U.S. patent application number 14/649973 was filed with the patent office on 2015-11-05 for methods and systems for methylotrophic production of organic compounds.
The applicant listed for this patent is GINKGO BIOWORKS, INC. Invention is credited to Curt P. FISCHER, Reshma P. SHETTY.
Application Number | 20150315599 14/649973 |
Document ID | / |
Family ID | 50884024 |
Filed Date | 2015-11-05 |
United States Patent
Application |
20150315599 |
Kind Code |
A1 |
SHETTY; Reshma P. ; et
al. |
November 5, 2015 |
Methods and Systems for Methylotrophic Production of Organic
Compounds
Abstract
The present disclosure identifies pathways, mechanisms, systems
and methods to confer production of carbon-based products of
interest, such as sugars, alcohols, chemicals, amino acids,
polymers, fatty acids and their derivatives, hydrocarbons,
isoprenoids, and intermediates thereof, in engineered and/or
evolved methylotrophs such that these organisms efficiently convert
C1 compounds, such as formate, formic acid, formaldehyde or
methanol, to organic carbon-based products of interest, and in
particular the use of organisms for the commercial production of
various carbon-based products of interest.
Inventors: |
SHETTY; Reshma P.; (Boston,
MA) ; FISCHER; Curt P.; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GINKGO BIOWORKS, INC |
Boston |
MA |
US |
|
|
Family ID: |
50884024 |
Appl. No.: |
14/649973 |
Filed: |
December 6, 2013 |
PCT Filed: |
December 6, 2013 |
PCT NO: |
PCT/US2013/073582 |
371 Date: |
June 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61734472 |
Dec 7, 2012 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/252.3; 435/253.6; 435/34 |
Current CPC
Class: |
C12P 7/40 20130101; C12N
15/52 20130101; C12N 1/36 20130101; C12P 7/16 20130101; C12N 15/74
20130101; Y02E 50/10 20130101; C12N 1/20 20130101 |
International
Class: |
C12N 15/52 20060101
C12N015/52; C12P 7/40 20060101 C12P007/40; C12P 7/16 20060101
C12P007/16; C12N 15/74 20060101 C12N015/74; C12N 1/20 20060101
C12N001/20 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under
contract number DE-AR0000091 awarded by U.S. Department of Energy,
Office of ARPA-E. The government has certain rights in the
invention.
Claims
1. An engineered cell for producing a carbon-based product,
comprising an at least partially engineered carbon product
biosynthetic pathway introduced into a methylotrophic organism,
wherein said engineered cell is capable of converting a C1 compound
into a carbon-based product of interest.
2. The engineered cell of claim 1, wherein the methylotrophic
organism is capable of converting the C1 compound into a central
metabolite.
3. The engineered cell of claim 1 or 2, wherein the C1 compound is
soluble in water, such as formate, formic acid, formaldehyde,
methanol, or any combination thereof.
4. The engineered cell of any one of claims 1-3, wherein the C1
compound is derived from electrolysis.
5. The engineered cell of any one of claims 1-4, wherein said
carbon-based product of interest is one or more of a sugar (for
example, glucose, fructose, sucrose, xylose, lactose, maltose,
pentose, rhamnose, galactose or arabinose), sugar phosphate (for
example, glucose-6-phosphate or fructose-6-phosphate), sugar
alcohol (for example, sorbitol), sugar derivative (for example,
ascorbate), alcohol (for example, ethanol, propanol, isopropanol or
butanol), fermentative product (for example, ethanol, butanol,
lactic acid, lactose or acetate), ethylene, propylene, 1-butene,
1,3-butadiene, acrylic acid, fatty acid (for example, O-cyclic
fatty acid), fatty acid intermediate or derivative (for example,
fatty acid alcohol, fatty acid ester, alkane, olegin or halogenated
fatty acid), amino acid or intermediate (for example, lysine,
glutamate, aspartate, shikimate, chorismate, phenylalanine,
tyrosine, tryptophan), phenylpropanoid, isoprenoid (for example,
hemiterpene, monoterpene, sesquiterpene, triterpene, tetraterpene,
polyterpene, isoprene, bisabolene, myrcene, amorpha-4,11-diene,
farnesene, taxadiene, squalene, lanosterol, .beta.-carotene,
.zeta.-carotene, lycopene, phytoene, limonene, or polyisoprene),
glycerol, 1,3-propanediol, 1,4-butanediol, 1,3-butadiene,
polyhydroxyalkanoate, polyhydroxybutyrate, lysine,
.gamma.-valerolactone, and acrylate.
6. The engineered cell of any one of claims 1-5, wherein when said
carbon product biosynthetic pathway is for fatty acid biosynthesis,
said carbon product biosynthetic pathway includes one or more of:
fatty acid synthase, acetyl-CoA carboxylase, fatty-acyl-CoA
reductase, aldehyde decarbonylase, lipase, thioesterase and
acyl-CoA synthase peptides; or when said carbon product
biosynthetic pathway is for branched chain fatty acid biosynthesis,
said carbon product biosynthetic pathway includes one or more of:
branched chain amino acid aminotransferase, branched chain
.alpha.-ketoacid dehydrogenase, dihydrolipoyl dehydrogenase,
beta-ketoacyl-ACP synthase, crotonyl-CoA reductase, isobutyryl-CoA
mutase, .beta.-ketoacyl-ACP synthase I, trans-2,cis-3-decenoyl-ACP
isomerase and trans-2-enoyl-ACP reductase II; or when said carbon
product biosynthetic pathway is for fatty alcohol biosynthesis,
said carbon product biosynthetic pathway includes one or more of:
fatty alcohol forming acyl-CoA reductase, fatty alcohol forming
acyl-CoA reductase, alcohol dehydrogenase and alcohol reductase; or
when said carbon product biosynthetic pathway is for fatty ester
biosynthesis, said carbon product biosynthetic pathway includes one
or more of: alcohol O-acetyltransferase, wax synthase, fatty acid
elongase, acyl-CoA reductase, acyltransferase, fatty acyl
transferase, diacylglycerol acyltransferase, acyl-CoA was alcohol
acyltransferase, bifunctional wax ester
synthase/acyl-CoA:diacylglycerol acyltransferase, and
.beta.-ketoacyl-ACP synthase I; or when said carbon product
biosynthetic pathway is for alkane biosynthesis, said carbon
product biosynthetic pathway includes one or more of: decarbonylase
and terminal alcohol oxidoreductase; or when said carbon product
biosynthetic pathway is for .omega.-cyclic fatty acid biosynthesis,
said carbon product biosynthetic pathway includes one or more of:
1-cyclohexenylcarbonyl CoA reductase,
5-enopyruvylshikimate-3-phosphate synthase, acyl-CoA dehydrogenase,
enoyl-(ACP) reductase, 2,4-dienoyl-CoA reductase, and acyl-CoA
isomerase; or when said carbon product biosynthetic pathway is for
halogenated fatty acid biosynthesis, said carbon product
biosynthetic pathway includes one or more of: fluorinase,
nucleotide phosphorylase, fluorometabolite-specific aldolase,
fluoroacetaldehyde dehydrogenase, and fluoroacetyl-CoA synthase; or
when said carbon product biosynthetic pathway is the deoxylylulose
5-phosphate (DXP) isoprenoid pathway, said carbon product
biosynthetic pathway includes one or more of:
1-deoxy-D-xylulose-5-phosphate synthase,
1-deoxy-D-xylulose-5-phosphate reductoisomerase,
4-diphosphocytidyl-2C-methyl-D-erythritol synthase,
4-diphosphocytidyl-2C-methyl-D-erythritol kinase,
2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase,
isopentyl/dimethylallyl diphosphate synthase and
4-hydroxy-3-methylbut-2-enyl diphosphate reductase; or when said
carbon product biosynthetic pathway is the mevalonate-dependent
(MEV) isoprenoid pathway, said carbon product biosynthetic pathway
includes one or more of: acetyl-CoA thiolase, HMG-CoA synthase,
HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase,
mevalonate pyrophosphate decarboxylase and isopentenyl
pyrophosphate isomerase; or when said carbon product biosynthetic
pathway is the glycerol/1,3-propanediol biosynthesis pathway, said
carbon product biosynthetic pathway includes one or more of:
sn-glycerol-3-P dehydrogenase, sn-glycerol-3-phosphatase, glycerol
dehydratase and 1,3-propanediol oxidoreductase; or when said carbon
product biosynthetic pathway is the 1,4-butanediol/1,3-butadiene
biosynthesis pathway, said carbon product biosynthetic pathway
includes one or more of: succinyl-CoA dehydrogenase,
4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase,
1,3-propanediol oxidoreductase and alcohol dehydratase; or when
said carbon product biosynthetic pathway is the polyhydroxybutyrate
biosynthesis pathway, said carbon product biosynthetic pathway
includes one or more of: acetyl-CoA:acetyl-CoA C-acetyltransferase,
(R)-3-hydroxyacyl-CoA:NADP.sup.+ oxidoreductase and
polyhydroxyalkanoate synthase; or when said carbon product
biosynthetic pathway is the lysine biosynthesis pathway, said
carbon product biosynthetic pathway includes one or more of:
aspartate aminotransferase, aspartate kinase, aspartate
semialdehyde dehydrogenase, dihydrodipicolinate synthase,
dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase,
N-succinyldiaminopimelate-aminotransferase,
N-succinyl-L-diaminopimelate desuccinylase, diaminopimelate
epimerase, diaminopimelate decarboxylase, L,L-diaminopimelate
aminotransferase, homocitrate synthase, homoaconitase,
homoisocitrate dehydrogenase, 2-aminoadipate transaminase,
2-aminoadipate reductase, aminoadipate semialdehyde-glutamate
reductase and lysine-2-oxoglutarate reductase; or when said carbon
product biosynthetic pathway is the chorismate biosynthesis
pathway, said carbon product biosynthetic pathway includes one or
more of: 2-dehydro-3-deoxyphosphoheptonate aldolase,
3-dehydroquinate synthase, 3-dehydroquinate dehydratase,
NADPH-dependent shikimate dehydrogenase, NAD(P)H-dependent
shikimate dehydrogenase, shikimate kinase,
3-phosphoshikimate-1-carboxyvinyltransferase and chorismate
synthase; or when said carbon product biosynthetic pathway is the
phenylalanine biosynthesis pathway, said carbon product
biosynthetic pathway includes one or more of: chorismate mutase,
prephenate dehydratase and phenylalanine transaminase; or when said
carbon product biosynthetic pathway is the tyrosine biosynthesis
pathway, said carbon product biosynthetic pathway includes one or
more of: chorismate mutase, prephenate dehydrogeanse and tyrosine
aminotransferase; or when said carbon product biosynthetic pathway
is the .gamma.-valerolactone biosynthesis pathway, said carbon
product biosynthetic pathway includes one or more of: propionyl-CoA
synthase, beta-ketothiolase, acetoacetyl-CoA reductase,
3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA
.DELTA.-isomerase, 4-hydroxybutyryl-CoA transferase and
1,4-lactonase; or when said carbon product biosynthetic pathway is
the butanol biosynthesis pathway, said carbon product biosynthetic
pathway includes one or more of: beta-ketothiolase, acetoacetyl-CoA
reductase, 3-hydroxybutyryl-CoA dehydrogenase, enoyl-CoA hydratase,
butyryl-CoA dehydrogenase, trans-enoyl-coenzyme A reductase,
butyrate CoA-transferase, aldehyde dehydrogenase, alcohol
dehydrogenase, acetyl-CoA acetyltransferase,
.beta.-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoA
dehydrogenase, CoA-acylating aldehyde dehydrogenase and
aldehyde-alcohol dehydrogenase; or when said carbon product
biosynthetic pathway is the acrylate biosynthesis pathway, said
carbon product biosynthetic pathway includes one or more of:
enoyl-CoA hydratase, propionyl-CoA synthase and acrylate
CoA-transferase.
7. The engineered cell of claim 6, wherein the
1-deoxy-D-xylulose-5-phosphate synthase is encoded by SEQ ID NO:1,
or a homolog thereof having at least 80% sequence identity; or the
isopentenyl pyrophosphate isomerase is encoded by SEQ ID NO:2, or a
homolog thereof having at least 80% sequence identity.
8. The engineered cell of any one of claims 1-7, wherein when said
carbon product biosynthetic pathway is the isoprene biosynthesis
pathway, said carbon product biosynthetic pathway includes isoprene
synthase.
9. The engineered cell of claim 8, wherein the isoprene synthase is
encoded by SEQ ID NO:3, or a homolog thereof having at least 80%
sequence identity.
10. The engineered cell of any one of claims 1-7, wherein when said
carbon product biosynthetic pathway is the bisabolene biosynthesis
pathway, said carbon product biosynthetic pathway includes
E-alpha-bisabolene synthase.
11. The engineered cell of claim 10, wherein the E-alpha-bisabolene
synthase is encoded by SEQ ID NO:4, or a homolog thereof having at
least 80% sequence identity.
12. The engineered cell of any one of claims 1-11, wherein the
methylotrophic organism is selected from the class
Alphaproteobacterium.
13. The engineered cell of any one of claims 1-12, wherein the
methylotrophic organism is selected from the genus Paracoccus.
14. The engineered cell of any one of claims 1-13, wherein the
methylotrophic organism is Paracoccus denitrificans, Paracoccus
versutus or Paracoccus zeaxanthinifaciens.
15. The engineered cell of any one of claims 1-14, further modified
to have a less reduced growth rate on electrolytically generated C1
compound relative to non-evolved methylotrophic organism, or a
substantially similar or enhanced growth rate on electrolytically
generated C1 compound relative to non-electrolytically generated C1
compound.
16. The engineered cell of any one of claims 1-15, further evolved
to have a less reduced growth rate on electrolytically generated C1
compound relative to non-evolved methylotrophic organism, or a
substantially similar or enhanced growth rate on electrolytically
generated C1 compound relative to non-electrolytically generated C1
compound.
17. An evolved methylotrophic organism, having a less reduced
growth rate on electrolytically generated C1 compound relative to
non-evolved methylotrophic organism, or having a substantially
similar or enhanced growth rate on electrolytically generated C1
compound relative to non-electrolytically generated C1
compound.
18. A method for selecting an evolved methylotrophic organism
having improved growth on a C1 compound, comprising: incubating
methylotrophic cells in a culture chamber with controlled
temperature, cell concentration, and medium inflow and outflow
rates, wherein a medium inflow includes a C1 compound; continuously
monitoring a concentration of biomass in the culture chamber; and
adjusting a flow rate of the C1 compound into the culture chamber
so as to continually maintain an environment that selects for an
improved growth rate.
19. The method of claim 18, further comprising adjusting the medium
inflow to be more permissive of growth or more suppressive of
growth, so as to provide an adaptive environment to select for a
fitness of the cells.
20. The method of any one of claims 18-19, wherein the C1 compound
is formate.
21. The method of any one of claims 18-20, wherein the C1 compound
is electrolytically generated.
22. The method of any one of claims 18-21, wherein the C1 compound
is soluble in water.
23. A method of introducing a conjugative plasmid into
methylotrophic host cells, comprising: incubating a mixture of
predetermined ratios of a donor culture and a recipient culture, at
temperatures between 4.degree. C. and 37.degree. C. for between 1
and 48 hours, wherein the donor culture comprises a conjugal donor
containing a conjugative plasmid having a first selectable trait,
and the recipient culture comprises methylotrophic host cell having
a second selectable trait; and subjecting the incubated mixture to
a dually selective condition where only plasmid-containing
transconjugants that have both the first selectable trait and the
second selectable trait can grow, wherein the method does not
include centrifugation or filtration of the mixture or incubated
mixture.
24. The method of claim 23, wherein the conjugal donor is an E.
coli strain such as E. coli S17-1, or an E. coli harboring plasmids
such as pRK2013 or pRK2073, or any E. coli strain expressing a tra
operon capable of mobilizing plasmids containing an RP4-derived
sequence.
25. The method of claim 23 or 24, wherein the conjugal donor is in
a different species or genus of the host cell.
26. The method of any one of claims 23-25, wherein the
transconjugated plasmid contains an RP4 or similar mob element.
27. The method of any one of claims 23-26, wherein the host cell is
from the class Alphaproteobacterium.
28. The method of any one of claims 23-27, wherein the host cell is
from the genus Paracoccus.
29. The method of any one of claims 23-28, wherein the host cell is
Paracoccus denitrificans, Paracoccus versutus or Paracoccus
zeaxanthinifaciens.
30. A composition for bacterial culture, formulated to provide
formate as the sole source of C1 compound and to enhance growth of
methylotrophic bacteria.
31. The composition of claim 30, comprising between 0 and 160 mM
sodium bicarbonate, between 0 and 16 mM sodium chloride, between 0
and 100 mM sodium nitrate, between 0 and 30 mM sodium thiosulfate,
and initially containing between 5 and 100 mM of a formate salt,
such as sodium formate and/or ammonium formate.
32. The composition of any one of claims 30-31, comprising 100 mM
sodium bicarbonate, 6 mM sodium chloride, 6 mM sodium nitrate, 11
mM sodium thiosulfate, and 26 mM sodium formate or ammonium
formate.
33. The composition of any one of claims 30-33, further comprising
a basal minimal medium.
34. The composition of claim 33, wherein the basal minimal medium
is MOPS minimal medium, M9 minimal medium, R medium or M63 medium,
or a medium substantially similar thereto.
35. A method for culturing methylotrophic bacteria, comprising
incubating methylotrophic bacteria in the composition of any one of
claims 30-34.
36. A composition of bacterial culture, formulated to provide
formate as the sole C1 compound and to enhance growth of
methylotrophic bacteria in a fed-batch bioreactor.
37. The composition of claim 36, comprising a medium initially
charged in the fed-batch bioreactor which comprises R medium
supplemented with between 1 and 100 micromolar sodium molybdate,
between 10 and 1000 nanomolar sodium selenite, between 0.01 to 1
mg/L of thiamine, and between 0.001 to 1 mg/L of cobalamin.
38. The composition of claim 37, wherein the medium comprises
between 5 and 20 micromolar sodium molbydate, between 50 and 200
nanomolar sodium selenite, between 0.05 to 2 mg/L of thiamine,
between 0.01 and 0.2 mg/L cobalamin.
39. The composition of claim 37 or 38, further comprising a feed
composition supplied to the fed-batch bioreactor comprising a
formate salt at supramolar concentration.
40. The composition of claim 39, wherein the formate salt is
ammonium formate and/or sodium formate.
41. The composition of claim 39 or 40, wherein feed composition
further comprises a supramolar concentration of nitrate salt.
42. The composition of claim 41, wherein the nitrate salt is sodium
nitrate.
43. The composition of any one of claims 39-42, wherein the nitrate
salt and the formate salt are provided in a molar ratio of 3.0:8 or
lower.
44. A method for culturing methylotrophic bacteria, comprising
incubating methylotrophic bacteria in the composition of any one of
claims 36-43 in a fed-batch bioreactor.
45. The method of claim 44, wherein a volumetric rate of C1
feedstock consumption in the fed-batch reactor exceeds 1.5
g*L.sup.-1 hr.sup.-1.
46. The method of claim 45, wherein said incubating is conducted
aerobically.
47. The method of any one of claims 44-46, wherein said incubating
is conducted in the presense of a nitrate salt as electron acceptor
and formate salt as electron donor.
48. The method of claim 47, wherein a molar ratio of the nitrate
salt to the formate salt is kept below 3.2:8 in the fed-batch
reactor.
49. The method of claim 47 or 48, wherein the nitrate salt and the
formate salt are provided to the fed-batch bioreactor in a feed
composition in supramolar concentrations in a molar ratio of 3.0:8
or lower.
50. The method of any one of claims 47-49, wherein the formate salt
is ammonium formate and/or sodium formate.
51. The method of any one of claims 47-50, wherein the nitrate salt
is sodium nitrate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Application No. 61/734,472 filed Dec. 7, 2012, the
disclosure of which is incorporated herein by its entirety.
TECHNICAL FIELD
[0003] The invention relates to systems, mechanisms and methods to
confer production of carbon-based products to a methylotroph or a
methylotrophic organism to efficiently convert C1 compounds into
various carbon-based products, and in particular the use of such
organism for the commercial production of various carbon-based
products of interest. The invention also relates to systems,
mechanisms and methods to confer additional and/or alternative
pathways for energy conversion, methylotrophy and/or carbon
fixation to a methylotroph.
BACKGROUND
[0004] Heterotrophs are biological organisms that utilize energy
from organic compounds for growth and reproduction. Commercial
production of various carbon-based products of interest generally
relies on heterotrophic organisms that ferment sugar from crop
biomass such as corn or sugarcane as their energy and carbon source
[Bai, 2008]. An alternative to fermentation-based bio-production is
the production of carbon-based products of interest from
photosynthetic organisms, such as plants, algae and cyanobacteria,
that derive their energy from sunlight and their carbon from carbon
dioxide to support growth [U.S. Pat. No. 7,981,647]. However, the
algae-based production of carbon-based products of interest relies
on the relatively inefficient process of photosynthesis to supply
the energy needed for production of organic compounds from carbon
dioxide [Larkum, 2010]. Moreover, commercial production of
carbon-based products of interest using photosynthetic organisms
relies on reliable and consistent exposure to light to achieve the
high productivities needed for economic feasibility; hence,
photobioreactor design remains a significant technical challenge
[Morweiser, 2010].
[0005] Methylotrophs are biological organisms that utilize energy
and/or carbon from C1 compounds containing no carbon-carbon bonds
such as formate, formic acid, formaldehyde, methanol, methane,
halogenated methanes, and methylated sulfur species to produce all
multi-carbon, organic compounds necessary for growth and
reproduction. Most existing, naturally-occuring methylotrophs are
poorly suited for industrial bio-processing and have therefore not
demonstrated commercial viability for this purpose. Such organisms
have long doubling times relative to industrialized heterotrophic
organisms such as Escherichia coli, reflective of low total
productivities. In addition, techniques for genetic manipulation
(homologous recombination, transformation or transfection of
nucleic acid molecules, and recombinant gene expression) are
inefficient, time-consuming, laborious or non-existent.
[0006] Thus, a need exists to develop engineered and/or evolved
methylotrops suitable for industrial uses. Accordingly, the ability
to endow a methylotroph with biosynthetic capability to produce
carbon-based products of interest, to grow the engineered and/or
evolved methylotroph at the high cell densities needed for
industrial bio-processing, and to efficiently provide the
engineered organism with C1 compounds would significantly enable
more energy- and carbon-efficient production of carbon-based
products of interest. In addition, the ability to add one or more
additional or alternative pathways for energy conversion,
methylotrophy and/or carbon fixation capability to the methylotroph
would enhance its ability to produce carbon-based products on
interest.
SUMMARY
[0007] Systems and methods of the present invention provide for
efficient production of renewable energy and other carbon-based
products of interest (e.g., fuels, sugars, chemicals) from C1
compounds. Furthermore, systems and methods of the present
invention can be used in the place of traditional methods of
producing chemicals such as olefins (e.g., ethylene, propylene),
which are traditionally derived from petroleum in a process that
generates toxic by-products that are recognized as hazardous waste
pollutants and harmful to the environment. As such, the present
invention can additionally avoid the use of petroleum and the
generation of such toxic by-products, and thus materially enhances
the quality of the environment by contributing to the maintenance
of basic life-sustaining natural elements such as air, water and/or
soil by avoiding the generation of hazardous waste pollutants in
the form of petroleum-derived by-products in the production of
various chemicals.
[0008] In certain aspect, the invention described herein provides a
methylotroph engineered to confer biosynthetic production of
various carbon-based products of interest from C1 compounds. The
engineered organism comprises one or more at least partially
engineered carbon product biosynthetic pathways that convert
central metabolites into desired products, such as carbon-based
products of interest. Carbon-based products of interest include but
are not limited to alcohols, fatty acids, fatty acid derivatives,
fatty alcohols, fatty acid esters, wax esters, hydrocarbons,
alkanes, polymers, fuels, commodity chemicals, specialty chemicals,
carotenoids, isoprenoids, sugars, sugar phosphates, central
metabolites, pharmaceuticals and pharmaceutical intermediates. For
example, the carbon-based products of interest can include one or
more of a sugar (for example, glucose, fructose, sucrose, xylose,
lactose, maltose, pentose, rhamnose, galactose or arabinose), sugar
phosphate (for example, glucose-6-phosphate or
fructose-6-phosphate), sugar alcohol (for example, sorbitol), sugar
derivative (for example, ascorbate), alcohol (for example, ethanol,
propanol, isopropanol or butanol), fermentative product (for
example, ethanol, butanol, lactic acid, lactose or acetate),
ethylene, propylene, 1-butene, 1,3-butadiene, acrylic acid, fatty
acid (for example, .omega.-cyclic fatty acid), fatty acid
intermediate or derivative (for example, fatty acid alcohol, fatty
acid ester, alkane, olegin or halogenated fatty acid), amino acid
or intermediate (for example, lysine, glutamate, aspartate,
shikimate, chorismate, phenylalanine, tyrosine, tryptophan),
phenylpropanoid, isoprenoid (for example, hemiterpene, monoterpene,
sesquiterpene, triterpene, tetraterpene, polyterpene, isoprene,
bisabolene, myrcene, amorpha-4,11-diene, farnesene, taxadiene,
squalene, lanosterol, .beta.-carotene, .zeta.-carotene, lycopene,
phytoene, limonene, or polyisoprene), glycerol, 1,3-propanediol,
1,4-butanediol, 1,3-butadiene, polyhydroxyalkanoate,
polyhydroxybutyrate, lysine, .gamma.-valerolactone, and acrylate.
In some embodiments, the carbon-based products of interest can be
carbon-based central metabolites.
[0009] The resulting engineered and/or evolved methylotroph of the
invention is capable of efficiently synthesizing carbon-based
products of interest from C1 compounds. The invention also provides
carbon product biosynthetic pathways for conferring biosynthetic
production of the carbon-based product of interest upon the host
organism where the organism lacks the ability to efficiently
produce carbon-based products of interest from C1 compounds. The
invention also provides methods for introducing the carbon product
biosynthetic pathways into the methylotroph. The invention also
provides methods and media compositions for culturing the
engineered and/or evolved methylotroph to support efficient
methylotrophic production of carbon-based products of interest.
[0010] In various embodiments, the invention provides for the C1
compound serving as a source of both energy and carbon for the
organism. In one embodiment, the C1 compound is soluble or miscible
in water. For example, the C1 compound can be one or more of
formate, formic acid, methanol and/or formaldehyde. C1 compounds
that dissolve at high concentration or are miscible in water, in
some instances, are preferable to less soluble or immiscible
chemical species, such as methane, because mass transfer and uptake
by the organism is more efficient. Similarly, soluble C1 compounds
are preferable to molecular hydrogen, carbon dioxide or carbon
monoxide, used in autotrophic production of carbon-based compounds
(see, e.g., Example 7). In some embodiments, the C1 compound can be
soluble in other solvents than water, depending on the composition
of the media used for growing the organism. For example, the
solubility of the C1 compound in the media may be enhanced by other
components therein. In some embodiments, the C1 compound can be
derived from electrolysis.
[0011] In certain embodiments, one or more of the following carbon
product biosynthetic pathways can be used: [0012] when said carbon
product biosynthetic pathway is for fatty acid biosynthesis, said
carbon product biosynthetic pathway includes one or more of: fatty
acid synthase, acetyl-CoA carboxylase, fatty-acyl-CoA reductase,
aldehyde decarbonylase, lipase, thioesterase and acyl-CoA synthase
peptides; or [0013] when said carbon product biosynthetic pathway
is for branched chain fatty acid biosynthesis, said carbon product
biosynthetic pathway includes one or more of: branched chain amino
acid aminotransferase, branched chain .alpha.-ketoacid
dehydrogenase, dihydrolipoyl dehydrogenase, beta-ketoacyl-ACP
synthase, crotonyl-CoA reductase, isobutyryl-CoA mutase,
.beta.-ketoacyl-ACP synthase I, trans-2,cis-3-decenoyl-ACP
isomerase and trans-2-enoyl-ACP reductase II; or [0014] when said
carbon product biosynthetic pathway is fatty alcohol biosynthesis,
said carbon product biosynthetic pathway includes one or more of:
fatty alcohol forming acyl-CoA reductase, fatty alcohol forming
acyl-CoA reductase, alcohol dehydrogenase and alcohol reductase; or
[0015] when said carbon product biosynthetic pathway is for fatty
ester biosynthesis, said carbon product biosynthetic pathway
includes one or more of: alcohol O-acetyltransferase, wax synthase,
fatty acid elongase, acyl-CoA reductase, acyltransferase, fatty
acyl transferase, diacylglycerol acyltransferase, acyl-CoA was
alcohol acyltransferase, bifunctional wax ester
synthase/acyl-CoA:diacylglycerol acyltransferase, and
.beta.-ketoacyl-ACP synthase I; or [0016] when said carbon product
biosynthetic pathway is for alkane biosynthesis, said carbon
product biosynthetic pathway includes one or more of: decarbonylase
and terminal alcohol oxidoreductase; or [0017] when said carbon
product biosynthetic pathway is for .omega.-cyclic fatty acid
biosynthesis, said carbon product biosynthetic pathway includes one
or more of: 1-cyclohexenylcarbonyl CoA reductase,
5-enopyruvylshikimate-3-phosphate synthase, acyl-CoA dehydrogenase,
enoyl-(ACP) reductase, 2,4-dienoyl-CoA reductase, and acyl-CoA
isomerase; or [0018] when said carbon product biosynthetic pathway
is for halogenated fatty acid biosynthesis, said carbon product
biosynthetic pathway includes one or more of: fluorinase,
nucleotide phosphorylase, fluorometabolite-specific aldolase,
fluoroacetaldehyde dehydrogenase, and fluoroacetyl-CoA synthase; or
[0019] when said carbon product biosynthetic pathway is the
deoxylylulose 5-phosphate (DXP) isoprenoid pathway, said carbon
product biosynthetic pathway includes one or more of:
1-deoxy-D-xylulose-5-phosphate synthase,
1-deoxy-D-xylulose-5-phosphate reductoisomerase,
4-diphosphocytidyl-2C-methyl-D-erythritol synthase,
4-diphosphocytidyl-2C-methyl-D-erythritol kinase,
2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase,
isopentyl/dimethylallyl diphosphate synthase and
4-hydroxy-3-methylbut-2-enyl diphosphate reductase; or [0020] when
said carbon product biosynthetic pathway is the
mevalonate-dependent (MEV) isoprenoid pathway, said carbon product
biosynthetic pathway includes one or more of: acetyl-CoA thiolase,
HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase,
phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase
and isopentenyl pyrophosphate isomerase; or [0021] when said carbon
product biosynthetic pathway is the glycerol/1,3-propanediol
biosynthesis pathway, said carbon product biosynthetic pathway
includes one or more of: sn-glycerol-3-P dehydrogenase,
sn-glycerol-3-phosphatase, glycerol dehydratase and 1,3-propanediol
oxidoreductase; or [0022] when said carbon product biosynthetic
pathway is the 1,4-butanediol/1,3-butadiene biosynthesis pathway,
said carbon product biosynthetic pathway includes one or more of:
succinyl-CoA dehydrogenase, 4-hydroxybutyrate dehydrogenase,
aldehyde dehydrogenase, 1,3-propanediol oxidoreductase and alcohol
dehydratase; or [0023] when said carbon product biosynthetic
pathway is the polyhydroxybutyrate biosynthesis pathway, said
carbon product biosynthetic pathway includes one or more of:
acetyl-CoA:acetyl-CoA C-acetyltransferase,
(R)-3-hydroxyacyl-CoA:NADP.sup.+ oxidoreductase and
polyhydroxyalkanoate synthase; or [0024] when said carbon product
biosynthetic pathway is the lysine biosynthesis pathway, said
carbon product biosynthetic pathway includes one or more of:
aspartate aminotransferase, aspartate kinase, aspartate
semialdehyde dehydrogenase, dihydrodipicolinate synthase,
dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase,
N-succinyldiaminopimelate-aminotransferase,
N-succinyl-L-diaminopimelate desuccinylase, diaminopimelate
epimerase, diaminopimelate decarboxylase, L,L-diaminopimelate
aminotransferase, homocitrate synthase, homoaconitase,
homoisocitrate dehydrogenase, 2-aminoadipate transaminase,
2-aminoadipate reductase, aminoadipate semialdehyde-glutamate
reductase and lysine-2-oxoglutarate reductase; or when said carbon
product biosynthetic pathway is the chorismate biosynthesis
pathway, said carbon product biosynthetic pathway includes one or
more of: 2-dehydro-3-deoxyphosphoheptonate aldolase,
3-dehydroquinate synthase, 3-dehydroquinate dehydratase,
NADPH-dependent shikimate dehydrogenase, NAD(P)H-dependent
shikimate dehydrogenase, shikimate kinase,
3-phosphoshikimate-1-carboxyvinyltransferase and chorismate
synthase; or [0025] when said carbon product biosynthetic pathway
is the phenylalanine biosynthesis pathway, said carbon product
biosynthetic pathway includes one or more of: chorismate mutase,
prephenate dehydratase and phenylalanine transaminase; or [0026]
when said carbon product biosynthetic pathway is the tyrosine
biosynthesis pathway, said carbon product biosynthetic pathway
includes one or more of: chorismate mutase, prephenate
dehydrogeanse and tyrosine aminotransferase; or [0027] when said
carbon product biosynthetic pathway is the .gamma.-valerolactone
biosynthesis pathway, said carbon product biosynthetic pathway
includes one or more of: propionyl-CoA synthase, beta-ketothiolase,
acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA dehydratase,
vinylacetyl-CoA .DELTA.-isomerase, 4-hydroxybutyryl-CoA transferase
and 1,4-lactonase; or [0028] when said carbon product biosynthetic
pathway is the butanol biosynthesis pathway, said carbon product
biosynthetic pathway includes one or more of: beta-ketothiolase,
acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA dehydrogenase,
enoyl-CoA hydratase, butyryl-CoA dehydrogenase,
trans-enoyl-coenzyme A reductase, butyrate CoA-transferase,
aldehyde dehydrogenase, alcohol dehydrogenase, acetyl-CoA
acetyltransferase, .beta.-hydroxybutyryl-CoA dehydrogenase,
crotonase, butyryl CoA dehydrogenase, CoA-acylating aldehyde
dehydrogenase and aldehyde-alcohol dehydrogenase; or [0029] when
said carbon product biosynthetic pathway is the acrylate
biosynthesis pathway, said carbon product biosynthetic pathway
includes one or more of: enoyl-CoA hydratase, propionyl-CoA
synthase and acrylate CoA-transferase.
[0030] In some embodiments, the 1-deoxy-D-xylulose-5-phosphate
synthase can be encoded by SEQ ID NO:1, or a homolog thereof having
at least 80% sequence identity; or the isopentenyl pyrophosphate
isomerase can be encoded by SEQ ID NO:2, or a homolog thereof
having at least 80% sequence identity. In one embodiment, when said
carbon product biosynthetic pathway is the isoprene biosynthesis
pathway, said carbon product biosynthetic pathway can include
isoprene synthase. The isoprene synthase can be encoded by SEQ ID
NO:3, or a homolog thereof having at least 80% sequence identity.
In another embodiment, when said carbon product biosynthetic
pathway is the bisabolene biosynthesis pathway, said carbon product
biosynthetic pathway can include E-alpha-bisabolene synthase. The
E-alpha-bisabolene synthase can be encoded by SEQ ID NO:4, or a
homolog thereof having at least 80% sequence identity.
[0031] In certain embodiments, the methylotrophic organism can be
selected from the class Alphaproteobacterium. The methylotrophic
organism may also be selected from the genus Paracoccus. For
example, the methylotrophic organism can be Paracoccus
denitrificans, Paracoccus versutus or Paracoccus
zeaxanthinifaciens.
[0032] In some embodiments, the engineered cell can be further
modified to have a less reduced growth rate on electrolytically
generated C1 compound relative to non-evolved methylotrophic
organism, or a substantially similar or enhanced growth rate on
electrolytically generated C1 compound relative to
non-electrolytically generated C1 compound. In certain embodiments,
the engineered cell can be further evolved to have a less reduced
growth rate on electrolytically generated C1 compound relative to
non-evolved methylotrophic organism, or a substantially similar or
enhanced growth rate on electrolytically generated C1 compound
relative to non-electrolytically generated C1 compound.
[0033] In another aspect, an evolved methylotrophic organism is
provided, having a less reduced growth rate on electrolytically
generated C1 compound relative to non-evolved methylotrophic
organism, or having a substantially similar or enhanced growth rate
on electrolytically generated C1 compound relative to
non-electrolytically generated C1 compound.
[0034] In a further aspect, a method for selecting an evolved
methylotrophic organism having improved growth on a C1 compound is
provided, comprising: incubating methylotrophic cells in a culture
chamber with controlled temperature, cell concentration, and medium
inflow and outflow rates, wherein the medium inflow includes a C1
compound; continuously monitoring a concentration of biomass in the
culture chamber; and adjusting a flow rate of the C1 compound into
the culture chamber so as to continually maintain an environment
that selects for an improved growth rate. In some embodiments, the
method can further include adjusting the medium inflow to be more
permissive of growth or more suppressive of growth, so as to
provide an adaptive environment to select for a fitness of the
cells. In certain examples, the C1 compound can be formate. The C1
compound may be electrolytically generated. The C1 compound can be
soluble in water.
[0035] In yet another aspect, a method of introducing a conjugative
plasmid into methylotrophic host cells is provided, comprising:
incubating a mixture of predetermined ratios of a donor culture and
a recipient culture, at temperatures between 4.degree. C. and
37.degree. C. for between 1 and 48 hours, wherein the donor culture
comprises a conjugal donor containing a conjugative plasmid having
a first selectable trait, and the recipient culture comprises
methylotrophic host cell having a second selectable trait; and
subjecting the incubated mixture to a dually selective condition
where only plasmid-containing transconjugants that have both the
first selectable trait and the second selectable trait can grow,
wherein the method does not include centrifugation or filtration of
the mixture or incubated mixture. In some embodiments, the conjugal
donor can be an E. coli strain such as E. coli S17-1, or an E. coli
harboring plasmids such as pRK2013 or pRK2073, or any E. coli
strain expressing a tra operon capable of mobilizing plasmids
containing an RP4-derived sequence. The conjugal donor can be in a
different species or genus of the host cell. In certain
embodiments, the transconjugated plasmid contains an RP4 or similar
mob element. In some embodiments, the host cell can be from the
class Alphaproteobacterium or from the genus Paracoccus. For
example, the host cell can be Paracoccus denitrificans, Paracoccus
versutus or Paracoccus zeaxanthinifaciens.
[0036] A further aspect of the invention relates to a composition
for bacterial culture, formulated to provide formate as the sole
source of C1 compound and to enhance the growth of methylotrophic
bacteria. The composition can contain between 0 and 160 mM sodium
bicarbonate, between 0 and 16 mM sodium chloride, between 0 and 100
mM sodium nitrate, between 0 and 30 mM sodium thio sulfate, and
initially containing between 5 and 100 mM of a formate salt, such
as sodium formate or ammonium formate. For example, the composition
can contain 100 mM sodium bicarbonate, 6 mM sodium chloride, 6 mM
sodium nitrate, 11 mM sodium thiosulfate, and 26 mM sodium formate
or ammonium formate. The composition can further include a basal
minimal medium. In some embodiments, the basal minimal medium can
be MOPS minimal medium, M9 minimal medium, R medium, M63 medium, or
a medium substantially similar to any of the foregoing.
[0037] In yet another aspect, a composition of bacterial culture is
provided, which is formulated to provide formate as the sole C1
compound and to enhance the growth of methylotrophic bacteria in a
fed-batch bioreactor. The composition can include a medium
initially charged in the fed-batch bioreactor which comprises R
medium supplemented with between 1 and 100 micromolar sodium
molybdate, between 10 and 1000 nanomolar sodium selenite, between
0.01 to 1 mg/L of thiamine, and between 0.001 to 1 mg/L of
cobalamin. For example, the medium can contain between 5 and 20
micromolar sodium molbydate, between 50 and 200 nanomolar sodium
selenite, between 0.05 to 2 mg/L of thiamine, and between 0.01 and
0.2 mg/L cobalamin. The composition can further include a feed
composition supplied to the fed-batch bioreactor comprising a
formate salt at supramolar concentration. The formate salt can be
ammonium formate and/or sodium formate. The feed composition can
further include a supramolar concentration of nitrate salt such as
sodium nitrate. The nitrate salt and the formate salt can be
provided in a molar ratio of 3.2:8, 3.0:8 or lower.
[0038] Also provided herein is a method for culturing
methylotrophic bacteria, comprising incubating methylotrophic
bacteria in any of the compositions described herein. In some
embodiments, the incubating can be conducted aerobically. The
incubating can take place in a fed-batch bioreactor. In some
embodiments, a volumetric rate of C1 feedstock consumption in the
fed-batch reactor can exceed 1.5 g*L.sup.-1 hr.sup.-1. In certain
embodiments, the incubating can be conducted in the presense of a
nitrate salt as electron acceptor and with a C1 feedstock as
electron donor. In some embodiments, the C1 feedstock is a formate
salt, such as sodium formate or ammonium formate. During
incubation, the molar ratio of the nitrate salt to the formate salt
can be kept below 3.2:8. The nitrate salt and the formate salt can
be provided to the fed-batch bioreactor in a feed composition in
supramolar concentrations in a molar ratio of 3.0:8 or lower. In
some embodiments, the formate salt can be ammonium formate and/or
sodium formate. The nitrate salt can be sodium nitrate.
[0039] In some aspects, growth of the methylotroph on C1 compounds
can be augmented by the addition of additional and/or alternative
pathways for energy conversion, methylotrophy and/or carbon
fixation. Exemplary energy conversion pathways and carbon fixation
pathways are described in U.S. Pat. No. 8,349,587, the entirety of
which is hereby incorporated herein by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIG. 1 depicts the metabolic reactions of the ribulose
monophosphate cycle [Strom, 1974]. Some methylotrophic organisms
such as Methylococcus capsulatus use this formaldehyde assimilation
pathway to make the central metabolites needed for growth. In
metabolite names, --P denotes phosphate. Each reaction is numbered.
Enzymes catalyzing each reaction are as follows: 1,
hexulose-6-phosphate synthase (E.C. 4.1.2.43);
2,6-phospho-3-hexuloisomerase (E.C. 5.3.1.27); 3,
phosphofructokinase (E.C. 2.7.1.11); 4, fructose bisphosphate
aldolase (E.C. 4.1.2.13); 5, transketolase (E.C. 2.2.1.1); 6,
transaldolase (E.C. 2.2.1.2); 7, transketolase (E.C. 2.2.1.1); 8,
ribose 5-phosphate isomerase (E.C. 5.3.1.6); 9,
ribulose-5-phosphate-3-epimerase (E.C. 5.1.3.1).
[0041] FIG. 2 depicts the metabolic reactions of the serine cycle.
Some methyltrophic organisms such as Hyphomicrobium methylovorum
GM2, Hyphomicrobium zavarzinii ZV580, Methylobacterium extorquens
AM1, Methylobacterium organophilum, Methylocystis parvus,
Methylosinus sporium and Methylosinus trichosporium use this
formaldehyde assimilation pathway to make the central metabolites
needed for growth. In metabolite names, --P denotes phosphate and
-CoA denotes coenzyme A. Enzymes catalyzing each reaction are as
follows: 1, spontaneous reaction; 2, serine
hydroxymethyltransferase (E.C. 2.1.2.1); 3, serine-glyoxylate
aminotransferase (E.C. 2.6.1.45); 4, hydroxypyruvate reductase
(E.C. 1.1.1.81); 5, glycerate 2-kinase (E.C. 2.7.1.165); 6, enolase
(E.C. 4.2.1.11); 7, phosphoenolpyruvate carboxylase (E.C.
4.1.1.31); 8, malate dehydrogenase (E.C. 1.1.1.37); 9, malate
thiokinase (E.C. 6.2.1.9); 10, malyl-CoA lyase (E.C. 4.1.3.24); 11,
the glyoxylate regeneration pathway.
[0042] FIG. 3 depicts the metabolic reactions of energy conversion
pathway(s) that oxidize C1 compounds in some methylotrophic
organisms such as Paracoccus species. Each reaction is numbered.
Enzymes catalyzing each reaction are as follows: 1, methanol
dehydrogenase (E.C. 1.1.2.7); 2, methylamine dehydrogenase (E.C.
1.4.9.1); 3, S-(hydroxymethyl) glutathione synthase (E.C.
4.4.1.22); 4, NAD- and glutathione-dependent formaldehyde
dehydrogenase (E.C. 1.1.1.284); 5, S-formylglutathione hydrolase
(E.C. 3.1.2.12); 6, formate dehydrogenase (E.C. 1.2.1.2).
[0043] FIG. 4 depicts the metabolic reactions of the
Calvin-Benson-Bassham cycle or the reductive pentose phosphate
(RPP) cycle [Bassham, 1954]. Some methylotrophic organisms such as
Paracoccus species use this carbon fixation pathway to reduce
carbon dioxide to central metabolites needed for growth. In
metabolite names, --P denotes phosphate. Each reaction is numbered.
Enzymes catalyzing each reaction are as follows: 1, ribulose
bisphosphate carboxylase (E.C. 4.1.1.39); 2, phosphoglycerate
kinase (E.C. 2.7.2.3); 3, glyceraldehyde-3P dehydrogenase
(phosphorylating) (E.C. 1.2.1.12 or E.C. 1.2.1.13); 4,
triose-phosphate isomerase (E.C. 5.3.1.1); 5, fructose-bisphosphate
aldolase (E.C. 4.1.2.13); 6, fructose-bisphosphatase (E.C.
3.1.3.11); 7, transketolase (E.C. 2.2.1.1); 8,
sedoheptulose-1,7-bisphosphate aldolase (E.C. 4.1.2.-); 9,
sedoheptulose bisphosphatase (E.C. 3.1.3.37); 10, transketolase
(E.C. 2.2.1.1); 11, ribose-5-phosphate isomerase (E.C. 5.3.1.6);
12, ribulose-5-phosphate-3-epimerase (E.C. 5.1.3.1); 13,
phosphoribulokinase (E.C. 2.7.1.19).
[0044] FIG. 5 depicts the metabolic reactions of the reductive
tricarboxylic acid cycle [Evans, 1966; Buchanan, 1990; Hiigler,
2011]. Some methylotrophic organisms such as Nautilia sp. strain
AmN use this carbon fixation pathway to reduce carbon dioxide to
central metabolites needed for growth. Each reaction is numbered.
For certain reactions, such as reaction 1 and 7, there are two
possible routes denoted by a and b, each of which is catalyzed by
different enzyme(s). Enzymes catalyzing each reaction are as
follows: 1a, ATP citrate lyase (E.C. 2.3.3.8); 1b, citryl-CoA
synthetase (E.C. 6.2.1.18) and citryl-CoA lyase (E.C. 4.1.3.34); 2,
malate dehydrogenase (E.C. 1.1.1.37); 3, fumarate dehydratase or
fumarase (E.C. 4.2.1.2); 4, fumarate reductase (E.C. 1.3.99.1); 5,
succinyl-CoA synthetase (E.C. 6.2.1.5); 6, 2-oxoglutarate synthase
or 2-oxoglutarate:ferredoxin oxidoreductase (E.C. 1.2.7.3); 7a,
isocitrate dehydrogenase (E.C. 1.1.1.41 or E.C. 1.1.1.42); 7b,
2-oxoglutarate carboxylase (E.C. 6.4.1.7) and oxalosuccinate
reductase (E.C. 1.1.1.41); 8, aconitate hydratrase (E.C. 4.2.1.3);
9, pyruvate synthase or pyruvate:ferredoxin oxidoreductase (E.C.
1.2.7.1); 10, phosphoenolpyruvate synthetase (E.C. 2.7.9.2); 11,
phosphoenolpyruvate carboxylase (E.C. 4.1.1.31).
[0045] FIG. 6 is a block diagram of a computing architecture.
[0046] FIG. 7 provides a schematic to convert succinate or
3-hydroxypropionate to various chemicals.
[0047] FIG. 8 provides a schematic of glutamate or itaconic acid
conversion to various chemicals.
[0048] FIG. 9 depicts the metabolic reactions of a galactose
biosynthetic pathway. In metabolite names, --P denotes phosphate.
Each reaction is numbered. Enzymes catalyzing each reaction are as
follows: 1, alpha-D-glucose-6-phosphate ketol-isomerase (E.C.
5.3.1.9); 2, D-mannose-6-phosphate ketol-isomerase (E.C. 5.3.1.8);
3, D-mannose 6-phosphate 1,6-phosphomutase (E.C. 5.4.2.8); 4,
mannose-1-phosphate guanylyltransferase (E.C. 2.7.7.22); 5,
GDP-mannose 3,5-epimerase (E.C. 5.1.3.18); 6, galactose-1-phosphate
guanylyltransferase (E.C. 2.7.n.n); 7, L-galactose 1-phosphate
phosphatase (E.C. 3.1.3.n).
[0049] FIG. 10 depicts different fermentation pathways from
pyruvate to ethanol. Each reaction is numbered. Enzymes catalyzing
each reaction are as follows: 1, pyruvate decarboxylase (E.C.
4.1.1.1); 2, alcohol dehydrogenase (E.C. 1.1.1.1); 3,
pyruvate-formate lyase (E.C. 2.3.1.54); 4, acetaldehyde
dehydrogenase (E.C. 1.2.1.10); 5, pyruvate synthase (E.C.
1.2.7.1).
[0050] FIG. 11 depicts the metabolic reactions of the
mevalonate-independent pathway (also known as the non-mevalonate
pathway or deoxyxylulose 5-phosphate (DXP) pathway) for production
of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl
pyrophosphate (DMAPP). In metabolite names, --P denotes phosphate.
Each reaction is numbered. Enzymes catalyzing each reaction are as
follows: 1, 1-deoxy-D-xylulose-5-phosphate synthase (E.C. 2.2.1.7);
2, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (E.C.
1.1.1.267); 3, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase
(E.C. 2.7.7.60); 4, 4-diphosphocytidyl-2C-methyl-D-erythritol
kinase (E.C. 2.7.1.148); 5, 2C-methyl-D-erythritol
2,4-cyclodiphosphate synthase (E.C. 4.6.1.12); 6,
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (E.C.
1.17.7.1); 7, isopentyl/dimethylallyl diphosphate synthase or
4-hydroxy-3-methylbut-2-enyl diphosphate reductase (E.C.
1.17.1.2).
[0051] FIG. 12 depicts the metabolic reactions of the mevalonate
pathway (also known as the HMG-CoA reductase pathway) for
production of isopentenyl pyrophosphate (IPP) and its isomer
dimethylallyl pyrophosphate (DMAPP). In metabolite names, --P
denotes phosphate. Each reaction is numbered. Enzymes catalyzing
each reaction are as follows: 1, acetyl-CoA thiolase; 2, HMG-CoA
synthase (E.C. 2.3.3.10); 3, HMG-CoA reductase (E.C. 1.1.1.34); 4,
mevalonate kinase (E.C. 2.7.1.36); 5, phosphomevalonate kinase
(E.C. 2.7.4.2); 6, mevalonate pyrophosphate decarboxylase (E.C.
4.1.1.33); 7, isopentenyl pyrophosphate isomerase (E.C.
5.3.3.2).
[0052] FIG. 13 depicts the metabolic reactions of the
glycerol/1,3-propanediol biosynthetic pathway for production of
glycerol or 1,3-propanediol. In metabolite names, --P denotes
phosphate. Each reaction is numbered. Enzymes catalyzing each
reaction are as follows: 1, sn-glycerol-3-P dehydrogenase (E.C.
1.1.1.8 or 1.1.1.94); 2, sn-glycerol-3-phosphatase (E.C. 3.1.3.21);
3, sn-glycerol-3-P glycerol dehydratase (E.C. 4.2.1.30); 4,
1,3-propanediol oxidoreductase (E.C. 1.1.1.202).
[0053] FIG. 14 depicts the metabolic reactions of the
polyhydroxybutyrate biosynthetic pathway. Each reaction is
numbered. Enzymes catalyzing each reaction are as follows: 1,
acetyl-CoA:acetyl-CoA C-acetyltransferase (E.C. 2.3.1.9); 2,
(R)-3-hydroxyacyl-CoA:NADP+oxidoreductase (E.C. 1.1.1.36); 3,
polyhydroxyalkanoate synthase (E.C. 2.3.1.-).
[0054] FIG. 15 depicts the metabolic reactions of one lysine
biosynthesis pathway. In metabolite names, --P denotes phosphate.
Each reaction is numbered. Enzymes catalyzing each reaction are as
follows: 1, aspartate aminotransferase (E.C. 2.6.1.1); 2, aspartate
kinase (E.C. 2.7.2.4); 3, aspartate semialdehyde dehydrogenase
(E.C. 1.2.1.11); 4, dihydrodipicolinate synthase (E.C. 4.2.1.52);
5, dihydrodipicolinate reductase (E.C. 1.3.1.26); 6,
tetrahydrodipicolinate succinylase (E.C. 2.3.1.117); 7,
N-succinyldiaminopimelate-aminotransferase (E.C. 2.6.1.17); 8,
N-succinyl-L-diaminopimelate desuccinylase (E.C. 3.5.1.18); 9,
diaminopimelate epimerase (E.C. 5.1.1.7); 10, diaminopimelate
decarboxylase (E.C. 4.1.1.20).
[0055] FIG. 16 depicts the metabolic reactions of the
.gamma.-valerolactone biosynthetic pathway. Each reaction is
numbered. Enzymes catalyzing each reaction are as follows: 1,
propionyl-CoA synthase (E.C. 6.2.1.-, E.C. 4.2.1.- and E.C.
1.3.1.-); 2, beta-ketothiolase (E.C. 2.3.1.16); 3, acetoacetyl-CoA
reductase (E.C. 1.1.1.36); 4, 3-hydroxybutyryl-CoA dehydratase
(E.C. 4.2.1.55); 5, vinylacetyl-CoA A-isomerase (E.C. 5.3.3.3); 6,
4-hydroxybutyryl-CoA transferase (E.C. 2.8.3.-); 7, 1,4-lactonase
(E.C. 3.1.1.25).
[0056] FIG. 17 depicts an example time course of formate usage,
formate accumulation, biomass formation and CO2 emission for a 1-L
aerobic, CSTR-type bioreactor initially charged with 0.5 L of
minimal medium containing Paracoccus versutus and fed ammonium
formate as a sole source of carbo and energy at a rate of 10 mM
hr.sup.-1. Over the course of the run the working volume changed
from 0.5 L to 0.785 L. The data for this run corresponds to formate
consumption rates of 1.6 g L.sup.-1 hr.sup.-1, maximum biomass
concentrations of 2.5 gDCW L.sup.-1, and carbon fixation fluxes of
8 mmol-C gDCW.sup.-1 L.sup.-1.
[0057] FIG. 18 depicts the required mass transfer coefficient
(K.sub.La) and required reactor volume for 0.5 t/d of fuel
production, as a function of maximum fuel productivity for
isooctanol, assuming fuel production from synthesis gas for an
ideal engineered organism. On the y axis, the typical range of
K.sub.La in large-scale stirred-tank bioreactors is denoted (A). On
the x axis, reported natural formate uptake rates at industrially
relevant culture densities are denoted (B).
DETAILED DESCRIPTION
[0058] The present invention relates to developing and using
engineered and/or evolved methylotrophs capable of utilizing C1
compounds to produce a desired product. The invention provides for
the engineering of a methylotroph, for example, Paracoccus
denitrificans, Paracoccus versutus or Paracoccus
zeaxanthinifaciens, or other organism suitable for commercial
large-scale production of fuels and chemicals, that can efficiently
utilize C1 compounds as a substrate for growth and for chemical
production provides cost-advantaged processes for manufacturing of
carbon based products of interest. The organisms can be optimized
and tested rapidly and at reasonable costs. The invention further
provides for the engineering of a methylotroph to include one or
more additional or alternative pathways for utilization of C1
compounds to produce central metabolites for growth and/or other
desired products.
[0059] C1 compounds represent an alternative feedstock to sugar or
light plus carbon dioxide for the production of carbon-based
products of interest. There exist non-biological routes to convert
C1 compounds to chemicals and fuels of interest. For example, the
Fischer-Tropsch process consumes carbon monoxide and hydrogen gas
generated from gasification of coal or biomass to produce methanol
or mixed hydrocarbons as fuels [U.S. Pat. No. 1,746,464]. The
drawbacks of Fischer-Tropsch processes are: 1) a lack of product
selectivity, which results in difficulties separating desired
products; 2) catalyst sensitivity to poisoning; 3) high energy
costs due to high temperatures and pressures required; and 4) the
limited range of products available at commercially competitive
costs. Without the advent of carbon sequestration technologies that
can operate at scale, the Fischer-Tropsch process is widely
considered to be an environmentally costly method for generating
liquid fuels. Alternatively, processes that rely on naturally
occurring microbes that convert synthesis gas or syngas, a mixture
of primarily molecular hydrogen and carbon monoxide that can be
obtained via gasification of any organic feedstock, such as coal,
coal oil, natural gas, biomass, or waste organic matter, to
products such as ethanol, acetate, methane, or molecular hydrogen
are available [Henstra, 2007]. However, these naturally occurring
microbes can produce only a very restricted set of products, are
limited in their efficiencies, lack established tools for genetic
manipulation, and are sensitive to their end products at high
concentrations. Finally, there is some work to introduce syngas
utilization into industrial microbial hosts [U.S. Pat. No.
7,803,589]; however, these processes have yet to be demonstrated at
commercial scale and are limited to using syngas as the
feedstock.
[0060] The present invention provides, in some aspects, engineered
or evolved methylotrophic organisms that are advantageous and/or
suitable for industrial uses. The invention also provides a source
of renewable energy. In some embodiments, the invention provides
for the use of a C1 compound, such as formate, formic acid,
formaldehyde, methanol or any combination thereof. In one
embodiment, the C1 compound can be derived from electrolysis. There
is tremendous commercial activity towards the goal of renewable
and/or carbon-neutral energy from solar voltaic, geothermal, wind,
nuclear, hydroelectric and more. However, most of these
technologies produce electricity and are thus limited in use to the
electrical grid [Whipple, 2010]. Furthermore, at least some of
these renewable energy sources such as solar and wind suffer from
being intermittent and unreliable. The lack of practical, large
scale electricity storage technologies limits how much of the
electricity demand can be shifted to renewable sources. The ability
to store electrical energy in chemical form, such as in
carbon-based products of interest, would both offer a means for
large-scale electricity storage and allow renewable electricity to
meet energy demand from the transportation sector. Renewable
electricity combined with electrolysis, such as the electrochemical
production of formate/formic acid from carbon dioxide [for example,
WO/2007/041872] or formaldehyde or methanol from carbon dioxide
[for example, WO/2010/088524, WO/2012/015909, WO/2012/015905],
opens the possibility of a sustainable, renewable supply of the C1
compound as one aspect of the present invention.
[0061] In some embodiments, the invention provides for the use of a
C1 compound, such as formaldehyde and/or methanol, derived from
waste streams. For example, formaldehyde is an oxidation product of
methanol or methane. Methanol can be prepared from synthesis gas
(the major product of gasification of coal, coal oil, natural gas,
and of carbonaceous materials such as biomass materials, including
agricultural crops and residues, and waste organic matter) or
reductive conversion of carbon dioxide and hydrogen by chemical
synthetic processes. Methane is a major component of natural gas
and can also be obtained from renewable biomass.
[0062] The invention provides for the expression of one or more
exogenous proteins or enzymes in the host cell, thereby conferring
biosynthetic pathway(s) to utilize central metabolites to produce
reduced organic compounds. The engineered cell can also be endowed
with one or more carbon product biosynthetic pathways that convert
central metabolites into desired products, such as carbon-based
products of interest.
[0063] 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
would 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.
DEFINITIONS
[0064] As used herein, the terms "nucleic acids," "nucleic acid
molecule" and "polynucleotide" may be used interchangeably and
include both single-stranded (ss) and double-stranded (ds) RNA, DNA
and RNA:DNA hybrids. As used herein the terms "nucleic acid",
"nucleic acid molecule", "polynucleotide", "oligonucleotide",
"oligomer" and "oligo" are used interchangeably and are intended to
include, but are not limited to, a polymeric form of nucleotides
that may have various lengths, including either
deoxyribonucleotides or ribonucleotides, or analogs thereof. For
example, oligos may be from 5 to about 200 nucleotides, from 10 to
about 100 nucleotides, or from 30 to about 50 nucleotides long.
However, shorter or longer oligonucleotides may be used. Oligos for
use in the present invention can be fully designed. A nucleic acid
molecule may encode a full-length polypeptide or a fragment of any
length thereof, or may be non-coding.
[0065] Nucleic acids can refer to naturally-occurring or synthetic
polymeric forms of nucleotides. The oligos and nucleic acid
molecules of the present invention may be formed from
naturally-occurring nucleotides, for example forming
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules.
Alternatively, the naturally-occurring oligonucleotides may include
structural modifications to alter their properties, such as in
peptide nucleic acids (PNA) or in locked nucleic acids (LNA). The
terms should be understood to include equivalents, analogs of
either RNA or DNA made from nucleotide analogs and as applicable to
the embodiment being described, single-stranded or double-stranded
polynucleotides. Nucleotides useful in the invention include, for
example, naturally-occurring nucleotides (for example,
ribonucleotides or deoxyribonucleotides), or natural or synthetic
modifications of nucleotides, or artificial bases. Modifications
can also include phosphorothioated bases for increased
stability.
[0066] Nucleic acid sequences that are "complementary" are those
that are capable of base-pairing according to the standard
Watson-Crick complementarity rules. As used herein, the term
"complementary sequences" means nucleic acid sequences that are
substantially complementary, as may be assessed by the nucleotide
comparison methods and algorithms set forth below, or as defined as
being capable of hybridizing to the polynucleotides that encode the
protein sequences.
[0067] As used herein, the term "gene" refers to a nucleic acid
that contains information necessary for expression of a
polypeptide, protein, or untranslated RNA (e.g., rRNA, tRNA,
anti-sense RNA). When the gene encodes a protein, it includes the
promoter and the structural gene open reading frame sequence (ORF),
as well as other sequences involved in expression of the protein.
When the gene encodes an untranslated RNA, it includes the promoter
and the nucleic acid that encodes the untranslated RNA.
[0068] As used herein, the term "genome" refers to the whole
hereditary information of an organism that is encoded in the DNA
(or RNA for certain viral species) including both coding and
non-coding sequences. In various embodiments, the term may include
the chromosomal DNA of an organism and/or DNA that is contained in
an organelle such as, for example, the mitochondria or chloroplasts
and/or extrachromosomal plasmid and/or artificial chromosome. A
"native gene" or "endogenous gene" refers to a gene that is native
to the host cell with its own regulatory sequences whereas an
"exogenous gene" or "heterologous gene" refers to any gene that is
not a native gene, comprising regulatory and/or coding sequences
that are not native to the host cell. In some embodiments, a
heterologous gene may comprise mutated sequences or part of
regulatory and/or coding sequences. In some embodiments, the
regulatory sequences may be heterologous or homologous to a gene of
interest. A heterologous regulatory sequence does not function in
nature to regulate the same gene(s) it is regulating in the
transformed host cell. "Coding sequence" refers to a DNA sequence
coding for a specific amino acid sequence. As used herein,
"regulatory sequences" refer to nucleotide sequences located
upstream (5' non-coding sequences), within, or downstream (3'
non-coding sequences) of a coding sequence, and which influence the
transcription, RNA processing or stability, or translation of the
associated coding sequence. Regulatory sequences may include
promoters, ribosome binding sites, translation leader sequences,
RNA processing site, effector (e.g., activator, repressor) binding
sites, stem-loop structures, and so on.
[0069] As described herein, a genetic element may be any coding or
non-coding nucleic acid sequence. In some embodiments, a genetic
element is a nucleic acid that codes for an amino acid, a peptide
or a protein. Genetic elements may be operons, genes, gene
fragments, promoters, exons, introns, regulatory sequences, or any
combination thereof. Genetic elements can be as short as one or a
few codons or may be longer including functional components (e.g.
encoding proteins) and/or regulatory components. In some
embodiments, a genetic element includes an entire open reading
frame of a protein, or the entire open reading frame and one or
more (or all) regulatory sequences associated therewith. One
skilled in the art would appreciate that the genetic elements can
be viewed as modular genetic elements or genetic modules. For
example, a genetic module can comprise a regulatory sequence or a
promoter or a coding sequence or any combination thereof. In some
embodiments, the genetic element includes at least two different
genetic modules and at least two recombination sites. In
eukaryotes, the genetic element can comprise at least three
modules. For example, a genetic module can be a regulator sequence
or a promoter, a coding sequence, and a polyadenlylation tail or
any combination thereof. In addition to the promoter and the coding
sequences, the nucleic acid sequence may comprises control modules
including, but not limited to a leader, a signal sequence and a
transcription terminator. The leader sequence is a non-translated
region operably linked to the 5' terminus of the coding nucleic
acid sequence. The signal peptide sequence codes for an amino acid
sequence linked to the amino terminus of the polypeptide which
directs the polypeptide into the cell's secretion pathway.
[0070] As generally understood, a codon is a series of three
nucleotides (triplets) that encodes a specific amino acid residue
in a polypeptide chain or for the termination of translation (stop
codons). There are 64 different codons (61 codons encoding for
amino acids plus 3 stop codons) but only 20 different translated
amino acids. The overabundance in the number of codons allows many
amino acids to be encoded by more than one codon. Different
organisms (and organelles) often show particular preferences or
biases for one of the several codons that encode the same amino
acid. The relative frequency of codon usage thus varies depending
on the organism and organelle. In some instances, when expressing a
heterologous gene in a host organism, it is desirable to modify the
gene sequence so as to adapt to the codons used and codon usage
frequency in the host. In particular, for reliable expression of
heterologous genes it may be preferred to use codons that correlate
with the host's tRNA level, especially the tRNA's that remain
charged during starvation. In addition, codons having rare cognate
tRNA's may affect protein folding and translation rate, and thus,
may also be used. Genes designed in accordance with codon usage
bias and relative tRNA abundance of the host are often referred to
as being "optimized" for codon usage, which has been shown to
increase expression level. Optimal codons also help to achieve
faster translation rates and high accuracy. In general, codon
optimization involves silent mutations that do not result in a
change to the amino acid sequence of a protein.
[0071] Genetic elements or genetic modules may derive from the
genome of natural organisms or from synthetic polynucleotides or
from a combination thereof. In some embodiments, the genetic
elements modules derive from different organisms. Genetic elements
or modules useful for the methods described herein may be obtained
from a variety of sources such as, for example, DNA libraries, BAC
(bacterial artificial chromosome) libraries, de novo chemical
synthesis, or excision and modification of a genomic segment. The
sequences obtained from such sources may then be modified using
standard molecular biology and/or recombinant DNA technology to
produce polynucleotide constructs having desired modifications for
reintroduction into, or construction of, a large product nucleic
acid, including a modified, partially synthetic or fully synthetic
genome. Exemplary methods for modification of polynucleotide
sequences obtained from a genome or library include, for example,
site directed mutagenesis; PCR mutagenesis; inserting, deleting or
swapping portions of a sequence using restriction enzymes
optionally in combination with ligation; in vitro or in vivo
homologous recombination; and site-specific recombination; or
various combinations thereof. In other embodiments, the genetic
sequences useful in accordance with the methods described herein
may be synthetic oligonucleotides or polynucleotides. Synthetic
oligonucleotides or polynucleotides may be produced using a variety
of methods known in the art.
[0072] In some embodiments, genetic elements share less than 99%,
less than 95%, less than 90%, less than 80%, less than 70% sequence
identity with a native or natural nucleic acid sequences. Identity
can each be determined by comparing a position in each sequence
which may be aligned for purposes of comparison. When an equivalent
position in the compared sequences is occupied by the same base or
amino acid, then the molecules are identical at that position; when
the equivalent site occupied by the same or a similar amino acid
residue (e.g., similar in steric and/or electronic nature), then
the molecules can be referred to as homologous (similar) at that
position. Expression as a percentage of homology, similarity, or
identity refers to a function of the number of identical or similar
amino acids at positions shared by the compared sequences.
Expression as a percentage of homology, similarity, or identity
refers to a function of the number of identical or similar amino
acids at positions shared by the compared sequences. Various
alignment algorithms and/or programs may be used, including FASTA,
BLAST, or ENTREZ FASTA and BLAST are available as a part of the GCG
sequence analysis package (University of Wisconsin, Madison, Wis.),
and can be used with, e.g., default settings. ENTREZ is available
through the National Center for Biotechnology Information, National
Library of Medicine, National Institutes of Health, Bethesda, Md.
In one embodiment, the percent identity of two sequences can be
determined by the GCG program with a gap weight of 1, e.g., each
amino acid gap is weighted as if it were a single amino acid or
nucleotide mismatch between the two sequences. Other techniques for
alignment are described [Doolittle, 1996]. Preferably, an alignment
program that permits gaps in the sequence is utilized to align the
sequences. The Smith-Waterman is one type of algorithm that permits
gaps in sequence alignments [Shpaer, 1997]. Also, the GAP program
using the Needleman and Wunsch alignment method can be utilized to
align sequences. An alternative search strategy uses MPSRCH
software, which runs on a MASPAR computer. MPSRCH uses a
Smith-Waterman algorithm to score sequences on a massively parallel
computer.
[0073] As used herein, 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. 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 would 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.
[0074] In contrast, as used herein, "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.
[0075] As used herein, 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 may 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 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.
[0076] 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 can 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.
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
would 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.
[0077] As used herein, the term "homolog" refers to any ortholog,
paralog, nonorthologous gene, or similar gene encoding an enzyme
catalyzing a similar or substantially similar metabolic reaction,
whether from the same or different species.
[0078] As used herein, the phrase "homologous recombination" refers
to the process in which nucleic acid molecules with similar
nucleotide sequences associate and exchange nucleotide strands. A
nucleotide sequence of a first nucleic acid molecule that is
effective for engaging in homologous recombination at a predefined
position of a second nucleic acid molecule can therefore have a
nucleotide sequence that facilitates the exchange of nucleotide
strands between the first nucleic acid molecule and a defined
position of the second nucleic acid molecule. Thus, the first
nucleic acid can generally have a nucleotide sequence that is
sufficiently complementary to a portion of the second nucleic acid
molecule to promote nucleotide base pairing. Homologous
recombination requires homologous sequences in the two recombining
partner nucleic acids but does not require any specific sequences.
Homologous recombination can be used to introduce a heterologous
nucleic acid and/or mutations into the host genome. Such systems
typically rely on sequence flanking the heterologous nucleic acid
to be expressed that has enough homology with a target sequence
within the host cell genome that recombination between the vector
nucleic acid and the target nucleic acid takes place, causing the
delivered nucleic acid to be integrated into the host genome. These
systems and the methods necessary to promote homologous
recombination are known to those of skill in the art.
[0079] It should be appreciated that the nucleic acid sequence of
interest or the gene of interest may be derived from the genome of
natural organisms. In some embodiments, genes of interest may be
excised from the genome of a natural organism or from the host
genome, for example E. coli. It has been shown that it is possible
to excise large genomic fragments by in vitro enzymatic excision
and in vivo excision and amplification. For example, the FLP/FRT
site specific recombination system and the Cre/loxP site specific
recombination systems have been efficiently used for excision large
genomic fragments for the purpose of sequencing [Yoon, 1998]. In
some embodiments, excision and amplification techniques can be used
to facilitate artificial genome or chromosome assembly. Genomic
fragments may be excised from the chromosome of a methylotroph and
altered before being inserted into the host cell artificial genome
or chromosome. In some embodiments, the excised genomic fragments
can be assembled with engineered promoters and/or other gene
expression elements and inserted into the genome of the host
cell.
[0080] As used herein, the term "polypeptide" refers to a sequence
of contiguous amino acids of any length. The terms "peptide,"
"oligopeptide," "protein" or "enzyme" may be used interchangeably
herein with the term "polypeptide". In certain instances, "enzyme"
refers to a protein having catalytic activities.
[0081] A "proteome" is the entire set of proteins expressed by a
genome, cell, tissue or organism. More specifically, it is the set
of expressed proteins in a given type of cells or an organism at a
given time under defined conditions. Transcriptome is the set of
all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding
RNA produced in one or a population of cells. Metabolome refers to
the complete set of small-molecule metabolites (such as metabolic
intermediates, hormones and other signaling molecules, and
secondary metabolites) to be found within a biological sample, such
as a single organism.
[0082] The term "fuse," "fused" or "link" refers to the covalent
linkage between two polypeptides in a fusion protein. The
polypeptides are typically joined via a peptide bond, either
directly to each other or via an amino acid linker. Optionally, the
peptides can be joined via non-peptide covalent linkages known to
those of skill in the art.
[0083] As used herein, unless otherwise stated, the term
"transcription" refers to the synthesis of RNA from a DNA template;
the term "translation" refers to the synthesis of a polypeptide
from an mRNA template. Translation in general is regulated by the
sequence and structure of the 5' untranslated region (5'-UTR) of
the mRNA transcript. One regulatory sequence is the ribosome
binding site (RBS), which promotes efficient and accurate
translation of mRNA. The prokaryotic RBS is the Shine-Dalgarno
sequence, a purine-rich sequence of 5'-UTR that is complementary to
the UCCU core sequence of the 3'-end of 16S rRNA (located within
the 30S small ribosomal subunit). Various Shine-Dalgarno sequences
have been found in prokaryotic mRNAs and generally lie about 10
nucleotides upstream from the AUG start codon. Activity of a RBS
can be influenced by the length and nucleotide composition of the
spacer separating the RBS and the initiator AUG. In eukaryotes, the
Kozak sequence A/GCCACCAUGG, which lies within a short 5'
untranslated region, directs translation of mRNA. An mRNA lacking
the Kozak consensus sequence may also be translated efficiently in
an in vitro systems if it possesses a moderately long 5'-UTR that
lacks stable secondary structure. While E. coli ribosome
preferentially recognizes the Shine-Dalgarno sequence, eukaryotic
ribosomes (such as those found in retic lysate) can efficiently use
either the Shine-Dalgarno or the Kozak ribosomal binding sites.
[0084] As used herein, the terms "promoter," "promoter element," or
"promoter sequence" refer to a DNA sequence which when ligated to a
nucleotide sequence of interest is capable of controlling the
transcription of the nucleotide sequence of interest into mRNA. A
promoter is typically, though not necessarily, located 5' (i.e.,
upstream) of a nucleotide sequence of interest whose transcription
into mRNA it controls, and provides a site for specific binding by
RNA polymerase and other transcription factors for initiation of
transcription.
[0085] One should appreciate that promoters have modular
architecture and that the modular architecture may be altered.
Bacterial promoters typically include a core promoter element and
additional promoter elements. The core promoter refers to the
minimal portion of the promoter required to initiate transcription.
A core promoter includes a Transcription Start Site, a binding site
for RNA polymerases and general transcription factor binding sites.
The "transcription start site" refers to the first nucleotide to be
transcribed and is designated +1. Nucleotides downstream the start
site are numbered +1, +2, etc., and nucleotides upstream the start
site are numbered -1, -2, etc. Additional promoter elements are
located 5' (i.e., typically 30-250 bp upstream of the start site)
of the core promoter and regulate the frequency of the
transcription. The proximal promoter elements and the distal
promoter elements constitute specific transcription factor site. In
prokaryotes, a core promoter usually includes two consensus
sequences, a -10 sequence or a -35 sequence, which are recognized
by sigma factors (see, for example, [Hawley, 1983]). The -10
sequence (10 bp upstream from the first transcribed nucleotide) is
typically about 6 nucleotides in length and is typically made up of
the nucleotides adenosine and thymidine (also known as the Pribnow
box). In some embodiments, the nucleotide sequence of the -10
sequence is 5'-TATAAT or may comprise 3 to 6 bases pairs of the
consensus sequence. The presence of this box is essential to the
start of the transcription. The -35 sequence of a core promoter is
typically about 6 nucleotides in length. The nucleotide sequence of
the -35 sequence is typically made up of the each of the four
nucleosides. The presence of this sequence allows a very high
transcription rate. In some embodiments, the nucleotide sequence of
the -35 sequence is 5'-TTGACA or may comprise 3 to 6 bases pairs of
the consensus sequence. In some embodiments, the -10 and the -35
sequences are spaced by about 17 nucleotides. Eukaryotic promoters
are more diverse than prokaryotic promoters and may be located
several kilobases upstream of the transcription starting site. Some
eukaryotic promoters contain a TATA box (e.g. containing the
consensus sequence TATAAA or part thereof), which is located
typically within 40 to 120 bases of the transcriptional start site.
One or more upstream activation sequences (UAS), which are
recognized by specific binding proteins can act as activators of
the transcription. Theses UAS sequences are typically found
upstream of the transcription initiation site. The distance between
the UAS sequences and the TATA box is highly variable and may be up
to 1 kb.
[0086] As used herein, the term "vector" refers to any genetic
element, such as a plasmid, phage, transposon, cosmid, chromosome,
artificial chromosome, episome, virus, virion, etc., capable of
replication when associated with the proper control elements and
which can transfer gene sequences into or between cells. The vector
may contain a marker suitable for use in the identification of
transformed or transfected cells. For example, markers may provide
antibiotic resistant, fluorescent, enzymatic, as well as other
traits. As a second example, markers may complement auxotrophic
deficiencies or supply critical nutrients not in the culture media.
Types of vectors include cloning and expression vectors. As used
herein, the term "cloning vector" refers to a plasmid or phage DNA
or other DNA sequence which is able to replicate autonomously in a
host cell and which is characterized by one or a small number of
restriction endonuclease recognition sites and/or sites for
site-specific recombination. A foreign DNA fragment may be spliced
into the vector at these sites in order to bring about the
replication and cloning of the fragment. The term "expression
vector" refers to a vector which is capable of expressing of a gene
that has been cloned into it. Such expression can occur after
transformation into a host cell, or in IVPS systems. The cloned DNA
is usually operably linked to one or more regulatory sequences,
such as promoters, activator/repressor binding sites, terminators,
enhancers and the like. The promoter sequences can be constitutive,
inducible and/or repressible.
[0087] As used herein, the term "host" or "host cell" refers to any
prokaryotic or eukaryotic (e.g., mammalian, insect, yeast, plant,
bacterial, archaeal, avian, animal, etc.) cell or organism. The
host cell can be a recipient of a replicable expression vector,
cloning vector or any heterologous nucleic acid molecule. In an
embodiment, the host cell is a methylotroph (e.g., naturally
exsisting or genetically engineered or metabolically evolved). Host
cells may be prokaryotic cells such as species of the genus
Paracoccus and Escherichia, or eukaryotic cells such as yeast,
insect, amphibian, or mammalian cells or cell lines. Cell lines
refer to specific cells that can grow indefinitely given the
appropriate medium and conditions. Cell lines can be mammalian cell
lines, insect cell lines or plant cell lines. Exemplary cell lines
can include tumor cell lines and stem cell lines. The heterologous
nucleic acid molecule may contain, but is not limited to, a
sequence of interest, a transcriptional regulatory sequence (such
as a promoter, enhancer, repressor, and the like) and/or an origin
of replication. As used herein, the terms "host," "host cell,"
"recombinant host" and "recombinant host cell" may be used
interchangeably. For examples of such hosts, see [Sambrook,
2001].
[0088] One or more nucleic acid sequences can be targeted for
delivery to target prokaryotic or eukaryotic cells via conventional
transformation or transfection techniques. As used herein, the
terms "transformation" and "transfection" are intended to refer to
a variety of art-recognized techniques for introducing an exogenous
nucleic acid sequence (e.g., DNA) into a target cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, conjugation,
electroporation, optoporation, injection and the like. Suitable
transformation or transfection media include, but are not limited
to, water, CaCl.sub.2, cationic polymers, lipids, and the like.
Suitable materials and methods for transforming or transfecting
target cells can be found in [Sambrook, 2001], and other laboratory
manuals. In certain instances, oligo concentrations of about 0.1 to
about 0.5 micromolar (per oligo) can be used for transformation or
transfection.
[0089] As used herein, the term "marker" or "reporter" refers to a
gene or protein that can be attached to a regulatory sequence of
another gene or protein of interest, so that upon expression in a
host cell or organism, the reporter can confer certain
characteristics that can be relatively easily selected, identified
and/or measured. Reporter genes are often used as an indication of
whether a certain gene has been introduced into or expressed in the
host cell or organism. Examples of commonly used reporters include:
antibiotic resistance genes, auxotropic markers,
.beta.-galactosidase (encoded by the bacterial gene lacZ),
luciferase (from lightning bugs), chloramphenicol acetyltransferase
(CAT; from bacteria), GUS (.beta.-glucuronidase; commonly used in
plants) and green fluorescent protein (GFP; from jelly fish).
Reporters or markers can be selectable or screenable. A selectable
marker (e.g., antibiotic resistance gene, auxotropic marker) is a
gene confers a trait suitable for artificial selection; typically
host cells expressing the selectable marker is protected from a
selective agent that is toxic or inhibitory to cell growth. A
screenable marker (e.g., gfp, lacZ) generally allows researchers to
distinguish between wanted cells (expressing the marker) and
unwanted cells (not expressing the marker or expressing at
insufficient level).
[0090] As used herein, the term "methylotroph" or "methylotrophic
organism" refers to organisms that produce complex organic
compounds from compounds that lack any carbon-carbon bonds, such as
formate, formic acid, formaldehyde, methane, methanol, methylamine,
halogenated methanes, and methylated sulfur species. Methylotrophs
often use C1 compounds as both a source of energy and carbon.
Example methylotrophic metabolic pathways for production of central
metabolites from C1 compounds include the ribulose monophosphate
cycle (FIG. 1) and the serine cycle (FIG. 2). "Autotrophs" or
"autotrophic organisms" refers to organisms that use simple,
inorganic carbon molecules, such as carbon dioxide, as its primary
carbon source for growth. Some but not all methylotrophs assimilate
C1 compounds via carbon dioxide and thus are also autotrophs. These
organisms oxidize C1 compounds such as methanol, methylamine,
formaldehyde or formate to carbon dioxide (see metabolic pathway
depicted in FIG. 3) and then reduce carbon dioxide to central
metabolites using carbon fixation cycles using, for example, the
Calvin-Benson-Bassham cycle (FIG. 4) or the reductive tricarboxlic
acid cycle (FIG. 5). In contrast, "heterotrophs" or "heterotrophic
organisms" refers to organisms that must use reduced, organic
carbon compounds with carbon-carbon bonds for growth because they
cannot use inorganic carbon as their primary carbon source.
Instead, heterotrophs obtain energy by breaking down the organic
molecules they consume. Organisms that can use a mix of different
sources of energy and carbon are mixotrophs or mixotrophic
organisms which can alternate, e.g., between autotrophy and
heterotrophy, between autotrophy and methylotrophy, between
heterotrophy and methylotrophy, between phototrophy and
chemotrophy, between lithotrophy and organotrophy, or a combination
thereof, depending on environmental conditions.
[0091] As used herein, the term "reducing cofactor" refers to
intracellular redox and energy carriers, such as NADH, NADPH,
ubiquinol, menaquinol, cytochromes, flavins and/or ferredoxin, that
can donate high energy electrons in reduction-oxidation reactions.
The terms "reducing cofacor", "reduced cofactor" and "redox
cofactor" can be used interchangeably.
[0092] As used herein, the term "C1 compound", "1C compound" or
"C.sub.1 compound" refers to chemical species that are reduced
species but contain no carbon-carbon bonds. C1 compounds may
contain either one carbon atom (e.g., formate, formic acid,
formamide, formaldehyde, methane, methanol, methylamine,
halogenated methanes, monomethyl sulfate) or multiple carbon atoms
(e.g., dimethyl ether, dimethylamine, dimethyl sulfide).
Furthermore, C1 compounds may be either inorganic (e.g., formate,
formic acid) or organic e.g., formaldehyde, methane, methanol). C1
compounds often serve as both a source of energy and a source of
carbon for methylotrophs.
[0093] As used herein, the term "central metabolite" refers to
organic carbon compounds, such as acetyl-coA, pyruvate, pyruvic
acid, 3-hydropropionate, 3-hydroxypropionic acid, glycolate,
glycolic acid, glyoxylate, glyoxylic acid, dihydroxyacetone
phosphate, glyceraldehyde-3-phosphate, malate, malic acid, lactate,
lactic acid, acetate, acetic acid, citrate and/or citric acid, that
can be converted into carbon-based products of interest by a host
cell or organism. Central metabolites are generally restricted to
those reduced organic compounds from which all or most cell mass
components can be derived in a given host cell or organism. In some
embodiments, the central metabolite is also the carbon product of
interest in which case no additional chemical conversion is
necessary.
[0094] Reference to a particular chemical species includes not only
that species but also water-solvated forms of the species, unless
otherwise stated. For example, carbon dioxide includes not only the
gaseous form (CO.sub.2) but also water-solvated forms, such as
bicarbonate ion.
[0095] As used herein, the term "biosynthetic pathway" or
"metabolic pathway" refers to a set of anabolic or catabolic
biochemical reactions for converting (transmuting) one chemical
species into another. Anabolic pathways involve constructing a
larger molecule from smaller molecules, a process requiring energy.
Catabolic pathways involve breaking down of larger molecules, often
releasing energy. As used herein, the term "energy conversion
pathway" refers to a metabolic pathway that transfers energy from a
C1 compound to a reducing cofactor. The term "carbon fixation
pathway" refers to a biosynthetic pathway that converts inorganic
carbon, such as carbon dioxide, bicarbonate or formate, to reduced
organic carbon, such as one or more carbon product precursors. The
term "methylotrophic pathway" refers to a biosynthetic pathway that
converts C1 compounds to compounds with carbon-carbon bonds, such
as one or more carbon product precursors. The term "carbon product
biosynthetic pathway" refers to a biosynthetic pathway that
converts one or more carbon product precursors to one or more
carbon based products of interest.
[0096] As used herein, the term "engineered methylotroph" or
"engineered methylotrophic organism" refers to organisms that have
been genetically engineered to convert C1 compounds, such as
formate, formic acid, formaldehyde, or methanol, to organic carbon
compounds. As used herein, an engineered methylotroph need not
derive its organic carbon compounds solely from C1 compounds. The
term engineered methylotroph may also be used to refer to
originally methylotrophic or mixotrophic organisms that have been
genetically engineered to include one or more energy conversion,
carbon fixation, methylotrophic and/or carbon product biosynthetic
pathways in addition or instead of its endogenous methylotrophic
capability. The term "engineer," "engineering" or "engineered," as
used herein, refers to genetic manipulation or modification of
biomolecules such as DNA, RNA and/or protein, or like technique
commonly known in the biotechnology art.
[0097] As used herein, the term "carbon based products of interest"
refers to a desired product containing carbon atoms and include,
but not limited to alcohols such as ethanol, propanol, isopropanol,
butanol, octanol, fatty alcohols, fatty acid esters, wax esters;
hydrocarbons and alkanes such as propane, octane, diesel, Jet
Propellant 8, polymers such as terephthalate, 1,3-propanediol,
1,4-butanediol, polyols, polyhydroxyalkanoates (PHAs),
polyhydroxybutyrates (PHBs), acrylate, adipic acid,
epsilon-caprolactone, isoprene, caprolactam, rubber; commodity
chemicals such as lactate, docosahexaenoic acid (DHA),
3-hydroxypropionate, .gamma.-valerolactone, lysine, serine,
aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid,
isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate,
1,3-butadiene, ethylene, propylene, succinate, citrate, citric
acid, glutamate, malate, 3-hydroxyprionic acid (HPA), lactic acid,
THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic
acid, levulinic acid, acrylic acid, malonic acid; specialty
chemicals such as carotenoids, isoprenoids, itaconic acid;
biological sugars such as glucose, fructose, lactose, sucrose,
starch, cellulose, hemicellulose, glycogen, xylose, dextrose,
galactose, uronic acid, maltose, polyketides, or glycerol; central
metabolites, such as acetyl-coA, pyruvate, pyruvic acid,
3-hydropropionate, 3-hydroxypropionic acid, glycolate, glycolic
acid, glyoxylate, glyoxylic acid, dihydroxyacetone phosphate,
glyceraldehyde-3-phosphate, malate, malic acid, lactate, lactic
acid, acetate, acetic acid, citrate and/or citric acid, from which
other carbon products can be made; pharmaceuticals and
pharmaceutical intermediates such as
7-aminodesacetoxycephalosporonic acid, cephalosporin, erythromycin,
polyketides, statins, paclitaxel, docetaxel, terpenes, peptides,
steroids, omega fatty acids and other such suitable products of
interest. Such products are useful in the context of biofuels,
industrial and specialty chemicals, as intermediates used to make
additional products, such as nutritional supplements,
neutraceuticals, polymers, paraffin replacements, personal care
products and pharmaceuticals.
[0098] As used herein, the term "hydrocarbon" referes a chemical
compound that consists of the elements carbon, hydrogen and
optionally, oxygen. "Surfactants" are substances capable of
reducing the surface tension of a liquid in which they are
dissolved. They are typically composed of a water-soluble head and
a hydrocarbon chain or tail. The water soluble group is hydrophilic
and can either be ionic or nonionic, and the hydrocarbon chain is
hydrophobic. The term "biofuel" is any fuel that derives from a
biological source.
[0099] The accession numbers provided throughout this description
are derived from the NCBI database (National Ceter for
Biotechnology Information) maintained by the National Institute of
Health, USA. The accession numbers are provided in the database on
Aug. 1, 2011. The Enzyme Classification Numbers (E.C.) provided
throughout this description are derived from the KEGG Ligand
database, maintained by the Kyoto Encyclopedia of Genes and
Genomics, sponsored in part by the University of Tokyo. The E.C.
numbers are provided in the database on Aug. 1, 2011.
[0100] Other terms used in the fields of recombinant nucleic acid
technology, microbiology, metabolic engineering, and molecular and
cell biology as used herein will be generally understood by one of
ordinary skill in the applicable arts.
Source of C1 Compounds
[0101] In some embodiments, suitable C1 compounds include, but not
limited to formate, formic acid, methanol and/or formaldehyde.
Formate, formic acid, formaldehyde and methanol can be produced via
the electrochemical reduction of CO.sub.2 [see, e.g., Hori,
2008].
[0102] In some instances, soluble, liquid feedstocks such as
formate, formic acid, formaldehyde or methanol can be preferable to
gaseous feedstocks, such as methane or synthesis gas. Methane is
generally known as a gas with low water solubility in water which
creates mass transfer limitations when using methane as the
feedstock for engineered and/or evolved methylotrophs (biological
systems are aqueous). Similarly, synthesis gas (composed of
molecular hydrogen and carbon monoxide) also has low water
solubility in water. At large reactor or fermentor scales, high
rates of mass transfer from the gas to liquid phases is
challenging. In contrast, formate, formaldehyde and methanol due to
their higher solubility/miscibility in H.sub.2O, do not have this
problem. Hence, when water is the solvent in the growth media, the
use of formate, formic acid, formaldehyde or methanol as the
feedstock can be more advantageous.
[0103] The energy efficiency of electrochemical conversion of
carbon dioxide impacts the overall energy efficiency of a
bio-manufacturing process using an engineered and/or evolved
methylotroph of the present invention. Electrolyzers achieve
overall energy efficiencies of 56-73% at current densities of
110-300 mA/cm.sup.2 (alkaline electrolyzers) or 800-1600
mA/cm.sup.2 (PEM electrolyzers) [Whipple, 2010]. In contrast,
electrochemical systems to date have achieved moderate energy
efficiencies or high current densities but not at the same time.
Hence, additional technology improvements are needed for
electrochemical production of formate, formic acid, formaldehyde
and methanol.
Organisms or Host Cells for Engineering or Evolution
[0104] The host cell or organism, as disclosed herein, may be
chosen from methylotrophic eukaryotic or prokaryotic systems, such
as bacterial cells (Gram-negative (e.g., Alphaproteobacterium) or
Gram-positive), archaea and yeast cells. Suitable cells and cell
lines can also include those commonly used in laboratories and/or
industrial applications. In some embodiments, host cells/organisms
can be selected from Bacillus species including Bacillus
methanolicus, Bilophila wadsworthia, Burkholderia species including
Burkholderia phymatum, Candida species including Candida boidinii,
Candida sonorensis, Cupravidus necator (formerly Alcaligenes
eutrophus and Ralstonia eutropha), Hyphomicrobium species including
Hyphomicrobium methylovorum, Hyphomicrobium zavarzinii,
Illethanococcus maripaludis, Methanomonas methanooxidans,
Methanosarcina species, Methylibium petroleiphilum, Methylobacillus
flagellatus, Methylobacillus flagellatum, Methylobacillus
fructoseoxidans, Methylobacillus glycogenes, Methylobacillus
viscogenes, Methylobacter bovis, Methylobacter capsulatus,
Methylobacter vinelandii, Methylobacterium species including
Methylobacterium dichloromethanicum, Methylobacterium extorquens,
Methylobacterium mesophilicum, Methylobacterium organophilum,
Methylobacterium rhodesianum, Methylococcus capsulatus,
Methylococcus minimus, Methylocystis species including
Methylocystis parvus, Methylomicrobium alcaliphilum, Methylomonas
species including Methylomonas agile, Methylomonas albus,
Methylomonas clara, Methylomonas methanica (formerly Bacillus
methanicus and Pseudomonas methanica), Methylomonas methanolica,
Methylomonas rosaceous, Methylomonas rubrum, Methylomonas
streptobacterium, Methylophilus methylotrophus, Methylosinus
species including Methylosinus sporium, Methylosinus trichosporium,
Methylosporovibrio methanica, Methyloversatilis universalis,
Methylovorus mays, Mycobacterium vaccae, Nautilia sp. strain AmN,
Nautilia lithotrophica, Nautilia profundicola, Paracoccus species
including Paracoccus denitrificans, Paracoccus versutus or
Paracoccus zeaxanthinifaciens, Picchia species including Picchia
angusta (formerly Hansenula polymorpha), Picchia guilliermondii,
Picchia pastoris, Protaminobacter ruber, Pseudomonas species
including Pseudomonas AM1, Pseudomonas methanitrificans, Schlegelia
plantiphila, Thermocrinus ruber, Verrucomicrobia species,
Xanthobacter species, or any modifications and/or derivatives
thereof. Those skilled in the art would understand that the genetic
modifications, including metabolic alterations exemplified herein,
are described with reference to a suitable host organism such as
Paracoccus denitrificans and their corresponding metabolic
reactions or a suitable source organism for desired nucleic acids
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 would readily be able to apply the teachings and guidance
provided herein to essentially all other methylotrophic host cells
and organisms. For example, the Paracoccus denitrificans metabolic
modifications 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
modifications include, for example, genetic alterations of species
homologs, in general, and in particular, orthologs, paralogs or
nonorthologous gene displacements.
[0105] In various aspects of the invention, the cells are
genetically engineered and/or metabolically evolved, for example,
for the purposes of optimized energy conversion, methylotrophy
and/or carbon fixation. The terms "metabolically evolved" or
"metabolic evolution" relates to growth-based selection (metabolic
evolution) of host cells that demonstrate improved growth (cell
yield).
[0106] Exemplary genomes and nucleic acids include full and partial
genomes of a number of organisms for which genome sequences are
publicly available and can be used with the disclosed methods, such
as, but not limited to, Aeropyrum pernix; Agrobacterium
tumefaciens; Anabaena; Anopheles gambiae; Apis mellifera; Aquifex
aeolicus; Arabidopsis thaliana; Archaeoglobus fulgidus; Ashbya
gossypii; Bacillus anthracis; Bacillus cereus; Bacillus halodurans;
Bacillus licheniformis; Bacillus subtilis; Bacteroides fragilis;
Bacteroides thetaiotaomicron; Bartonella henselae; Bartonella
quintana; Bdellovibrio bacteriovirus; Bifidobacterium longum;
Blochmannia floridanus; Bordetella bronchiseptica; Bordetella
parapertussis; Bordetella pertussis; Borrelia burgdorferi;
Bradyrhizobium japonicum; Brucella melitensis; Brucella suis;
Buchnera aphidicola; Burkholderia mallei; Burkholderia
pseudomallei; Caenorhabditis briggsae; Caenorhabditis elegans;
Campylobacter jejuni; Candida glabrata; Canis familiaris;
Caulobacter crescentus; Chlamydia muridarum; Chlamydia trachomatis;
Chlamydophila caviae; Chlamydophila pneumoniae; Chlorobium tepidum;
Chromobacterium violaceum; Ciona intestinalis; Clostridium
acetobutylicum; Clostridium perfringens; Clostridium tetania
Corynebacterium diphtheriae; Corynebacterium efficiens; Coxiella
burnetii; Cryptosporidium hominis; Cryptosporidium parvum;
Cyanidioschyzon merolae; Debaryomyces hansenii; Deinococcus
radiodurans; Desulfotalea psychrophile; Desulfovibrio vulgaris;
Drosophila melanogaster; Encephalitozoon cuniculi; Enterococcus
faecalis; Erwinia carotovora; Escherichia coli; Fusobacterium
nucleaturn; Gallus gallus; Geobacter sulfurreducens; Gloeobacter
violaceus; Guillardia theta; Haemophilus ducreyi; Haemophilus
influenzae; Halobacterium; Helicobacter hepaticus; Helicobacter
pylori; Homo sapiens; Kluyveromyces waltii; Lactobacillus
johnsonii; Lactobacillus plantarum; Legionella pneumophila;
Leifsonia xyli; Lactococcus lactis; Leptospira interrogans;
Listeria innocua; Listeria monocytogenes; Magnaporthe grisea;
Mannheimia succiniciproducens; Mesoplasma florum; Mesorhizobium
loti; Methanobacterium thermoautotrophicum; Methanococcoides
burtonii; Methanococcus jannaschii; Methanococcus maripaludis;
Methanogenium frigidum; Methanopyrus kandleri; Methanosarcina
acetivorans; Methanosarcina mazei; Methylococcus capsulatus; Mus
musculus; Mycobacterium bovis; Mycobacterium leprae; Mycobacterium
paratuberculosis; Mycobacterium tuberculosis; Mycoplasma
gallisepticum; Mycoplasma genitalium; Mycoplasma mycoides;
Mycoplasma penetrans; Mycoplasma pneumoniae; Mycoplasma pulmonis;
Mycoplasma mobile; Nanoarchaeum equitans; Neisseria meningitidis;
Neurospora crassa; Nitrosomonas europaea; Nocardia farcinica;
Oceanobacillus iheyensis; Onions yellows phytoplasma; Oryza sativa;
Pan troglodytes; Paracoccus denitrificans; Paracoccus versutus;
Paracoccus zeaxanthinifaciens; Pasteurella multocida; Phanerochaete
chrysosporium; Photorhabdus luminescens; Picrophilus torridus;
Plasmodium falciparum; Plasmodium yoelii yoelii; Populus
trichocarpa; Porphyromonas gingivalis Prochlorococcus marinus;
Propionibacterium acnes; Protochlamydia amoebophila; Pseudomonas
aeruginosa; Pseudomonas putida; Pseudomonas syringae; Pyrobaculum
aerophilum; Pyrococcus abyssi; Pyrococcus furiosus; Pyrococcus
horikoshii; Pyrolobus fumarii; Ralstonia solanacearum; Rattus
norvegicus; Rhodopirellula baltica; Rhodopseudomonas palustris;
Rickettsia conorii; Rickettsia typhi; Rickettsia prowazekii;
Rickettsia sibirica; Saccharomyces cerevisiae; Saccharomyces
bayanus; Saccharomyces boulardii; Saccharopolyspora erythraea;
Schizosaccharomyces pombe; Salmonella enterica; Salmonella
typhimurium; Schizosaccharomyces pornbe; Shewanella oneidensis;
Shigella flexneria; Sinorhizobium meliloti; Staphylococcus aureus;
Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus
mutans; Streptococcus pneumoniae; Streptococcus pyogenes;
Streptococcus thermophilus; Streptomyces avermitilis; Streptomyces
coelicolor; Sulfolobus solfataricus; Sulfolobus tokodaii;
Synechococcus; Synechoccous elongates; Synechocystis; Takifugu
rubripes; Tetraodon nigroviridis; Thalassiosira pseudonana;
Thermoanaerobacter tengcongensis; Thermoplasma acidophilum;
Thermoplasma volcanium; Thermosynechococcus elongatus; Thermotagoa
maritima; Thermus thermophilus; Treponema denticola; Treponema
pallidum; Tropheryma whipplei; Ureaplasma urealyticum; Vibrio
cholerae; Vibrio parahaemolyticus; Vibrio vulnificus;
Wigglesworthia glossinidia; Wolbachia pipientis; Wolinella
succinogenes; Xanthomonas axonopodis; Xanthomonas campestris;
Xylella fastidiosa; Yarrowia lipolytica; Yersinia
pseudotuberculosis; and Yersinia pestis nucleic acids.
[0107] In certain embodiments, sources of encoding nucleic acids
for enzymes for a biosynthetic pathway can include, for example,
any species where the encoded gene product is capable of catalyzing
the referenced reaction. Exemplary species for such sources
include, for example, Aeropyrum pernix; Aquifex aeolicus; Aquifex
pyrophilus; Candidatus Arcobacter sulfidicus; Candidatus Endoriftia
persephone; Candidatus Nitrospira defluvii; Chlorobium limicola;
Chlorobium tepidum; Clostridium pasteurianum; Desulfobacter
hydrogenophilus; Desulfurobacterium thermolithotrophum; Geobacter
metallireducens; Halobacterium sp. NRC-1; Hydrogenimonas
thermophila; Hydrogenivirga strain 128-5-R1; Hydrogenobacter
thermophilus; Hydrogenobaculum sp. Y04AAS1; Lebetimonas acidiphila
Pd55.sup.T; Leptospirillum ferriphilum; Leptospirillum
ferrodiazotrophum; Leptospirillum rubarum; Magnetococcus marinus;
Magnetospirillum magneticum; Mycobacterium bovis; Mycobacterium
tuberculosis; Methylobacterium nodulans; Nautilia lithotrophica;
Nautilia profundicola; Nautilia sp. strain AmN; Nitratifractor
salsuginis; Nitratiruptor sp. strain SB155-2; Paracoccus
denitrificans; Paracoccus versutus; Paracoccus zeaxanthinifaciens;
Persephonella marina; Rimcaris exoculata episymbiont; Streptomyces
avermitilis; Streptomyces coelicolor; Sulfolobus avermitilis;
Sulfolobus solfataricus; Sulfolobus tokodaii; Sulfurihydrogenibium
azorense; Sulfurihydrogenibium sp. Y03AOP1; Sulfurihydrogenibium
yellowstonense; Sulfurihydrogenibium subterraneum; Sulfurimonas
autotrophica; Sulfurimonas denitrificans; Sulfurimonas
paralvinella; Sulfurovum lithotrophicum; Sulfurovum sp. strain
NBC37-1; Thermocrinis ruber; Thermovibrio ammonificans;
Thermovibrio ruber; Thioreductor micatisoli; Nostoc sp. PCC 7120;
Acidithiobacillus ferrooxidans; Allochromatium vinosum; Aphanothece
halophytica; Oscillatoria limnetica; Rhodobacter capsulatus;
Thiobacillus denitrificans; Cupriavidus necator (formerly Ralstonia
eutropha), Methanosarcina barkeri; Methanosarcia mazei;
Methanococcus maripaludis; Mycobacterium smegmatis; Burkholderia
stabilis; Candida boidinii; Candida methylica; Pseudomonas sp. 101;
Methylcoccus capsulatus; Mycobacterium gastri; Cenarchaeum
symbiosum; Chloroflexus aurantiacus; Erythrobacter sp. NAP1;
Metallosphaera sedula; gamma proteobacterium NOR51-B; marine gamma
proteobacterium HTCC2080; Nitrosopumilus maritimus; Roseiflexus
castenholzii; Synechococcus elongatus; and the like, as well as
other exemplary species disclosed herein or available as source
organisms for corresponding genes. However, with the complete
genome sequence publicly available for now more than 4400 species
(including viruses), including 1701 microbial genomes and a variety
of yeast, fungi, plant, and mammalian genomes, the identification
of genes encoding the requisite energy conversion, methylotrophic,
carbon fixation or carbon product biosynthetic activity for one or
more genes in related or distant species, including for example,
homologs, orthologs, paralogs and nonorthologous gene displacements
of known genes, and the replacement of gene homolog either within a
particular engineered and/or evolved methylotroph or between
different host cells for the engineered and/or evolved methylotroph
is routine and well known in the art. Accordingly, the metabolic
modifications enabling methylotrophic growth and production of
carbon-based products described herein with reference to a
particular organism such as Paracoccus denitrificans can be readily
applied to other methylotrophic microorganisms, including
prokaryotic and eukaryotic organisms alike. Given the teachings and
guidance provided herein, those skilled in the art would know that
a metabolic modification exemplified in one organism can be applied
equally to other organisms.
[0108] In some instances, such as when an alternative energy
conversion, carbon fixation, methylotrophic or carbon product
biosynthetic pathway exists in an unrelated species, enhanced
methylotrophic growth and production of carbon-based products 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 would 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 would
understand that the teachings and methods of the invention can be
applied to all microbial organisms using the cognate metabolic
modifications to those exemplified herein to construct a microbial
organism in a species of interest that would produce carbon-based
products of interest from C1 compounds.
[0109] It should be noted that various engineered strains and/or
mutations of the organisms or cell lines discussed herein can also
be used.
Methods for Identification and Selection of Candidate Enzymes for a
Metabolic Activity of Interest
[0110] In one aspect, the present invention provides a method for
identifying candidate proteins or enzymes of interest capable of
performing a desired metabolic activity. Leveraging the exponential
growth of gene and genome sequence databases and the availability
of commercial gene synthesis at reasonable cost, Bayer and
colleagues adopted a synthetic metagenomics approach to
bioinformatically search sequence databases for homologous or
similar enzymes, computationally optimize their encoding gene
sequences for heterologous expression, synthesize the designed gene
sequence, clone the synthetic gene into an expression vector and
screen the resulting enzyme for a desired function in E. coli or
yeast [Bayer, 2009]. However, depending on the metabolic activity
or protein of interest, there can be thousands of putative homologs
in the publicly available sequence databases. Thus, it can be
experimentally challenging or in some cases infeasible to
synthesize and screen all possible homologs at reasonable cost and
within a reasonable timeframe. To address this challenge, in one
aspect, this invention provides an alternate method for identifying
and selecting candidate protein sequences for a metabolic activity
of interest. The method comprises the following steps. First, for a
desired metabolic activity, such as an enzyme-catalyzed step in an
energy conversion, methylotrophic, carbon fixation or carbon
product biosynthetic pathway, one or more enzymes of interest are
identified. Typically, the enzyme(s) of interest have been
previously experimentally validated to perform the desired
activity, for example in the published scientific literature. In
some embodiments, one or more of the enzymes of interest has been
heterologously expressed and experimentally demonstrated to be
functional. Second, a bioinformatic search is performed on protein
classification or grouping databases, such as Clusters of
Orthologous Groups (COGs) [Tatusov, 1997; Tatusov, 2003], Entrez
Protein Clusters (ProtClustDB) [Klimke, 2009] and/or InterPro
[Zdobnov, 2001], to identify protein groupings that contain one or
more of the enzyme(s) of interest (or closely related enzymes). If
the enzyme(s) of interest contain multiple subunits, then the
protein corresponding to a single subunit, for example the
catalytic subunit or the largest subunit, is selected as being
representative of the enzyme(s) of interest for the purposes of
bioinformatic analysis. Third, a systematic, expert-guided search
is then performed to identify which database groupings are likely
to contain a majority of members whose metabolic activity is the
same or similar as the protein(s) of interest. Fourth, the list of
NCBI Protein accession numbers corresponding to every members of
each selected database grouping is then compiled and the
corresponding protein sequences are downloaded from the sequence
databases. Protein sequences available from sources other than the
public sequence databases may be added to this set. Fifth,
optionally, one or more outgroup protein sequences are identified
and added to the set. Outgroup proteins are proteins which may
share some functional, structural, or sequence similarities to the
model enzyme(s) but lack an essential feature of the enzyme(s) of
interest or desired metabolic activity. For example, the enzyme
flavocytochrome c (E.C. 1.8.2.3) is similar to sulfide-quinone
oxidoreductase (E.C. 1.8.5.4) in that it oxidizes hydrogen sulfide
but it reduces cytochrome c instead of ubiquinone and thus offers a
useful outgroup during bioinformatic analysis of sulfide-quinone
oxidoreductases. Sixth, the complete set of protein sequences are
aligned with an sequence alignment program capable of aligning
large numbers of sequences, such as MUSCLE [Edgar, 2004a; Edgar,
2004b]. Seventh, a tree is drawn based on the resulting MUSCLE
alignment via methods known to those skilled in the art, such as
neighbor joining [Saitou, 1987] or UPGMA [Sokal, 1958; Murtagh,
1984]. Eighth, different clades are selected from the tree so that
the number of clades equals the desired number of proteins for
screening. Finally, one protein from each Glade is selected for
gene synthesis and functional screening based on the following
heuristics [0111] Preference is given to proteins that have been
heterologously expressed and experimentally demonstrated to have
the desired metabolic activity. [0112] Preference is given to
proteins that have been biochemically characterized to have the
desired metabolic activity previously. [0113] Preference is given
to proteins from source organisms for which there is strong
experimental or genomic evidence that the organism has the desired
metabolic activity. [0114] Preference is given to proteins in which
the key catalytic, binding and/or other signature residues are
conserved with respect to the protein(s) of interest. [0115]
Preference is given to protein from source organisms whose optimal
growth temperature is similar to that of the host cell or organism.
For example, if the host cell is a mesophile, then the source
organism is also a mesophile.
[0116] Therefore, in constructing the engineered and/or evolved
methylotroph of the invention, those skilled in the art would
understand that by applying the teaching and guidance provided
herein, it is possible to replace or augment particular genes
within a metabolic pathway, such as an energy conversion pathway, a
carbon fixation pathway, a methylotrophic pathway and/or a carbon
product biosynthetic pathway, with homologs identified using the
methods described here, whose gene products catalyze a similar or
substantially similar metabolic reaction. Such modifications can be
done, for example, to increase flux through a metabolic pathway
(for example, flux of energy or carbon), to reduce accumulation of
toxic intermediates, to improve the kinetic properties of the
pathway, and/or to otherwise optimize the engineered and/or evolved
methylotroph.
Methods for Design of Nucleic Acids Encoding Enzymes for
Heterologous Expression
[0117] In one aspect, the present invention provides a computer
program product for designing a nucleic acid that encodes a protein
or enzyme of interest that is codon optimized for the host cell or
organism (the target species). The program can reside on a hardware
computer readable storage medium and having a plurality of
instructions which, when executed by a processor, cause the
processor to perform operations. The program comprises the
following operations. At each amino acid position of the protein of
interest, the codon is selected in which the rank order codon usage
frequency of that codon in the target species is the same as the
rank order codon usage frequency of the codon that occurs at that
position in the source species gene. To select the desired codon at
each amino acid position, both the genetic code (the mapping of
codons to amino acids [Jukes, 1993]) and codon frequency table (the
frequency with which each synonymous codon occurs in a genome or
genome [Grantham, 1980]) for both the source and target species are
needed. For source species for which a complete genome sequence is
available, the usage frequency for each codon may be calculate
simply by summing the number of instances of that codon in all
annotated coding sequences, dividing by the total number of codons
in that genome, and then multiplying by 1000. For source species
for which no complete genome is available, the usage frequency can
be computed based on any available coding sequences or by using the
codon frequency table of a closely related organism. The program
then preferably standardizes the start codon to ATG, the stop codon
to TAA, and the second and second last codons to one of twenty
possible codons (one per amino acid). The program then subjects the
codon optimized nucleic acid sequence to a series of checks to
improve the likelihood that the sequence can be synthesized via
commercial gene synthesis and subsequently manipulated via
molecular biology [Sambrook, 2001] and DNA assembly methods
[WO/2010/070295]. These checks comprise identifying if key
restriction enzyme recognition sites used in a DNA assembly
standard or DNA assembly method are present; if hairpins whose GC
content exceeds a threshold percentage, such as 60%, and whose
length exceeds a threshold number of base pairs, such as 10, are
present; if sequence repeats are present; if any subsequence
between 100 and 150 nucleotides in length exceeds a threshold GC
content, such as 65%; if G or C homopolymers greater than 5
nucleotides in length are present; and, optionally, if any sequence
motifs are present that might give rise to spurious transposon
insertion sites, transcriptional or translational initiation or
termination, mRNA secondary structure, RNase cleavage, and/or
transcription factor binding. If the codon optimized nucleic acid
sequence fails any of these checks, the program then iterates
through all possible synonymous mutations and designs a new nucleic
acid sequence that both passes all checks and minimizes the
difference in codon frequencies between the original and new
nucleic acid sequence.
[0118] Various implementations of the systems and techniques
described here can be realized in digital electronic circuitry,
integrated circuitry, specially designed ASICs
(application-specific integrated circuits), computer hardware,
firmware, software, and/or combinations thereof. These various
implementations can include one or more computer programs that are
executable and/or interpretable on a programmable system including
at least one programmable processor, which may be special or
general purpose, coupled to receive data and instructions from, and
to transmit data and instructions to, a storage system, at least
one input device, and at least one output device. Such computer
programs (also known as programs, software, software applications
or code) may include machine instructions for a programmable
processor, and may be implemented in any form of programming
language, including high-level procedural and/or object-oriented
programming languages, and/or in assembly/machine languages. A
computer program may be deployed in any form, including as a
stand-alone program, or as a module, component, subroutine, or
other unit suitable for use in a computing environment. A computer
program may be deployed to be executed or interpreted on one
computer or on multiple computers at one site, or distributed
across multiple sites and interconnected by a communication
network.
[0119] A computer program may, in an embodiment, be stored on a
computer readable storage medium. A computer readable storage
medium stores computer data, which data can include computer
program code that is executed and/or interpreted by a computer
system or processor. By way of example, and not limitation, a
computer readable medium may comprise computer readable storage
media, for tangible or fixed storage of data, or communication
media for transient interpretation of code-containing signals.
Computer readable storage media, may refer to physical or tangible
storage (as opposed to signals) and may include without limitation
volatile and non-volatile, removable and non-removable media
implemented in any method or technology for the tangible storage of
information such as computer-readable instructions, data
structures, program modules or other data. Computer readable
storage media includes, but is not limited to, RAM, ROM, EPROM,
EEPROM, flash memory or other solid state memory technology,
CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic
tape, magnetic disk storage or other magnetic storage devices, or
any other physical or material medium which can be used to tangibly
store the desired information or data or instructions and which can
be accessed by a computer or processor.
[0120] FIG. 6 shows a block diagram of a generic processing
architecture, which may execute software applications and
processes. Computer processing device 200 may be coupled to display
202 for graphical output. Processor 204 may be a computer processor
capable of executing software. Typical examples of processor 204
are general-purpose computer processors (such as Intel.RTM. or
AMD.RTM. processors), ASICs, microprocessors, any other type of
processor, or the like. Processor 204 may be coupled to memory 206,
which may be a volatile memory (e.g. RAM) storage medium for
storing instructions and/or data while processor 204 executes.
Processor 204 may also be coupled to storage device 208, which may
be a non-volatile storage medium such as a hard drive, FLASH drive,
tape drive, DVDROM, or similar device. Program 210 may be a
computer program containing instructions and/or data, and may be
stored on storage device 208 and/or in memory 206, for example. In
a typical scenario, processor 204 may load some or all of the
instructions and/or data of program 210 into memory 206 for
execution.
[0121] Program 210 may be a computer program capable of performing
the processes and functions described above. Program 210 may
include various instructions and subroutines, which, when loaded
into memory 206 and executed by processor 204 cause processor 204
to perform various operations, some or all of which may effectuate
the methods, processes, and/or functions associated with the
presently disclosed embodiments.
[0122] Although not shown, computer processing device 200 may
include various forms of input and output. The I/O may include
network adapters, USB adapters, Bluetooth radios, mice, keyboards,
touchpads, displays, touch screens, LEDs, vibration devices,
speakers, microphones, sensors, or any other input or output device
for use with computer processing device 200.
Methods for Expression of Heterologous Enzymes
[0123] Composite nucleic acids can be constructed to include one or
more energy conversion, methylotrophic, carbon fixation and/or
carbon product biosynthetic pathway encoding nucleic acids as
exemplified herein. The composite nucleic acids can subsequently be
transformed or transfected into a suitable host organism for
expression of one or more proteins of interest. Composite nucleic
acids can be constructed by operably linking nucleic acids encoding
one or more standardized genetic parts with protein(s) of interest
encoding nucleic acids that have also been standardized.
Standardized genetic parts are nucleic acid sequences that have
been refined to conform to one or more defined technical standards,
such as an assembly standard [Knight, 2003; Shetty, 2008; Shetty,
2011]. Standardized genetic parts can encode transcriptional
initiation elements, transcriptional termination elements,
translational initiation elements, translational termination
elements, protein affinity tags, protein degradation tags, protein
localization tags, selectable markers, replication elements,
recombination sites for integration onto the genome, and more.
Standardized genetic parts have the advantage that their function
can be independently validated and characterized [Kelly, 2009] and
then readily combined with other standardized parts to produce
functional nucleic acids [Canton, 2008]. By mixing and matching
standardized genetic parts encoding different expression control
elements with nucleic acids encoding proteins of interest,
transforming the resulting nucleic acid into a suitable host cell
and functionally screening the resulting engineered cell, the
process of both achieving soluble expression of proteins of
interest and validing the function of those proteins is made
dramatically faster. For example, the set of standardized parts
might comprise constitutive promoters of varying strengths [Davis,
2011], ribosome binding sites of varying strengths [Anderson, 2007]
and protein degradation of tags of varying strengths [Andersen,
1998].
[0124] For exogenous expression in Paracoccus or other prokaryotic
cells, some nucleic acids encoding proteins of interest can be
modified to introduce solubility tags onto the protein of interest
to ensure soluble expression of the protein of interest. For
example, addition of the maltose binding protein to a protein of
interest has been shown to enhance soluble expression in E. coli
[Sachdev, 1998; Kapust, 1999; Sachdev, 2000]. Either alternatively
or in addition, chaperone proteins, such as DnaK, DnaJ, GroES and
GroEL may be either co-expressed or overexpressed with the proteins
of interest, such as RuBisCO [Greene, 2007], to promote correct
folding and assembly [Martinez-Alonso, 2009; Martinez-Alonso,
2010].
[0125] For exogenous expression in Parococcus 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, 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.
[0126] Exemplary, optimized methods for introduction of exogenous
nucleic acids into the methylotrophic bacteria Paracoccus versutus
and Paracoccus denitrificans via conjugative plasmid transfer are
described in detail herein in Example 2.
Production of Central Metabolites as the Carbon-Based Products of
Interest
[0127] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces the central
metabolites, including but not limited to citrate, malate,
succinate, fumarate, dihydroxyacetone, dihydroxyacetone phosphate,
3-hydroxypropionate, pyruvate, as the carbon-based products of
interest. The engineered and/or evolved methylotroph produces
central metabolites as an intermediate or product of the carbon
fixation or methylotrophic pathway or as a intermediate or product
of host metabolism. In such cases, one or more transporters may be
expressed in the engineered and/or evolved methylotroph to export
the central metabolite from the cell. For example, one or more
members of a family of enzymes known as C4-dicarboxylate carriers
serve to export succinate from cells into the media [Janausch,
2002; Kim, 2007]. These central metabolites can be converted to
other products (FIG. 7).
[0128] In some embodiments, the engineered and/or evolved
methylotroph may interconvert between different central metabolites
to produce alternate carbon-based products of interest. In one
embodiment, the engineered and/or evolved methylotroph produces
aspartate by expressing one or more aspartate aminotransferase
(E.C. 2.6.1.1), such as Escherichia coli AspC, to convert
oxaloacetate and L-glutamate to L-aspartate and 2-oxoglutarate.
[0129] In another embodiment, the engineered and/or evolved
methylotroph produces dihydroxyacetone phosphate by expressing one
or more dihydroxyacetone kinases (E.C. 2.7.1.29), such as C.
freundii DhaK, to convert dihydroxyacetone and ATP to
dihydroxyacetone phosphate.
[0130] In another embodiment, the engineered and/or evolved
methylotroph produces serine as the carbon-based product of
interest. The metabolic reactions necessary for serine biosynthesis
include: phosphoglycerate dehydrogenase (E.C. 1.1.1.95),
phosphoserine transaminase (E.C. 2.6.1.52), phosphoserine
phosphatase (E.C. 3.1.3.3). Phosphoglycerate dehydrogenase, such as
E. coli SerA, converts 3-phospho-D-glycerate and NAD.sup.+ to
3-phosphonooxypyruvate and NADH. Phosphoserine transaminase, such
as E. coli SerC, interconverts between
3-phosphonooxypyruvate+L-glutamate and
O-phospho-L-serine+2-oxoglutarate. Phosphoserine phosphatase, such
as E. coli SerB, converts O-phospho-L-serine to L-serine.
[0131] In another embodiment, the engineered and/or evolved
methylotroph produces glutamate as the carbon-based product of
interest. The metabolic reactions necessary for glutamate
biosynthesis include glutamate dehydrogenase (E.C. 1.4.1.4; e.g.,
E. coli GdhA) which converts .alpha.-ketoglutarate, NH.sub.3 and
NADPH to glutamate. Glutamate can subsequently be converted to
various other carbon-based products of interest, e.g., according to
the scheme presented in FIG. 8.
[0132] In another embodiment, the engineered and/or evolved
methylotroph produces itaconate as the carbon-based product of
interest. The metabolic reactions necessary for itaconate
biosynthesis include aconitate decarboxylase (E.C. 4.1.1.6; such as
that from A. terreus) which converts cis-aconitate to itaconate and
CO.sub.2. Itaconate can subsequently be converted to various other
carbon-based products of interest, e.g., according to the scheme
presented in FIG. 8.
Production of Sugars as the Carbon-Based Products of Interest
[0133] Industrial production of chemical products from biological
organisms is often accomplished using a sugar source, such as
glucose or fructose, as the feedstock. Hence, in certain
embodiments, the engineered and/or evolved methylotroph of the
present invention produces sugars including glucose and fructose or
sugar phosphates including triose phosphates (such as
3-phosphoglyceraldehyde and dihydroxyacetone-phosphate) as the
carbon-based products of interest. Sugars and sugar phosphates may
also be interconverted. For example, glucose-6-phosphate isomerase
(E.C. 5.3.1.9; e.g., E. coli Pgi) may interconvert between
D-fructose 6-phosphate and D-glucose-6-phosphate.
Phosphoglucomutase (E.C. 5.4.2.2; e.g., E. coli Pgm) converts
D-.alpha.-glucose-6-P to D-.alpha.-glucose-1-P.
Glucose-1-phosphatase (E.C. 3.1.3.10; e.g., E. coli Agp) converts
D-.alpha.-glucose-1-P to D-.alpha.-glucose. Aldose 1-epimerase
(E.C. 5.1.3.3; e.g., E. coli GalM) D-.beta.-glucose to
D-.alpha.-glucose. The sugars or sugar phosphates may optionally be
exported from the engineered and/or evolved methylotroph into the
culture medium.
[0134] Sugar phosphates may be converted to their corresponding
sugars via dephosphorylation that occurs either intra- or
extracellularly. For example, phosphatases such as a
glucose-6-phosphatase (E.C. 3.1.3.9) or glucose-1-phosphatase (E.C.
3.1.3.10) can be introduced into the engineered and/or evolved
methylotroph of the present invention. Exemplary phosphatases
include Homo sapiens glucose-6-phosphatase G6PC (P35575),
Escherichia coli glucose-1-phosphatase Agp (P19926), E. cloacae
glucose-1-phosphatase AgpE (Q6EV19) and Escherichia coli acid
phosphatase YihX (POA8Y3).
[0135] Sugar phosphates can be exported from the engineered and/or
evolved methylotroph into the culture media via transporters.
Transporters for sugar phosphates generally act as anti-porters
with inorganic phosphate. An exemplary triose phosphate transporter
includes A. thaliana triose-phosphate transporter APE2 (Genbank
accession AT5G46110.4). Exemplary glucose-6-phosphate transporters
include E. coli sugar phosphate transporter UhpT
(NP.sub.--418122.1), A. thaliana glucose-6-phosphate transporter
GPT1 (AT5G54800.1), A. thaliana glucose-6-phosphate transporter
GPT2, or homologs thereof. Dephosphorylation of glucose-6-phosphate
can also be coupled to glucose transport, such as Genbank accession
numbers AAA16222, AAD19898, 043826.
[0136] Sugars can be diffusively effluxed from the engineered
and/or evolved methylotroph into the culture media via permeases.
Exemplary permeases include H. sapiens glucose transporter GLUT-1,
-3, or -7 (P11166, P11169, Q6PXP3), S. cerevisiae hexose
transporter HXT-1, -4, or -6 (P32465, P32467, P39003), Z. mobilis
glucose uniporter Glf (P21906), Synechocystis sp. 1148
glucose/fructose:H.sup.+ symporter G1 cP (T.C. 2.A.1.1.32; P15729)
[Zhang, 1989], Streptomyces lividans major glucose (or
2-deoxyglucose) uptake transporter G1 cP (T.C. 2.A.1.1.35; Q7BEC4)
[van Wezel, 2005], Plasmodium falciparum hexose (glucose and
fructose) transporter PfHT1 (T.C. 2.A.1.1.24; 097467), or homologs
thereof. Alternatively, to enable active efflux of sugars from the
engineered and/or evolved methylotroph, one or more active
transporters may be introduced to the cell. Exemplary transporters
include mouse glucose transporter GLUT 1 (AAB20846) or homologs
thereof.
[0137] In some embodiments, to prevent buildup of other storage
polymers from sugars or sugar phosphates, the engineered and/or
evolved methylotrophs of the present invention are attenuated in
their ability to build other storage polymers such as glycogen,
starch, sucrose, and cellulose using one or more of the following
enzymes: cellulose synthase (UDP forming) (E.C. 2.4.1.12), glycogen
synthase e.g. glgA1, glgA2 (E.C. 2.4.1.21), sucrose phosphate
synthase (E.C. 2.4.1.14), sucrose phosphorylase (E.C. 3.1.3.24),
alpha-1,4-glucan lyase (E.C. 4.2.2.13), glycogen synthase (E.C.
2.4.1.11), 1,4-alpha-glucan branching enzyme (E.C. 2.4.1.18).
[0138] The invention also provides engineered and/or evolved
methylotrophs that produce other sugars such as sucrose, xylose,
lactose, maltose, pentose, rhamnose, galactose and arabinose
according to the same principles. A pathway for galactose
biosynthesis is shown (FIG. 9). The metabolic reactions in the
galactose biosynthetic pathway are catalyzed by the following
enzymes: alpha-D-glucose-6-phosphate ketol-isomerase (E.C. 5.3.1.9;
e.g., Arabidopsis thaliana PGI1), D-mannose-6-phosphate
ketol-isomerase (E.C. 5.3.1.8; e.g., Arabidopsis thaliana DINS),
D-mannose 6-phosphate 1,6-phosphomutase (E.C. 5.4.2.8; e.g.,
Arabidopsis thaliana ATPMM), mannose-1-phosphate
guanylyltransferase (E.C. 2.7.7.22; e.g., Arabidopsis thaliana
CYT), GDP-mannose 3,5-epimerase (E.C. 5.1.3.18; e.g., Arabidopsis
thaliana GME), galactose-1-phosphate guanylyltransferase (E.C.
2.7.n.n; e.g., Arabidopsis thaliana VTC2), L-galactose 1-phosphate
phosphatase (E.C. 3.1.3.n; e.g., Arabidopsis thaliana VTC4). In one
embodiment, the invention provides an engineered and/or evolved
methylotroph comprising one or more exogenous proteins from the
galactose biosynthetic pathway.
[0139] The invention also provides engineered and/or evolved
methylotrophs that produce sugar alcohols, such as sorbitol, as the
carbon-based product of interest. In certain embodiments, the
engineered and/or evolved methylotroph produces D-sorbitol from
D-.alpha.-glucose and NADPH via the enzyme polyol dehydrogenase
(E.C. 1.1.1.21; e.g., Saccharomyces cerevisiae GRE3).
[0140] The invention also provides engineered and/or evolved
methylotrophs that produce sugar derivatives, such as ascorbate, as
the carbon-based product of interest. In certain embodiments, the
engineered and/or evolved methylotroph produces ascorbate from
galactose via the enzymes L-galactose dehydrogenase (E.C.
1.1.1.122; e.g., Arabidopsis thaliana At4G33670) and
L-galactonolactone oxidase (E.C. 1.3.3.12; e.g., Saccharomyes
cerevisiae ATGLDH). Optionally, a catalase (E.C. 1.11.1.6; e.g., E.
coli KatE) may be included to convert the waste produce hydrogen
peroxide to molecular oxygen.
[0141] The fermentation products according to the above aspect of
the invention are sugars, which are exported into the media as a
result of C1 metabolism during methylotrophy. The sugars can also
be reabsorbed later and fermented, directly separated, or utilized
by a co-cultured organism. This approach has several advantages.
First, the total amount of sugars the cell can handle is not
limited by maximum intracellular concentrations because the
end-product is exported to the media. Second, by removing the
sugars from the cell, the equilibria of methylotrophic reactions
are pushed towards creating more sugar. Third, during
methylotrophy, there is no need to push carbon flow towards
glycolysis. Fourth, the sugars are potentially less toxic than the
fermentation products that would be directly produced.
[0142] Methylotrophy may be followed by flux of carbon compounds to
the creation and maintenance of biomass and to the storage of
retrievable carbon in the form of glycogen, cellulose and/or
sucrose. Glycogen is a polymer of glucose composed of linear alpha
1,4-linkages and branched alpha 1,6-linkages. The polymer is
insoluble at degree of polymerization (DP) greater than about
60,000 and forms intracellular granules. Glycogen in synthesized in
vivo via a pathway originating from glucose 1-phosphate. Its
hydrolysis can proceed through phosphorylation to glucose
phosphates; via the internal cleavage of polymer to maltodextrins;
via the successive exo-cleavage to maltose; or via the concerted
hydrolysis of polymer and maltodextrins to maltose and glucose.
Hence, an alternative biosynthetic route to glucose and/or maltose
is via the hydrolysis of glycogen which can optionally be exported
from the cell as described above. There are a number of potential
enzyme candidates for glycogen hydrolysis (Table 1).
[0143] In addition to the above, another mechanism is described to
produce glucose biosynthetically. In certain embodiments, the
present invention provides for cloned genes for glycogen
hydrolyzing enzymes to hydrolyze glycogen to glucose and/or maltose
and transport maltose and glucose from the cell. Exemplary enzymes
are set forth below in Table 1. Glucose is transported from the
engineered and/or evolved methylotroph by a glucose/hexose
transporter. This alternative allows the cell to accumulate
glycogen naturally but adds enzyme activities to continuously
return it to maltose or glucose units that can be collected as a
carbon-based product.
TABLE-US-00001 TABLE 1 Enzymes for hydrolysis of glycogen E.C.
Enzyme number Function .alpha.-amylase 3.2.1.1 endohydrolysis of
1,4-.alpha.-D-glucosidic linkages in polysaccharides .beta.-amylase
3.2.1.2 hydrolysis of 1,4-.alpha.-D-glucosidic linkages in
polysaccharides so as to remove successive maltose units from the
non-reducing ends of the chains .gamma.-amylase 3.2.1.3 hydrolysis
of terminal 1,4-linked .alpha.-D-glucose residues successively from
non-reducing ends of the chains with release of .beta.-D-glucose
glucoamylase 3.2.1.3 hydrolysis of terminal 1,4-linked
.alpha.-D-glucose residues successively from non-reducing ends of
the chains with release of .beta.-D-glucose isoamylase 3.2.1.68
hydrolysis of (1->6)-.alpha.-D-glucosidic branch linkages in
glycogen, amylopectin and their beta-limit dextrins pullulanase
3.2.1.41 hydrolysis of (1->6)-.alpha.-D-glucosidic linkages in
pullulan [a linear polymer of .alpha.-(1->6)-linked maltotriose
units] and in amylopectin and glycogen, and the .alpha.- and
.beta.-limit dextrins of amylopectin and glycogen amylomaltase
2.4.1.25 transfers a segment of a 1,4-.alpha.-D-glucan to a new
position in an acceptor, which may be glucose or a
1,4-.alpha.-D-glucan (part of yeast debranching system)
amylo-.alpha.-1,6- 3.2.1.33 debranching enzyme; hydrolysis of
(1->6)-.alpha.-D-glucosidic branch linkages in glucosidase
glycogen phosphorylase limit dextrin phosphorylase 2.7.11.19 2 ATP
+ phosphorylase b = 2 ADP + phosphorylase a kinase phosphorylase
2.4.1.1 (1,4-.alpha.-D-glucosyl).sub.n + phosphate =
(1,4-.alpha.-D-glucosyl).sub.n-1 +
.alpha.-D-glucose-1-phosphate
Production of Fermentative Products as the Carbon-Based Products of
Interest
[0144] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces alcohols such as
ethanol, propanol, isopropanol, butanol and fatty alcohols as the
carbon-based products of interest.
[0145] In some embodiments, the engineered and/or evolved
methylotroph of the present invention is engineered to produce
ethanol via pyruvate fermentation. Pyruvate fermentation to ethanol
is well know to those in the art and there are several pathways
including the pyruvate decarboxylase pathway, the pyruvate synthase
pathway and the pyruvate formate-lyase pathway (FIG. 10). The
reactions in the pyruvate decarboxylase pathway are catalyzed by
the following enzymes: pyruvate decarboxylase (E.C. 4.1.1.1) and
alcohol dehydrogenase (E.C. 1.1.1.1 or E.C. 1.1.1.2). The reactions
in the pyruvate synthase pathway are catalyzed by the following
enzymes: pyruvate synthase (E.C. 1.2.7.1), acetaldehyde
dehydrogenase (E.C. 1.2.1.10 or E.C. 1.2.1.5), and alcohol
dehydrogenase (E.C. 1.1.1.1 or E.C. 1.1.1.2). The reactions in the
pyruvate formate-lyase pathway are catalyzed by the following
enzymes: pyruvate formate-lyase (E.C. 2.3.1.54), acetaldehyde
dehydrogenase (E.C. 1.2.1.10 or E.C. 1.2.1.5), and alcohol
dehydrogenase (E.C. 1.1.1.1 or E.C. 1.1.1.2).
[0146] In some embodiments, the engineered and/or evolved
methylotroph of the present invention is engineered to produce
lactate via pyruvate fermentation. Lactate dehydrogenase (E.C.
1.1.1.28) converts NADH and pyruvate to D-lactate. Exemplary
enzymes include E. coli ldhA.
[0147] Currently, fermentative products such as ethanol, butanol,
lactic acid, formate, acetate produced in biological organisms
employ a NADH-dependent processes. However, depending on the
metabolism of the engineered and/or evolved methylotroph, the cell
may produce NADPH or reduced ferredoxin as the reducing cofactor.
NADPH is used mostly for biosynthetic operations in biological
organisms, e.g., cell for growth, division, and for building up
chemical stores, such as glycogen, sucrose, and other
macromolecules. Using natural or engineered enzymes that utilize
NADPH or reduced ferredoxin as a source of reducing power instead
of NADH would allow direct use of methylotrophic reducing power
towards formation of normally fermentative byproducts. Accordingly,
the present invention provides methods for producing fermentative
products such as ethanol by expressing NADP.sup.+-dependent or
ferredoxin-dependent enzymes. NADP.sup.+-dependent enzymes include
alcohol dehydrogenase [NADP.sup.+] (E.C. 1.1.1.2) and acetaldehyde
dehydrogenase [NAD(P).sup.+] (E.C. 1.2.1.5). Exemplary
NADP.sup.+-dependent alcohol dehydrogenases include Moorella sp.
HUC22-1 AdhA (YP.sub.--430754) [Inokuma, 2007], and homologs
thereof.
[0148] In addition to providing exogenous genes or endogenous genes
with novel regulation, the optimization of ethanol production in
engineered and/or evolved methylotrophs sometimes requires the
elimination or attenuation of certain host enzyme activities. These
include, but are not limited to, pyruvate oxidase (E.C. 1.2.2.2),
D-lactate dehydrogenase (E.C. 1.1.1.28), acetate kinase (E.C.
2.7.2.1), phosphate acetyltransferase (E.C. 2.3.1.8), citrate
synthase (E.C. 2.3.3.1), phosphoenolpyruvate carboxylase (E.C.
4.1.1.31). The extent to which these manipulations are necessary is
determined by the observed byproducts found in the bioreactor or
shake-flask. For instance, observation of acetate would suggest
deletion of pyruvate oxidase, acetate kinase, and/or
phosphotransacetylase enzyme activities. In another example,
observation of D-lactate would suggest deletion of D-lactate
dehydrogenase enzyme activities, whereas observation of succinate,
malate, fumarate, oxaloacetate, or citrate would suggest deletion
of citrate synthase and/or PEP carboxylase enzyme activities.
Production of Ethylene, Propylene, 1-Butene, 1,3-Butadiene, Acrylic
Acid, Etc. as the Carbon-Based Products of Interest
[0149] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces ethylene, propylene,
1-butene, 1,3-butadiene and acrylic acid as the carbon-based
products of interest. Ethylene and/or propylene may be produced by
either (1) the dehydration of ethanol or propanol (E.C. 4.2.1.-),
respectively or (2) the decarboxylation of acrylate or crotonate
(E.C. 4.1.1.-), respectively. While many dehydratases exist in
nature, none has been shown to convert ethanol to ethylene (or
propanol to propylene, propionic acid to acrylic acid, etc.) by
dehydration. Genes encoding enzymes in the 4.2.1.x or 4.1.1.x group
can be identified by searching databases such as GenBank using the
methods described above, expressed in any desired host (such as
Escherichia coli, for simplicity), and that host can be assayed for
the the appropriate enzymatic activity. A high-throughput screen is
especially useful for screening many genes and variants of genes
generated by mutagenesis (i.e., error-prone PCR, synthetic
libraries, chemical mutagenesis, etc.).
[0150] The ethanol dehydratase gene, after development to a
suitable level of activity, can then be expressed in an
ethanologenic organism to enable that organism to produce ethylene.
For instance, coexpress native or evolved ethanol dehydratase gene
into an organism that already produces ethanol, then test a culture
by GC analysis of offgas for ethylene production that is
significantly higher than without the added gene or via a
high-throughput assay adapted from a colorimetric test [Larue,
1973]. It may be desirable to eliminate ethanol-export proteins
from the production organism to prevent ethanol from being secreted
into the medium and preventing its conversion to ethylene.
[0151] Alternatively, acryloyl-CoA can be produced as described
above, and acryloyl-CoA hydrolases (E.C. 3.1.2.-), such as the acuN
gene from Halomonas sp. HTNK1, can convert acryloyl-CoA into
acrylate, which can be thermally decarboxylated to yield
ethylene.
[0152] Alternatively, genes encoding ethylene-forming enzyme
activities (EfE, E.C. 1.14.17.4) from various sources are
expressed. Exemplary enzymes include Pseudomonas syringae pv.
Phaseolicola (BAA02477), P. syringae pv. Pisi (AAD16443), Ralstonia
solanacearum (CAD18680). Optimizing production may require further
metabolic engineering (improving production of alpha-ketogluterate,
recycling succinate as two examples).
[0153] In some embodiments, the engineered and/or evolved
methylotroph of the present invention is engineered to produce
ethylene from methionine. The reactions in the ethylene
biosynthesis pathway are catalyzed by the following enzymes:
methionine adenosyltransferase (E.C. 2.5.1.6),
1-aminocyclopropane-1-carboxylate synthase (E.C. 4.4.1.14) and
1-aminocyclopropane-1-carboxylate oxidase (E.C. 1.14.17.4).
[0154] In some embodiments, the engineered and/or evolved
methylotroph of the present invention is engineered to produce
propylene as the carbon-based product of interest. In one
embodiment, the engineered and/or evolved methylotroph is
engineered to express one or more of the following enzymes:
propionyl-CoA synthase (E.C. 6.2.1.-, E.C. 4.2.1.- and E.C.
1.3.1.-), propionyl-CoA transferase (E.C. 2.8.3.1), aldehyde
dehydrogenase (E.C. 1.2.1.3 or E.C. 1.2.1.4), alcohol dehydrogenase
(E.C. 1.1.1.1 or E.C. 1.1.1.2), and alcohol dehydratase (E.C.
4.2.1.-). Propionyl-CoA synthase is a multi-functional enzyme that
converts 3-hydroxypropionate, ATP and NADPH to propionyl-CoA.
Exemplary propionyl-CoA synthases include AAL47820, and homologs
thereof. The present invention provides nucleic acids each
comprising or consisting of a sequence which is a codon optimized
version of the wild-type propionyl-CoA synthase gene. In another
embodiment, the invention provides a nucleic acid encoding a
polypeptide having the amino acid sequence of SEQ ID NO:5, or a
sequence having 70%, %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higher identity
thereto. Propionyl-CoA transferase converts propionyl-CoA and
acetate to acetyl-CoA and propionate. Exemplary enzymes include
Ralstonia eutropha pct and homologs thereof. Aldehyde dehydrogenase
converts propionate and NADPH to propanal. Alcohol dehydrogenase
converts propanal and NADPH to 1-propanol. Alcohol dehydratase
converts 1-propanol to propylene.
[0155] In another embodiment, E. coli thiolase atoB (E.C. 2.3.1.9)
converts 2 acetyl-CoA into acetoacetyl-CoA, and C. acetobutylicum
hbd (E.C. 1.1.1.157) converts acetoacetyl-CoA and NADH into
3-hydroxybutyryl-CoA. E. coli tesB (EC 3.1.2.20) or C.
acetobutylicum ptb and buk (E.C. 2.3.1.19 and 2.7.2.7 respectively)
convert 3-hydroxybutyryl-CoA into 3-hydroxybutyrate, which can be
simultaneously decarboxylated and dehydrated to yield propylene.
Optionally, the 3-hydroxybutyryl-CoA is polymerized to form
poly(3-hydroxybutyrate), a solid compound which can be extracted
from the fermentation medium and simultaneously depolymerizied,
hydrolyzed, dehydrated, and decarboxyated to yield propylene (U.S.
patent application Ser. No. 12/527,714, 2008).
Production of Fatty Acids, their Intermediates and Derivatives as
the Carbon-Based Products of Interest
[0156] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces fatty acids, their
intermediates and their derivatives as the carbon-based products of
interest. The engineered and/or evolved methylotrophs of the
present invention can be modified to increase the production of
acyl-ACP or acyl-CoA, to reduce the catabolism of fatty acid
derivatives and intermediates, or to reduce feedback inhibition at
specific points in the biosynthetic pathway used for fatty acid
products. In addition to modifying the genes described herein,
additional cellular resources can be diverted to over-produce fatty
acids. For example the lactate, succinate and/or acetate pathways
can be attenuated and the fatty acid biosynthetic pathway
precursors acetyl-CoA and/or malonyl-CoA can be overproduced.
[0157] In one embodiment, the engineered and/or evolved
methylotrophs of the present invention can be engineered to express
certain fatty acid synthase activities (FAS), which is a group of
peptides that catalyze the initiation and elongation of acyl chains
[Marrakchi, 2002a]. The acyl carrier protein (ACP) and the enzymes
in the FAS pathway control the length, degree of saturation and
branching of the fatty acids produced, which can be attenuated or
over-expressed. Such enzymes include accABCD, FabD, FabH, FabG,
FabA, FabZ, Fabl, FabK, FabL, FabM, FabB, FabF, and homologs
thereof.
[0158] In another embodiment, the engineered and/or evolved
methylotrophs of the present invention form fatty acid byproducts
through ACP-independent pathways, for example, the pathway
described recently by [Dellomonaco, 2011] involving reversal of
beta oxidation. Enzymes involved in these pathways include such
genes as atoB, fadA, fadB, fadD, fadE, fad I, fadK, fadJ, paaZ,
ydiO, yfcY, yfcZ, ydiD, and homologs thereof.
[0159] In one aspect, the fatty acid biosynthetic pathway
precursors acetyl-CoA and malonyl-CoA can be overproduced in the
engineered and/or evolved methylotroph of the present invention.
Several different modifications can be made, either in combination
or individually, to the host cell to obtain increased acetyl
CoA/malonyl CoA/fatty acid and fatty acid derivative production. To
modify acetyl-CoA and/or malonyl-CoA production, the expression of
acetyl-CoA carboxylase (E.C. 6.4.1.2) can be modulated. Exemplary
genes include accABCD (AAC73296) or homologs thereof. To increase
acetyl CoA production, the expression of several genes may be
altered including pdh, panK, aceEF, (encoding the Elp dehydrogenase
component and the E2p dihydrolipoamide acyltransferase component of
the pyruvate and 2-oxoglutarate dehydrogenase complexes),
fabH/fabD/fabG/acpP/fabF, and in some examples additional nucleic
acid encoding fatty-acyl-CoA reductases and aldehyde
decarbonylases. Exemplary enzymes include pdh (BAB34380, AAC73227,
AAC73226), panK (also known as coaA, AAC76952), aceEF (AAC73227,
AAC73226), fabH (AAC74175), fabD (AAC74176), fabG (AAC74177), acpP
(AAC74178), fabF (AAC74179).
[0160] Genes to be knocked-out or attenuated include fadE, gpsA,
ldhA, pflb, adhE, pta, poxB, ackA, and/or ackB. Exemplary enzymes
include fadE (AAC73325), gspA (AAC76632), ldhA (AAC74462), pflb
(AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA
(AAC75356), ackB (BAB81430), and homologs thereof.
[0161] Additional potential modifications include the following. To
achieve fatty acid overproduction, lipase (E.C. 3.1.1.3) which
produce triacylglyerides from fatty acids and glycerol and in some
cases serves as a suppressor of fabA can be included in the
engineered and/or evolved methylotroph of the present invention.
Exemplary enzymes include Saccharomyces cerevisiae LipA (CAA89087),
Saccharomyces cerevisiae TGL2 CAA98876, and homologs thereof. To
remove limitations on the pool of acyl-CoA, the D311E mutation in
plsB (AAC77011) can be introduced.
[0162] To engineer an engineered and/or evolved methylotroph for
the production of a population of fatty acid derivatives with
homogeneous chain length, one or more endogenous genes can be
attenuated or functionally deleted and one or more thioesterases
can be expressed. Thioesterases (E.C. 3.1.2.14) generate acyl-ACP
from fatty acid and ACP. For example, C10 fatty acids can be
produced by attenuating endogenous C18 thioesterases (for example,
E. coli tesA AAC73596 and POADA1, and homologs thereof), which uses
C18:1-ACP, and expressing a C10 thioesterase, which uses C10-ACP,
thus, resulting in a relatively homogeneous population of fatty
acids that have a carbon chain length of 10. In another example,
C14 fatty acid derivatives can be produced by attenuating
endogenous thioesterases that produce non-C14 fatty acids and
expressing the C14 thioesterase, which uses C14-ACP. In yet another
example, C12 fatty acid derivatives can be produced by expressing
thioesterases that use C12-ACP and attenuating thioesterases that
produce non-C12 fatty acids. Exemplary C8:0 to C10:0 thioesterases
include Cuphea hookeriana fatB2 (AAC49269) and homologs thereof.
Exemplary C12:0 thioesterases include Umbellularia california fatB
(Q41635) and homologs thereof. Exemplary C14:0 thioesterases
include Cinnamonum camphorum fatB (Q39473). Exemplary C14:0 to
C16:0 thioesterases include Cuphea hookeriana fatB3 (AAC49269).
Exemplary C16:0 thioesterases include Arabidopsis thaliana fatB
(CAA85388), Cuphea hookeriana fatB1 (Q39513) and homologs thereof.
Exemplary C18:1 thioesterases include Arabidopsis thaliana fatA
(NP.sub.--189147, NP.sub.--193041), Arabidopsis thaliana fatB
(CAA85388), Bradyrhizobium japonicum fatA (CAC39106), Cuphea
hookeriana fatA (AAC72883), Escherichia coli tesA (NP.sub.--415027)
and homologs thereof. Acetyl CoA, malonyl CoA, and fatty acid
overproduction can be verified using methods known in the art, for
example by using radioactive precursors, HPLC, and GC-MS subsequent
to cell lysis.
[0163] In yet another aspect, fatty acids of various lengths can be
produced in the engineered and/or evolved methylotroph by
expressing or overexpressing acyl-CoA synthase peptides (E.C.
2.3.1.86), which catalyzes the conversion of fatty acids to
acyl-CoA. Some acyl-CoA synthase peptides, which are non-specific,
accept other substrates in addition to fatty acids.
[0164] In yet another aspect, branched chain fatty acids, their
intermediates and their derivatives can be produced in the
engineered and/or evolved methylotroph as the carbon-based products
of interest. By controlling the expression of endogenous and
heterologous enzymes associated with branched chain fatty acid
biosynthesis, the production of branched chain fatty acid
intermediates including branched chain fatty acids can be enhanced.
Branched chain fatty acid production can be achieved through the
expression of one or more of the following enzymes [Kaneda, 1991]:
branched chain amino acid aminotransferase to produce
.alpha.-ketoacids from branched chain amino acids such as
isoleucine, leucine and valine (E.C. 2.6.1.42), branched chain
.alpha.-ketoacid dehydrogenase complexes which catalyzes the
oxidative decarboxylation of .alpha.-ketoacids to branched chain
acyl-CoA (bkd, E.C. 1.2.4.4) [Denoya, 1995], dihydrolipoyl
dehydrogenase (E.C. 1.8.1.4), beta-ketoacyl-ACP synthase with
branched chain acyl CoA specificity (E.C. 2.3.1.41) [Li, 2005],
crotonyl-CoA reductase (E.C. 1.3.1.8, 1.3.1.85 or 1.3.1.86) [Han,
1997], and isobutyryl-CoA mutase (large subunit E.C. 5.4.99.2 and
small subunit E.C. 5.4.99.13). Exemplary branched chain amino acid
aminotransferases include E. coli ilvE (YP.sub.--026247),
Lactococcus lactis ilvE (AAF34406), Pseudomonas putida ilvE
(NP.sub.--745648), Streptomyces coelicolor ilvE (NP.sub.--629657),
and homologs thereof. Branched chain .alpha.-ketoacid dehydrogenase
complexes consist of E1.alpha./.beta. (decarboxylase), E2
(dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase)
subunits. The industrial host E. coli has only the E3 component as
a part of its pyruvate dehydrogenase complex (lpd, E.C. 1.8.1.4,
NP.sub.--414658) and so it requires the E1.alpha./.beta. and E2 bkd
proteins. Exemplary .alpha.-ketoacid dehydrogenase complexes
include Streptomyces coelicolor bkdA1 (NP.sub.--628006) E1.alpha.
(decarboxylase component), S. coelicolor bkdB2 (NP.sub.--628005)
E1.beta. (decarboxylase component), S. coelicolor bkdA3
(NP.sub.--638004) E2 (dihydrolipoyl transacylase); or S. coelicolor
bkdA2 (NP.sub.--733618) E1.alpha. (decarboxylase component), S.
coelicolor bkdB2 (NP.sub.--628019) E1.beta. (decarboxylase
component), S. coelicolor bkdC2 (NP.sub.--628018) E2 (dihydrolipoyl
transacylase); or S. avermitilis bkdA (BAC72074) E1.alpha.
(decarboxylase component), S. avermitilis bkdB (BAC72075) E1.beta.
(decarboxylase component), S. avermitilis bkdC (BAC72076) E2
(dihydrolipoyl transacylase); S. avermitilis bkdF (E.C.1.2.4.4,
BAC72088) E1.alpha. (decarboxylase component), S. avermitilis bkdG
(BAC72089) E1.beta. (decarboxylase component), S. avermitilis bkdH
(BAC72090) E2 (dihydrolipoyl transacylase); B. subtilis bkdAA
(NP.sub.--390288) E1.alpha. (decarboxylase component), B. subtilis
bkdAB (NP.sub.--390288) E1.beta. (decarboxylase component), B.
subtilis bkdB (NP.sub.--390288) E2 (dihydrolipoyl transacylase); or
P. putida bkdA1 (AAA65614) Ela (decarboxylase component), P. putida
bkdA2 (AAA65615) E1.beta. (decarboxylase component), P. putida bkdC
(AAA65617) E2 (dihydrolipoyl transacylase); and homologs thereof.
An exemplary dihydrolipoyl dehydrogenase is E. coli lpd
(NP.sub.--414658) E3 and homologs thereof. Exemplary
beta-ketoacyl-ACP synthases with branched chain acyl CoA
specificity include Streptomyces coelicolor fabH1
(NP.sub.--626634), ACP (NP.sub.--626635) and fabF
(NP.sub.--626636); Streptomyces avermitilis fabH3
(NP.sub.--823466), fabC3 (NP.sub.--823467), fabF (NP.sub.--823468);
Bacillus subtilis fabH_A (NP.sub.--389015), fabH_B
(NP.sub.--388898), ACP (NP.sub.--389474), fabF (NP.sub.--389016);
Stenotrophomonas maltophilia SmalDRAFT.sub.--0818
(ZP.sub.--01643059), SmalDRAFT.sub.--0821 (ZP.sub.--01643063),
SmalDRAFT.sub.--0822 (ZP.sub.--01643064); Legionella pneumophila
fabH (YP.sub.--123672), ACP (YP.sub.--123675), fabF
(YP.sub.--123676); and homologs thereof. Exemplary crotonyl-CoA
reductases include Streptomyces coelicolor ccr (NP.sub.--630556),
Streptomyces cinnamonensis ccr (AAD53915), and homologs thereof.
Exemplary isobutyryl-CoA mutases include Streptomyces coelicolor
icmA & icmB (NP.sub.--629554 and NP.sub.--630904), Streptomyces
cinnamonensis icmA and icmB (AAC08713 and AJ246005), and homologs
thereof. Additionally or alternatively, endogenous genes that
normally lead to straight chain fatty acids, their intermediates,
and derivatives may be attenuated or deleted to eliminate competing
pathways. Enzymes that interfere with production of branched chain
fatty acids include .beta.-ketoacyl-ACP synthase II (E.C. 2.3.1.41)
and .beta.-ketoacyl-ACP synthase III (E.C. 2.3.1.41) with straight
chain acyl CoA specificity. Exemplary enzymes for deletion include
E. coli fabF (NP.sub.--415613) and fabH (NP.sub.--415609).
[0165] In yet another aspect, fatty acids, their intermediates and
their derivatives with varying degrees of saturation can be
produced in the engineered and/or evolved methylotroph as the
carbon-based products of interest. In one aspect, hosts are
engineered to produce unsaturated fatty acids by over-expressing
.beta.-ketoacyl-ACP synthase I (E.C. 2.3.1.41), or by growing the
host at low temperatures (for example less than 37.degree. C.).
FabB has preference to cis-.delta..sup.3decenoyl-ACP and results in
unsaturated fatty acid production in E. coli. Over-expression of
FabB results in the production of a significant percentage of
unsaturated fatty acids [de Mendoza, 1983]. These unsaturated fatty
acids can then be used as intermediates in hosts that are
engineered to produce fatty acids derivatives, such as fatty
alcohols, esters, waxes, olefins, alkanes, and the like.
Alternatively, the repressor of fatty acid biosynthesis, E. coli
FabR (NP.sub.--418398), can be deleted, which can also result in
increased unsaturated fatty acid production in E. coli [Zhang,
2002]. Further increase in unsaturated fatty acids is achieved by
over-expression of heterologous trans-2, cis-3-decenoyl-ACP
isomerase and controlled expression of trans-2-enoyl-ACP reductase
II [Marrakchi, 2002b], while deleting E. coli FabI
(trans-2-enoyl-ACP reductase, E.C. 1.3.1.9, NP.sub.--415804) or
homologs thereof in the host organism. Exemplary
.beta.-ketoacyl-ACP synthase I include Escherichia coli fabB
(BAA16180) and homologs thereof. Exemplary trans-2,
cis-3-decenoyl-ACP isomerase include Streptococcus mutans UA159
FabM (DAA05501) and homologs thereof. Exemplary trans-2-enoyl-ACP
reductase II include Streptococcus pneumoniae R6 FabK
(NP.sub.--357969) and homologs thereof. To increase production of
monounsaturated fatty acids, the sfa gene, suppressor of FabA, can
be over-expressed [Rock, 1996]. Exemplary proteins include AAN79592
and homologs thereof. One of ordinary skill in the art would
appreciate that by attenuating fabA, or over-expressing fabB and
expressing specific thioesterases (described above), unsaturated
fatty acids, their derivatives, and products having a desired
carbon chain length can be produced.
[0166] In some examples the fatty acid or intermediate is produced
in the cytoplasm of the cell. The cytoplasmic concentration can be
increased in a number of ways, including, but not limited to,
binding of the fatty acid to coenzyme A to form an acyl-CoA
thioester. Additionally, the concentration of acyl-CoAs can be
increased by increasing the biosynthesis of CoA in the cell, such
as by over-expressing genes associated with pantothenate
biosynthesis (panD) or knocking out the genes associated with
glutathione biosynthesis (glutathione synthase).
Production of Fatty Alcohols as the Carbon-Based Products of
Interest
[0167] In yet further aspects, hosts cells are engineered to
convert acyl-CoA to fatty alcohols by expressing or overexpressing
a fatty alcohol forming acyl-CoA reductase (FAR, E.C. 1.1.1.*), or
an acyl-CoA reductases (E.C. 1.2.1.50) and alcohol dehydrogenase
(E.C. 1.1.1.1) or a combination of the foregoing to produce fatty
alcohols from acyl-CoA. Hereinafter fatty alcohol forming acyl-CoA
reductase (FAR, E.C. 1.1.1.*), acyl-CoA reductases (E.C. 1.2.1.50)
and alcohol dehydrogenase (E.C. 1.1.1.1) are collectively referred
to as fatty alcohol forming peptides. Some fatty alcohol forming
peptides are non-specific and catalyze other reactions as well: for
example, some acyl-CoA reductase peptides accept other substrates
in addition to fatty acids. Exemplary fatty alcohol forming
acyl-CoA reductases include Acinetobacter baylyi ADP1 acrl
(AAC45217), Simmondsia chinensis jjfar (AAD38039), Mus musculus
mfar1 (AAH07178), Mus musculus mfar2 (AAH55759), Acinetobacter sp.
M1 acrM1, Homo sapiens hfar (AAT42129), and homologs thereof. Fatty
alcohols can be used as surfactants.
[0168] Many fatty alcohols are derived from the products of fatty
acid biosynthesis. Hence, the production of fatty alcohols can be
controlled by engineering fatty acid biosynthesis in the engineered
and/or evolved methylotroph. The chain length, branching and degree
of saturation of fatty acids and their intermediates can be altered
using the methods described herein, thereby affecting the nature of
the resulting fatty alcohols.
[0169] As mentioned above, through the combination of expressing
genes that support brFA synthesis and alcohol synthesis, branched
chain alcohols can be produced. For example, when an alcohol
reductase such as Acrl from Acinetobacter baylyi ADP1 is
coexpressed with a bkd operon, E. coli can synthesize isopentanol,
isobutanol or 2-methyl butanol. Similarly, when Acrl is coexpressed
with ccr/icm genes, E. coli can synthesize isobutanol.
Production of Fatty Esters as the Carbon-Based Products of
Interest
[0170] In another aspect, engineered and/or evolved methylotrophs
produce various lengths of fatty esters (biodiesel and waxes) as
the carbon-based products of interest. Fatty esters can be produced
from acyl-CoAs and alcohols. The alcohols can be provided in the
fermentation media, produced by the engineered and/or evolved
methylotroph itself or produced by a co-cultured organism.
[0171] In some embodiments, one or more alcohol
O-acetyltransferases is expressed in the engineered and/or evolved
methylotroph to produce fatty esters as the carbon-based product of
interest. Alcohol O-acetyltransferase (E.C. 2.3.1.84) catalyzes the
reaction of acetyl-CoA and an alcohol to produce CoA and an acetic
ester. In some embodiments, the alcohol O-acetyltransferase
peptides are co-expressed with selected thioesterase peptides, FAS
peptides and fatty alcohol forming peptides to allow the carbon
chain length, saturation and degree of branching to be controlled.
In other embodiments, the bkd operon can be co-expressed to enable
branched fatty acid precursors to be produced.
[0172] Alcohol O-acetyltransferase peptides catalyze other
reactions such that the peptides accept other substrates in
addition to fatty alcohols or acetyl-CoA thioester. Other
substrates include other alcohols and other acyl-CoA thioesters.
Modification of such enzymes and the development of assays for
characterizing the activity of a particular alcohol
O-acetyltransferase peptides are within the scope of a skilled
artisan. Engineered O-acetyltransferases and O-acyltransferases can
be created that have new activities and specificities for the donor
acyl group or acceptor alcohol moiety.
[0173] Alcohol acetyl transferases (AATs, E.C. 2.3.1.84), which are
responsible for acyl acetate production in various plants, can be
used to produce medium chain length waxes, such as octyl octanoate,
decyl octanoate, decyl decanoate, and the like. Fatty esters,
synthesized from medium chain alcohol (such as C6, C8) and medium
chain acyl-CoA (or fatty acids, such as C6 or C8) have a relative
low melting point. For example, hexyl hexanoate has a melting point
of -55.degree. C. and octyl octanoate has a melting point of -18 to
-17.degree. C. The low melting points of these compounds make them
good candidates for use as biofuels. Exemplary alcohol
acetyltransferases include Fragaria x ananassa SAAT (AAG13130)
[Aharoni, 2000], Saccharomyces cerevisiae Atfpl (NP.sub.--015022),
and homologs thereof.
[0174] In some embodiments, one or more wax synthases (E.C.
2.3.1.75) is expressed in the engineered and/or evolved
methylotroph to produce fatty esters including waxes from acyl-CoA
and alcohols as the carbon-based product of interest. Wax synthase
peptides are capable of catalyzing the conversion of an
acyl-thioester to fatty esters. Some wax synthase peptides can
catalyze other reactions, such as converting short chain acyl-CoAs
and short chain alcohols to produce fatty esters. Methods to
identify wax synthase activity are provided in U.S. Pat. No.
7,118,896, which is herein incorporated by reference. Medium-chain
waxes that have low melting points, such as octyl octanoate and
octyl decanoate, are good candidates for biofuel to replace
triglyceride-based biodiesel. Exemplary wax synthases include
Acinetobacter baylyi ADP1 wsadp1, Acinetobacter baylyi ADP1
wax-dgaT (AA017391) [Kalscheuer, 2003], Saccharomyces cerevisiae
Eeb1 (NP.sub.--015230), Saccharomyces cerevisiae YMR210w
(NP.sub.--013937), Simmondsia chinensis acyltransferase (AAD38041),
Mus musculus Dgat214 (Q6E1M8), and homologs thereof.
[0175] In other aspects, the engineered and/or evolved
methylotrophs are modified to produce a fatty ester-based biofuel
by expressing nucleic acids encoding one or more wax ester
synthases in order to confer the ability to synthesize a saturated,
unsaturated, or branched fatty ester. In some embodiments, the wax
ester synthesis proteins include, but are not limited to: fatty
acid elongases, acyl-CoA reductases, acyltransferases or wax
synthases, fatty acyl transferases, diacylglycerol
acyltransferases, acyl-coA wax alcohol acyltransferases,
bifunctional wax ester synthase/acyl-CoA: diacylglycerol
acyltransferase selected from a multienzyme complex from Simmondsia
chinensis, Acinetobacter sp. strain ADP1 (formerly Acinetobacter
calcoaceticus ADP1), Pseudomonas aeruginosa, Fundibacter jadensis,
Arabidopsis thaliana, or Alkaligenes eutrophus. In one embodiment,
the fatty acid elongases, acyl-CoA reductases or wax synthases are
from a multienzyme complex from Alkaligenes eutrophus and other
organisms known in the literature to produce wax and fatty acid
esters.
[0176] Many fatty esters are derived from the intermediates and
products of fatty acid biosynthesis. Hence, the production of fatty
esters can be controlled by engineering fatty acid biosynthesis in
the engineered and/or evolved methylotroph. The chain length,
branching and degree of saturation of fatty acids and their
intermediates can be altered using the methods described herein,
thereby affecting the nature of the resulting fatty esters.
[0177] Additionally, to increase the percentage of unsaturated
fatty acid esters, the engineered and/or evolved methylotroph can
also overexpress Sfa which encodes a suppressor of fabA (AAN79592,
AAC44390), .beta.-ketoacyl-ACP synthase I (E.C. 2.3.1.41,
BAA16180), and secG null mutant suppressors (cold shock proteins)
gnsA and gnsB (ABD18647 and AAC74076). In some examples, the
endogenous fabF gene can be attenuated, thus, increasing the
percentage of palmitoleate (C 16:1) produced.
[0178] Optionally a wax ester exporter such as a member of the FATP
family is used to facilitate the release of waxes or esters into
the extracellular environment from the engineered and/or evolved
methylotroph. An exemplary wax ester exporter that can be used is
fatty acid (long chain) transport protein CG7400-PA, isoform A from
D. melanogaster (NP.sub.--524723), or homologs thereof.
[0179] The centane number (CN), viscosity, melting point, and heat
of combustion for various fatty acid esters have been characterized
in for example, [Knothe, 2005]. Using the teachings provided herein
the engineered and/or evolved methylotroph can be engineered to
produce any one of the fatty acid esters described in [Knothe,
2005].
Production of Alkanes as the Carbon-Based Products of Interest
[0180] In another aspect, engineered and/or evolved methylotrophs
produce alkanes of various chain lengths (hydrocarbons) as the
carbon-based products of interest. Many alkanes are derived from
the products of fatty acid biosynthesis. Hence, the production of
alkanes can be controlled by engineering fatty acid biosynthesis in
the engineered and/or evolved methylotroph. The chain length,
branching and degree of saturation of fatty acids and their
intermediates can be altered using the methods described herein.
The chain length, branching and degree of saturation of alkanes can
be controlled through their fatty acid biosynthesis precursors.
[0181] In certain aspects, fatty aldehydes can be converted to
alkanes and CO in the engineered and/or evolved methylotroph via
the expression of decarbonylases [Cheesbrough, 1984; Dennis, 1991].
Exemplary enzymes include Arabidopsis thaliana cerl
(NP.sub.--171723), Oryza sativacer1 CER1 (AAD29719) and homologs
thereof.
[0182] In another aspect, fatty alcohols can be converted to
alkanes in the engineered and/or evolved methylotroph via the
expression of terminal alcohol oxidoreductases as in Vibrio
furnissii M1 [Park, 2005].
Production of Olefins as the Carbon-Based Products of Interest
[0183] In another aspect, engineered and/or evolved methylotrophs
produce olefins (hydrocarbons) as the carbon-based products of
interest. Olefins are derived from the intermediates and products
of fatty acid biosynthesis. Hence, the production of olefins can be
controlled by engineering fatty acid biosynthesis in the engineered
and/or evolved methylotroph. Introduction of genes affecting the
production of unsaturated fatty acids, as described above, can
result in the production of olefins. Similarly, the chain length of
olefins can be controlled by expressing, overexpressing or
attenuating the expression of endogenous and heterologous
thioesterases which control the chain length of the fatty acids
that are precursors to olefin biosynthesis. Also, by controlling
the expression of endogenous and heterologous enzymes associated
with branched chain fatty acid biosynthesis, the production of
branched chain olefins can be enhanced. Methods for controlling the
chain length and branching of fatty acid biosynthesis intermediates
and products are described above. Olefins can be obtained by
downstreaming processing of 3-hydroxy alkanoates as taught by
Fischer et al. [Ind Eng Chem Res, 2011, 50(8):4420-4424, DOI:
10.1021/ie1023386]. Accordingly, the fermentation product for
methylotrophic production of olefins need not be an olefin
itself.
Production of .omega.-Cyclic Fatty Acids and their Derivatives as
the Carbon-Based Products of Interest
[0184] In another aspect, the engineered and/or evolved
methylotroph of the present invention produces co-cyclic fatty
acids (cyFAs) as the carbon-based product of interest. To
synthesize co-cyclic fatty acids (cyFAs), several genes need to be
introduced and expressed that provide the cyclic precursor
cyclohexylcarbonyl-CoA [Cropp, 2000]. The genes (fabH, ACP and
fabF) can then be expressed to allow initiation and elongation of
co-cyclic fatty acids. Alternatively, the homologous genes can be
isolated from microorganisms that make cyFAs and expressed in E.
coli. Relevant genes include bkdC, lpd, fabH, ACP, fabF, fabH1,
ACP, fabF, fabH3, fabC3, fabF, fabH_A, fabH_B, ACP.
[0185] Expression of the following genes are sufficient to provide
cyclohexylcarbonyl-CoA in E. coli: ansJ, ansK, ansL, chcA
(1-cyclohexenylcarbonyl CoA reductase) and ansM from the
ansatrienin gene cluster of Streptomyces collinus [Chen, 1999] or
plmJK (5-enolpyruvylshikimate-3-phosphate synthase), plmL (acyl-CoA
dehydrogenase), chcA (enoyl-(ACP) reductase) and plmM
(2,4-dienoyl-CoA reductase) from the phoslactomycin B gene cluster
of Streptomyces sp. HK803 [Palaniappan, 2003] together with the
acyl-CoA isomerase (chcB gene) [Patton, 2000] from S. collinus, S.
avermitilis or S. coelicolor. Exemplary ansatrienin gene cluster
enzymes include AAC44655, AAF73478 and homologs thereof. Exemplary
phoslactomycin B gene cluster enzymes include AAQ84158, AAQ84159,
AAQ84160, AAQ84161 and homologs thereof. Exemplary chcB enzymes
include NP.sub.--629292, AAF73478 and homologs thereof.
[0186] The genes (fabH, ACP and fabF) are sufficient to allow
initiation and elongation of co-cyclic fatty acids, because they
can have broad substrate specificity. In the event that
coexpression of any of these genes with the ansJKLM/chcAB or
pmlJKLM/chcAB genes does not yield cyFAs, fabH, ACP and/or fabF
homologs from microorganisms that make cyFAs can be isolated (e.g.,
by using degenerate PCR primers or heterologous DNA probes) and
coexpressed.
Production of Halogenated Derivatives of Fatty Acids
[0187] Genes are known that can produce fluoroacetyl-CoA from
fluoride ion. In one embodiment, the present invention allows for
production of fluorinated fatty acids by combining expression of
fluoroacetate-involved genes (e.g., fluorinase, nucleotide
phosphorylase, fluorometabolite-specific aldolases,
fluoroacetaldehyde dehydrogenase, and fluoroacetyl-CoA
synthase).
Transport/Efflux/Release of Fatty Acids and their Derivatives
[0188] Also disclosed herein is a system for continuously producing
and exporting hydrocarbons out of recombinant host microorganisms
via a transport protein. Many transport and efflux proteins serve
to excrete a large variety of compounds and can be evolved to be
selective for a particular type of fatty acid. Thus, in some
embodiments an ABC transporter can be functionally expressed by the
engineered and/or evolved methylotroph, so that the organism
exports the fatty acid into the culture medium. In one example, the
ABC transporter is an ABC transporter from Caenorhabditis elegans,
Arabidopsis thalania, Alkaligenes eutrophus or Rhodococcus
erythropolis or homologs thereof. Exemplary transporters include
AAU44368, NP.sub.--188746, NP.sub.--175557, AAN73268 or homologs
thereof.
[0189] The transport protein, for example, can also be an efflux
protein selected from: AcrAB (NP.sub.--414996.1,
NP.sub.--414995.1), ToIC (NP.sub.--417507.2) and AcrEF
(NP.sub.--417731.1, NP.sub.--417732.1) from E. coli, or t111618
(NP.sub.--682408), t111619 (NP.sub.--682409), t110139
(NP.sub.--680930), H11619 and U10139 from Thermosynechococcus
elongatus BP-I or homologs thereof.
[0190] In addition, the transport protein can be, for example, a
fatty acid transport protein (FATP) selected from Drosophila
melanogaster, Caenorhabditis elegans, Mycobacterium tuberculosis or
Saccharomyces cerevisiae, Acinetobacter sp. H01-N, any one of the
mammalian FATPs or homologs thereof. The FATPs can additionally be
resynthesized with the membranous regions reversed in order to
invert the direction of substrate flow. Specifically, the sequences
of amino acids composing the hydrophilic domains (or membrane
domains) of the protein can be inverted while maintaining the same
codons for each particular amino acid. The identification of these
regions is well known in the art.
Production of Isoprenoids as the Carbon-Based Products of
Interest
[0191] In one aspect, the engineered and/or evolved methylotroph of
the present invention produces isoprenoids or their precursors
isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl
pyrophosphate (DMAPP) as the carbon-based products of interest.
There are two known biosynthetic pathways that synthesize IPP and
DMAPP. Prokaryotes, with some exceptions, use the
mevalonate-independent or deoxyxylulose 5-phosphate (DXP) pathway
to produce IPP and DMAPP separately through a branch point (FIG.
11). Eukaryotes other than plants use the mevalonate-dependent
(MEV) isoprenoid pathway exclusively to convert acetyl-coenzyme A
(acetyl-CoA) to IPP, which is subsequently isomerized to DMAPP
(FIG. 12). In general, plants use both the MEV and DXP pathways for
IPP synthesis.
[0192] The reactions in the DXP pathway are catalyzed by the
following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (E.C.
2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (E.C.
1.1.1.267), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase
(E.C. 2.7.7.60), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase
(E.C. 2.7.1.148), 2C-methyl-D-erythritol 2,4-cyclodiphosphate
synthase (E.C. 4.6.1.12), (E)-4-hydroxy-3-methylbut-2-enyl
diphosphate synthase (E.C. 1.17.7.1), isopentyl/dimethylallyl
diphosphate synthase or 4-hydroxy-3-methylbut-2-enyl diphosphate
reductase (E.C. 1.17.1.2). In one embodiment, the engineered and/or
evolved methylotroph of the present invention expresses one or more
enzymes from the DXP pathway. For example, one or more exogenous
proteins can be selected from 1-deoxy-D-xylulose-5-phosphate
reductoisomerase, 4-diphosphocytidyl-2C-methyl-D-erythritol
synthase, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase,
2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase, and
4-hydroxy-3-methylbut-2-enyl diphosphate reductase. The host
organism can also express two or more, three or more, four or more,
and the like, including up to all the protein and enzymes that
confer the DXP pathway. Exemplary 1-deoxy-D-xylulose-5-phosphate
synthases include E. coli Dxs (AAC46162); P. putida KT2440 Dxs
(AAN66154); Salmonella enterica Paratyphi, see ATCC 9150 Dxs
(AAV78186); Rhodobacter sphaeroides 2.4.1 Dxs (YP.sub.--353327);
Rhodopseudomonas palustris CGA009 Dxs (NP.sub.--946305); Xylella
fastidiosa Temeculal Dxs (NP.sub.--779493); Arabidopsis thaliana
Dxs (NP.sub.--001078570 and/or NP.sub.--196699); and homologs
thereof. SEQ ID NO:1 represents the Paracoccus codon optimized
coding sequence for the E. coli dxs gene of the present invention.
In one aspect, the invention provides nucleic acid molecules and
homologs, variants and derivatives of SEQ ID NO:1. The nucleic acid
sequences can have 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higher
identity to SEQ ID NO:1. The present invention provides nucleic
acids each comprising or consisting of a sequence which is a codon
optimized version of one of the wild-type dxs gene. In another
embodiment, the invention provides nucleic acids each encoding a
polypeptide having the amino acid sequence of one of AAC46162, YP
353327, AAV78186, YP.sub.--353327, NP.sub.--946305,
NP.sub.--779493, NP.sub.--001078570, NP.sub.--196699, or homologs
thereof having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higher identity
thereto. Exemplary 1-deoxy-D-xylulose-5-phosphate reductoisomerases
include E. coli Dxr (BAA32426); Arabidopsis thaliana DXR
(AAF73140); Pseudomonas putida KT2440 Dxr (NP.sub.--743754 and/or
Q88MH4); Streptomyces coelicolor A3(2) Dxr (NP.sub.--629822);
Rhodobacter sphaeroides 2.4.1 Dxr (YP.sub.--352764); Pseudomonas
fluorescens PfO-1 Dxr (YP.sub.--346389); and homologs thereof.
Exemplary 4-diphosphocytidyl-2C-methyl-D-erythritol synthases
include E. coli IspD (AAF43207); Rhodobacter sphaeroides 2.4.1 IspD
(YP.sub.--352876); Arabidopsis thaliana ISPD (NP.sub.--565286); P.
putida KT2440 IspD (NP.sub.--743771); and homologs thereof.
Exemplary 4-diphosphocytidyl-2C-methyl-D-erythritol kinases include
E. coli IspE (AAF29530); Rhodobacter sphaeroides 2.4.1 IspE
(YP.sub.--351828); and homologs thereof. Exemplary
2C-methyl-D-erythritol 2,4-cyclodiphosphate synthases include E.
coli IspF (AAF44656); Rhodobacter sphaeroides 2.4.1 IspF
(YP.sub.--352877); P. putida KT2440 IspF (NP.sub.--743775); and
homologs thereof. Exemplary (E)-4-hydroxy-3-methylbut-2-enyl
diphosphate synthase include E. coli IspG (AAK53460); P. putida
KT2440 IspG (NP.sub.--743014); Rhodobacter sphaeroides 2.4.1 IspG
(YP.sub.--353044); and homologs thereof. Exemplary
4-hydroxy-3-methylbut-2-enyl diphosphate reductases include E. coli
IspH (AAL38655); P. putida KT2440 IspH (NP.sub.--742768); and
homologs thereof.
[0193] The reactions in the MEV pathway are catalyzed by the
following enzymes: acetyl-CoA thiolase, HMG-CoA synthase (E.C.
2.3.3.10), HMG-CoA reductase (E.C. 1.1.1.34), mevalonate kinase
(E.C. 2.7.1.36), phosphomevalonate kinase (E.C. 2.7.4.2),
mevalonate pyrophosphate decarboxylase (E.C. 4.1.1.33), isopentenyl
pyrophosphate isomerase (E.C. 5.3.3.2). In one embodiment, the
engineered and/or evolved methylotroph of the present invention
expresses one or more enzymes from the MEV pathway. For example,
one or more exogenous proteins can be selected from acetyl-CoA
thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase,
phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase
and isopentenyl pyrophosphate isomerase. The host organism can also
express two or more, three or more, four or more, and the like,
including up to all the protein and enzymes that confer the MEV
pathway. Exemplary acetyl-CoA thiolases include NC.sub.--000913
REGION: 232413 L.2325315, E. coli; D49362, Paracoccus
denitrificans; L20428, S. cerevisiae; and homologs thereof.
Exemplary HMG-CoA synthases include NC.sub.--001145 complement
19061 . . . 20536, S. cerevisiae; X96617, S. cerevisiae; X83882, A.
thaliana; AB037907, Kitasatospora griseola; BT007302, H. sapiens;
NC.sub.--002758, Locus tag SAV2546, GeneID 1 122571, S. aureus; and
homlogs thereof. Exemplary HMG-CoA reductases include
NM.sub.--206548, D. melanogaster; NC.sub.--002758, Locus tag
SAV2545, GeneID 1122570, S. aureus; NM.sub.--204485, Gallus gallus;
AB015627, Streptomyces sp. KO 3988; AF542543, Nicotiana attenuata;
AB037907, Kitasatospora griseola; AX128213, providing the sequence
encoding a truncated HMGR, S. cerevisiae; NC001145: complement
115734 . . . 1 18898, S. cerevisiae; and homologs thereof.
Exemplary mevalonate kinases include L77688, A. thaliana; X55875,
S. cerevisiae; and homologs thereof. Exemplary phosphomevalonate
kinases include AF429385, Hevea brasiliensis; NM.sub.--006556, H.
sapiens; NC.sub.--001145 complement 712315 . . . 713670, S.
cerevisiae; and homologs thereof. Exemplary mevalonate
pyrophosphate decarboxylase include include X97557, S. cerevisiae;
AF290095, E. faecium; U49260, H. sapiens; and homologs thereof.
Exemplary isopentenyl pyrophosphate isomerases include
NP.sub.--417365, E. coli Idi; AAC32209, Haematococcus pluvialis
Idi; and homologs thereof. SEQ ID NO:2 represents the Paracoccus
codon optimized coding sequence for the E. coli idi gene of the
present invention. In one aspect, the invention provides nucleic
acid molecules and homologs, variants and derivatives of SEQ ID
NO:2. The nucleic acid sequences can have 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or
even higher identity to SEQ ID NO:2. The present invention provides
nucleic acids each comprising or consisting of a sequence which is
a codon optimized version of one of the wild-type idi gene. In
another embodiment, the invention provides nucleic acids each
encoding a polypeptide having the amino acid sequence of one of
NP.sub.--417365, AAC32209, or homologs thereof having 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81-85%, 90-95%,
96-98%, 99%, 99.9% or even higher identity thereto.
[0194] In some embodiments, the host cell produces IPP via the MEV
pathway, either exclusively or in combination with the DXP pathway.
In other embodiments, a host cell's DXP pathway is functionally
disabled so that the host cell produces IPP exclusively through a
heterologously introduced MEV pathway. The DXP pathway can be
functionally disabled by disabling gene expression or inactivating
the function of one or more of the DXP pathway enzymes.
[0195] In some embodiments, the host cell produces IPP via the DXP
pathway, either exclusively or in combination with the MEV pathway.
In other embodiments, a host cell's MEV pathway is functionally
disabled so that the host cell produces IPP exclusively through a
heterologously introduced DXP pathway. The MEV pathway can be
functionally disabled by disabling gene expression or inactivating
the function of one or more of the MEV pathway enzymes.
[0196] Provided herein is a method to produce isoprenoids in
engineered and/or evolved methylotrophs engineered with the
isopentenyl pyrophosphate pathway enzymes. Some examples of
isoprenoids include: hemiterpenes (derived from 1 isoprene unit)
such as isoprene; monoterpenes (derived from 2 isoprene units) such
as myrcene or limonene; sesquiterpenes (derived from 3 isoprene
units) such as amorpha-4,11-diene, bisabolene or farnesene;
diterpenes (derived from four isoprene units) such as taxadiene;
sesterterpenes (derived from 5 isoprene units); triterpenes
(derived from 6 isoprene units) such as squalene; sesquarterpenes
(derived from 7 isoprene units); tetraterpenes (derived from 8
isoprene units) such as f3-carotene or lycopene; and polyterpenes
(derived from more than 8 isoprene units) such as polyisoprene. The
production of isoprenoids is also described in some detail in the
published PCT applications WO2007/139925 and WO/2007/140339.
[0197] In another embodiment, the engineered and/or evolved
methylotroph of the present invention produces isoprene as the
carbon-based product of interest via the isopentenyl pyrophosphate
pathway enzymes and isoprene synthase (E.C. 4.2.3.27) which
converts to dimethylallyl diphosphate to isoprene. Exemplary
enzymes include Populus nigra IspS (CAL69918) and homologs thereof.
SEQ ID NO:3 represents the Paracoccus codon optimized coding
sequence for the P. nigra ispS gene of the present invention. In
one aspect, the invention provides nucleic acid molecules and
homologs, variants and derivatives of SEQ ID NO:3. The nucleic acid
sequences can have 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higher
identity to SEQ ID NO:3. The present invention provides nucleic
acids each comprising or consisting of a sequence which is a codon
optimized version of one of the wild-type ispS gene. In another
embodiment, the invention provides nucleic acids each encoding a
polypeptide having the amino acid sequence of CAL69918, or homologs
thereof having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81-85%, 90-95%, 96-98%, 99%, 99.9.degree. A or even higher
identity thereto.
[0198] In another embodiment, the engineered and/or evolved
methylotroph of the present invention produces bisabolene as the
carbon-based product of interest via the isopentenyl pyrophosphate
pathway enzymes and E-alpha-bisabolene synthase (E.C. 4.2.3.38)
which converts to farnesyl diphosphate to bisabolene. Exemplary
enzymes include Picea abies TPS-bis (AAS47689) and homologs
thereof. SEQ ID NO:4 represents the Paracoccus codon optimized
coding sequence for the P. abies tps-bis gene of the present
invention. In one aspect, the invention provides nucleic acid
molecules and homologs, variants and derivatives of SEQ ID NO:4.
The nucleic acid sequences can have 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9.degree.
A or even higher identity to SEQ ID NO:4. The present invention
provides nucleic acids each comprising or consisting of a sequence
which is a codon optimized version of one of the wild-type tps-bis
gene. In another embodiment, the invention provides nucleic acids
each encoding a polypeptide having the amino acid sequence of
AAS47689, or homologs thereof having 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even
higher identity thereto.
[0199] In another embodiment, the engineered and/or evolved
methylotroph of the present invention produces rubber as the
carbon-based product of interest via the isopentenyl pyrophosphate
pathway enzymes and cis-polyprenylcistransferase (E.C. 2.5.1.20)
which converts isopentenyl pyrophosphate to rubber. The enzyme
cis-polyprenylcistransferase may come from, for example, Hevea
brasiliensis.
[0200] In another embodiment, the engineered and/or evolved
methylotroph of the present invention produce isopentanol as the
carbon-based product of interest via the isopentenyl pyrophosphate
pathway enzymes and isopentanol dikinase.
[0201] In another embodiment, the engineered and/or evolved
methylotroph produces squalene as the carbon-based product of
interest via the isopentenyl pyrophosphate pathway enzymes, geranyl
diphosphate synthase (E.C. 2.5.1.1), farnesyl diphosphate synthase
(E.C. 2.5.1.10) and squalene synthase (E.C. 2.5.1.21). Geranyl
diphosphate synthase converts dimethylallyl pyrophosphate and
isopentenyl pyrophosphate to geranyl diphosphate. Farnesyl
diphosphate synthase converts geranyl diphosphate and isopentenyl
diphosphate to farnesyl diphosphate. A bifunctional enzyme carries
out the conversion of dimethylallyl pyrophosphate and two
isopentenyl pyrophosphate to farnesyl pyrophosphate. Exemplary
enzymes include Escherichia coli IspA (NP.sub.--414955) and
homologs thereof. Squalene synthase converts two farnesyl
pyrophosphate and NADPH to squalene. In another embodiment, the
engineered and/or evolved methylotroph produces lanosterol as the
carbon-based product of interest via the above enzymes, squalene
monooxygenase (E.C. 1.14.99.7) and lanosterol synthase (E.C.
5.4.99.7). Squalene monooxygenase converts squalene, NADPH and
O.sub.2 to (S)-squalene-2,3-epoxide. Exemplary enzymes include
Saccharomyces cerevisiae Erg1 (NP.sub.--011691) and homologs
thereof. Lanosterol synthase converts (S)-squalene-2,3-epoxide to
lanosterol. Exemplary enzymes include Saccharomyces cerevisiae Erg?
(NP.sub.--011939) and homologs thereof.
[0202] In another embodiment, the engineered and/or evolved
methylotroph of the present invention produces lycopene as the
carbon-based product of interest via the isopentenyl pyrophosphate
pathway enzymes, geranyl diphosphate synthase (E.C. 2.5.1.21,
described above), farnesyl diphosphate synthase (E.C. 2.5.1.10,
described above), geranylgeranyl pyrophosphate synthase (E.C.
2.5.1.29), phytoene synthase (E.C. 2.5.1.32), phytoene
oxidoreductase (E.C. 1.14.99.n) and .zeta.-carotene oxidoreductase
(E.C. 1.14.99.30). Geranylgeranyl pyrophosphate synthase converts
isopentenyl pyrophosphate and farnesyl pyrophosphate to (all
trans)-geranylgeranyl pyrophosphate. Exemplary geranylgeranyl
pyrophosphate synthases include Synechocystis sp. PCC6803 crtE
(NP.sub.--440010) and homologs thereof. Phytoene synthase converts
2 geranylgeranyl-PP to phytoene. Exemplary enzymes include
Synechocystis sp. PCC6803 crtB (P37294). Phytoene oxidoreductase
converts phytoene, 2 NADPH and 2 O.sub.2 to .zeta.-carotene.
Exemplary enzymes include Synechocystis sp. PCC6803 crtI and
Synechocystis sp. PCC6714 crtI (P21134). .zeta.-carotene
oxidoreductase converts .zeta.-carotene, 2 NADPH and 2 O.sub.2 to
lycopene. Exemplary enzymes include Synechocystis sp. PCC6803
crtQ-2 (NP.sub.--441720).
[0203] In another embodiment, the engineered and/or evolved
methylotroph of the present invention produces limonene as the
carbon-based product of interest via the isopentenyl pyrophosphate
pathway enzymes, geranyl diphosphate synthase (E.C. 2.5.1.21,
described above) and one of (R)-limonene synthase (E.C. 4.2.3.20)
and (4S)-limonene synthase (E.C. 4.2.3.16) which convert geranyl
diphosphate to a limonene enantiomer. Exemplary (R)-limonene
synthases include that from Citrus limon (AAM53946) and homologs
thereof. Exemplary (4S)-limonene synthases include that from Mentha
spicata (AAC37366) and homologs thereof.
Production of Glycerol or 1,3-Propanediol as the Carbon-Based
Products of Interest
[0204] In one aspect, the engineered and/or evolved methylotroph of
the present invention produces glycerol or 1,3-propanediol as the
carbon-based products of interest (FIG. 13). The reactions in the
glycerol pathway are catalyzed by the following enzymes:
sn-glycerol-3-P dehydrogenase (E.C. 1.1.1.8 or E.C. 1.1.1.94) and
sn-glycerol-3-phosphatase (E.C. 3.1.3.21). To produce
1,3,-propanediol, the following enzymes are also included:
sn-glycerol-3-P. glycerol dehydratase (E.C. 4.2.1.30) and
1,3-propanediol oxidoreductase (E.C. 1.1.1.202). Exemplary
sn-glycerol-3-P dehydrogenases include Saccharomyces cerevisiae
darl and homologs thereof. Exemplary sn-glycerol-3-phosphatases
include Saccharomyces cerevisiae gpp2 and homologs thereof.
Exemplary sn-glycerol-3-P. glycerol dehydratases include K.
pneumoniae dhaB1-3. Exemplary 1,3-propanediol oxidoreductase
include K. pneumoniae dhaT.
Production of 1,4-Butanediol or 1,3-Butadiene as the Carbon-Based
Products of Interest
[0205] In one aspect, the engineered and/or evolved methylotroph of
the present invention produces 1,4-butanediol or 1,3-butanediene as
the carbon-based products of interest. The metabolic reactions in
the 1,4-butanediol or 1,3-butadiene pathway are catalyzed by the
following enzymes: succinyl-CoA dehydrogenase (E.C. 1.2.1.n; e.g.,
C. kluyveri SucD), 4-hydroxybutyrate dehydrogenase (E.C. 1.1.1.2;
e.g., Arabidopsis thaliana GHBDH), aldehyde dehydrogenase (E.C.
1.1.1.n; e.g., E. coli A1dH), 1,3-propanediol oxidoreductase (E.C.
1.1.1.202; e.g., K. pneumoniae DhaT), and optionally alcohol
dehydratase (E.C. 4.2.1.-). Succinyl-CoA dehydrogenase converts
succinyl-CoA and NADPH to succinic semialdehyde and CoA.
4-hydroxybutyrate dehydrogenase converts succinic semialdehyde and
NADPH to 4-hydroxybutyrate. Aldehyde dehydrogenase converts
4-hydroxybutyrate and NADH to 4-hydroxybutanal. 1,3-propanediol
oxidoreductase converts 4-hydroxybutanal and NADH to
1,4-butanediol. Alcohol dehydratase converts 1,4-butanediol to
1,3-butadiene.
Production of Polyhydroxybutyrate as the Carbon-Based Products of
Interest
[0206] In one aspect, the engineered and/or evolved methylotroph of
the present invention produces polyhydroxybutyrate as the
carbon-based products of interest (FIG. 14). The reactions in the
polyhydroxybutyrate pathway are catalyzed by the following enzymes:
acetyl-CoA:acetyl-CoA C-acetyltransferase (E.C. 2.3.1.9),
(R)-3-hydroxyacyl-CoA:NADP+oxidoreductase (E.C. 1.1.1.36) and
polyhydroxyalkanoate synthase (E.C. 2.3.1.-). Exemplary
acetyl-CoA:acetyl-CoA C-acetyltransferases include Ralstonia
eutropha phaA. Exemplary (R)-3-hydroxyacyl-CoA:NADP+oxidoreductases
include Ralstonia eutropha phaB. Exemplary polyhydroxyalkanoate
synthase include Ralstonia eutropha phaC. In the event that the
host organism also has the capacity to degrade polyhydroxybutyrate,
the corresponding degradation enzymes, such as
poly[(R)-3-hydroxybutanoate] hydrolase (E.C. 3.1.1.75), may be
inactivated. Hosts that lack the ability to naturally synthesize
polyhydroxybutyrate generally also lack the capacity to degrade it,
thus leading to irreversible accumuation of polyhydroxybutyrate if
the biosynthetic pathway is introduced. Some methylotrophic
bacteria can naturally make poly(3-hydroxybutyrate) or
poly(3-hydroxybutyrate-co-3-hydroxyvalerate), such as Paracoccus
denitrificans [Appl Environ Microbiol, 1996, 62(2):380-384].
[0207] Intracellular polyhydroxybutyrate can be measured by solvent
extraction and esterification of the polymer from whole cells.
Typically, lyophilized biomass is extracted with
methanol-chloroform with 10% HCl as a catalyst. The chloroform
dissolves the polymer, and the methanol esterifies it in the
presence of HCl. The resulting mixture is extracted with water to
remove hydrophilic substances and the organic phase is analyzed by
GC.
Production of Lysine as the Carbon-Based Products of Interest
[0208] In one aspect, the engineered and/or evolved methylotroph of
the present invention produces lysine as the carbon-based product
of interest. There are several known lysine biosynthetic pathways.
One lysine biosynthesis pathway is depicted in FIG. 15. The
reactions in one lysine biosynthetic pathway are catalyzed by the
following enzymes: aspartate aminotransferase (E.C. 2.6.1.1; e.g.
E. coli AspC), aspartate kinase (E.C. 2.7.2.4; e.g., E. coli LysC),
aspartate semialdehyde dehydrogenase (E.C. 1.2.1.11; e.g., E. coli
Asd), dihydrodipicolinate synthase (E.C. 4.2.1.52; e.g., E. coli
DapA), dihydrodipicolinate reductase (E.C. 1.3.1.26; e.g., E. coli
DapB), tetrahydrodipicolinate succinylase (E.C. 2.3.1.117; e.g., E.
coli DapD), N-succinyldiaminopimelate-aminotransferase (E.C.
2.6.1.17; e.g., E. coli ArgD), N-succinyl-L-diaminopimelate
desuccinylase (E.C. 3.5.1.18; e.g., E. coli DapE), diaminopimelate
epimerase (E.C. 5.1.1.7; E. coli DapF), diaminopimelate
decarboxylase (E.C. 4.1.1.20; e.g., E. coli LysA). In one
embodiment, the engineered and/or evolved methylotroph of the
present invention expresses one or more enzymes from a lysine
biosynthetic pathway. For example, one or more exogenous proteins
can be selected from aspartate aminotransferase, aspartate kinase,
aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase,
dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase,
N-succinyldiaminopimelate-aminotransferase,
N-succinyl-L-diaminopimelate desuccinylase, diaminopimelate
epimerase, diaminopimelate decarboxylase, L,L-diaminopimelate
aminotransferase (E.C. 2.6.1.83; e.g., Arabidopsis thaliana
At4g33680), homocitrate synthase (E.C. 2.3.3.14; e.g.,
Saccharomyces cerevisiae LYS21), homoaconitase (E.C. 4.2.1.36;
e.g., Saccharomyces cerevisiae LYS4, LYS3), homoisocitrate
dehydrogenase (E.C. 1.1.1.87; e.g., Saccharomyces cerevisiae LYS12,
LYS11, LYS10), 2-aminoadipate transaminase (E.C. 2.6.1.39; e.g.,
Saccharomyces cerevisiae ARO8), 2-aminoadipate reductase (E.C.
1.2.1.31; e.g., Saccharomyces cerevisiae LYS2, LYS5), aminoadipate
semialdehyde-glutamate reductase (E.C. 1.5.1.10; e.g.,
Saccharomyces cerevisiae LYS9, LYS13), lysine-2-oxoglutarate
reductase (E.C. 1.5.1.7; e.g., Saccharomyces cerevisiae LYS1). The
host organism can also express two or more, three or more, four or
more, and the like, including up to all the protein and enzymes
that confer lysine biosynthesis.
Production of Aromatic Compounds as the Carbon-Based Products of
Interest
[0209] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces aromatic amino
acids, their intermediates or their derivatives, including but not
limited to shikimate, chorismate, prephenate, phenylalanine,
tyrosine, tryptophan, or phenylpropranoids, as the carbon-based
products of interest. The engineered and/or evolved methylotroph
produces aromatic compounds as an intermediate or product of the
methylotrophic or carbon fixation pathway or as a intermediate or
product of host metabolism. In such cases, one or more transporters
may be expressed in the engineered and/or evolved methylotroph to
export the aromatic compound from the cell. These aromatic
metabolites can be converted to other products.
[0210] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces chorismate as the
carbon-based product of interested or as a central metabolite
precursor to an aromatic carbon-based product of interest. There
are multiple pathways for chorismate biosynthesis. The reactions in
one chorismate biosynthesis pathway are catalyzed by the following
enzymes: 2-dehydro-3-deoxyphosphoheptonate aldolase (E.C. 2.5.1.54,
e.g., E. coli AroG, AroH, AroF), 3-dehydroquinate synthase (E.C.
4.2.3.4, e.g., E. coli AroB), 3-dehydroquinate dehydratase (E.C.
4.2.1.10, e.g., E. coli AroD), NADPH-dependent shikimate
dehydrogenase (E.C. 1.1.1.25, e.g., E. coli AroE),
NAD(P)H-dependent shikimate dehydrogenase (E.C. 1.1.1.282, e.g., E.
coli YdiB), shikimate kinase (E.C. 2.7.1.71, e.g., E. coli AroL or
AroK), 3-phosphoshikimate-1-carboxyvinyltransferase (E.C. 2.5.1.19,
e.g., E. coli AroA) and chorismate synthase (E.C. 4.2.3.5, e.g., E.
coli AroC). In one embodiment, the engineered and/or evolved
methylotroph of the present invention expresses one or more enzymes
from a chorismate biosynthetic pathway. For example, one or more
exogenous proteins can be selected from
2-dehydro-3-deoxyphosphoheptonate aldolase, 3-dehydroquinate
synthase, 3-dehydroquinate dehydratase, NADPH-dependent shikimate
dehydrogenase, NAD(P)H-dependent shikimate dehydrogenase, shikimate
kinase, 3-phosphoshikimate-1-carboxyvinyltransferase and chorismate
synthase. The host organism can also express two or more, three or
more, four or more, and the like, including up to all the protein
and enzymes that confer chorismate biosynthesis. Chorismate serves
as an intermediate to several aromatic compounds including
phenylalanine, tyrosine, tryptophan and the phenylpropranoids.
[0211] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces phenylalanine as the
carbon-based product of interested or as a precursor to an aromatic
carbon-based product of interest. There are multiple pathways for
phenylalanine biosynthesis. The reactions in one phenylalanine
biosynthesis pathway are catalyzed by the following enzymes:
chorismate mutase (E.C. 5.4.99.5, e.g., E. coli PheA or TyrA),
prephenate dehydratase (E.C. 4.2.1.51, e.g., E. coli PheA),
phenylalanine transaminase (E.C. 2.6.1.57, e.g., E. coli IlvE). In
one embodiment, the engineered and/or evolved methylotroph of the
present invention expresses one or more enzymes from a
phenylalanine biosynthetic pathway. For example, one or more
exogenous proteins can be selected from chorismate mutase,
prephenate dehydratase and phenylalanine transaminase. The host
organism can also express two or more, three or more, and the like,
including up to all the protein and enzymes that confer
phenylalanine biosynthesis. Mutants of the methylotrophic
Paracoccus denitrificans have been isolated with high
aminotransferase (transaminase) activity [Appl Microbiol
Biotechnol, 1989, 30(3):243-246, DOI: 10.1007/BF00256212].
[0212] In certain embodiments, the engineered and/or evolved
methylotroph of the present invention produces tyrosine as the
carbon-based product of interested or as a precursor to an aromatic
carbon-based product of interest. There are multiple pathways for
tyrosine biosynthesis. The reactions in one tyrosine biosynthesis
pathway are catalyzed by the following enzymes: chorismate mutase
(E.C. 5.4.99.5, e.g., E. coli PheA or TyrA), prephenate
dehydrogenase (E.C. 1.3.1.12, e.g., E. coli TyrA), tyrosine
aminotransferase (E.C. 2.6.1.57, e.g., E. coli AspC or TyrB). In
one embodiment, the engineered and/or evolved methylotroph of the
present invention expresses one or more enzymes from a tyrosine
biosynthetic pathway. For example, one or more exogenous proteins
can be selected from chorismate mutase, prephenate dehydrogeanse
and tyrosine aminotransferase. The host organism can also express
two or more, three or more, and the like, including up to all the
protein and enzymes that confer tyrosine biosynthesis.
Production of .gamma.-Valerolactone as the Carbon-Based Product of
Interest
[0213] In some embodiments, the engineered and/or evolved
methylotroph of the present invention is engineered to produce
.gamma.-valerolactone as the carbon-based product of interest. One
example .gamma.-valerolactone biosynthetic pathway is shown in FIG.
16. In one embodiment, the engineered and/or evolved methylotroph
is engineered to express one or more of the following enzymes:
propionyl-CoA synthase (E.C. 6.2.1.-, E.C. 4.2.1.- and E.C.
1.3.1.-), beta-ketothiolase (E.C. 2.3.1.16; e.g., Ralstonia
eutropha BktB), acetoacetyl-CoA reductase (E.C. 1.1.1.36; e.g.,
Ralstonia eutropha PhaB), 3-hydroxybutyryl-CoA dehydratase (E.C.
4.2.1.55; e.g., X axonopodis Crt), vinylacetyl-CoA A-isomerase
(E.C. 5.3.3.3; e.g., C. difficile AbfD), 4-hydroxybutyryl-CoA
transferase (E.C. 2.8.3.-; e.g., C. kluyveri OrfZ), 1,4-lactonase
(E.C. 3.1.1.25; e.g., that from R. norvegicus). Propionyl-CoA
synthase is a multi-functional enzyme that converts
3-hydroxypropionate, ATP and NADPH to propionyl-CoA. Exemplary
propionyl-CoA synthases include AAL47820, and homologs thereof. In
another embodiment, the invention provides a nucleic acid encoding
a polypeptide having the amino acid sequence of SEQ ID NO:5, or a
sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81-85%, 90-95%, 96-98%, 99%, 99.9% or even higher identity
thereto.
Integration of Metabolic Pathways into Host Metabolism
[0214] The engineered and/or evolved methylotrophs 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
carbon product biosynthetic pathways. Depending on the host
methylotroph chosen, nucleic acids for some or all of particular
metabolic pathways can be expressed. For example, if a chosen host
methylotroph is deficient in one or more enzymes or proteins for
desired metabolic pathways, 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
methylotroph 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 production of
desired carbon products from C1 compounds. Thus, an engineered
and/or evolved methylotroph of the invention can be produced by
introducing exogenous enzyme or protein activities to obtain
desired metabolic pathways or desired metabolic pathways 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 reduced cofactors,
central metabolites and/or carbon-based products of interest.
[0215] Depending on the metabolic pathway constituents of a
selected host methylotroph, the engineered and/or evolved
methylotrophs of the invention can include at least one exogenously
expressed metabolic pathway-encoding nucleic acid and up to all
encoding nucleic acids for one or more energy conversion, carbon
fixation, methylotrophic and/or carbon-based product pathways. For
example, a RuMP-derived carbon fixation pathway 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 a metabolic 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 carbon fixation pathway
derived from the 3-HPA bicycle can be included, such as the
acetyl-CoA carboxylase, malonyl-CoA reductase, propionyl-CoA
synthase, propionyl-CoA carboxylase, methylmalonyl-CoA epimerase,
methylmalonyl-CoA mutase, succinyl-CoA:(S)-malate CoA transferase,
succinate dehydrogenase, fumarate hydratase,
(S)-malyl-CoA/.beta.-methylmalyl-CoA/(S)-citramalyl-CoA lyase,
mesaconyl-C1-CoA hydratase, mesaconyl-CoA C1-C4 CoA transferase,
and mesaconyl-C4-CoA hydratase. Given the teachings and guidance
provided herein, those skilled in the art would understand that the
number of encoding nucleic acids to introduce in an expressible
form can, at least, parallel the metabolic pathway deficiencies of
the selected methylotroph.
Genetic Engineering Methods for Optimization of Metabolic
Pathways
[0216] In some embodiments, the engineered and/or evolved
methylotrophs of the invention also can include other genetic
modifications that facilitate or optimize production of a
carbon-based product from C1 compounds or that confer other useful
functions onto the host organism.
[0217] In one aspect, the expression levels of the proteins of
interest of the energy conversion pathways, carbon fixation
pathways, methylotrophic pathways and/or carbon product
biosynthetic pathways can be either increased or decreased by, for
example, replacing or altering the expression control sequences
with alternate expression control sequences encoded by standardized
genetic parts. The exogenous standardized genetic parts can
regulate the expression of either heterologous or endogenous genes
of the metabolic pathway. Altered expression of the enzyme or
enzymes and/or protein or proteins of a metabolic pathway can
occur, for example, through changing gene position or gene order
[Smolke, 2002b], altered gene copy number [Smolke, 2002a],
replacement of a endogenous, naturally occurring regulated
promoters with constitutive or inducible synthetic promoters,
mutation of the ribosome binding sites [Wang, 2009], or
introduction of RNA secondary structural elements and/or cleavage
sites [Smolke, 2000; Smolke, 2001].
[0218] In another aspect, some engineered and/or evolved
methylotrophs of the present invention may require specific
transporters to facilitate uptake of C1 compounds. In some
embodiments, the engineered and/or evolved methylotrophs use
formate as a C1 compound. If formate uptake is limiting for either
growth or production of carbon-based products of interest, then
expression of one or more formate transporters in the engineered
and/or evolved methylotroph of the present invention can alleviate
this bottleneck. The formate transporters may be heterologous or
endogenous to the host organism. Exemplary formate transporters
include NP.sub.--415424 and NP.sub.--416987, and homologs thereof.
The present invention provides nucleic acids each comprising or
consisting of a sequence which is a codon optimized version of one
of the wild-type formate transporter genes. In another embodiment,
the invention provides nucleic acids each encoding a polypeptide
having the amino acid sequence of one of NP.sub.--415424 and
NP.sub.--416987.
[0219] In addition, the invention provides an engineered and/or
evolved methylotroph comprising a genetic modification conferring
to the engineered and/or evolved methylotrophic microorganism an
increased efficiency of using C1 compounds to produce carbon-based
products of interest relative to the microorganism in the absence
of the genetic modification. The genetic modification comprises one
or more gene disruptions, whereby the one or more gene disruptions
increase the efficiency of producing carbon-based products of
interest from C1 compounds. In one aspect, the one or more gene
disruptions target genes encoding competing reactions for C1
compounds, reduced cofactors, and/or central metabolites. In
another aspect, the one or more gene disruptions target genes
encoding competing reactions for intermediates or products of the
energy conversion, methylotrophic, carbon fixation, and/or carbon
product biosynthetic pathways of interest. The competing reactions
usually, but not exclusively, arise from metabolism endogenous to
the host cell or organism. Methods for introducing unmarked
mutations into the genome of methylotrophic bacteria such as
Paracoccus denitrificans have been shown previously [J Bacteriol,
1991, 173(21):6962-6970].
[0220] A combination of different approaches may be used to
identify candidate genetic modifications. Such approaches include,
for example, metabolomics (which may be used to identify
undesirable products and metabolic intermediates that accumulate
inside the cell), metabolic modeling and isotopic labeling (for
determining the flux through metabolic reactions contributing to
hydrocarbon production), and conventional genetic techniques (for
eliminating or substantially disabling unwanted metabolic
reactions). For example, metabolic modeling provides a means to
quantify fluxes through the cell's metabolic pathways and determine
the effect of elimination of key metabolic steps. In addition,
metabolomics and metabolic modeling enable better understanding of
the effect of eliminating key metabolic steps on production of
desired products.
[0221] To predict how a particular manipulation of metabolism
affects cellular metabolism and synthesis of the desired product, a
theoretical framework was developed to describe the molar fluxes
through all of the known metabolic pathways of the cell. Several
important aspects of this theoretical framework include: (i) a
relatively complete database of known pathways, (ii) incorporation
of the growth-rate dependence of cell composition and energy
requirements, (iii) experimental measurements of the amino acid
composition of proteins and the fatty acid composition of membranes
at different growth rates and dilution rates and (iv) experimental
measurements of side reactions which are known to occur as a result
of metabolism manipulation. These new developments allow
significantly more accurate prediction of fluxes in key metabolic
pathways and regulation of enzyme activity [Keasling, 1999a;
Keasling, 1999b; Martin, 2002; Henry, 2006].
[0222] Such types of models have been applied, for example, to
analyze metabolic fluxes in organisms responsible for enhanced
biological phosphorus removal in wastewater treatment reactors and
in filamentous fungi producing polyketides [Pramanik, 1997;
Pramanik, 1998a; Pramanik, 1998b; Pramanik, 1998c].
[0223] In another aspect, some engineered and/or evolved
methylotrophs of the present invention may require alterations to
the pool of intracellular reducing cofactors for efficient growth
and/or production of the carbon-based product of interest from C1
compounds. In some embodiments, the total pool of NAD.sup.+/NADH in
the engineered and/or evolved methylotroph is increased or
decreased by adjusting the expression level of nicotinic acid
phosphoribosyltransferase (E.C. 2.4.2.11). Over-expression of
either the E. coli or Salmonella gene pncB which encodes nicotinic
acid phosphoribosyltransferase has been shown to increase total
NAD.sup.+/NADH levels in E. coli [Wubbolts, 1990; Berrios-River,
2002; San, 2002]. In another embodiment, the availability of
intracellular NADPH can be also altered by modifying the engineered
and/or evolved methylotroph to express an NADH:NADPH
transhydrogenase [Sauer, 2004; Chin, 2011]. In another embodiment,
the total pool of ubiquinone in the engineered and/or evolved
methylotroph is increased or decreased by adjusting the expression
level of ubiquinone biosynthetic enzymes, such as
p-hydroxybenzoate-polyprenyl pyrophosphate transferase and
polyprenyl pyrophosphate synthetase. Overexpression of the
corresponding E. coli genes ubiA and ispB increased the ubiquinone
pool in E. coli [Zhu, 1995]. In the methylotroph Paracoccus
denitrificans, p-hydroxybenzoate and mevalonate have been shown to
be limiting in production of ubiquinone-10 under anaerobic
conditions [Appl Microbiol Biotechnol, 1983, 17(2):85-89, DOI:
10.1007/BF00499856]. In another embodiment, the level of the redox
cofactor ferredoxin in the engineered and/or evolved methylotroph
can be increased or decreased by changing the expression control
sequences that regulate its expression.
[0224] In another aspect, in addition to a C1 compound, some
engineered and/or evolved methylotrophs may require a specific
nutrients or vitamin(s) for growth and/or production of
carbon-based products of interest. For example, hydroxocobalamin, a
vitamer of vitamin B12, is a cofactor for particular enzymes of the
present invention, such as methylmalonyl-CoA mutase (E.C.
5.4.99.2). Required nutrients are generally supplemented to the
growth media during bench scale propagation of such organisms.
However, such nutrients can be prohibitively expensive in the
context of industrial scale bio-processing. In one embodiment of
the present invention, the host cell is selected from a
methylotroph that naturally produces the required nutrient(s), such
as Protaminobacter ruber or Methylobacterium extorquens, which
naturally produces hydroxocobalamin. In an alternate embodiment,
the need for a vitamin is obviated by modifying the engineered
and/or evolved methylotroph to express a vitamin biosynthesis
pathway [Roessner, 1995]. An exemplary biosynthesis pathway for
hydroxocobalamin comprises the following enzymes: uroporphyrin-III
C-methyltransferase (E.C. 2.1.1.107), precorrin-2 cobaltochelatase
(E.C. 4.99.1.3), cobalt-precorrin-2 (C.sup.20)-methyltransferase
(E.C. 2.1.1.151), cobalt-precorrin-3 (C.sup.17)-methyltransferase
(E.C. 2.1.1.131), cobalt precorrin-4 (C.sup.11)-methyltransferase
(E.C. 2.1.1.133), cobalt-precorrin 5A hydrolase (E.C. 3.7.1.12),
cobalt-precorrin-5B (C.sup.1)-methyltransferase (E.C. 2.1.1.195),
cobalt-precorrin-6A reductase, cobalt-precorrin-6V
(C.sup.5)-methyltransferase (E.C. 2.1.1.-), cobalt-precorrin-7
(C.sup.15)-methyltransferase (decarboxylating) (E.C. 2.1.1.196),
cobalt-precorrin-8X methylmutase, cobyrinate A,C-diamide synthase
(E.C. 6.3.5.11), cob(II)yrinate a,c-diamide reductase (E.C.
1.16.8.1), cob(I)yrinic acid a,c-diamide adenosyltransferase (E.C.
2.5.1.17), adenosyl-cobyrate synthase (E.C. 6.3.5.10),
adenosylcobinamide phosphate synthase (E.C. 6.3.1.10),
GTP:adenosylcobinamide-phosphate guanylyltransferase (E.C.
2.7.7.62), nicotinate-nucleotide dimethylbenzimidazole
phosphoribosyltransferase (E.C. 2.4.2.21),
adenosylcobinamide-GDP:.alpha.-ribazole-5-phosphate
ribazoletransferase (E.C. 2.7.8.26) and
adenosylcobalamine-5'-phosphate phosphatase (E.C. 3.1.3.73). In
addition, to allow for cobalt uptake and incorporation into vitamin
B12, the genes encoding the cobalt transporter are overexpressed.
The exemplary cobalt transporter protein found in Salmonella
enterica is overexpressed and is encoded by proteins ABC-type
Co.sup.2+ transport system, permease component (CbiM,
NP.sub.--460968), ABC-type cobalt transport system, periplasmic
component (CbiN, NP.sub.--460967), and ABC-type cobalt transport
system, permease component (CbiQ, NP.sub.--461989).
[0225] In some embodiments, the intracellular concentration (e.g.,
the concentration of the intermediate in the engineered and/or
evolved methylotroph) of the metabolic pathway intermediate can be
increased to further boost the yield of the final product. For
example, by increasing the intracellular amount of a substrate
(e.g., a primary substrate) for an enzyme that is active in the
metabolic pathway, and the like.
[0226] In another aspect, the carbon-based products of interest are
or are derived from the intermediates or products of fatty acid
biosynthesis. To increase the production of waxes/fatty acid
esters, and fatty alcohols, one or more of the enzymes of fatty
acid biosynthesis can be over expressed or mutated to reduce
feedback inhibition. Additionally, enzymes that metabolize the
intermediates to make nonfatty-acid based products (side reactions)
can be functionally deleted or attenuated to increase the flux of
carbon through the fatty acid biosynthetic pathway thereby
enhancing the production of carbon-based products of interest.
Growth-Based Selection Methods for Optimization of Engineered
Carbon-Fixing Strains
[0227] Selective pressure provides a valuable means for testing and
optimizing the engineered methylotrophs of the present invention.
Alternatively, an evolved methylotroph having selected
functionality after such selection can be further engineered to
include additional or altered functionality. In some embodiments,
the engineered methylotrophs of the invention can be evolved under
selective pressure to optimize production of a carbon-based product
from a C1 compound or that confer other useful functions onto the
host organism. The ability of an optimized engineered methylotroph
to replicate more rapidly than unmodified counterparts confirms the
utility of the optimization. Similarly, the ability to survive and
replicate in media lacking a required nutrient, such as vitamin
B12, confirms the successful implementation of a nutrient
biosynthetic module. In some embodiments, the engineered
methylotrophs can be cultured in the presence of a limiting amount
of C1 compound in order to select for evolved strains that more
efficiently utilize the C1 compound. In some embodiments, the
engineered methylotrophs of the invention can be evolved to grow
despite the presence of inhibitory compounds in the C1 feedstock
(see, e.g., Example 5).
[0228] Evolution can occur as a result of either spontaneous,
natural mutation or by addition of mutagenic agents or conditions
to live cells. If desired, additional genetic variation can be
introduced prior to or during selective pressure by treatment with
mutagens, such as ultra-violet light, alkylators [e.g., ethyl
methanesulfonate (EMS), methyl methane sulfonate (MMS),
diethylsulfate (DES), and nitrosoguanidine (NTG, NG, MMG)], DNA
intercalcators (e.g., ethidium bromide), nitrous acid, base
analogs, bromouracil, transposonsm and the like. Alternatively,
genetic variation may be introduced via untargeted genetic
mutagenesis techniques such as transposon insertion. Transposable
elements have been used previously to generate phenotypic diversity
in methylotrophic Paracoccus strains [PLoS ONE, 2012, 7(2):e32277,
DOI: 10.1371/journal.pone.0032277]. The engineered methylotrophs
can be propagated either in serial batch culture or in a
turbidostat as a controlled growth rate.
[0229] Alternately or in addition to selective pressure, pathway
activity can be monitored following growth under permissive (i.e.,
non-selective) conditions by measuring specific product output via
various metabolic labeling studies (including radioactivity),
biochemical analyses (Michaelis-Menten), gas chromatography-mass
spectrometry (GC/MS), mass spectrometry, matrix assisted laser
desorption ionization time-of-flight mass spectrometry (MALDI-TOF),
capillary electrophoresis (CE), and high pressure liquid
chromatography (HPLC).
[0230] To generate engineered methylotrophs with improved yield of
central metabolites and/or carbon-based products of interest,
metabolic modeling can be utilized to guide strain optimization.
Modeling analysis allows reliable predictions of the effects on
cell growth of shifting the metabolism towards more efficient
production of central metabolites or products derived from central
metabolites. Modeling can also be used to design gene knockouts
that additionally optimize utilization of the energy conversion,
methylotrophic, carbon fixation and carbon product biosynthetic
pathways. In some embodiments, modeling is used to select growth
conditions that create selective pressure towards uptake and
utilization of C1 compound(s). An in silico stoichiometric model of
host organism metabolism and the metabolic pathway(s) of interest
can be constructed (see, for example, a model of the E. coli
metabolic network [Edwards, 2002]). The resulting model can be used
to compute phenotypic phase planes for the engineered methylotrophs
of the present invention. A phenotypic phase plane is a portrait of
the accessible growth states of an engineered methylotroph as a
function of imposed substrate uptake rates. A particular engineered
methylotroph, at particular uptake rates for limiting nutrients,
may not grow as well as the phenotypic phase plane predicts, but no
strain should be able to grow better than indicated by the
phenotypic phase plane. Under a variety of circumstances, it has
been shown the modified E. coli strains evolve towards, and then
along, the phenotypic phase plane, always in the direction of
increasing growth rates [Fong, 2004]. Thus, a phenotypic phase
plane can be viewed as a landscape of selective pressure. Strains
in an environment where a given nutrient uptake is positively
correlated with growth rate are predicted to evolve towards
increased nutrient uptake. Conversely, strains in an environment
where nutrient uptake are inversely correlated with growth rate are
predicted to evolve away from nutrient uptake.
Fermentation Conditions
[0231] The engineered and/or evolved methylotrophs of the present
invention are cultured in a medium comprising C1 compound(s) and
any required nutrients. The culture conditions can include, for
example, liquid culture procedures as well as fermentation and
other large scale culture procedures. In one embodiment, the
engineered and/or evolved methylotroph is grown in a minimal salts
medium containing a C1 feedstock, such as formate, formic acid,
formaldehyde, or methanol. The medium composition can be optimized
for enhanced growth and production of carbon-based products of
interest (see, e.g., Example 1). In one embodiment, the medium
composition is 100 mM sodium bicarbonate, 6 mM sodium chloride, 6
mM sodium nitrate, 11 mM sodium thiosulfate, and 26 mM sodium
formate in addition to standard MOPS minimal medium components.
[0232] The production and isolation of carbon-based products of
interest can be enhanced by employing specific fermentation
techniques. One method for maximizing production while reducing
costs is increasing the percentage of the carbon that is converted
to carbon-based products of interest. During normal cellular
lifecycles carbon is used in cellular functions including producing
lipids, saccharides, proteins, organic acids, and nucleic acids.
Reducing the amount of carbon necessary for growth-related
activities can increase the efficiency of carbon source conversion
to output. This can be achieved by first growing engineered and/or
evolved methylotrophs to a desired density, such as a density
achieved at the peak of the log phase of growth. At such a point,
replication checkpoint genes can be harnessed to stop the growth of
cells. Specifically, quorum sensing mechanisms [Camilli, 2006;
Venturi, 2006; Reading, 2006] can be used to activate genes such as
p53, p21, or other checkpoint genes. Genes that can be activated to
stop cell replication and growth in E. coli include umuDC genes,
the over-expression of which stops the progression from stationary
phase to exponential growth [Murli, 2000]. UmuC is a DNA polymerase
that can carry out translesion synthesis over non-coding
lesions--the mechanistic basis of most UV and chemical mutagenesis.
The umuDC gene products are used for the process of translesion
synthesis and also serve as a DNA damage checkpoint. UmuDC gene
products include UmuC, UmuD, umuD', UmuD'.sub.2C, UmuD'.sub.2 and
UmUD.sub.2. Simultaneously, the carbon product biosynthetic pathway
genes are activated, thus minimizing the need for replication and
maintenance pathways to be used while the carbon-based product of
interest is being made.
[0233] Alternatively, cell growth and product production can be
achieved simultaneously. In this method, cells are grown in
bioreactors with a continuous supply of inputs and continuous
removal of product. Batch, fed-batch, and continuous fermentations
are common and well known in the art and examples can be found in
[Brock, 1989; Deshpande, 1992].
[0234] In one embodiment, the engineered and/or evolved
methylotroph is engineered such that the final product is released
from the cell. In embodiments where the final product is released
from the cell, a continuous process can be employed. In this
approach, a reactor with organisms producing desirable products can
be assembled in multiple ways. In one embodiment, the reactor is
operated in bulk continuously, with a portion of media removed and
held in a less agitated environment such that an aqueous product
can self-separate out with the product removed and the remainder
returned to the fermentation chamber. In embodiments where the
product does not separate into an aqueous phase, media is removed
and appropriate separation techniques (e.g., chromatography,
distillation, etc.) are employed.
[0235] In an alternate embodiment, the product is not secreted by
the engineered and/or evolved methylotrophs. In this embodiment, a
batch-fed fermentation approach is employed. In such cases, cells
are grown under continued exposure to inputs (C1 compounds) as
specified above until the reaction chamber is saturated with cells
and product. A significant portion to the entirety of the culture
is removed, the cells are lysed, and the products are isolated by
appropriate separation techniques (e.g., chromatography,
distillation, filtration, centrifugation, etc.).
[0236] In certain embodiments, the engineered and/or evolved
methylotrophs of the invention can be sustained, cultured or
fermented under anaerobic or substantially anaerobic conditions.
Briefly, anaerobic conditions 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. It is highly desirable to maintain anaerobic conditions
in the fermenter to reduce the cost of the overall process.
[0237] 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 C1 feedstock uptake rate by
monitoring carbon source depletion over time.
[0238] In another embodiment, the engineered and/or evolved
methylotrophs can be cultured in the presence of an electron
acceptor, for example, nitrate, in particular under substantially
anaerobic conditions. It is understood that an appropriate amount
of nitrate can be added to a culture to achieve a desired increase
in biomass, for example, 1 mM to 100 mM nitrate, or lower or higher
concentrations, as desired, so long as the amount added provides a
sufficient amount of electron acceptor for the desired increase in
biomass. Such amounts include, but are not limited to, 5 mM, 10 mM,
15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, as appropriate to achieve
a desired increase in biomass. In one embodiment, the engineered
and/or evolved methylotroph is a denitrifier that can use nitrate
as a terminal electron acceptor and reduce nitrate to nitrogen gas.
In some embodiments, the engineered and/or evolved methylotroph is
derived from methylotrophic denitrifers, such as Paracoccus
denitrificans. Other electron acceptors include fumarate,
trimethylammonium oxide, ferricyanide, or dimethyl sulfoxide.
[0239] In some embodiments, the engineered and/or evolved
methylotrophs of the present invention are initially grown in
culture conditions with a limiting amount of multi-carbon compounds
to facilitate growth. Then, once the supply of organic carbon is
exhausted, the engineered and/or evolved methylotrophs transition
from heterotrophic to methylotrophic growth relying on energy from
a C1 compounds in order to produce carbon-based products of
interest. The organic carbon can be, for example, a carbohydrate
source. Such sources include, for example, sugars such as glucose,
xylose, arabinose, galactose, mannose, fructose and starch. Other
sources of carbohydrate include, for example, renewable feedstocks
and biomass. Exemplary types of biomasses that can be used as
feedstocks in the methods of the invention 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 would understand that renewable feedstocks and biomass other
than those exemplified above also can be used for culturing the
engineered and/or evolved methylotrophs of the invention. In some
embodiments, the engineered and/or evolved methylotrophs are
optimized for a two stage fermentation by regulating the expression
of the carbon product biosynthetic pathway.
[0240] In one aspect, the percentage of input carbon atoms
converted to hydrocarbon products is an efficient and inexpensive
process. Typical efficiencies in the literature are .about.<5%.
Engineered and/or evolved methylotrophs which produce hydrocarbon
products can have greater than 1, 3, 5, 10, 15, 20, 25, and 30%
efficiency. In one example engineered and/or evolved methylotrophs
can exhibit an efficiency of about 10% to about 25%. In other
examples, such microorganisms can exhibit an efficiency of about
25% to about 30%, and in other examples such engineered and/or
evolved methylotrophs can exhibit >30% efficiency.
[0241] In some examples where the final product is released from
the cell, a continuous process can be employed. In this approach, a
reactor with engineered and/or evolved methylotrophs producing for
example, fatty acid derivatives, can be assembled in multiple ways.
In one example, a portion of the media is removed and allowed to
separate. Fatty acid derivatives are separated from the aqueous
layer, which can in turn, be returned to the fermentation
chamber.
[0242] In another example, the fermentation chamber can enclose a
fermentation that is undergoing a continuous reduction. In this
instance, a stable reductive environment can be created. The
electron balance would be maintained by the release of oxygen.
Efforts to augment the NAD/H and NADP/H balance can also facilitate
in stabilizing the electron balance.
Consolidated Methylotrophic Fermentation
[0243] The above aspect of the invention is an alternative to
directly producing final carbon-based product of interest as a
result of methylotrophic metabolism. In this approach, carbon-based
products of interest would be produced by leveraging other
organisms that are more amenable to making any one particular
product while culturing the engineered and/or evolved methylotroph
for its carbon source. Consequently, fermentation and production of
carbon-based products of interest can occur separately from carbon
source production in a bioreactor.
[0244] In one aspect, the methods of producing such carbon-based
products of interest include two steps. The first-step includes
using engineered and/or evolved methylotrophs to convert C1
compound(s) to central metabolites or sugars such as glucose. The
second-step is to use the central metabolites or sugars as a carbon
source for cells that produce carbon-based products of interest. In
one embodiment, the two-stage approach comprises a bioreactor
comprising engineered and/or evolved methylotrophs; a second
reactor comprising cells capable of fermentation; wherein the
engineered and/or evolved methylotrophs provides a carbon source
such as glucose for cells capable of fermentation to produce a
carbon-based product of interest. The second reactor may comprise
more than one type of microorganism. The resulting carbon-based
products of interest are subsequently separated and/or
collected.
[0245] In some embodiments, the two steps are combined into a
single-step process whereby the engineered and/or evolved
methylotrophs convert C1 compound(s) and directly into central
metabolites or sugars such as glucose and such organisms are
capable of producing a variety of carbon-based products of
interest.
[0246] The present invention also provides methods and compositions
for sustained glucose production in engineered and/or evolved
methylotrophs wherein these or other organisms that use the sugars
are cultured using C1 compound(s) for use as a carbon source to
produce carbon-based products of interest. In such embodiments, the
host cells are capable of secreting the sugars, such as glucose
from within the cell to the culture media in continuous or
fed-batch in a bioreactor.
[0247] Certain changes in culture conditions of engineered and/or
evolved methylotrophs for the production of sugars can be optimized
for growth. For example, conditions are optimized for C1
compound(s) and their concentration(s), electron acceptor(s) and
their concentrations, addition of supplements and nutrients. As
would be apparent to those skilled in the art, the conditions
sufficient to achieve optimum growth can vary depending upon
location, climate, and other environmental factors, such as the
temperature, oxygen concentration and humidity. Other adjustments
may be required, for example, an organism's ability for carbon
uptake.
[0248] Advantages of consolidated methylotrophic fermentation
include a process where there is separation of chemical end
products, e.g., glucose, spatial separation between end products
(membranes) and time. Additionally, unlike traditional or
cellulosic biomass to biofuels production, pretreatment,
saccharification and crop plowing are obviated.
[0249] The consolidated methylotrophic fermentation process
produces continuous products. In some embodiments, the process
involves direct conversion of C1 compound(s) to product from
engineered front-end organisms to produce various products without
the need to lyse the organisms. For instance, the organisms can
utilize 3PGAL to make a desired fermentation product, e.g.,
ethanol. Such end products can be readily secreted as opposed to
intracellular products such as oil and cellulose. In yet other
embodiments, organisms produce sugars, which are secreted into the
media and such sugars are used during fermentation with the same or
different organisms or a combination of both.
Processing and Separation of Carbon-Based Products of Interest
[0250] The carbon-based products produced by the engineered and/or
evolved methylotrophs during fermentation can be separated from the
fermentation media. Known techniques for separating fatty acid
derivatives from aqueous media can be employed. One exemplary
separation process provided herein is a two-phase (bi-phasic)
separation process. This process involves fermenting the
genetically-engineered production hosts under conditions sufficient
to produce for example, a fatty acid, allowing the fatty acid to
collect in an organic phase and separating the organic phase from
the aqueous fermentation media. This method can be practiced in
both a batch and continuous fermentation setting.
[0251] Bi-phasic separation uses the relative immisciblity of fatty
acid to facilitate separation. A skilled artisan would appreciate
that by choosing a fermentation media and the organic phase such
that the fatty acid derivative being produced has a high log P
value, even at very low concentrations the fatty acid can separate
into the organic phase in the fermentation vessel.
[0252] When producing fatty acids by the methods described herein,
such products can be relatively immiscible in the fermentation
media, as well as in the cytoplasm. Therefore, the fatty acid can
collect in an organic phase either intracellularly or
extracellularly. The collection of the products in an organic phase
can lessen the impact of the fatty acid derivative on cellular
function and allows the production host to produce more
product.
[0253] The fatty alcohols, fatty acid esters, waxes, and
hydrocarbons produced as described herein allow for the production
of homogeneous compounds with respect to other compounds wherein at
least 50%, 60%, 70%, 80%, 90%, or 95% of the fatty alcohols, fatty
acid esters, waxes and hydrocarbons produced have carbon chain
lengths that vary by less than 4 carbons, or less than 2 carbons.
These compounds can also be produced so that they have a relatively
uniform degree of saturation with respect to other compounds, for
example at least 50%, 60%, 70%, 80%, 90%, or 95% of the fatty
alcohols, fatty acid esters, hydrocarbons and waxes are mono-, di-,
or tri-unsaturated.
Detection and Analysis
[0254] Generally, the carbon-based products of interest produced
using the engineered and/or evolved methylotrophs described herein
can be analyzed by any of the standard analytical methods, e.g.,
gas chromatography (GC), mass spectrometry (MS) gas
chromatography-mass spectrometry (GCMS), and liquid
chromatography-mass spectrometry (LCMS), high performance liquid
chromatography (HPLC), capillary electrophoresis, Matrix-Assisted
Laser Desorption Ionization time-of-flight mass spectrometry
(MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared
(NIR) spectroscopy, viscometry [Knothe, 1997; Knothe, 1999],
titration for determining free fatty acids [Komers, 1997],
enzymatic methods [Bailer, 1991], physical property-based methods,
wet chemical methods, etc.
Sequences Provided by the Invention
[0255] Table 4 provides a summary of SEQ ID NOs:1-5 disclosed
herein.
TABLE-US-00002 TABLE 4 Sequences SEQ ID NO Sequence 1 Codon
optimized Escherichia coli DXS gene 2 Codon optimized Escherichia
coli IDI gene 3 Codon optimized Populus nigra IspS gene 4 Codon
optimized Picea abies TPS-bis gene 5 Chloroflexus aurantiacus PCS
amino acid sequence
EXAMPLES
[0256] The examples below are provided herein for illustrative
purposes and are not intended to be restrictive.
Example 1
Optimization of Growth Medium for Paracoccus sp. when Using Formate
as the C1 Compound
[0257] Paracoccus zeaxanthinifaciens ATCC 21588, Paracoccus
versutus ATCC 25364, and Paracoccus denitrificans ATCC 13534 were
obtained from the American Type Culture Collection (ATCC).
[0258] Strains were tested for the ability to grow aerobically on
sodium formate as a sole source of carbon and/or energy using MOPS
minimal medium (Teknova, Inc.) with sodium formate as a sole carbon
source at 37C. Unlike previous media used to evaluate the
formate-dependent growth of Paracoccus, this medium contains
defined levels of trace elements molybdenum, boron, copper, zinc,
manganese, and other trace metals.
[0259] Growth was conducted in various high-throughput machinery
capable of monitoring growth by light scattering at 600 nm,
including a Gemini SpectraNax plate reader (Molecular Devices,
Inc.), a Tecan M3000 plate reader (Tecan, Inc.), and a BioLector
device (m2p-labs, Inc.). For the BioLector, the CO.sub.2 gas
content in the culture headspace was controllable, as was the
humidity.
[0260] Paracoccus zeaxanthinifaciens ATCC 21588 was incapable of
growth on formate as a sole carbon or energy source. The other two
Paracoccus strains were capable of growth on formate as a sole
carbon source, as has been previously reported [Microbiology, 1979,
114(1):1-13, DOI: 10.1099/00221287-114-1-1; Arch Microbiol, 1978,
118(1):21-26, DOI: 10.1007/BF00406069].
[0261] The effect on growth rate of changes in temperature (T, in
degrees Celsius), the partial pressure of CO.sub.2 in the culture
headspace gas (pCO.sub.2, in percent by volume), shaking speed (in
rpm), and/or the concentrations (in mM) of sodium formate (HCOONa),
sodium nitrate (NaNO.sub.3), sodium thiosulfate
(Na.sub.2S.sub.2O.sub.3), sodium chloride (NaCl), and sodium
bicarbonate (NAHCO.sub.3) added to the Basal MOPS minimal medium
were systematically evaluated. Combinations examined are shown in
Table 2. Paracoccus strains are labelled according to their ATCC
number. For each measurement, the instrument used (BioLector;
SpectraMax; Tecan), plate type (Flower, Flower Plate; 96 transp,
96-well transparent microtiter plate; 96 opaque, 96-well opaque
microtiter plate), use of plate lid, use of humidity control and
total culture volume in uL is indicated. N/A indicates that a
particular experimental condition is not applicable for the
instrument used.
TABLE-US-00003 TABLE 2 Tested growth conditions for each Paracoccus
strain Shaking Humidity ATCC NaHCO.sub.3 NaCl NaNO.sub.3
Na.sub.2S.sub.2O.sub.3 HCOONa T speed Instrument Plate Lid Control
pCO.sub.2 Vol 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower
N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector
Flower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 37.0 1200
BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0 37.0
1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0 50.0
37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0
20.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0
0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 150.0
0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534
0.0 60.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300
13534 0.0 0.0 60.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5%
1300 13534 0.0 0.0 0.0 80.0 50.0 37.0 1200 BioLector Flower N/A
TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 20.0 37.0 1200 BioLector Flower
N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 50.0 34.0 900 BioLector
Flower N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 34.0 900
BioLector Flower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 34.0
900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0
34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0
50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0
0.0 20.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0
0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 150.0
0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534
0.0 60.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300
13534 0.0 0.0 60.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5%
1300 13534 0.0 0.0 0.0 80.0 50.0 34.0 900 BioLector Flower N/A TRUE
5% 1300 13534 0.0 0.0 0.0 0.0 20.0 34.0 900 BioLector Flower N/A
TRUE 5% 1300 25364 10.7 4.3 4.3 40.0 45.7 36.6 1157 BioLector
Flower N/A TRUE 5% 1300 13534 75.0 4.3 4.3 5.7 45.7 36.6 1157
BioLector Flower N/A TRUE 5% 1300 13534 10.7 4.3 30.0 5.7 45.7 36.6
1157 BioLector Flower N/A TRUE 5% 1300 25364 42.9 17.1 17.1 0.0
32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 13534 0.0 17.1
17.1 22.9 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 13534
42.9 17.1 0.0 22.9 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300
25364 101.0 6.1 6.1 11.4 26.7 34.4 937 BioLector Flower N/A TRUE 5%
1300 25364 101.0 6.1 6.1 0.0 26.7 34.4 937 BioLector Flower N/A
TRUE 5% 1300 13534 122.4 29.4 14.7 16.3 67.8 34.4 937 BioLector
Flower N/A TRUE 5% 1300 13534 122.4 29.4 14.7 0.0 67.8 34.4 937
BioLector Flower N/A TRUE 5% 1300 21588 0.0 0.0 0.0 0.0 50.0 31.0
unknown Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0
31.0 unknown Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0
50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 21588 0.0 0.0
0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 25364
0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150
13534 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A
N/A 150 21588 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp
TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96
transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 N/A
SpectraMax 96 transp TRUE N/A N/A 150 21588 0.0 0.0 0.0 0.0 50.0
31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0
0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150
13534 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE
N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96
opaque TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear
(8.5) Tecan 96 opaque TRUE N/A N/A 100 13534 0.0 0.0 0.0 0.0 50.0
37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 13534 0.0 0.0
0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100
25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE
FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque
TRUE FALSE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector
96 opaque TRUE FALSE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200
BioLector 96 opaque TRUE FALSE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0
37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 25364 0.0 0.0
0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100
13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE
N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96
opaque TRUE N/A N/A 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 800
BioLector 96 opaque TRUE TRUE FALSE 150 25364 0.0 0.0 0.0 0.0 50.0
37.0 800 BioLector 96 opaque TRUE TRUE FALSE 100 13534 0.0 0.0 0.0
0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 150 13534 0.0
0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 100
25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE
FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque
FALSE TRUE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector
96 opaque FALSE TRUE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200
BioLector 96 opaque FALSE TRUE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0
37.0 1200 BioLector Flower N/A TRUE FALSE 1300 13534 0.0 0.0 0.0
0.0 50.0 37.0 1200 BioLector Flower N/A TRUE FALSE 1300
[0262] The particular values for salt, bicarbonate, formate,
thiosulfate, or nitrate concentration, as well as temperature, were
chosen by implementing a Nelder-Mead simplex optimization algorithm
(as described in Chapter 18 of Chemometrics: a textbook ISBN:
0444426604) using the starting simplices with points chosen from
the following possibilities: temperature, 34.degree. C. or
37.degree. C.; sodium bicarbonate, 150 mM or 0 mM; sodium chloride,
60 mM or 0 mM; sodium formate, 20 mM or 50 mM; sodium nitrate, 60
mM or 0 mM; sodium thiosulfate, 0 mM or 80 mM; shaking speed, 1200
rpm or 900 rpm. Growth was evaluated for both 25364 and 13534 at
each chosen medium condition. For each strain and medium condition,
a score indicative of the growth was calculated as the time (in
hours) to 50% of the maximum growth attained in the entire
experiment minus the time to 5% of the maximum growth. This metric
is easy to compute and avoids penalizing conditions with longer lag
phases.
[0263] From the scores, new medium conditions were calculated
according to the Nelder-Mead simplex algorithm. These medium
conditions were tested as well. The growth under the new condition
as well as the old ones was used to define the points of a new
simplex, and the process repeated.
[0264] After several rounds of the medium optimization process, a
satisfactory medium condition, allowing for faster growth than the
initially chosen medium conditions, was obtained. In total, 68
different unique medium/strain/temperature/shaking conditions were
examined. The fastest growth on formate as a sole carbon source was
obtained for ATCC strain 25364. Under the optimal conditions, the
medium consisted of 100 mM sodium bicarbonate, 6 mM sodium
chloride, 6 mM sodium nitrate, 11 mM sodium thio sulfate, and 26 mM
sodium formate in addition to standard MOPS minimal medium
components. The optimal growth temperature was 34.degree. C. Under
these conditions, ATCC strain 25364 had a growth rate of >0.7
hr.sup.-1, corresponding to a doubling time of 0.95 hr.
[0265] ATCC strain 25364 was found capable of growth at rates in
excess of 0.4 hr.sup.-1 using a simpler medium composition of MOPS
minimal medium plus 50 mM sodium formate.
[0266] Large concentrations of thiosulfate were found to give
slowed, biphasic growth curves for both strains of Paracoccus
tested. In general, moderate concentrations of sodium nitrate
showed improved growth. Growth at 34.degree. C. was slightly better
than growth at 37.degree. C., and both of these temperatures were
significantly better than growth at 30.degree. C.
Example 2
Automatable Protocol for Conjugative Transfer of Plasmids from E.
coli Donors to Paracoccus sp
[0267] E. coli strain 517-1 was obtained from the Yale E. coli
Genetic Stock Center. Paracoccus denarificans PD1222 was obtained
from Stephen Spiro (University of Texas at Dallas). E. coli S17-1
strain is tra+, meaning it is able to mobilize for conjugative
transfer those plasmids harboring a mob+genotype. Plasmid pDIY313K,
obtained from Dariusz Bartosik (University of Warsaw, Poland) and
described by his laboratory [J Microbiol Methods, 2011,
86(2):166-74, DOI: 10.1016/j.mimet.2011.04.016], and introduced
into E. coli 517-1 by standard methods.
[0268] E. coli S17-1 was grown on Luria broth with carbenicillin
overnight. Paracoccus versutus ATCC 25364 was grown overnight on
MOPS minimal medium (Teknova, Inc.) with 50 mM sucrose and 40 mM
sodium nitrate.
[0269] The next day, E. coli S17-1 was subcultured in
antibiotic-free Luria broth for >4 hr. Paracoccus strains were
subcultured in identical MOPS/sucrose/nitrate medium. After
cultures of both E. coli and Paracoccus had reach log phase, with
optimal density greater than 1.0 cm.sup.-1, cultures were mixed in
equal volumes in wells of a standard, SBS-format 96-well plate. No
effort was made to pellet the cells, to immobilize cells on porous
filters, to culture the cells on solid media, or to otherwise
manipulate the mixtures. Cultures were simply mixed in equal
volumes and incubated overnight at 37.degree. C. without
agitation.
[0270] After overnight incubation, the mixed cultures were diluted
in PBS and dilutions were plated on MOPS/sucrose/kanamycin agar. E.
coli cannot use sucrose as a carbon source, and only strains
carrying pDIY313K can grow in the presence of kanamycin. Thus on
these plates only transconjugants--strains of Paracoccus containing
plasmid DNA and expressing plasmid-derived kanamycin resistance
genes--can grow. In parallel we plated the same dilutions on
MOPS/sucrose agar without kanamycin, in order to calculate the cell
concentration of total Paracoccus cells used in the experiment and
to calculate the transconjugation frequency (colonies of
plasmid-bearing Paracoccus isolated per colony of recipient
Paracoccus cell).
[0271] Using this simple technique we were able to demonstrate
conjugation frequencies of 10.sup.-5 using Paracoccus denitrificans
PD1222 and 2.times.10.sup.-7 using Paracoccus versutus. It should
be emphasized that this frequency was determined via a protocol
which did not require non-selective growth on soft medium, the use
of filters, or any centrifugation steps. These steps are required
in protocols for conjugation frequently taught in the literature.
For example, Bartosik [J Microbiol Methods, 2011, 86(2):166-74,
DOI: 10.1016/j.mimet.2011.04.016] teaches that cells must be grown,
pelleted by centrifugation, washed, resuspendend, mixed,
immobilized on a porous filter, grown under non-selective agar
overnight, removed from the filter by washing, pelleted, and
finally plated on selective medium. The lack of any such laborious
cell manipulation procedures in our protocol is essential for
conduction of the protocol on a robotics-based liquid-handling
platform, where centrifugation and resuspension operations are much
more error-prone, hard to implement, and/or unreliable in
comparison with simple liquid handling steps.
Example 3
Genome Sequencing of Paracoccus Strains
[0272] Genomic DNA was isolated from Paracoccus zeaxanthinifaciens
ATCC 21588, Paracoccus versutus ATCC 25364, and Paracoccus
denitrificans ATCC 13534 using a Wizard Genomic DNA Isolation Kit
(Promega, Inc.). The resulting DNA samples were fragmented and
converted to paired-end libraries for whole-genome shotgun
sequencing on a 454 pyrosequencing platform (Roche, Inc.).
[0273] For Paracoccus denitrificans, 37,585,886 paired reads, each
100 nt in length, were obtained. This represents approximately 3.7
gigabases of sequence data, or approximately 730-fold coverage of
the 5.2 megabase genome of Paracoccus denitrificans PD1222 (Genbank
accession numbers CP000489, CP000490, and CP000491 for chromosome
1, chromosome 2, and a 653,815 bp megaplasmid, respectively). Reads
were assembled first by de-novo assembly and second by mapping the
de-novo contigs to the published PD1222 genome.
[0274] The resulting reads could be assembled into a crude
whole-genome assembly of 351 scaffolds comprising 21,972,742 total
reads. The maximum scaffold was 7974 nt and minimum-length scaffold
2004 nt.
Example 4
Analysis of Methylerythritol Pathway in Paracoccus
[0275] The Paracoccus denitrificans PD1222 genome has been
published. Through manual inspection and BLAST searching we found
homologs to all but one member of the methylerythritol pathway for
isoprenoid biosynthesis. The Paraoccus gene homologs are shown in
Table 3. Gene names refer to standard names given to E. coli genes
(see accession Genbank accession U000096 for more information).
Names for Paracoccus genes correspond to the nomenclature annotated
as part of the Paracoccus PD 1222 genome sequence, available at
Genbank accession numbers CP000489 for chromosome 1 and CP000490
for chromosome 2.
TABLE-US-00004 TABLE 3 Methylerythritol pathway gene homologs in
Paracoccus denitrificans PD122 P. denitrificans PD1222 E. coli gene
PD1222 gene chromosome # dxs Pden_0400 1 dxr Pden_3997 2 ispD
Pden_3667 (KEGG); none (Metacyc) 2 ispE Pden_0423 1 ispF Pden_3667
2 ispG Pden_1820 1 ispH Pden_3619 2 idi no type I or type II
homologues ? ispA Pden_0399 1
[0276] The sole member of the methylerythritol pathway missing a
homolog from the P. denitrificans PD1222 genome is the gene idi,
encoding isopentenyl-diphosphate .DELTA.-isomerase (E.C. 5.3.3.2).
It is responsible for interconverting isopentenyl diphosphate
(IPPP) and dimethylallyl diphosphate (DMAP). However, several
studies have shown that this gene is not required for pathway
activity, since the preceding pathway step, coded for in E. coli by
the ispH gene that shares homology with predicted Paracoccus gene
Pden.sub.--3619, generates both IPPP and DMAP to some degree
[Lipids, 2008, 43(12):1095-1107, DOI:
10.1007/s11745-008-3261-7].
[0277] P. denitrificans is known to contain prenylated quinones as
constituents of its cell membrane [Biochem Eng J, 2003,
16(2):183-190, DOI: 10.1016/51369-703X(03)00035-4]. These compounds
are indicative of terpene production. The presence of gene homologs
for the methylerythritol pathway indicate that this pathway is
responsible for formation of terpenoids in this organism.
Example 5
Selection for Populations of Paracoccus Versutus with Improved
Doubling Times on Electrolytically Generated Formate
[0278] A computer-controlled continuous culture device that can be
operated as a chemostat or a turbidostat was constructed. The
device has a working volume between 20 and 50 mL (not yet tested
above 50 mL). The device uses air pressure to move liquids
throughout the fluidics system, and an array of solenoid valves to
direct fluid flow. The culture is mixed and aerated by the
turbulence created by sparging with air at a flow rate of >10
vvm. The valves are controlled by an Arduino Mega 2560
microprocessor. The Arduino also interfaces with the sensors and
control mechanisms of the device. The optical density (OD) of the
culture is determined by an infrared LED-photodiode pair which
measures the transmittance of light across the culture vessel.
[0279] From Edward Rode at DNV, Inc. (formerly Det Norske Veritas),
we obtained two samples of formate generated by DNV from
electricity and carbon dioxide by electrolysis. The first solution
received from DNV contained 0.5 M potassium chloride (electrolyte),
0.5 M potassium formate, and 2.5 M sodium bicarbonate. The second
solution as received from DNV contained approximately 0.5 M
potassium chloride (electrolyte), and 0.56 M potassium formate.
This solution was directly obtained from the cathodic chamber of
DNV's electrolysis reactor without any upgrading or purifying, and
thus it may contain other uncharacterized contaminants or other
agents which inhibit the growth of bacteria. These may arise from
metals or plastics leaching into solution, from uncharacterized
electrochemical reactions going on in parallel with the cathodic
reduction of bicarbonate (i.e. dissolved CO.sub.2) to formate
salts, or from other processes. The ability for engineered cells to
operate directly on such solutions would be of interest for the
development of low-cost electricity-to-chemicals bioconversion
processes.
[0280] We verified that electrolytically generated formate reduces
the growth rate of Paracoccus versutus. Overnight cultures of MOPS
minimal formate medium were inoculated into MOPS minimal medium
containing either various dilutions of sodium formate
(Sigma-Aldrich) or various dilutions of formate sourced from DNV's
first sample. Growth was uninhibited by pure sodium formate at the
highest concentration tested, 50 mM. In contrast growth was
strongly inhibited by electrolytically generated formate, with no
growth observed above 20 mM formate concentration, and only weak
growth at concentrations above 10 mM. However at lower
concentrations, electrolytic formate supported Paracoccus growth at
rates similar to pure sodium formate.
[0281] In an effort to select for strains with an increased ability
to thrive on electrolytically generated formate, Paracoccus
versutus was inoculated into the culture device. MOPS minimal
medium with commercial (Sigma-Aldrich) sodium formate at 50 mM
flowed through the device at flow rates controlled by the Arduino
in order to maintain with a target OD setpoint between 0.2 and 0.3
cm.sup.-1. In practice this flow rate was between 6 to 9 mL
hr.sup.-1. The working volume of the device was 24 mL, meaning the
dilution rate was between 0.25 hr.sup.-1 and 0.3 hr.sup.-1.
Periodically throughout the continuous culture, samples of the
culture were taken and preserved as a glycerol stock at -80.degree.
C.
[0282] After 48 generations of growth, the medium feed was changed
to be a mixture of 75 volume % MOPS minimal medium with 50 mM
sodium formate, and 25 volume % MOPS minimal medium with 50 mM
electrolytic formate (from DNV's second sample). The culture was
incubated continuously for 32 more generations of growth.
[0283] After the conclusion of the experiment, glycerol stocks
previously taken from the reactor population and reserved at
-80.degree. C. were revived and cultured in MOPS formate minimal
medium to determine if any improvements in growth on formate had
taken place. We found that the P. versutus population sampled from
the turbidostat zero generations of growth had much lower growth
rates on 50 mM electrolytic formate (MOPS-EF) medium than on 50 mM
pure sodium formate (MOPS-PF) medium (0.32 hr.sup.-1 for MOPS-PF
vs. 0.26 hr.sup.-1 for MOPS-EF). Populations sampled from the
reactor at later times had faster growth rates, as shown in Table
4. Clones from this population can be used as hosts for production
of fuels or other carbon products of interest because of their
ability to better tolerate solutions of electrolytic formate as
their sole source of carbon and energy, and their ability to grow
more quickly than wild-type P. versutus under these conditions.
TABLE-US-00005 TABLE 4 Growth rates of evolved Paraoccus strains
Generations of Generations of Selection on MOPS-EF Growth MOPS-PF
Growth Selection, total MOPS-EF Rate, hr.sup.-1 rate, hr.sup.-1 0 0
0.25 0.32 35 0 0.32 0.43 80 32 0.41 0.41
Example 6
High Intensity Bioreactor Cultivation of Paracoccus on Formate
Salts
[0284] To our knowledge, the high-cell density bioreactor
cultivation of industrially relevant, genetically tractable
microbes using formate as the sole source of carbon and energy has
not been reported. We sought to demonstrate the high-cell density
bioreactor cultivation of Paracoccus versutus under
process-relevant conditions.
[0285] In a series of two experiments comprising 12 different
fed-batch runs, the reactors were initially charged with 0.5 L of a
formate minimal medium based on the recipe of R minimal medium, but
with emendations of sodium molybdate, sodium selenite, thiamin, and
cobalamin. The reactors were inoculated with overnight flask
cultures of Paracoccus versutus. After the initial charge of
formate in the reactor was consumed, supplemental feeding was begun
by flowing 8.0 M ammonium formate to the reactors. Over the course
of 12 experiments, we studied the effect of initial inoculum size,
feeding rate, and aerobicity on rates of formate consumption and of
biomass and CO.sub.2 formation. We monitored formate consumption by
HPLC, CO.sub.2 emission through IR-based off-gas measurement, and
biomass formation by total insoluble solids.
[0286] Initially, initial biomass concentration corresponding to OD
0.1 was used; however, biomass measurement by weight was unreliable
due to mineral precipitation during fermentation. Subsequently,
initial biomass concentration was OD1.0 and we used a dilute-acid
wash during the processing of biomass samples in order to remove
inorganic precipitates. We assumed that biomass contained 0.5 g-C
g.sup.-1. This assumption allowed us to close carbon balances
around the aerobic, high-inoculum runs to within 10%, indicating
that the assumption was reasonable and constituting a consistency
check on our HPLC and off-gas measurements.
[0287] FIG. 17 depicts sample fermentation data for an aerobic
fermentation feed of 10 mM hr.sup.-1. From the beginning of the
formate feed at 3.45 hr post-inoculation to near the end of the run
at 50 hr, the formate level remained below detection limits. The 75
mmol-C of biomass formed resulted in a final biomass concentration
of 2.6 g L.sup.-1, implying that specific formate consumption rates
were at or above 0.51 g formate gDCW.sup.-1 hr.sup.-1 throughout
the fermentation. In this run, 91% of the formate consumed was
converted to CO.sub.2 that left the reactor and 8.9% was accounted
for by biomass formation. The total of biomass formation and
CO.sub.2 emisson accounted for 100.0% of the carbon used, a figure
which varied between 100 and 103% across other runs. These rates
correspond to a specific carbon fixation flux of 8 mmol C
gDCW.sup.-1 hr.sup.-1 or a volumetric carbon fixation flux of
>1.68 mmol L.sup.-1 hr.sup.-1.
[0288] Nominal feed rates of 10, 30, and 100 mM hr.sup.-1 were
studied for aerobic fermentations. Only the 100 mM hr.sup.-1
condition showed any evidence of formate accumulation, although
this feed was only tried with a low-inoculum size condition
(OD0.1). The 10 and 30 mM hr.sup.-1 feed rates did not show any
formate accumulation until the end of the fermentations, indicating
that the capacity of the culture for formate utilization was
greater than 30 mM hr.sup.-1 under the densities used for
cultivation.
[0289] Respiration of formic acid or formate is a proton-consuming,
i.e. pH increasing process. In these experiments, the medium pH was
held constant at 7.0 by the addition of concentrated phosphoric
acid. Ammonium formate was chosen as a formate source because it
provides an additional means of pH control (ammonium formate
solutions are neutral in pH) and because it was hoped that as
formate was consumed, ammonium would not accumulate due to the
potential for offgassing of ammonia. We measured ammonium
accumulation in the reactors by ion chromatography. At pH 7.0,
ammonium offgassing did not occur to an appreciable extent, because
86-102% of the fed ammonium formate accumulated as ammonium in the
medium Ammonium accumulation likely limited the end-point biomass
titer attained in most of the fermentations, as it accumulated to
supra-molar concentrations in many of the vessels.
[0290] Paracoccus versutus can grow anaerobically using nitrate as
an electron acceptor. We carried out nitrate-based anaerobic
formate bioconversion using feeds that contained 8.0 M ammonium
formate and 3.1 M sodium nitrate. When feeding was begun, the
reactors were brought under anaerobiosis by sparging with nitrogen.
Anaerobic fermentations also consumed formate at high rates, up to
0.67 g L.sup.-1 hr.sup.-1 in the experiments described here.
Maximal biomass attained under anaerobic conditions was 1.3 gDCW
L.sup.-1. The low nitrogen sparging flow rates were incompatible
with CO.sub.2 measurement in the off-gas, so carbon balances are
not available.
[0291] Initial results indicated that nitrate was converted to
nitrite and that nitrite accumulated stoichiometrically in the
reactor and was not further reduced. We successfully eliminated
nitrite accumulation in the reactor by doubling the amount of
copper in the medium and reducing the level of nitrate from 3.1 M
to 3.0 M. These results demonstrate that Paracoccus is capable of
anaerobic formate consumption with complete nitrate respiration to
dinitrogen gas Ammonium, formate and nitrate reached levels of
1100, 1500 and 540 mmol L.sup.-1, respectively.
Example 7
Computing Mass Transfer Limitations of Synthesis Gas Versus Formate
as a Feedstock
[0292] The mass tranfer limitations of synthesis gas (composed of
molecular hydrogen and carbon monoxide) from the gas to liquid
phase is illustrated here. For the purpose of this analysis, an
ideal engineered organism that has an unlimited capacity to (i)
metabolize dissolved aqueous-phase synthesis gas and (ii) convert
it to a desired fuel at 100% of the theoretical yield is assumed.
Under these conditions, the rate of fuel production per unit of
reactor volume can depend solely on the rate at which synthesis gas
can be transferred from the gas phase to the liquid phase.
[0293] Fuel productivity Pin units of gL.sup.-1h.sup.-1 can be
expressed as the product of fuel molecular weight m.sub.F, fuel
molar yield on synthesis gas Y.sub.F/S, the biomass concentration
in a bioreactor X, and the specific cellular uptake rate of
synthesis gas q.sub.S, as shown in the equation below.
P=M.sub.FY.sub.F/SXq.sub.S
[0294] At steady state, the bulk hydrogen uptake rate Xq.sub.S is
equal to the rate of synthesis gas transfer from gas to liquid,
meaning the productivity can be expressed as in the equation below,
where C* is the liquid-phase solubility of synthesis gas, C.sub.L
is the liquid-phase concentration of synthesis gas, and K.sub.La is
the mass transfer coefficient for synthesis gas transport from the
gas phase (e.g., as bubbles sparged into the reactor) to the
liquid. K.sub.La is a complex function of reactor geometry, bubble
size, superficial gas velocity, impeller speed, etc. and is best
regarded as an empirical parameter that needs to be determined for
a given bioreactor setup.
P=m.sub.FY.sub.F/SK.sub.La(C*-C.sub.L)
[0295] Again, as a best-case scenario, an ideal engineered organism
capable of maintaining rapid synthesis gas uptake rates even at
vanishingly low synthesis gas concentrations (i.e. that q.sub.S is
not a function of C.sub.L even as C.sub.L tends to zero) is
assumed. This assumption maximizes the fuel productivity at
P=m.sub.FY.sub.F/SK.sub.LaC*.
[0296] For a fixed production target t, say 0.5 t d.sup.-1
(equivalent to 20800 g h.sup.-1), the productivity P determines the
required reactor volume V because V=t/P. Thus, both fuel
productivity and reactor volumes, even assuming "perfect"
organisms, are bounded by achieveable K.sub.La values, as shown in
the equations below.
P = ( m F Y F / S C * ) K L a ##EQU00001## V = t ( m F Y F / S C *
) K L a ##EQU00001.2##
[0297] Maximal productivity corresponds to minimal reaction
volumes, and occurs at maximal values of
M.sub.FY.sub.F/SC*K.sub.La. The fuel yield cannot exceed the
stoichiometric maximal yield. For the fuel isooctanol, the
stoichiometric maximal yield is determined from the balanced
chemical equation 8 CO+16 H.sub.2.fwdarw.C.sub.8H.sub.18O+7
H.sub.2O, which shows that 16 moles of H.sub.2 and 8 moles of CO
are required for each mole of isooctanol produced. At atmospheric
pressure, C* is unlikely to greatly exceed 0.75 mM, the solubility
of H.sub.2 in pure water (CO has approximately the same solubility
as H.sub.2). Using these representative values for representative
values for m.sub.F, Y.sub.F/S, C* and t, the relationships between
K.sub.La and P as well as between K.sub.La and t are shown (FIG.
18).
[0298] Alternative electron donors have the potential to solve both
the safety problem and the mass transfer problem presented by
hydrogen. An ideal non-synthesis gas vector for carrying electrical
energy would have (a) a highly negative standard reduction
potential and (b) established high-efficiency technology to for
converting electricity into the vector. Unlike synthesis gas,
however, it would (c) have a low propensity to explode when mixed
with air, and (d) have high water solubility under bio-compatible
conditions. Formic acid, HCOOH, or its salts, satisfies these
conditions. Formic acid is stoichiometrically equivalent to
H.sub.2+CO.sub.2, and formate has as standard reduction potential
nearly identical to that of hydrogen. Since both formic acid and
formate salts are highly soluble in water, the mass transfer
limitations discussed above for hydrogen do not apply. However, a
modified form of the fuel productivity equation, written for formic
acid (A) instead of hydrogen (H), still applies, as shown
below.
P=m.sub.FY.sub.F/AXq.sub.A
[0299] Unlike hydrogen-powered electrofuels bioproduction, limits
on formate-powered fuel productivity P stem only from the
attainable yield, the biomass concentration in the reactor, and the
specific uptake rate. We assume Y.sub.F/A, the molar yield of fuel
on formic acid, is the stoichiometric maximum, whose value is the
same as for hydrogen, 0.0467 mol isooctanol (mol HCOOH).sup.-1. For
high-cell density cultivations of E. coli, biomass concentrations
of X=50 gDCW L.sup.-1 are attainable, although these values have
not been observed for growth on formate or in minimal medium. For
Paracoccus versutus, naturally capable of growing on formate,
observed values of were 0.0368 mol formate. gDCW.sup.-1h.sup.-1
[Kelly, 1979]. The representative values for q.sub.A and X imply a
maximal isooctanol productivity on formate of about 10
gL.sup.-1h.sup.-1.
[0300] On the .gamma.-axis of FIG. 18, the range of reported
K.sub.La attainable in large-scale stirred-tank bioreactors is
shown. Although there are many reports of higher K.sub.La values in
laboratory-scale reactors, during scale up the inevitable increase
in volume-to-surface area ratios means that maintaining high
K.sub.La values is for practical purposes impossible. The maximum
of the indicated range of 10-800 h.sup.-1 translates to a best-case
productivity of 4 gL.sup.-1h.sup.-1, which implies a best-case
reactor volume of 6,400 L. The best-case productivity on formate is
10 gL.sup.-1h.sup.-1, implying a reactor volume less than half as
large would be required to achieve the same production. Most
sources that give K.sub.La values for large scale reactors have
values much closer to 100 h.sup.-1, meaning the best-case
productivity using formate as the energy source would be more than
15 times larger than on synthesis gas.
Example 8
Engineered Organisms Producing Butanol
[0301] The enzyme beta-ketothiolase (R. eutropha PhaA or E. coli
AtoB) (E.C. 2.3.1.16) converts 2 acetyl-CoA to acetoacetyl-CoA and
CoA. Acetoacetyl-CoA reductase (R. eutropha PhaB) (E.C. 1.1.1.36)
generates R-3-hydroxybutyryl-CoA from acetoacetyl-CoA and NADPH.
Alternatively, 3-hydroxybutyryl-CoA dehydrogenase (C.
acetobutylicum Hbd) (E.C. 1.1.1.30) generates
S-3-hydroxybutyryl-CoA from acetoacetyl-CoA and NADH. Enoyl-CoA
hydratase (E. coli MaoC or C. acetobutylicum Crt) (E.C. 4.2.1.17)
generates crotonyl-CoA from 3-hydroxybutyryl-CoA. Butyryl-CoA
dehydrogenase (C. acetobutylicum Bcd) (E.C. 1.3.99.2) generates
butyryl-CoA and NAD(P)H from crotonyl-CoA. Alternatively,
trans-enoyl-coenzyme A reductase (Treponema denticola Ter) (E.C.
1.3.1.86) generates butyryl-CoA from crotonyl-CoA and NADH.
Butyrate CoA-transferase (R. eutropha Pct) (E.C. 2.8.3.1) generates
butyrate and acetyl-CoA from butyryl-CoA and acetate. Aldehyde
dehydrogenase (E. coli AdhE) (E.C. 1.2.1. {3,4}) generates butanal
from butyrate and NADH. Alcohol dehydrogenase (E. coli adhE) (E.C.
1.1.1. {1,2}) generates 1-butanol from butanal and NADH, NADPH.
Production of 1-butanol is conferred by the engineered host cell by
expression of the above enzyme activities.
[0302] To create butanol-producing cells, host cells can be further
engineered to express acetyl-CoA acetyltransferase (atoB) from E.
coli K12, .beta.-hydroxybutyryl-CoA dehydrogenase from Butyrivibrio
fibrisolvens, crotonase from Clostridium beijerinckii, butyryl CoA
dehydrogenase from Clostridium beijerinckii, CoA-acylating aldehyde
dehydrogenase (ALDH) from Cladosporium fulvum, and adhE encoding an
aldehyde-alcohol dehydrogenase of Clostridium acetobutylicum (or
homologs thereof).
Example 9
Engineered Organisms Producing Acrylate
[0303] Enoyl-CoA hydratase (E. coli paaF) (E.C. 4.2.1.17) converts
3-hydroxypropionyl-CoA to acryloyl-CoA. Propionyl-CoA synthase
(E.C. 6.2.1.-, E.C. 4.2.1.- and E.C. 1.3.1.-) also converts
3-hydroxypropionyl-CoA to acryloyl-CoA (AAL47820, SEQ ID NO:5).
Acrylate CoA-transferase (R. eutropha pct) (E.C. 2.8.3.n) generates
acrylate+acetyl-CoA from acryloyl-CoA and acetate.
OTHER EMBODIMENTS
[0304] The examples have focused on Paracoccus. Nevertheless, the
key concept of using genetically engineering to confer production
of carbon-based products of interest to a methylotroph is
extensible to other methylotrophs such as other prokaryotes or
eukaryotic single cell organisms such as methylotrophic yeast.
Alternatively, the energy conversion and/or carbon fixation
pathways described in U.S. Pat. No. 8,349,587 may be used to
enhance or augment the methylotrophic capability of an organism
that is natively methylotrophic; U.S. Pat. No. 8,349,587 is hereby
incorporated by reference in its entirety.
[0305] Various aspects of the present invention may be used alone,
in combination, or in a variety of arrangements not specifically
discussed in the embodiments described in the foregoing and is
therefore not limited in its application to the details and
arrangement of components set forth in the foregoing description or
illustrated in the drawings. For example, aspects described in one
embodiment may be combined in any manner with aspects described in
other embodiments.
[0306] Use of ordinal terms such as "first," "second," "third,"
etc., in the claims to modify a claim element does not by itself
connote any priority, precedence, or order of one claim element
over another or the temporal order in which acts of a method are
performed, but are used merely as labels to distinguish one claim
element having a certain name from another element having a same
name (but for use of the ordinal term) to distinguish the claim
elements.
[0307] Also, the phraseology and terminology used herein is for the
purpose of description and should not be regarded as limiting. The
use of "including," "comprising," or "having," "containing,"
"involving," and variations thereof herein, is meant to encompass
the items listed thereafter and equivalents thereof as well as
additional items.
EQUIVALENTS
[0308] The present invention provides among other things novel
methods and systems for synthetic biology. While specific
embodiments of the subject invention have been discussed, the above
specification is illustrative and not restrictive. Many variations
of the invention will become apparent to those skilled in the art
upon review of this specification. The full scope of the invention
should be determined by reference to the claims, along with their
full scope of equivalents, and the specification, along with such
variations.
INCORPORATION BY REFERENCE
[0309] The Sequence Listing filed as an ASCII text file via EFS-Web
(file name: "010401SequenceListing"; date of creation: Dec. 2,
2013; size: 25,461 bytes) at the U.S. Patent and Trademark Office
as the Receiving Office is hereby incorporated by reference in its
entirety.
[0310] All publications, patents and patent applications referenced
in this specification are incorporated herein by reference in their
entirety for all purposes to the same extent as if each individual
publication, patent or patent application were specifically
indicated to be so incorporated by reference.
REFERENCES CITED
[0311] Aharoni A, Keizer L C, Bouwmeester H J, Sun Z,
Alvarez-Huerta M, Verhoeven H A, Blaas J, van Houwelingen A M, De
Vos R C, van der Voet H, Jansen R C, Guis M, Mol J, Davis R W,
Schena M, van Tunen A J, O'Connell A R Identification of the SAAT
gene involved in strawberry flavor biogenesis by use of DNA
microarrays. Plant Cell. 2000 May; 12(5):647-62. [0312] Andersen J
B, Sternberg C, Poulsen L K, Bjorn S P, Givskov M, Molin S. New
unstable variants of green fluorescent protein for studies of
transient gene expression in bacteria. Appl Environ Microbiol. 1998
June; 64(6):2240-6. [0313] Anderson J C, Voigt C A, Arkin A R
Environmental signal integration by a modular AND gate. Mol Syst
Biol. 2007; 3:133. [0314] Bai F W, Anderson W A, Moo-Young M.
Ethanol fermentation technologies from sugar and starch feedstocks.
Biotechnol Adv. 2008 January-February; 26(1):89-105. [0315] Bailer
J, de Hueber K. Determination of saponifiable glycerol in
"bio-diesel." Fresenius J Anal Chem. 1991; 340(3):186. [0316] Bas
sham J A, Benson A A, Kay L D, Harris A Z, Wilson A T, Calvin M.
The path of carbon in photosynthesis. XXI. The cyclic regeneration
of carbon dioxide acceptor. J Am Chem Soc. 1954; 76:1760-70. [0317]
Bayer T S, Widmaier D M, Temme K, Mirsky E A, Santi D V, Voigt C A.
Synthesis of methyl halides from biomass using engineered microbes.
J Am Chem Soc. 2009 May 13; 131(18):6508-15. [0318] Brock T.
Biotechnology: A Textbook of Industrial Microbiology. Second
Edition. Sinauer Associates, Inc. Sunderland, Mass. 1989. [0319]
Buchanan B B, Amon D I. A reverse KREBS cycle in photosynthesis:
consensus at last. Photosynth Res. 1990; 24:47-53. [0320] Camilli
A, Bassler B L. Bacterial small-molecule signaling pathways.
Science. 2006 Feb. 24; 311(5764):1113-6. [0321] Canton B, Labno A,
Endy D. Refinement and standardization of synthetic biological
parts and devices. Nat Biotechnol. 2008 July; 26(7):787-93. [0322]
Cheesbrough T M, Kolattukudy P E. Alkane biosynthesis by
decarbonylation of aldehydes catalyzed by a particulate preparation
from Pisum sativum. Proc Natl Acad Sci USA. 1984 November;
81(21):6613-7. [0323] Chen S, von Bamberg D, Hale V, Breuer M,
Hardt B, Milller R, Floss H G, Reynolds K A, Leistner E.
Biosynthesis of ansatrienin (mycotrienin) and naphthomycin.
Identification and analysis of two separate biosynthetic gene
clusters in Streptomyces collinus Tu 1892. Eur J Biochem. 1999
April; 261(1):98-107. [0324] Chin J W, Cirino P C. Improved NADPH
supply for xylitol production by engineered Escherichia coli with
glycolytic mutations. Biotechnol Prog. 2011 March-April;
27(2):333-41. [0325] Cropp T A, Wilson D J, Reynolds K A.
Identification of a cyclohexylcarbonyl CoA biosynthetic gene
cluster and application in the production of doramectin. Nat
Biotechnol. 2000 September; 18(9):980-3. [0326] Davis J H, Rubin A
J, Sauer R T. Design, construction and characterization of a set of
insulated bacterial promoters. Nucleic Acids Res. 2011 February;
39(3):1131-41. [0327] de Mendoza D, Klages Ulrich A, Cronan J E Jr.
Thermal regulation of membrane fluidity in Escherichia coli.
Effects of overproduction of beta-ketoacyl-acyl carrier protein
synthase I. J Biol Chem. 1983 Feb. 25; 258(4):2098-101. [0328]
Dellomonaco C, Clomburg J M, Miller E N, Gonzalez R. Engineered
reversal of the f3-oxidation cycle for the synthesis of fuels and
chemicals. Nature. 2011 Aug. 10; 476(7360):355-9. [0329] Dennis M
W, Kolattukudy P E. Alkane biosynthesis by decarbonylation of
aldehyde catalyzed by a microsomal preparation from Botryococcus
braunii. Arch Biochem Biophys. 1991 June; 287(2):268-75. [0330]
Denoya C D, Fedechko R W, Hafner E W, McArthur H A, Morgenstern M
R, Skinner D D, Stutzman-Engwall K, Wax R G, Wernau W C. A second
branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH)
from Streptomyces avermitilis: its relationship to avermectin
biosynthesis and the construction of a bkdF mutant suitable for the
production of novel antiparasitic avermectins. J Bacteriol. 1995
June; 177(12):3504-11. [0331] Deshpande M V. Ethanol production
from cellulose by coupled saccharification/fermentation using
Saccharomyces cerevisiae and cellulase complex from Sclerotium
rolfsii U V-8 mutant. Appl Biochem Biotechnol. 1992 September;
36(3):227-34. [0332] Doolittle, R F (Editor). Computer Methods for
Macromolecular Sequence Analysis. Methods in Enzymology. 1996;
266:3-711. [0333] Edgar R C. MUSCLE: multiple sequence alignment
with high accuracy and high throughput. Nucleic Acids Res. 2004
Mar. 19; 32(5):1792-7. (a) [0334] Edgar R C. MUSCLE: a multiple
sequence alignment method with reduced time and space complexity.
BMC Bioinformatics. 2004 Aug. 19; 5:113. (b) [0335] Edwards J S,
Ramakrishna R, Palsson B O. Characterizing the metabolic phenotype:
a phenotype phase plane analysis. Biotechnol Bioeng. 2002 Jan. 5;
77(1):27-36. [0336] Evans M C, Buchanan B B, Arnon D I. A new
ferredoxin-dependent carbon reduction cycle in a photosynthetic
bacterium. Proc Natl Acad Sci USA. 1966 April; 55(4):928-34. [0337]
Fong S S, Palsson B O. Metabolic gene-deletion strains of
Escherichia coli evolve to computationally predicted growth
phenotypes. Nat Genet. 2004 October; 36(10):1056-8. [0338] Grantham
R, Gautier C, Gouy M, Mercier R, Pave A. Codon catalog usage and
the genome hypothesis. Nucleic Acids Res. 1980 Jan. 11;
8(1):r49-r62. [0339] Greene D N, Whitney S M, Matsumura I.
Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit
improvements in folding and catalytic efficiency. Biochem J. 2007
Jun. 15; 404(3):517-24. [0340] Han L, Reynolds K A. A novel
alternate anaplerotic pathway to the glyoxylate cycle in
streptomycetes. J Bacteriol. 1997 August; 179(16):5157-64. [0341]
Hawley D K, McClure W R. Compilation and analysis of Escherichia
coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr. 25;
11(8):2237-55. [0342] Henry C S, Jankowski M D, Broadbelt L J,
Hatzimanikatis V. Genome-scale thermodynamic analysis of
Escherichia coli metabolism. Biophys J. 2006 Feb. 15;
90(4):1453-61. [0343] Henstra A M, Sipma J, Rinzema A, Stams A J.
Microbiology of synthesis gas fermentation for biofuel production.
Curr Opin Biotechnol. 2007 June; 18(3):200-6. [0344] Hoffrneister
M, Piotrowski M, Nowitzki U, Martin W. Mitochondrial
trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena
gracilis defines a new family of enzymes involved in lipid
synthesis. J Biol Chem. 2005 Feb. 11; 280(6):4329-38. [0345] Hugler
M, Huber H, Molyneaux S J, Vetriani C, Sievert S M. Autotrophic C
O.sub.2 fixation via the reductive tricarboxylic acid cycle in
different lineages within the phylum Aquificae: evidence for two
ways of citrate cleavage. Environ Microbiol. 2007 January;
9(1):81-92. [0346] Inokuma K, Nakashimada Y, Akahoshi T, Nishio N.
Characterization of enzymes involved in the ethanol production of
Moorella sp. HUC22-1. Arch Microbiol. 2007 July; 188(1):37-45.
[0347] Ivlev A A. Carbon isotope effects (.sup.13C/.sup.12C) in
biological systems. Separation Sci Technol. 2010; 36:1819-1914.
[0348] Janausch I G, Zientz E, Tran Q H, Kroger A, Unden G.
C4-dicarboxylate carriers and sensors in bacteria. Biochim Biophys
Acta. 2002 Jan. 17; 1553(1-2):39-56. [0349] Jukes T H, Osawa S.
Evolutionary changes in the genetic code. Comp Biochem Physiol B.
1993 November; 106(3):489-94. [0350] Kalscheuer R, Steinbilchel A.
A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol
acyltransferase mediates wax ester and triacylglycerol biosynthesis
in Acinetobacter calcoaceticus ADP1. J Biol Chem. 2003 Mar. 7;
278(10):8075-82. [0351] Kaneda T. Iso- and anteiso-fatty acids in
bacteria: biosynthesis, function, and taxonomic significance.
[0352] Microbiol Rev. 1991 June; 55(2):288-302. [0353] Kapust R B,
Waugh D S. Escherichia coli maltose-binding protein is uncommonly
effective at promoting the solubility of polypeptides to which it
is fused. Protein Sci. 1999 August; 8(8):1668-74. [0354] Keasling J
D, Jones K L, Van Dien S J. New Tools for Metabolic Engineering of
Escherichica coli. Chapter 5 in Metabolic Engineering. Marcel
Dekker. New York, N.Y. 1999. (a) [0355] Keasling J D.
Gene-expression tools for the metabolic engineering of bacteria.
Trends Biotechnol. 1999 November; 17(11):452-60. (b) [0356] Kelly D
P, Wood P, Gottschal J C, Kuenen J G. Autotrophic metabolism of
formate by Thiobacillus strain A2. J Gen Microbiol. 1979; 114:1-13.
[0357] Kelly J R, Rubin A J, Davis J H, Ajo-Franklin C M, Cumbers
J, Czar M J, de Mora K, Glieberman A L, Monie D D, Endy D.
Measuring the activity of BioBrick promoters using an in vivo
reference standard. J Biol Eng. 2009 Mar. 20; 3:4. [0358] Kim O B,
Unden G. The L-tartrate/succinate antiporter TtdT (YgjE) of
L-tartrate fermentation in Escherichia coli. J Bacteriol. 2007
March; 189(5):1597-603. [0359] Klimke W, Agarwala R, Badretdin A,
Chetvernin S, Ciufo S, Fedorov B, Kiryutin B, O'Neill K, Resch W,
Resenchuk S, Schafer S, Tolstoy I, Tatusova T. The National Center
for Biotechnology Information's Protein Clusters Database. Nucleic
Acids Res. 2009 January; 37(Database issue):D216-23. [0360] Knight
T. Idempotent Vector Design for Standard Assembly of Biobricks.
DOI: 1721.1/21168. [0361] Knight T. BBF RFC10: Draft Standard for
BioBrick.TM. biological parts. DOI: 1721.1/45138. [0362] Larkum A
W. Limitations and prospects of natural photosynthesis for
bioenergy production. Curr Opin Biotechnol. 2010 June; 21(3):271-6.
[0363] Knothe G, Dunn R O, Bagby M O. Biodiesel: The use of
vegetable oils and their derivatives as alternative diesel fuels.
Am Chem Soc Symp Series. 1997; 666:172-208. [0364] Knothe G. Rapid
monitoring of transesterification and assessing biodiesel fuel
quality by NIR spectroscopy using a fiber-optic probe. J Am Oil
Chem Soc. 1999; 76(7):795-800. [0365] Knothe G. Dependence of
biodiesel fuel properties on the structure of fatty acid alkyl
esters. Fuel Process Technol. 2005; 86:1059-1070. [0366] Komers K,
Skopal F, Stloukal R. Determination of the neutralization number
for biodiesel fuel production. Fett/Lipid. 1997; 99(2):52-54.
[0367] Larue T A, Kurz W G. Estimation of nitrogenase using a
colorimetric determination for ethylene. Plant Physiol. 1973 June;
51(6):1074-5. [0368] Li Y, Florova G, Reynolds K A. Alteration of
the fatty acid profile of Streptomyces coelicolor by replacement of
the initiation enzyme 3-ketoacyl acyl carrier protein synthase III
(FabH). J Bacteriol. 2005 June; 187(11):3795-9. [0369] Marrakchi H,
Zhang Y M, Rock C O. Mechanistic diversity and regulation of Type I
I fatty acid synthesis. Biochem Soc Trans. 2002 November; 30(Pt
6):1050-5. (a) [0370] Marrakchi H, Choi K H, Rock C O. A new
mechanism for anaerobic unsaturated fatty acid formation in
Streptococcus pneumoniae. J Biol Chem. 2002 Nov. 22;
277(47):44809-16. (b) Martin V J J, Smolke C, Keasling J D.
Redesigning cells for production of complex organic molecules. ASM
News. 2002; 68:336-343. [0371] Martinez-Alonso M, Toledo-Rubio V,
Noad R, Unzueta U, Ferrer-Miralles N, Roy P, Villaverde A.
Rehosting of bacterial chaperones for high-quality protein
production. Appl Environ Microbiol. 2009 December; 75(24):7850-4.
[0372] Martinez-Alonso M, Garcia-FruitOs E, Ferrer-Miralles N,
Rinas U, Villaverde A. Side effects of chaperone gene co-expression
in recombinant protein production. Microb Cell Fact. 2010 Sep. 2;
9:64. [0373] Minshull J, Stemmer W P. Protein evolution by
molecular breeding. Curr Opin Chem Biol. 1999 June; 3(3):284-90.
[0374] Morweiser M, Kruse 0, Hankamer B, Posten C. Developments and
perspectives of photobioreactors for biofuel production. Appl
Microbiol Biotechnol. 2010 July; 87(4):1291-301. [0375] Murli S,
Opperman T, Smith B T, Walker G C. A role for the umuDC gene
products of Escherichia coli in increasing resistance to DNA damage
in stationary phase by inhibiting the transition to exponential
growth. J Bacteriol. 2000 February; 182(4): 1127-35. [0376]
Murtagh, F. Complexities of Hierarchic Clustering Algorithms: the
State of the Art. Computational Statistics Quarterly. 1984;
1:101-13. Nature Genetics. 1999; 21(1):1-60. [0377] Palaniappan N,
Kim B S, Sekiyama Y, Osada H, Reynolds K A. Enhancement and
selective production of phoslactomycin B, a protein phosphatase Ha
inhibitor, through identification and engineering of the
corresponding biosynthetic gene cluster. J Biol Chem. 2003 Sep. 12;
278(37):35552-7. [0378] Park M O. New pathway for long-chain
n-alkane synthesis via 1-alcohol in Vibrio furnissii M1. J
Bacteriol. 2005 February; 187(4):1426-9. [0379] Patton S M, Cropp T
A, Reynolds K A. A novel delta(3),delta(2)-enoyl-CoA isomerase
involved in the biosynthesis of the cyclohexanecarboxylic
acid-derived moiety of the polyketide ansatrienin A. Biochemistry.
2000 Jun. 27; 39(25):7595-604. [0380] Pramanik J, Keasling J D.
Stoichiometric model of Escherichia coli metabolism: incorporation
of growth-rate dependent biomass composition and mechanistic energy
requirements. Biotechnol Bioeng. 1997 Nov. 20; 56(4):398-421.
[0381] Pramanik J, Keasling J D. Effect of Escherichia coli biomass
composition on central metabolic fluxes predicted by a
stoichiometric model. Biotechnol Bioeng. 1998 Oct. 20; 60(2):230-8.
(a) [0382] Pramanik J, Trelstad P L, Keasling J D. A flux-based
stoichiometric model of enhanced biological phosphorus removal
metabolism. Wat Sci Technol. 1998; 37(4-5):609-13. (b) [0383]
Pramanik J, Trelstad P L, Schuler A J, Jenkins D, Keasling J D.
Development and validation of a flux-based stoichiometric model for
enhanced biological phosphorus removal metabolism. Water Res. 1998;
33(2):462-76. (c). [0384] Reading N C, Sperandio V. Quorum sensing:
the many languages of bacteria. FEMS Microbiol Lett. 2006 January;
254(1):1-11. [0385] Rock C O, Tsay J T, Heath R, Jackowski S.
Increased unsaturated fatty acid production associated with a
suppressor of the fabA6(Ts) mutation in Escherichia coli. J
Bacteriol. 1996 September; 178(18):5382-7. [0386] Roessner C A,
Spencer J B, Ozaki S, Min C, Atshaves B P, Nayar P, Anousis N,
Stolowich N J, Holderman M T, [0387] Scott A I. Overexpression in
Escherichia coli of 12 vitamin B12 biosynthetic enzymes. Protein
Expr Purif. 1995 April; 6(2):155-63. [0388] Sachdev D, Chirgwin J
M. Solubility of proteins isolated from inclusion bodies is
enhanced by fusion to maltose-binding protein or thioredoxin.
Protein Expr Purif. 1998 February; 12(1):122-32. [0389] Sachdev D,
Chirgwin J M. Fusions to maltose-binding protein: control of
folding and solubility in protein purification. Methods Enzymol.
2000; 326:312-21. [0390] Saitou N, Nei M. The neighbor-joining
method: a new method for reconstructing phylogenetic trees. Mol
Biol Evol. 1987 July; 4(4):406-25. [0391] Sambrook, J, Russell, D.
Molecular Cloning: A Laboratory Manual, Third Edition. CSHL Press.
Cold Spring Harbor, N.Y. 2001. [0392] San K Y, Bennett G N,
Berrios-Rivera S J, Vadali R V, Yang Y T, Horton E, Rudolph F B,
Sariyar B, Blackwood K. Metabolic engineering through cofactor
manipulation and its effects on metabolic flux redistribution
in
Escherichia coli. Metab Eng. 2002 April; 4(2):182-92. [0393] Sauer
U, Canonaco F, Heri S, Perrenoud A, Fischer E. The soluble and
membrane-bound transhydrogenases UdhA and PntAB have divergent
functions in NADPH metabolism of Escherichia coli. J Biol Chem.
2004 Feb. 20; 279(8):6613-9. [0394] Shetty R P, Endy D, Knight T F
Jr. Engineering BioBrick vectors from BioBrick parts. J Biol Eng.
2008 Apr. 14;2:5. [0395] Shetty R, Lizarazo M, Rettberg R, Knight T
F. Assembly of BioBrick standard biological parts using three
antibiotic assembly. Methods Enzymol. 2011; 498:311-26. [0396]
Shpaer E G. GeneAssist. Smith-Waterman and other database
similarity searches and identification of motifs. Methods Mol Biol.
1997; 70:173-87. [0397] Smolke C D, Carrier T A, Keasling J D.
Coordinated, differential expression of two genes through directed
mRNA cleavage and stabilization by secondary structures. Appl
Environ Microbiol. 2000 December; 66(12):5399-405. [0398] Smolke C
D, Martin V J, Keasling J D. Controlling the metabolic flux through
the carotenoid pathway using directed mRNA processing and
stabilization. Metab Eng. 2001 October; 3(4):313-21. [0399] Smolke
C D, Keasling J D. Effect of copy number and mRNA processing and
stabilization on transcript and protein levels from an engineered
dual-gene operon. Biotechnol Bioeng. 2002 May 20; 78(4):412-24. (a)
[0400] Smolke C D, Keasling J D. Effect of gene location, mRNA
secondary structures, and RNase sites on expression of two genes in
an engineered operon. Biotechnol Bioeng. 2002 Dec. 30;
80(7):762-76. (b) [0401] Sokal R, Michener, C. A Statistical Method
for Evaluating Systematic Relationships. University of Kansas
Science Bulletin. 1958; 38:1409-38. [0402] Strom T, Ferenci T, and
Quayle J R. The carbon assimilation pathways of Methylococcus
capsulatus, Pseudomonas methanica and Methylosinus trichosporium (O
B3B) during growth on methane. Biochem J 1974 December; 144(3)
465-76. [0403] Tatusov R L, Koonin E V, Lipman D J. A genomic
perspective on protein families. Science. 1997 Oct. 24;
278(5338):631-7. [0404] Tatusov R L, Fedorova N D, Jackson J D,
Jacobs A R, Kiryutin B, Koonin E V, Krylov D M, Mazumder R,
Mekhedov S L, Nikolskaya A N, Rao B S, Smirnov S, Sverdlov A V,
Vasudevan S, Wolf Y I, Yin J J, Natale D A. The COG database: an
updated version includes eukaryotes. BMC Bioinformatics. 2003 Sep.
11; 4:41. [0405] van Wezel G P, Mahr K, Konig M, Traag B A,
Pimentel-Schmitt E F, Willimek A, Titgemeyer F. G1 cP constitutes
the major glucose uptake system of Streptomyces coelicolorA3(2).
Mol Microbiol. 2005 January; 55(2):624-36. [0406] Venturi V.
Regulation of quorum sensing in Pseudomonas. FEMS Microbiol Rev.
2006 March; 30(2):274-91. [0407] Wubbolts M G, Terpstra P, van
Beilen J B, Kingma J, Meesters H A, Witholt B. Variation of
cofactor levels in Escherichia coli. Sequence analysis and
expression of the pncB gene encoding nicotinic acid
phosphoribosyltransferase. J Biol Chem. 1990 Oct. 15;
265(29):17665-72. [0408] Yoon Y G, Cho J H, Kim S C.
Cre/loxP-mediated excision and amplification of large segments of
the Escherichia coli genome. Genet Anal. 1998 January; 14(3):89-95.
[0409] Zdobnov E M, Apweiler R. InterProScan--an integration
platform for the signature-recognition methods in InterPro.
Bioinformatics. 2001 September; 17(9):847-8. [0410] Zhang C C,
Durand M C, Jeanjean R, Joset F. Molecular and genetical analysis
of the fructose-glucose transport system in the cyanobacterium
Synechocystis PCC6803. Mol Microbiol. 1989 September; 3(9):1221-9.
[0411] Zhang Y M, Marrakchi H, Rock C O. The FabR (YijC)
transcription factor regulates unsaturated fatty acid biosynthesis
in Escherichia coli. J Biol Chem. 2002 May 3; 277(18):15558-65.
[0412] Zhu X, Yuasa M, Okada K, Suzuki K, Nakagawa T, Kawamukai M,
Matsuda H. Production of ubiquinone in Escherichia coli by
expression of various genes responsible for ubiquinone
biosynthesis. J Ferm Bioeng. 1995; 79(5):493-5.
Sequence CWU 1
1
511902DNAArtificial SequenceSynthetic DNA 1atggacctac tctcaataca
ggatccgagc ttcctcaaga acatgtctat cgacgagctc 60gaaaagctct ccgacgagat
tcggcagttc ctcatcactt ccctctctgc ttctggcggc 120cacattggcc
ccaacctcgg ggttgtagaa ctaacagtgg cgctgcataa agagttcaac
180tcgccgaagg ataagttcct ctgggacgta ggccatcaga gctatgttca
taaactgctg 240actggacgcg gaaaggagtt cgccacgcta cgccagtaca
agggtctatg cggattcccc 300aaacgttccg agtcggaaca cgacgtgtgg
gagaccggtc actcgagcac aagtctgtca 360ggcgccatgg gaatggcagc
tgcgcgggac atcaagggaa cggacgagta tatcatcccg 420attatcgggg
atggcgccct gaccggcggg atggccttgg aggcgctaaa ccacattggc
480gatgaaaaga aggatatgat cgttattcta aatgacaatg agatgtccat
cgcccccaac 540gttggggcga tccacagtat gttgggacgt ttgcgcacag
ccggtaagta ccagtgggtt 600aaggacgaac tagagtacct tttcaagaaa
atcccggcag tgggtggcaa actagcggcg 660acggccgagc gtgttaagga
ttcgctgaag tacatgttgg tttctggaat gttctttgaa 720gaattggggt
tcacgtatct tggccccgtc gatggacata gttatcatga actgatcgaa
780aatctacaat acgcgaagaa gacgaagggc cctgtgctac tgcacgttat
cacgaagaag 840ggtaaaggtt acaagccggc tgaaaccgac acgatcggta
cttggcatgg gaccggaccc 900tataagatca ataccgggga tttcgtaaaa
ccgaaggcgg cagctcctag ctggtcgggg 960ctagtttcgg gaacagtcca
gaggatggcc cgcgaagatg gacgcatcgt agcgatcacg 1020ccggctatgc
ctgttgggtc aaaactagag ggctttgcaa aagagtttcc tgatcgtatg
1080tttgatgtag gaattgcaga gcagcatgcg gcaactatgg ctgctgctat
ggcaatgcag 1140gggatgaaac cgttccttgc catctactca acctttctgc
aaagagcata tgatcaagtg 1200gtgcatgata tttgccgcca aaacgctaat
gtcttcatcg gaatcgatcg ggctggattg 1260gtgggcgctg acggagaaac
tcatcaaggc gtcttcgaca tcgccttcat gcgccacatc 1320cccaacatgg
ttctcatgat gccgaaggat gagaatgagg gccagcacat ggtgcatact
1380gcactatcgt atgatgaggg cccgatagca atgcggttcc cgcgcggaaa
cggattgggc 1440gtaaagatgg acgagcagct taagacgatc ccgattggga
cgtgggaagt cctgcggccc 1500ggtaacgacg ctgttattct cacttttggc
actactattg agatggccat cgaggcagcg 1560gaggaactgc aaaaggaggg
cctatctgtc cgcgtggtca atgcccggtt catcaaaccg 1620atcgacgaga
aaatgatgaa atccatttta aaggagggcc ttcccatcct cactatcgag
1680gaggccgttc tcgagggcgg gtttggatcg agcatcctcg agtttgctca
tgaccaaggg 1740gagtatcata caccgatcga tcgaatgggg atacctgacc
gtttcatcga gcacggatcc 1800gtaactgccc tactagaaga aatcggactg
actaagcagc aggtcgcaaa tcggatccga 1860cttctgatgc ccccgaaaac
tcacaaggga atcggatcgt ag 19022549DNAArtificial SequenceSynthetic
DNA 2atgcagacgg agcacgttat cctccttaat gcacagggag tgccaacggg
gacgctggag 60aaatatgcgg cacacacggc agatacccgc ctccatttgg ccttttcttc
ctggctgttc 120aatgcgaagg gacaactcct cgtgacccgc cgcgcactgt
cgaagaaggc atggcctggc 180gtctggacaa acagcgtgtg tggtcacccc
caactgggag agtcgaacga ggatgcagtc 240attcgccggt gccggtatga
actaggcgtc gagatcacgc ctcctgagag tatttatcct 300gatttccgct
accgcgcgac cgacccgtcc ggcatcgtcg agaatgaggt ctgtccggta
360ttcgcggcac gcaccacatc cgccctccag attaatgacg acgaggtcat
ggactatcaa 420tggtgtgacc tcgcagacgt actccacggg atcgacgcga
cgccgtgggc cttttccccg 480tggatggtca tgcaggccac taatcgcgag
gcgcgaaaga ggctcagtgc attcacccag 540ctaaagtaa 54931788DNAArtificial
SequenceSynthetic DNA 3atggccacag agctcctttg ccttcaccgg cccatttcac
tgactcacaa gctttttcga 60aatccacttc ctaaggttat tcaggcgaca ccactcacac
ttaagttgcg atgtagtgta 120tcgactgaga acgtttcgtt tactgagact
gagactgaga cccgaagaag tgcgaattat 180gagcccaatt cgtgggacta
tgactatctt ctgagtagcg atacagatga gagcatcgag 240gtatacaagg
ataaagcgaa gaaactggaa gctgaagtcc gacgagaaat caacaatgag
300aaagcagaat ttcttacact gcctgagctg atagacaatg ttcaaagact
cggactcggg 360taccgttttg aatccgatat aagacgagcg ctagaccgat
tcgtgagttc aggaggattc 420gacgctgtga ctaagacatc gctacatgct
acagctctat cgtttagact attgcgacag 480catggcttcg aagttagtca
agaggccttt tcgggattta aagaccaaaa tggcaatttt 540cttaagaacc
taaaagaaga tattaaagca atattatcgt tatatgaggc ttcattccta
600gcgctcgagg gagagaatat tcttgacgaa gcgaaagtct tcgcaatatc
acatttaaag 660gaattgtcgg aggagaaaat cggaaaggat ctggcggagc
aggtcaatca tgcacttgaa 720ctaccccttc ataggagaac gcaacgatta
gaggctgtgt ggtcgatcga ggcataccgg 780aagaaagagg acgcagacca
agtactgtta gaactagcta tacttgatta caacatgatc 840caatcagtat
accaacgaga cctacgcgaa acttcaagat ggtggagacg ggtcggtcta
900gcaactaaac ttcatttcgc tcgagataga ctcatcgagt cgttctactg
ggcagtggga 960gtggccttcg agcctcaata ctccgactgc cggaatagtg
tagcaaagat gttcagcttc 1020gtaactatta tcgacgacat ttatgacgtg
tatgggacac tggacgaact tgaattattc 1080actgacgctg tggaacgatg
ggacgtgaat gcgattgacg acctaccgga ctatatgaaa 1140ttgtgctttt
tagctttgta taacacaatt aatgaaatag cttatgacaa tctgaaagat
1200aaaggtgaga acatcctacc ctacttaact aaggcctggg cagacctctg
caatgcattt 1260ttacaagagg caaagtggct ttacaataaa tctactccca
ctttcgacga gtattttgga 1320aatgcatgga agtcatcttc aggtcctcta
caattagtgt tcgcgtactt cgcggtggtg 1380caaaacatta aaaaggaaga
aatcgacaac ctccaaaaat atcatgacat tatttccaga 1440ccttctcaca
ttttccggct atgcaacgat cttgcttcag caagcgctga aatagcccga
1500ggggagaccg ccaatagtgt atcatgctac atgcggacta agggcatcag
tgaagaacta 1560gctacagagt ctgtaatgaa tcttattgat gagacctgga
agaaaatgaa caaggagaaa 1620ctagggggca gtctgttcgc aaagcctttc
gttgagactg ctatcaacct agcaaggcaa 1680tctcattgca catatcacaa
cggagacgcc catacatcac ccgacgaatt gacaagaaag 1740cgggttctgt
cagtaattac tgaacctatc ttaccattcg aacgctag 178842424DNAArtificial
SequenceSynthetic DNA 4atgacctcgg tgagtgtgga gtcaggaact gtcagttgtc
tttcttccaa caaccttatt 60aggagaacgg cgaatcctca tcctaatatt tgggggtatg
acttcgtcca tagtctaaag 120agcccttaca cacatgacag ttcctaccga
gagcgggccg aaactctaat ctcggaaatc 180aaagtcatgc taggaggggg
agagctcatg atgacaccca gtgcttatga tactgcatgg 240gtagccagag
tcccatctat cgacggcagt gcttgcccgc aattcccaca aactgtggag
300tggatcctta agaatcagct caaggacggg tcttggggta cagaaagcca
cttcttactg 360tctgatcgcc tgttagctac actaagttgt gtcctagcct
tgctaaagtg gaaggtggca 420gacgtccaag tagaacaggg catcgagttt
ataaaacgaa atttacaagc aataaaagat 480gagcgagacc aagattccct
tgtaactgat ttcgaaatta ttttcccttc tcttttaaag 540gaggcccaaa
gtttgaactt gggactacca tacgatctgc cttatatacg gctgctgcaa
600acgaagcgtc aggaacgact cgcgaatttg tcaatggata agatccatgg
gggaactctc 660ctttcaagtc ttgaaggcat acaagacatc gttgaatggg
agactataat ggacgtgcaa 720tcgcaagacg gaagttttct ttcgtcacct
gctagtacag cgtgcgtttt tatgcacaca 780ggagatatga agtgcctaga
ttttcttaac aacgtcttga caaaattcgg ctccagtgtg 840ccctgcctgt
atcccgtcga cctgctggag cgcttgctta ttgtcgacaa tgtggagcgc
900ctaggaattg accgacactt cgagaaagag attaaagagg ctttagacta
tgtgtacaga 960cattggaatg accgaggaat cggttggggc cgactgtccc
caatagcaga cctagaaacc 1020acagctcttg gattccgtct gctaagactg
catcgttaca acgtaagtcc cgttgtgctt 1080gataacttta aggacgcgga
cggtgagttt ttctgctcca ccgggcaatt taacaaggac 1140gtagcatcga
tgctgtcgct atatcgagct tctcagttgg cattcccaga ggagtcgatc
1200cttgacgagg ctaagtcgtt ttccacacaa tatcttcgag aggcgctaga
aaagtccgaa 1260acattttcgt catggaacca ccgacaatcg ttatcggagg
aaatcaagta cgccctgaaa 1320acaagttggc atgcgtccgt tccacgagtc
gaggcaaaac gatactgtca agtctatcgc 1380caagactatg cacacctcgc
aaagtcggtg tacaaactgc caaaggtcaa caatgagaaa 1440atccttgaac
tggcaaaact tgattttaac atcattcagt ctattcacca aaaggaaatg
1500aaaaatgtta cctcgtggtt ccgagacagc ggtcttcctc tgtttacctt
cgctcgtgaa 1560agaccgctgg agttttactt tctcatagcc ggtggtacgt
atgaaccaca gtatgcgaag 1620tgccgatttt tgttcactaa ggtcgcctgt
cttcagacag tgctggacga catgtatgac 1680acttatggaa ccccttccga
gcttaaatta tttacagaag ctgtccgacg atgggatttg 1740agtttcactg
agaacctacc cgattatatg aagttatgtt acaagattta ttatgatata
1800gtgcacgagg tcgcttggga agtagaaaaa gagcaaggtc gggagcttgt
gtcgttcttt 1860cgaaaaggat gggaagacta tctactgggg tattacgaag
aagctgagtg gctcgctgct 1920gaatatgtcc ctacacttga tgaatacata
aaaaatggta ttacatctat tgggcaacgg 1980atactactgc tgtccggagt
ccttataatg gagggtcaat tgttgagcca agaagccctt 2040gaaaaggtag
actatcccgg acgacgggtg ttgactgaac tgaatagttt gatcagtcgt
2100ctggccgacg atactaagac ttataaagct gaaaaagctc ggggagagct
tgcttcttcg 2160atcgagtgtt acatgaagga tcatcctgga tgtcaagagg
aggaagcttt gaatcacatt 2220tatggcatcc tggaaccggc agtaaaagaa
ctgacgagag aatttctgaa agcagatcat 2280gttccctttc cgtgcaaaaa
aatgctattc gacgaaaccc gagtcactat ggtcatattt 2340aaagatggag
acggcttcgg catctctaag ctcgaggtca aagaccacat taaggaatgt
2400ctgattgagc ccttgcccct ctag 242451822PRTChloroflexus aurantiacus
5Met Ile Asp Thr Ala Pro Leu Ala Pro Pro Arg Ala Pro Arg Ser Asn 1
5 10 15 Pro Ile Arg Asp Arg Val Asp Trp Glu Ala Gln Arg Ala Ala Ala
Leu 20 25 30 Ala Asp Pro Gly Ala Phe His Gly Ala Ile Ala Arg Thr
Val Ile His 35 40 45 Trp Tyr Asp Pro Gln His His Cys Trp Ile Arg
Phe Asn Glu Ser Ser 50 55 60 Gln Arg Trp Glu Gly Leu Asp Ala Ala
Thr Gly Ala Pro Val Thr Val 65 70 75 80 Asp Tyr Pro Ala Asp Tyr Gln
Pro Trp Gln Gln Ala Phe Asp Asp Ser 85 90 95 Glu Ala Pro Phe Tyr
Arg Trp Phe Ser Gly Gly Leu Thr Asn Ala Cys 100 105 110 Phe Asn Glu
Val Asp Arg His Val Thr Met Gly Tyr Gly Asp Glu Val 115 120 125 Ala
Tyr Tyr Phe Glu Gly Asp Arg Trp Asp Asn Ser Leu Asn Asn Gly 130 135
140 Arg Gly Gly Pro Val Val Gln Glu Thr Ile Thr Arg Arg Arg Leu Leu
145 150 155 160 Val Glu Val Val Lys Ala Ala Gln Val Leu Arg Asp Leu
Gly Leu Lys 165 170 175 Lys Gly Asp Arg Ile Ala Leu Asn Met Pro Asn
Ile Met Pro Gln Ile 180 185 190 Tyr Tyr Thr Glu Ala Ala Lys Arg Leu
Gly Ile Leu Tyr Thr Pro Val 195 200 205 Phe Gly Gly Phe Ser Asp Lys
Thr Leu Ser Asp Arg Ile His Asn Ala 210 215 220 Gly Ala Arg Val Val
Ile Thr Ser Asp Gly Ala Tyr Arg Asn Ala Gln 225 230 235 240 Val Val
Pro Tyr Lys Glu Ala Tyr Thr Asp Gln Ala Leu Asp Lys Tyr 245 250 255
Ile Pro Val Glu Thr Ala Gln Ala Ile Val Ala Gln Thr Leu Ala Thr 260
265 270 Leu Pro Leu Thr Glu Ser Gln Arg Gln Thr Ile Ile Thr Glu Val
Glu 275 280 285 Ala Ala Leu Ala Gly Glu Ile Thr Val Glu Arg Ser Asp
Val Met Arg 290 295 300 Gly Val Gly Ser Ala Leu Ala Lys Leu Arg Asp
Leu Asp Ala Ser Val 305 310 315 320 Gln Ala Lys Val Arg Thr Val Leu
Ala Gln Ala Leu Val Glu Ser Pro 325 330 335 Pro Arg Val Glu Ala Val
Val Val Val Arg His Thr Gly Gln Glu Ile 340 345 350 Leu Trp Asn Glu
Gly Arg Asp Arg Trp Ser His Asp Leu Leu Asp Ala 355 360 365 Ala Leu
Ala Lys Ile Leu Ala Asn Ala Arg Ala Ala Gly Phe Asp Val 370 375 380
His Ser Glu Asn Asp Leu Leu Asn Leu Pro Asp Asp Gln Leu Ile Arg 385
390 395 400 Ala Leu Tyr Ala Ser Ile Pro Cys Glu Pro Val Asp Ala Glu
Tyr Pro 405 410 415 Met Phe Ile Ile Tyr Thr Ser Gly Ser Thr Gly Lys
Pro Lys Gly Val 420 425 430 Ile His Val His Gly Gly Tyr Val Ala Gly
Val Val His Thr Leu Arg 435 440 445 Val Ser Phe Asp Ala Glu Pro Gly
Asp Thr Ile Tyr Val Ile Ala Asp 450 455 460 Pro Gly Trp Ile Thr Gly
Gln Ser Tyr Met Leu Thr Ala Thr Met Ala 465 470 475 480 Gly Arg Leu
Thr Gly Val Ile Ala Glu Gly Ser Pro Leu Phe Pro Ser 485 490 495 Ala
Gly Arg Tyr Ala Ser Ile Ile Glu Arg Tyr Gly Val Gln Ile Phe 500 505
510 Lys Ala Gly Val Thr Phe Leu Lys Thr Val Met Ser Asn Pro Gln Asn
515 520 525 Val Glu Asp Val Arg Leu Tyr Asp Met His Ser Leu Arg Val
Ala Thr 530 535 540 Phe Cys Ala Glu Pro Val Ser Pro Ala Val Gln Gln
Phe Gly Met Gln 545 550 555 560 Ile Met Thr Pro Gln Tyr Ile Asn Ser
Tyr Trp Ala Thr Glu His Gly 565 570 575 Gly Ile Val Trp Thr His Phe
Tyr Gly Asn Gln Asp Phe Pro Leu Arg 580 585 590 Pro Asp Ala His Thr
Tyr Pro Leu Pro Trp Val Met Gly Asp Val Trp 595 600 605 Val Ala Glu
Thr Asp Glu Ser Gly Thr Thr Arg Tyr Arg Val Ala Asp 610 615 620 Phe
Asp Glu Lys Gly Glu Ile Val Ile Thr Ala Pro Tyr Pro Tyr Leu 625 630
635 640 Thr Arg Thr Leu Trp Gly Asp Val Pro Gly Phe Glu Ala Tyr Leu
Arg 645 650 655 Gly Glu Ile Pro Leu Arg Ala Trp Lys Gly Asp Ala Glu
Arg Phe Val 660 665 670 Lys Thr Tyr Trp Arg Arg Gly Pro Asn Gly Glu
Trp Gly Tyr Ile Gln 675 680 685 Gly Asp Phe Ala Ile Lys Tyr Pro Asp
Gly Ser Phe Thr Leu His Gly 690 695 700 Arg Ser Asp Asp Val Ile Asn
Val Ser Gly His Arg Met Gly Thr Glu 705 710 715 720 Glu Ile Glu Gly
Ala Ile Leu Arg Asp Arg Gln Ile Thr Pro Asp Ser 725 730 735 Pro Val
Gly Asn Cys Ile Val Val Gly Ala Pro His Arg Glu Lys Gly 740 745 750
Leu Thr Pro Val Ala Phe Ile Gln Pro Ala Pro Gly Arg His Leu Thr 755
760 765 Gly Ala Asp Arg Arg Arg Leu Asp Glu Leu Val Arg Thr Glu Lys
Gly 770 775 780 Ala Val Ser Val Pro Glu Asp Tyr Ile Glu Val Ser Ala
Phe Pro Glu 785 790 795 800 Thr Arg Ser Gly Lys Tyr Met Arg Arg Phe
Leu Arg Asn Met Met Leu 805 810 815 Asp Glu Pro Leu Gly Asp Thr Thr
Thr Leu Arg Asn Pro Glu Val Leu 820 825 830 Glu Glu Ile Ala Ala Lys
Ile Ala Glu Trp Lys Arg Arg Gln Arg Met 835 840 845 Ala Glu Glu Gln
Gln Ile Ile Glu Arg Tyr Arg Tyr Phe Arg Ile Glu 850 855 860 Tyr His
Pro Pro Thr Ala Ser Ala Gly Lys Leu Ala Val Val Thr Val 865 870 875
880 Thr Asn Pro Pro Val Asn Ala Leu Asn Glu Arg Ala Leu Asp Glu Leu
885 890 895 Asn Thr Ile Val Asp His Leu Ala Arg Arg Gln Asp Val Ala
Ala Ile 900 905 910 Val Phe Thr Gly Gln Gly Ala Arg Ser Phe Val Ala
Gly Ala Asp Ile 915 920 925 Arg Gln Leu Leu Glu Glu Ile His Thr Val
Glu Glu Ala Met Ala Leu 930 935 940 Pro Asn Asn Ala His Leu Ala Phe
Arg Lys Ile Glu Arg Met Asn Lys 945 950 955 960 Pro Cys Ile Ala Ala
Ile Asn Gly Val Ala Leu Gly Gly Gly Leu Glu 965 970 975 Phe Ala Met
Ala Cys His Tyr Arg Val Ala Asp Val Tyr Ala Glu Phe 980 985 990 Gly
Gln Pro Glu Ile Asn Leu Arg Leu Leu Pro Gly Tyr Gly Gly Thr 995
1000 1005 Gln Arg Leu Pro Arg Leu Leu Tyr Lys Arg Asn Asn Gly Thr
Gly 1010 1015 1020 Leu Leu Arg Ala Leu Glu Met Ile Leu Gly Gly Arg
Ser Val Pro 1025 1030 1035 Ala Asp Glu Ala Leu Glu Leu Gly Leu Ile
Asp Ala Ile Ala Thr 1040 1045 1050 Gly Asp Gln Asp Ser Leu Ser Leu
Ala Cys Ala Leu Ala Arg Ala 1055 1060 1065 Ala Ile Gly Ala Asp Gly
Gln Leu Ile Glu Ser Ala Ala Val Thr 1070 1075 1080 Gln Ala Phe Arg
His Arg His Glu Gln Leu Asp Glu Trp Arg Lys 1085 1090 1095 Pro Asp
Pro Arg Phe Ala Asp Asp Glu Leu Arg Ser Ile Ile Ala 1100 1105 1110
His Pro Arg Ile Glu Arg Ile Ile Arg Gln Ala His Thr Val Gly 1115
1120 1125 Arg Asp Ala Ala Val His Arg Ala Leu Asp Ala Ile Arg Tyr
Gly 1130 1135 1140 Ile Ile His Gly Phe Glu Ala Gly Leu Glu His Glu
Ala Lys Leu 1145 1150 1155 Phe Ala Glu Ala Val Val Asp Pro Asn Gly
Gly Lys Arg Gly Ile 1160 1165 1170 Arg Glu Phe Leu Asp Arg Gln Ser
Ala Pro Leu Pro Thr Arg Arg 1175 1180 1185 Pro Leu Ile Thr Pro Glu
Gln Glu Gln Leu Leu Arg Asp Gln Lys 1190 1195 1200 Glu Leu Leu Pro
Val Gly Ser Pro Phe Phe Pro Gly Val Asp Arg 1205 1210 1215 Ile Pro
Lys Trp Gln Tyr Ala Gln Ala Val Ile Arg Asp Pro Asp 1220
1225 1230 Thr Gly Ala Ala Ala His Gly Asp Pro Ile Val Ala Glu Lys
Gln 1235 1240 1245 Ile Ile Val Pro Val Glu Arg Pro Arg Ala Asn Gln
Ala Leu Ile 1250 1255 1260 Tyr Val Leu Ala Ser Glu Val Asn Phe Asn
Asp Ile Trp Ala Ile 1265 1270 1275 Thr Gly Ile Pro Val Ser Arg Phe
Asp Glu His Asp Arg Asp Trp 1280 1285 1290 His Val Thr Gly Ser Gly
Gly Ile Gly Leu Ile Val Ala Leu Gly 1295 1300 1305 Glu Glu Ala Arg
Arg Glu Gly Arg Leu Lys Val Gly Asp Leu Val 1310 1315 1320 Ala Ile
Tyr Ser Gly Gln Ser Asp Leu Leu Ser Pro Leu Met Gly 1325 1330 1335
Leu Asp Pro Met Ala Ala Asp Phe Val Ile Gln Gly Asn Asp Thr 1340
1345 1350 Pro Asp Gly Ser His Gln Gln Phe Met Leu Ala Gln Ala Pro
Gln 1355 1360 1365 Cys Leu Pro Ile Pro Thr Asp Met Ser Ile Glu Ala
Ala Gly Ser 1370 1375 1380 Tyr Ile Leu Asn Leu Gly Thr Ile Tyr Arg
Ala Leu Phe Thr Thr 1385 1390 1395 Leu Gln Ile Lys Ala Gly Arg Thr
Ile Phe Ile Glu Gly Ala Ala 1400 1405 1410 Thr Gly Thr Gly Leu Asp
Ala Ala Arg Ser Ala Ala Arg Asn Gly 1415 1420 1425 Leu Arg Val Ile
Gly Met Val Ser Ser Ser Ser Arg Ala Ser Thr 1430 1435 1440 Leu Leu
Ala Ala Gly Ala His Gly Ala Ile Asn Arg Lys Asp Pro 1445 1450 1455
Glu Val Ala Asp Cys Phe Thr Arg Val Pro Glu Asp Pro Ser Ala 1460
1465 1470 Trp Ala Ala Trp Glu Ala Ala Gly Gln Pro Leu Leu Ala Met
Phe 1475 1480 1485 Arg Ala Gln Asn Asp Gly Arg Leu Ala Asp Tyr Val
Val Ser His 1490 1495 1500 Ala Gly Glu Thr Ala Phe Pro Arg Ser Phe
Gln Leu Leu Gly Glu 1505 1510 1515 Pro Arg Asp Gly His Ile Pro Thr
Leu Thr Phe Tyr Gly Ala Thr 1520 1525 1530 Ser Gly Tyr His Phe Thr
Phe Leu Gly Lys Pro Gly Ser Ala Ser 1535 1540 1545 Pro Thr Glu Met
Leu Arg Arg Ala Asn Leu Arg Ala Gly Glu Ala 1550 1555 1560 Val Leu
Ile Tyr Tyr Gly Val Gly Ser Asp Asp Leu Val Asp Thr 1565 1570 1575
Gly Gly Leu Glu Ala Ile Glu Ala Ala Arg Gln Met Gly Ala Arg 1580
1585 1590 Ile Val Val Val Thr Val Ser Asp Ala Gln Arg Glu Phe Val
Leu 1595 1600 1605 Ser Leu Gly Phe Gly Ala Ala Leu Arg Gly Val Val
Ser Leu Ala 1610 1615 1620 Glu Leu Lys Arg Arg Phe Gly Asp Glu Phe
Glu Trp Pro Arg Thr 1625 1630 1635 Met Pro Pro Leu Pro Asn Ala Arg
Gln Asp Pro Gln Gly Leu Lys 1640 1645 1650 Glu Ala Val Arg Arg Phe
Asn Asp Leu Val Phe Lys Pro Leu Gly 1655 1660 1665 Ser Ala Val Gly
Val Phe Leu Arg Ser Ala Asp Asn Pro Arg Gly 1670 1675 1680 Tyr Pro
Asp Leu Ile Ile Glu Arg Ala Ala His Asp Ala Leu Ala 1685 1690 1695
Val Ser Ala Met Leu Ile Lys Pro Phe Thr Gly Arg Ile Val Tyr 1700
1705 1710 Phe Glu Asp Ile Gly Gly Arg Arg Tyr Ser Phe Phe Ala Pro
Gln 1715 1720 1725 Ile Trp Val Arg Gln Arg Arg Ile Tyr Met Pro Thr
Ala Gln Ile 1730 1735 1740 Phe Gly Thr His Leu Ser Asn Ala Tyr Glu
Ile Leu Arg Leu Asn 1745 1750 1755 Asp Glu Ile Ser Ala Gly Leu Leu
Thr Ile Thr Glu Pro Ala Val 1760 1765 1770 Val Pro Trp Asp Glu Leu
Pro Glu Ala His Gln Ala Met Trp Glu 1775 1780 1785 Asn Arg His Thr
Ala Ala Thr Tyr Val Val Asn His Ala Leu Pro 1790 1795 1800 Arg Leu
Gly Leu Lys Asn Arg Asp Glu Leu Tyr Glu Ala Trp Thr 1805 1810 1815
Ala Gly Glu Arg 1820
* * * * *