobacter autotrophicus. Changes to a single amino
acid near the active site alters the substrate specificity of the
enzyme (Ratnatilleke et al., supra, 1999; Rohwerder et al., supra,
2006), so directed engineering of similar enzymes at this site,
such as methylmalonyl-CoA mutase or isobutryryl-CoA mutase
described previously, can be used to achieve the desired
reactivity.
TABLE-US-00213 Gene GenBank ID GI Number Organism Mpe B0541
YP_001023546.1 124263076 Methylibium petroleiphilum PM1
Rsphl7029_3657 YP_001045519.1 126464406 Rhodobacter sphaeroides
Xaut_5021 YP_001409455.1 154243882 Xanthobacter autotrophicus
Py2
[0685] D) 2-Hydroxyisobutyryl-CoA Dehydratase.
[0686] Step D involves 2-hydroxyisobutyryl-CoA dehydratase. The
dehydration of 2-hydroxyacyl-CoA such as 2-hydroxyisobutyryl-CoA
can be catalyzed by a special class of oxygen-sensitive enzymes
that dehydrate 2-hydroxyacyl-CoA derivatives via a
radical-mechanism (Buckel and Golding, Annu. Rev. Microbiol.
60:27-49 (2006); Buckel et al., Curr. Opin. Chem. Biol. 8:462-467
(2004); Buckel et al., Biol. Chem. 386:951-959 (2005); Kim et al.,
FEBS 272:550-561 (2005); Kim et al., FEMS Microbiol. Rev.
28:455-468 (2004); Zhang et al., Microbiology 145 (Pt 9):2323-2334
(1999)). Exemplary enzymes are the following.
TABLE-US-00214 GenBank Gene Accession No. GI No. Organism hgdA
P11569 296439332 Acidaminococcus fermentans hgdB P11570 296439333
Acidaminococcus fermentans hgdC P11568 2506909 Acidaminococcus
fermentans hadB YP_001086863 126697966 Clostridium difficile hadC
YP_001086864 126697967 Clostridium difficile hadI YP_001086862
126697965 Clostridium difficile lcdB AJ276553 7242547 Clostridium
propionicum
[0687] E) Methacrylyl-CoA Synthetase, Hydrolase, or Transferase,
and F) 2-Hydroxyisobutyryl-CoA Synthetase, Hydrolase, or
Transferase.
[0688] Steps E and F involve a transferase (EC 2.8.3.-), hydrolase
(EC 3.1.2.-), or synthetase (EC 6.2.1.-) with activity on a
methacrylic acid or 2-hydroxyisobutyric acid, respectively. Direct
conversion of methacrylyl-CoA to MAA or 2-hydroxyisobutyryl-CoA to
2-hydrioxyisobutyrate can be accomplished by a CoA transferase,
synthetase or hydrolase. Pathway energetics are most favorable if a
CoA transferase or a CoA synthetase is employed to accomplish this
transformation. In the transferase family, the enzyme
acyl-CoA:acetate-CoA transferase, also known as acetate-CoA
transferase, is a suitable candidate to catalyze the desired
2-hydroxyisobutyryl-CoA or methacryl-CoA transferase activity due
to its broad substrate specificity that includes branched acyl-CoA
substrates (Matthies and Schink, Appl. Environ. Microbiol.
58:1435-1439 (1992); Vanderwinkel et al., Biochem. Biophys. Res.
Commun. 33:902-908 (1968)). ADP-forming acetyl-CoA synthetase (ACD)
is a promising enzyme in the CoA synthetase family operating on
structurally similar branched chain compounds (Brasen and
Schonheit, Arch. Microbiol. 182:277-287 (2004); Musfeldt and
Schonheit, J. Bacteriol. 184:636-644 (2002)). In the CoA-hydrolase
family, the enzyme 3-hydroxyisobutyryl-CoA hydrolase has been shown
to operate on a variety of branched chain acyl-CoA substrates
including 3-hydroxyisobutyryl-CoA, methylmalonyl-CoA, and
3-hydroxy-2-methylbutanoyl-CoA (Hawes et al., Methods Enzymol.
324:218-228 (2000); Hawes et al., J. Biol. Chem. 271:26430-26434
(1996); Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)).
Additional exemplary gene candidates for CoA transferases,
synthetases, and hydrolases are discussed elsewhere above.
Example XII
Attenuation or Disruption of Endogenous Enzymes
[0689] This example provides endogenous enzyme targets for
attenuation or disruption that can be used for enhancing carbon
flux through methanol dehydrogenase and formaldehyde assimilation
pathways.
DHA Kinase
[0690] Methylotrophic yeasts typically utilize a cytosolic DHA
kinase to catalyze the ATP-dependent activation of DHA to DHAP.
DHAP together with G3P is combined to form
fructose-1,6-bisphosphate (FBP) by FBP aldolase. FBP is then
hydrolyzed to F6P by fructose bisphosphatase. The net conversion of
DHA and G3P to F6P by this route is energetically costly (1 ATP) in
comparison to the F6P aldolase route, described above and shown in
FIG. 1. DHA kinase also competes with F6P aldolase for the DHA
substrate. Attenuation of endogenous DHA kinase activity will thus
improve the energetics of formaldehyde assimilation pathways, and
also increase the intracellular availability of DHA for DHA
synthase. DHA kinases of Saccharomyces cerevisiae, encoded by DAK1
and DAK2, enable the organism to maintain low intracellular levels
of DHA (Molin et al, J Biol Chem 278:1415-23 (2003)). In
methylotrophic yeasts DHA kinase is essential for growth on
methanol (Luers et al, Yeast 14:759-71 (1998)). The DHA kinase
enzymes of Hansenula polymorpha and Pichia pastoris are encoded by
DAK (van der Klei et al, Curr Genet 34:1-11 (1998); Luers et al,
supra). DAK enzymes in other organisms can be identified by
sequence similarity to known enzymes.
TABLE-US-00215 Protein GenBank ID GI Number Organism DAK1
NP_013641.1 6323570 Saccharomyces cerevisiae DAK2 NP_116602.1
14318466 Saccharomyces cerevisiae DAK AAC27705.1 3171001 Hansenula
polymorpha DAK AAC39490.1 3287486 Pichia pastoris DAK2 XP_505199.1
50555582 Yarrowia lipolytica
Methanol Oxidase
[0691] Attenuation of redox-inefficient endogenous methanol
oxidizing enzymes, combined with increased expression of a
cytosolic NADH-dependent methanol dehydrogenase, will enable
redox-efficient oxidation of methanol to formaldehyde in the
cytosol. Methanol oxidase, also called alcohol oxidase (EC
1.1.3.13), catalyzes the oxygen-dependent oxidation of methanol to
formaldehyde and hydrogen peroxide. In eukaryotic organisms,
alcohol oxidase is localized in the peroxisome. Exemplary methanol
oxidase enzymes are encoded by AOD of Candida boidinii (Sakai and
Tani, Gene 114:67-73 (1992)); and AOX of H. polymorpha, P.
methanolica and P. pastoris (Ledeboer et al, Nucl Ac Res 13:3063-82
(1985); Koutz et al, Yeast 5:167-77 (1989); Nakagawa et al, Yeast
15:1223-1230 (1999)).
TABLE-US-00216 Protein GenBank ID GI Number Organism AOX2
AAF02495.1 6049184 Pichia methanolica AOX1 AAF02494.1 6049182
Pichia methanolica AOX1 AAB57849.1 2104961 Pichia pastoris AOX2
AAB57850.1 2104963 Pichia pastoris AOX P04841.1 113652 Hansenula
polymorpha AOD1 Q00922.1 231528 Candida boidinii AOX1 AAQ99151.1
37694459 Ogataea pini
PQQ-Dependent Methanol Dehydrogenase
[0692] PQQ-dependent methanol dehydrogenase from M. extorquens
(mxaIF) uses cytochrome as an electron Gather (Nunn et al, Nucl
Acid Res 16:7722 (1988)). Methanol dehydrogenase enzymes of
methanotrophs such as Methylococcus capsulatis function in a
complex with methane monooxygenase (MMO) (Myronova et al, Biochem
45:11905-14 (2006)). Note that of accessory proteins, cytochrome CL
and PQQ biosynthesis enzymes are needed for active methanol
dehydrogenase. Attenuation of one or more of these required
accessory proteins, or retargeting the enzyme to a different
cellular compartment, would also have the effect of attenuating
PQQ-dependent methanol dehydrogenase activity.
TABLE-US-00217 Protein GenBank ID GI Number Organism MCA0299
YP_112833.1 53802410 Methylococcus capsulatis MCA0782 YP_113284.1
53804880 Methylococcus capsulatis mxaI YP_002965443.1 240140963
Methylohacterium extorquens mxaF YP_002965446.1 240140966
Methylobacterium extorquens
DHA Synthase and Other Competing Formaldehyde Assimilation and
Dissimilation Pathways
[0693] Carbon-efficient formaldehyde assimilation can be improved
by attenuation of competing formaldehyde assimilation and
dissimilation pathways. Exemplary competing assimilation pathways
in eukaryotic organisms include the peroxisomal dissimilation of
formaldehyde by DHA synthase, and the DHA kinase pathway for
converting DHA to F6P, both described herein. Exemplary competing
endogenous dissimilation pathways include one or more of the
enzymes shown in FIG. 1.
[0694] Methylotrophic yeasts normally target selected methanol
assimilation and dissimilation enzymes to peroxisomes during growth
on methanol, including methanol oxidase, DHA synthase and
S-(hydroxymethyl)-glutathione synthase (see review by Yurimoto et
al, supra). The peroxisomal targeting mechanism comprises an
interaction between the peroxisomal targeting sequence and its
corresponding peroxisomal receptor (Lametschwandtner et al, J Biol
Chem 273:33635-43 (1998)). Peroxisomal methanol pathway enzymes in
methylotrophic organisms contain a PTS1 targeting sequence which
binds to a peroxisomal receptor, such as Pex5p in Candida boidinfi
(Horiguchi et al, J Bacteriol 183:6372-83 (2001)). Disruption of
the PTS1 targeting sequence, the Pex5p receptor and/or genes
involved in peroxisomal biogenesis would enable cytosolic
expression of DHA synthase, S-(hydroxymethyl)-glutathione synthase
or other methanol-inducible peroxisomal enzymes. PTS1 targeting
sequences of methylotrophic yeast are known in the art (Horiguchi
et al, supra). Identification of peroxisomal targeting sequences of
unknown enzymes can be predicted using bioinformatic methods (eg.
Neuberger et al, J Moi Biol 328:581-92 (2003))).
Example XIII
Methanol Assimilation Via Methanol Dehydrogenase and the Ribulose
Monophosphate Pathway
[0695] This example shows that co-expression of an active methanol
dehydrogenase (MeDH) and the enzymes of the Ribulose Monophosphate
(RuMP) pathway can effectively assimilate methanol derived
carbon.
[0696] An experimental system was designed to test the ability of a
MeDH in conjunction with the enzymes H6P synthase (HPS) and
6-phospho-3-hexuloisomerase (PHI) of the RuMP pathway to assimilate
methanol carbon into the glycolytic pathway and the TCA cycle.
Escherichia coli strain ECh-7150 (.DELTA.lacIA, .DELTA.pflB,
.DELTA.ptsI, .DELTA.PpckA(pckA), .DELTA.Pglk(glk), glk::glfB,
.DELTA.hycE, .DELTA.frmR, .DELTA.frmA, .DELTA.frmB) was constructed
to remove the glutathione-dependent formaldehyde detoxification
capability encoded by the FrmA and FrmB enzyme. This strain was
then transformed with plasmid pZA23S variants that either contained
or lacked gene 2616A encoding a fusion of the HPS and PHI enzymes.
These two transformed strains were then each transformed with
pZS*13S variants that contained gene 2315L (encoding an active
MeDH), or gene 2315 RIP2 (encoding a catalytically inactive MeDH),
or no gene insertion. Genes 2315 and 2616 are internal
nomenclatures for NAD-dependent methanol dehydrogenase from
Bacillus methanolicus MGA3 and 2616 is a fused phs-hpi constructs
as described in Orita et al. (2007) Appl Microbiol Biotechnol
76:439-45.
[0697] The six resulting strains were aerobically cultured in
quadruplicate, in 5 ml minimal medium containing 1% arabinose and
0.6 M 13C-methanol as well as 100 ug/ml carbenicillin and 25
.mu.g/mlkanamycin to maintain selection of the plasmids, and 1 mM
IPTG to induce expression of the methanol dehydrogenase and HPS-PHI
fusion enzymes. After 18 hours incubation at 37.degree. C., the
cell density was measured spectrophotometrically at 600 nM
wavelength and a clarified sample of each culture medium was
submitted for analysis to detect evidence of incorporation of the
labeled methanol carbon into TCA-cycle derived metabolites. The
label can be further enriched by deleting the gene araD that
competes with ribulose-5-phosphate.
[0698] .sup.13C carbon derived from labeled methanol provided in
the experiment was found to be significantly enriched in the
metabolites pyruvate, lactate, succinate, fumarate, malate,
glutamate and citrate, but only in the strain expressing both
catalytically active MeDH 2315L and the HPS-PHI fusion 2616A
together (data not shown). Moreover, this strain grew significantly
better than the strain expressing catalytically active MeDH but
lacking expression of the HPS-PHI fusion (data not shown),
suggesting that the HPS-PHI enzyme is capable of reducing growth
inhibitory levels of formaldehyde that cannot be detoxified by
other means in this strain background. These results show that
co-expression of an active MeDH and the enzymes of the RUMP pathway
can effectively assimilate methanol derived carbon and channel it
into TCA-cycle derived products.
[0699] Throughout this application various publications have been
referenced. The disclosures of these publications in their
entireties, including GenBank and GI number publications, are
hereby incorporated by reference in this application in order to
more fully describe the state of the art to which this invention
pertains. Although the invention has been described with reference
to the examples provided above, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
* * * * *