U.S. patent application number 12/278957 was filed with the patent office on 2010-09-30 for production of fatty acids & derivatives thereof.
This patent application is currently assigned to LS9, Inc. Invention is credited to David Berry, Shane Brubaker, George Church, Stephen B. del Cardayre, Lisa Friedman, Zhihao Hu, Jay D. Keasling, Andreas Schirmer, Chris Sommerville.
Application Number | 20100242345 12/278957 |
Document ID | / |
Family ID | 42782392 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100242345 |
Kind Code |
A1 |
Keasling; Jay D. ; et
al. |
September 30, 2010 |
PRODUCTION OF FATTY ACIDS & DERIVATIVES THEREOF
Abstract
Genetically engineered microorganisms are provided that produce
products from the fatty acid biosynthetic pathway (fatty acid
derivatives), as well as methods of their use.
Inventors: |
Keasling; Jay D.; (Berkeley,
CA) ; Hu; Zhihao; (So. San Francisco, CA) ;
Sommerville; Chris; (So. San Francisco, CA) ; Church;
George; (So. San Francisco, CA) ; Berry; David;
(So. San Francisco, CA) ; Friedman; Lisa; (So. San
Francisco, CA) ; Schirmer; Andreas; (So. San
Francisco, CA) ; Brubaker; Shane; (So. San Francisco,
CA) ; del Cardayre; Stephen B.; (So. San Francisco,
CA) |
Correspondence
Address: |
LS9, Inc.
600 Gateway Boulevard
South San Francisco
CA
94080
US
|
Assignee: |
LS9, Inc
So. San Francisco
CA
|
Family ID: |
42782392 |
Appl. No.: |
12/278957 |
Filed: |
May 18, 2007 |
PCT Filed: |
May 18, 2007 |
PCT NO: |
PCT/US07/11923 |
371 Date: |
April 20, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60801995 |
May 19, 2006 |
|
|
|
60908547 |
Mar 28, 2007 |
|
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60802016 |
May 19, 2006 |
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Current U.S.
Class: |
44/388 ; 435/134;
435/243; 435/245; 435/252.3; 435/252.31; 435/252.32; 435/252.33;
435/254.11; 435/254.3; 435/254.5; 554/224 |
Current CPC
Class: |
C07C 33/02 20130101;
C10L 1/328 20130101; C12P 7/6463 20130101; C10L 1/026 20130101;
C12P 7/649 20130101; C12P 7/6436 20130101; C07C 31/125 20130101;
C12P 7/04 20130101; Y02E 50/10 20130101 |
Class at
Publication: |
44/388 ; 435/243;
435/245; 554/224; 435/252.33; 435/252.31; 435/252.32; 435/252.3;
435/134; 435/254.11; 435/254.5; 435/254.3 |
International
Class: |
C12N 1/00 20060101
C12N001/00; C12N 1/36 20060101 C12N001/36; C07C 57/03 20060101
C07C057/03; C12N 1/21 20060101 C12N001/21; C12P 7/64 20060101
C12P007/64; C10L 1/19 20060101 C10L001/19; C12N 1/19 20060101
C12N001/19 |
Claims
1. A microorganism comprising: (a) one or more exogenous nucleic
acid sequences encoding at least one peptide selected from accA (EC
6.4.1.2), accB (EC 6.4.1.2), accC (EC 6.4.1.2), accD (EC 6.4.1.2),
aceE (EC 1.2.4.1, 2.3.1.61, 2.3.1.12), aceF (EC 1.2.4.1, 2.3.4.16,
2.3.1.12), acpP (AAC74178), fadD (EC 2.3.1.86), cer1 (EC 4.1.99.5),
fabA (EC4.2.1.60), fabB (EC 2.3.1.41), fabD (EC 2.3.1.39), fabG (EC
1.1.1.100), fabH (EC 2.3.1.180), fabI (EC 1.3.1.9), fabZ(EC
4.2.1.-), lipase (EC 3.1.1.3), malonyl-CoA decarboxylase (EC
4.1.1.9, 4.1.1.41), panD (EC 4.1.1.11), panK (EC 2.7.1.33), pdh (EC
1.2.4.1), and udhA (EC 1.6.1.1); (b) a nucleic acid sequence
encoding a wax synthase (EC 2.3.1.75) or an alcohol
acetyltransferase (EC 2.3.1.84).
2-4. (canceled)
5. The microorganism of claim 1, wherein ackA (EC 2.7.2.1), ackB
(EC 2.7.2.1), adhE (EC 1.1.1.1, 1.2.1.10), fabF (EC 2.3.1.179),
fabR (accession NP.sub.--418398), fadE (EC 1.3.99.3, 1.3.99.-), GST
(EC 6.3.2.3), gpsA (EC 1.1.1.94), ldhA (EC 1.1.1.28), pflB (EC
2.3.1.54), plsB (EC 2.3.1.15), poxB (EC 1.2.2.2), pta (EC 2.3.1.8),
or glutathione synthase (EC 6.3.2.3) is attenuated.
6-10. (canceled)
11. The microorganism of claim 1, wherein the microorganism
additionally comprises: (a) an exogenous nucleic acid sequence
encoding a thioesterase (EC 3.1.2.-, 3.1.1.-); (b) at least one
exogenous nucleic acid sequence encoding an enzyme selected from
one or more components of the branch chain keto acid dehydrogenase
complex (EC 1.2.4.4), llve (EC 2.6.1.42), lpd (EC 1.8.1.4), Ccr
(EC1.1.19), IcmA (EC5.4.99.2), IcmB (5.4.99.13), fabH (EC
2.3.1.180), fabF (EC 2.3.1.179), fabH3 (EC 2.3.1.180), fabC3(NP
823468), beta-ketoacyl-ACP synthase II (EC 2.3.1.180), enoyl-CoA
reductase (EC 1.3.1.34), enoyl-CoA isomerase (EC 4.2.1.-), and
combinations thereof; (c) at least one exogenous nucleic acid
sequence encoding an enzyme selected from FabB (EC 2.3.1.41), FabK
(EC 1.2.1.9), FabL (EC 1.2.1.9), FabM (EC 5.3.3.14), FadE (EC
1.3.99.3, 1.3.99.-), and combinations thereof; (d) an exogenous
nucleic acid sequence encoding ACP, Sfa, or combinations thereof;
or (e) an exogenous nucleic acid sequence encoding an enzyme
selected from FadA (EC 2.3.1.16), FadI (EC 2.3.1.16), FadB (EC
1.1.1.35), FadJ (EC 4.2.1.17, EC 5.1.2.3, EC 5.3.3.8, EC 1.1.1.35),
and combinations thereof.
12-15. (canceled)
16. The microorganism of claims 1, wherein accA, accB, accC, accD,
or fadD is over-expressed.
17. The microorganism of claims 1, wherein the microorganism is in
a vessel comprising a fermentation broth comprising at least 10
mg/L fatty acid ester or at least 10 mg/L wax.
18. A fatty acid derivative produced by the microorganism of claim
1, wherein the fatty acid derivative comprises: (a) from about 1 to
about 5 double bonds, a carbon chain length of from about 8 to
about 30, about 1 to about 5 branch points, or between 1 and 10
cyclopropyl moieties: (b) an A side and a B side, wherein the A
side, the B side, or both the A side and the B side, are produced
by the microorganism; (c) an A side and a B side, wherein the A
side, the B side, or both the A side and the B side, comprise from
about 1 to about 5 double bonds; (d) an A side and a B side,
wherein the A side, the B side, or both the A side and the B side,
comprise a carbon chain length of from about 1 to about 26; (e) an
A side and a B side, wherein the A side, the B side, or both the A
side and the B side, comprise from about 1 to about 5 branch
points; or (f) an A side and a B side, wherein the A side, the B
side, or both the A side and the B side, comprise between 1 and 5
cyclopropyl moieties.
19-26. (canceled)
27. The microorganism of claims 1, wherein the microorganism is an
E. coli, Arthrobacter sp., Bacillus sp., Botryococcus braunii,
Chromatium sp., Cladosporium resina (ATCC22711), Clostridium
pasteurianum VKM, Clostridium tenanomorphum, Clostridium
acidiurici, Corynebacterium species, cyanobacterial species (Nostoc
muscorum, Anacystis (Synechococcus) nidulans, Phormidium luridum,
Chlorogloea fritschii, Trichodesmium erythaeum, Oscillatoria
williamsii, Microcoleus chthonoplaseis, Coccochloris elabens,
Agmenellum quadruplicatum, Plectonema terebrans, M vaginatus, and
C. scopulorum), Desulfovibrio desulfuricans (ATCC29577),
Kineococcus radiotolerans (BAA-149), Micrococcus luteus (FD533,
ATCC 272, 381, 382, ISU, 540, 4698, 7468, 27141), Micrococcus sp.
(ATCC 146, 398, 401, 533), Micrococcus roseus (ATCC 412, 416, 516),
Micrococcus lysodeikticus, Mycobacterium species, Penicillium sp.,
Aspergillus sp., Trichoderma virida, Pullularia pullulans,
Jeotgalicoccus sp. (M. candicans)(ATCC 8456), Rhodopseudomonas
spheroids Chlorobium sp., Rhodospirillium rubrum (ATCC11170),
Rhodomicrobium vannielii, Stenotrophomonas maltophilia (ATCC 13637,
17444, 17445, 17666, 17668, 17673, 17674, 17679, 17677),
Saccharomycodes ludwigii (ATCC 22711), Saccharomyces sp.
(oviformus,ludwiggi, tropicalis), Vibrio furnissii M1, Vibrio
marinus MP-1, Vibrio ponticus, Serratia marinorubra, Ustilago
maydis, Ustilago nuda, Urocystis agropyri, Sphacelotheca reiliana,
or Tilletia sp. (foetida, caries, controversa).
28. A method of producing a fatty acid derivative comprising: (a)
culturing the microorganism of claim 1 under conditions sufficient
to produce a fatty acid derivative; and (b) separating the fatty
acid derivative.
29. The method of claim 28, wherein the microorganism further
comprises: (a) an exogenous nucleic acid sequence encoding a
thioesterase (EC 3.1.2.-, 3.1.1.-); (b) at least one exogenous
nucleic acid sequence encoding an enzyme selected from one or more
components of the branch chain keto acid dehydrogenase complex (EC
1.2.4.4), llve (EC 2.6.1.42), lpd (EC 1.8.1.4), Ccr (EC1.1.19),
IcmA (EC5.4.99.2), IcmB (5.4.99.13), fabH (EC 2.3.1.180), fabF (EC
2.3.1.179), fabH3 (EC 2.3.1.180), fabC3(NP 823468),
beta-ketoacyl-ACP synthase II (EC 2.3.1.180), enoyl-CoA reductase
(EC 1.3.1.34), enoyl-CoA isomerase (EC 4.2.1.-), and combinations
thereof; (c) at least one exogenous nucleic acid sequence encoding
an enzyme selected from FabB (EC 2.3.1.41), FabK (EC 1.2.1.9), FabL
(EC 1.2.1.9), FabM (EC 5.3.3.14), FadE (EC 1.3.99.3, 1.3.99.-), and
combinations thereof; (d) an exogenous nucleic acid sequence
encoding ACP, Sfa, or combinations thereof; or (e) an exogenous
nucleic acid sequence encoding an enzyme selected from FadA (EC
2.3.1.16), FadI (EC 2.3.1.16), FadB (EC 1.1.1.35), FadJ (EC
4.2.1.17, EC 5.1.2.3, EC 5.3.3.8, EC 1.1.1.35), and combinations
thereof.
30. The method of claim 28, wherein ackA (EC 2.7.2.1), ackB (EC
2.7.2.1), adhE (EC 1.1.1.1, 1.2.1.10), fabF (EC 2.3.1.179), fabR
(accession NP 418398), fadE (EC 1.3.99.3, 1.3.99.-), GST (EC
6.3.2.3), gpsA (EC 1.1.1.94), ldhA (EC 1.1.1.28), pflB (EC
2.3.1.54), plsB (EC 2.3.1.15), poxB (EC 1.2.2.2), pta (EC 2.3.1.8),
or glutathione synthase (EC 6.3.2.3) of the microorganism is
attenuated.
31. The microorganism of claim 1, which produces an increased
amount of fatty acid derivatives as compared to the wild-type
microorganism.
32-36. (canceled)
37. The method of claim 28, wherein separating the fatty acid
derivative comprises: allowing the fatty acid derivative to
separate into an organic phase; and purifying the fatty acid
derivative from the organic phase.
38. (canceled)
39. A biofuel composition, comprising: at least about 11% or at
least about 17% of a fatty acid derivative, wherein the fatty acid
derivative comprises a carbon chain selected from the group
consisting of 8:0, 10:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1,
18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 22:0, 22:1 or 22:3; and at
least about 80% conventional diesel fuel.
40. The biofuel composition of claim 39 wherein the-fatty acid
derivative has .delta..sup.13C of from about -10.9 to about -15.4,
or a fraction of modern carbon of at least about 1.003.
41. (canceled)
42. The biofuel composition of claim 40, wherein the fatty acid
derivative accounts for at least about 85% of biosourced fatty
acid-derived material in the composition.
43-46. (canceled)
47. The biofuel composition of claim 39, further comprising a lower
alcohol, a surfactant, or a microemulsion in the biofuel
composition.
48-57. (canceled)
58. The biofuel composition of claim 39, wherein the biofuel
comprises less than 0.1% glycerin, or less than 0.1%
transesterification catalyst.
59. (canceled)
60. A fatty acid derivative produced by the method of claim 28.
61. A biofuel composition comprising the fatty acid derivative of
claim 60.
62. The biofuel composition of claim 61, comprising at least about
11%, or at least about 17% of the fatty acid derivative, and at
least about 80% of conventional diesel fuel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/802,016 filed May 19, 2006, U.S. Provisional
Application No. 60/801,995 filed May 19, 2006, U.S. Provisional
Application 60/908,547 filed Mar. 28, 2007 and PCT application
number PCT/US2007/003736 filed Feb. 13, 2007, all of which are
herein incorporated by reference.
FIELD
[0002] Genetically engineered microorganisms are provided that
produce products from the fatty acid biosynthetic pathway (fatty
acid derivatives), as well as methods of their use.
BACKGROUND
[0003] Developments in technology have been accompanied by an
increased reliance on fuel sources and such fuel sources are
becoming increasingly limited and difficult to acquire. With the
burning of fossil fuels taking place at an unprecedented rate, it
has likely that the world's fuel demand will soon outweigh the
current fuel supplies.
[0004] As a result, efforts have been directed toward harnessing
sources of renewable energy, such as sunlight, water, wind, and
biomass. The use of biomasses to produce new sources of fuel which
are not derived from petroleum sources, (i.e. biofuel) has emerged
as one alternative option. Biofuel (biodiesel) is a biodegradable,
clean-burning combustible fuel made of king chain alkanes and
esters. Biodiesel can be used in most internal combustion diesel
engines in either a pure form, which is referred to as "neat"
biodiesel, or as a mix in any concentration with regular petroleum
diesel. Current methods of making biodiesel involve
transesterification of triacylglycerides (mainly vegetable oil)
which leads to a mixture of fatty acid esters and the unwanted side
product glycerin, thus, providing a product that is heterogeneous
and a waste product that causes economic inefficiencies.
SUMMARY
[0005] Disclosed herein are recombinant microorganisms that are
capable of synthesizing products derived from the fatty acid
biosynthetic pathway (fatty acid derivatives), and optionally
releasing such products into the fermentation broth. Such fatty
acid derivatives are useful, inter alia, as biofuels and specialty
chemicals. These biofuels and specialty chemicals can be used to
make additional products, such as nutritional supplements,
polymers, paraffin replacements, and personal care products.
[0006] The recombinant microorganisms disclosed herein can be
engineered to yield various fatty acid derivatives including, but
not limited to, short chain alcohols such as ethanol, propanol
isopropanol and butanol, fatty alcohols, fatty acid esters,
hydrocarbons and wax esters.
[0007] In one example, the disclosure provides a method for
modifying a microorganism so that it produces, and optionally
releases, fatty acid derivatives generated from a renewable carbon
source. Such microorganisms are genetically engineered, for
example, by introducing an exogenous DNA sequence encoding one or
more proteins capable of metabolizing a renewable carbon source to
produce, and in some examples secrete, a fatty acid derivative. The
modified microorganisms can then be used in a fermentation process
to produce useful fatty acid derivatives using the renewable carbon
source (biomass) as a starting material. In some examples, an
existing genetically tractable microorganism is used because of the
ease of engineering its pathways for controlling growth, production
and reducing or eliminating side reactions that reduce biosynthetic
pathway efficiencies. In addition, such modified microorganisms can
be used to consume renewable carbon sources in order to generate
fuels that can be directly used as biofuels, without the need for
special methods for storage, or transportation. In other examples,
microorganisms that naturally produce hydrocarbons are engineered
to overproduce hydrocarbons by expressing exogenous nucleic acid
sequences that increase fatty acid production.
[0008] Provided herein are microorganisms that produce fatty acid
derivatives having defined carbon chain length, branching, and
saturation levels. In particular examples, the production of
homogeneous products decreases the overall cost associated with
fermentation and separation. In some examples microorganisms
including one or more exogenous nucleic acid sequences encoding at
least one thioesterase (EC 3.1.2.14), and at least one wax synthase
(EC 2.3.1.75) are provided. In other examples microorganisms are
provided that include one or more exogenous nucleic acid sequences
encoding at least one thioesterase (EC 3.1.2.14) and at least one
alcohol acetyltransferase (2.3.1.84). In yet other examples,
microorganisms including one or more exogenous nucleic acid
sequences encoding at least one thioesterase (EC 3.1.2.14), at
least one acyl-CoA reductase (EC 1.2.1.50) and at least one alcohol
dehydrogenase (EC 1.1.1.1) are provided. Microorganisms expressing
one or more exogenous nucleic acid sequences encoding at least one
thioesterase (EC 3.1.2.14) and at least one fatty alcohol forming
acyl-CoA reductase (1.1.1.*) are also provided. The thioesterase
peptides encoded by the exogenous nucleic acid sequences can be
chosen to provide homogeneous products.
[0009] In some examples the microorganism that is engineered to
produce the fatty acid derivative is E. coli, Z. mobilis,
Rhodococcus opacus, Ralstonia eutropha, Vibrio furnissii,
Saccharomyces cerevisiae, Lactococcus lactis, Streptomycetes,
Stenotrophomonas maltophila, Pseudomonas or Micrococus leuteus and
their relatives.
[0010] In other examples microorganisms that produce hydrocarbons
endogenously can be engineered to overproduce hydrocarbons by
optimizing the fatty acid biosynthetic pathway as described herein.
Exemplary microorganisms that are known to produce hydrocarbons and
can be engineered to over-produce hydrocarbons using the teachings
provided herein include Arthrobacter sp., Bacillus sp.,
Botryococcus braunii, Chromatium sp., Cladosporium resina
(ATCC22711), Clostridium pasteurianum VKM, Clostridium
tenanomorphum, Clostridium acidiurici, Corynebacterium species,
cyanobacterial species (Nostoc muscorum, Anacystis (Synechococcus)
nidulans, Phormidium luridum, Chlorogloea Trichodesmium erythaeum,
Oscillatoria williamsii, Microcoleus chthonoplaseis, Coccochloris
elabens, Agmenellum quadruplicatum, Plectonema terebrans, M
vaginatus, and C. scopulorum), Desulfovibrio desulfuricans
(ATCC29577), Kineococcus radiotolerans (BAA-149), Micrococcus
luteus (FD533, ATCC 272, 381, 382, ISU, 540, 4698, 7468, 27141),
Micrococcus sp. (ATCC 146, 398, 401, 533), Micrococcus roseus (ATCC
412, 416, 516), Micrococcus lysodeikticus, Mycobacterium species,
Penicillium sp., Aspergillus sp., Trichoderma virida, Pullularia
pullulans, Jeotgalicoccus sp. (M. candicans)(ATCC 8456),
Rhodopseudomonas spheroids Chlorobium sp., Rhodospirillium rubrum
(ATCC11170), Rhodomicrobium vannielii, Stenotrophomonas maltophilia
(ATCC 13637, 17444, 17445, 17666, 17668, 17673, 17674, 17679,
17677), Saccharomycodes ludwigii (ATCC 22711), Saccharomyces sp.
(oviformus,ludwiggi, tropicalis), Vibrio furnissii M1, Vibrio
marinus MP-1, Vibrio ponticus, Serratia marinorubra, Ustilago
maydis, Ustilago nuda, Urocystis agropyri, Sphacelotheca reiliana,
and Tilletia sp. (foetida, caries, controversa).
[0011] In addition to being engineered to express exogenous nucleic
acid sequences that allow for the production of fatty acid
derivatives, the microorganism can additionally have one or more
endogenous genes functionally deleted or attenuated. For example,
ackA (EC 2.7.2.1), ackB (EC 2.7.2.1), adhE. (EC 1.1.1.1, 1.2.1.10),
fabF (EC 2.3.1.179), fabR (accession NP.sub.--418398), fadE (EC
1.3.99.3, 1.3.99.-), GST (EC 6.3.2.3), gpsA (EC 1.1.1.94), ldhA (EC
1.1.1.28), pflB (EC 2.3.1.54), plsB (EC 2.3.1.15), poxB (EC
1.2.2.2), pta (EC 2.3.1.8), glutathione synthase (EC 6.3.2.3) and
combinations thereofcan be attenuated.
[0012] In addition to being engineered to express exogenous nucleic
acid sequences that allow for the production of fatty acid
derivatives, the microorganism can additionally have one or more
additional genes over-expressed. For example, pdh, panK, aceEF
(encoding the E1p dehydrogenase component and the E2p
dihydrolipoamide acyltransferase component of the pyruvate and
2-oxoglutarate dehydrogenase complexes, Accessions:
NP.sub.--414656, NP.sub.--414657, EC: 1.2.4.1. 2.3.1.61, 2.3.1.12),
accABCD/fabH/fabD/fabG/acpP/fabF (encoding FAS, Accessions:
CAD85557, CAD85558, NP.sub.--842277, NP.sub.--841683,
NP.sub.--415613, EC: 2.3.1.180, 2.3.1.39, 1.1.1.100, 1.6.5.3,
2.3.1.179), genes encoding fatty-acyl-coA reductases (Accessions:
AAC45217, EC 1.2.1.-), UdhA or similar genes (encoding pyridine
nucleotide transhydrogenase, Accession: CAA46822 , EC:1.6.1.1) and
genes encoding fatty-acyl-coA reductases (Accessions: AAC45217, EC
1.2.1.-).
[0013] In some examples, the microorganisms described herein
produce at least 1 mg of fatty acid derivative per liter
fermentation broth. In other examples the microorganisms produce at
least 100 mg/L, 500 mg/L, 1 g/L, 5 g/L, 10 g/L, 20 g/L, 25 g/L, 30
g/L, 35 g/L, 40 g/L, 50 g/L, 100 g/L, or 120 g/L of fatty acid
derivative per liter fermentation broth. In some examples, the
fatty acid derivative is produced and released from the
microorganism and in yet other examples the microorganism is lysed
prior to separation of the product.
[0014] In some examples, the fatty acid derivative includes a
carbon chain that is at least 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, or 34 carbons long. In some examples at least 50%,
60%, 70%, 80%, 85%, 90%, or 95% of the fatty acid derivative
product made contains a carbon chain that is 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, or 34 carbons long. In yet other
examples, at least 60%, 70%, 80%, 85%, 90%, or 95% of the fatty
acid derivative product contain 1, 2, 3, 4, or 5, points of
unsaturation
[0015] Also provided are methods of producing fatty acid
derivatives. These methods include culturing the microorganisms
described herein and separating the product from the fermentation
broth.
[0016] These and other examples are described further in the
following detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the FAS biosynthetic pathway.
[0018] FIG. 2 shows biosynthetic pathways that produce waxes. Waxes
can be produced in a host cell using alcohols produced within the
host cell or they can be produced by adding exogenous alcohols in
the medium. A microorganism designed to produce waxes will produce
wax synthase enzymes (EC 2.3.1.75) using exogenous nucleic acid
sequences as well as thioesterase (EC 3.1.2.14) sequences. Other
enzymes that can be also modulated to increase the production of
waxes include enzymes involved in fatty acid synthesis (FAS enzymes
EC 2.3.1.85), acyl-CoA synthase (EC 2.3.1.86), fatty alcohol
forming acyl-CoA reductase (EC 1.1.1.*), acyl-CoA reductase
(1.2.1.50) and alcohol dehydrogenase (EC 1.1.1.1).
[0019] FIG. 3 shows biosynthetic pathways that produce fatty
alcohols. Fatty alcohols having defined carbon chain lengths can be
produced by expressing exogenous nucleic acid sequences encoding
thioesterases (EC 3.1.2.14), and combinations of acyl-CoA
reductases (EC 1.2.1.50), alcohol dehydrogenases (EC 1.1.1.1) and
fatty alcohol forming acyl-CoA reductases (FAR, EC 1.1.1*). Other
enzymes that can be also modulated to increase the production of
fatty alcohols include enzymes involved in fatty acid synthesis
(FAS enzymes EC 2.3.1.85), and acyl-CoA synthase (EC 2.3.1.86).
[0020] FIG. 4 shows biosynthetic pathways that produce fatty acids
esters. Fatty acids esters having defined carbon chain lengths can
be produced by exogenously expressing various thioesterases (EC
3.1.2.14), combinations of acyl-CoA reductase (1.2.1.50), alcohol
dehydrogenases (EC 1.1.1.1), and fatty alcohol forming Acyl-CoA
reductase (FAR, EC 1.1.1*), as well as, acetyl transferase (EC
2.3.1.84). Other enzymes that can be modulated to increase the
production of fatty acid esters include enzymes involved in fatty
acid synthesis (FAS enzymes EC 2.3.1.85), and acyl-CoA synthase (EC
2.3.1.86).
[0021] FIG. 5 shows fatty alcohol production by the strain
described in Example 4, co-transformed with pCDFDuet-1-fadD-acr1
and plasmids containing various thioesterase genes. The strains
were grown aerobically at 25.degree. C. in M9 mineral medium with
0.4% glucose in shake flasks. Saturated C10, C12, C14, C16 and C18
fatty alcohol were identified. Small amounts of C16:1 and C18:1
fatty alcohols were also detected in some samples. Fatty alcohols
were extracted from cell pellets using ethyl acetate and
derivatized with N-trimethylsilyl (TMS) imidazole to increase
detection.
[0022] FIG. 6 shows the release of fatty alcohols from the
production strain. Approximately 50% of the fatty alcohol produced
was released from the cells when they were grown at 37.degree.
C.
[0023] FIGS. 7A-7D show GS-MS spectrum of octyl octanoate (C8C8)
produced by a production hosts expressing alcohol acetyl
transferase (AATs, EC 2.3.1.84) and production hosts expressing wax
synthase (EC 2.3.1.75). FIG. 7A shows acetyl acetate extract of
strain C41(DE3, .DELTA.fadE/pHZ1.43)/pRSET B+pAS004.114B) wherein
the pHZ1.43 plasmid expressed ADP1 (wax synthase). FIG. 7B shows
acetyl acetate extract of strain C41(DE3,
.DELTA.fadE/pHZ1.43)/pRSET B+pAS004.114B) wherein the pHZ1.43
plasmid expressed SAAT. FIG. 7C shows acetyl acetate extract of
strain C41(DE3, .DELTA.fadE/pHZ1.43)/pRSET B+pAS004.114B) wherein
the pHZ1.43 plasmid did not contain ADP1 (wax synthase) or SAAT.
FIG. 7D shows the mass spectrum and fragmentation pattern of C8C8
produced by C41(DE3, .DELTA.fadE/pHZ1.43)/pRSET B+pAS004.114B)
wherein the pHZ1.43 plasmid expressed SAAT).
[0024] FIG. 8 shows the distribution of ethyl esters made when the
wax synthase from A. baylyi ADP1 (WSadp1) was co-expressed with
thioesterase gene from Cuphea hookeriana in a production host.
[0025] FIGS. 9A and 9B show chromatograms of GC/MS analysis. FIG.
9A shows a chromatogram of the ethyl extract of the culture of E.
coil LS9001 strain transformed with plasmids
pCDFDuet-1-fadD-WSadp1, pETDuet-1-'tesA. Ethanol was fed to
fermentations. FIG. 9B shows a chromatogram of ethyl hexadecanoate
and ethyl oleate used as reference.
[0026] FIG. 10 shows a table that identifies various genes that can
be over-expressed or attenuated to increase fatty acid derivative
production. The table also identifies various genes that can be
modulated to alter the structure of the fatty acid derivative
product. One of ordinary skill in the art will appreciate that some
of the genes that are used to alter the structure of the fatty acid
derivative will also increase the production of fatty acid
derivatives.
ABBREVIATIONS AND TERMS
[0027] The following explanations of terms and methods are provided
to better describe the present disclosure and to guide those of
ordinary skill in the art in the practice of the present
disclosure. As used herein, "comprising" means "including" and the
singular forms "a" or "an" or "the" include plural references
unless the context clearly dictates otherwise. For example,
reference to "comprising a cell" includes one or a plurality of
such cells, and reference to "comprising the thioesterase" includes
reference to one or more thioesterase peptides and equivalents
thereof known to those of ordinary skill in the art, and so forth.
The term "or" refers to a single element of stated alternative
elements or a combination of two or more elements, unless the
context clearly indicates otherwise. For example, the phrase
"thioesterase activity or fatty alcohol-forming acyl-CoA reductase
activity" refers to thioesterase activity, fatty alcohol forming
acyl-CoA reductase activity, or a combination of both fatty alcohol
forming acyl-CoA reductase activity, and thioesterase activity.
[0028] Unless explained otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only
and not intended to be limiting. Other features of the disclosure
are apparent from the following detailed description and the
claims.
[0029] Accession Numbers: The accession numbers throughout this
description are derived from the NCBI database (National Center for
Biotechnology Information) maintained by the National Institute of
Health, U.S.A. The accession numbers are as provided in the
database on Mar. 27, 2007.
[0030] Enzyme Classification Numbers (EC): The EC numbers 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 EC
numbers are as provided in the database on Mar. 27, 2007.
[0031] Attenuate: To lessen the impact, activity or strength of
something. In one example, the sensitivity of a particular enzyme
to-feedback inhibition or inhibition caused by a composition that
is not a product or a reactant (non-pathway specific feedback) is
lessened such that the enzyme activity is not impacted by the
presence of a compound. For example, the fabH gene and its
corresponding amino acid sequence are temperature sensitive and can
be altered to decrease the sensitivity to temperature fluctuations.
The attenuation of the fabH gene can be used when branched amino
acids are desired. In another example, an enzyme that has been
altered to be less active can be referred to as attenuated.
[0032] A functional deletion of an enzyme can be used to attenuate
an enzyme. A functional deletion is a mutation, partial or complete
deletion, insertion, or other variation made to a gene sequence or
a sequence controlling the transcription of a gene sequence, which
reduces or inhibits production of the gene product, or renders the
gene product non-functional (i.e. the mutation described herein for
the plsB gene). For example, functional deletion of fabR in E. coli
reduces the repression of the fatty acid biosynthetic pathway and
allows E. coli to produce more unsaturated fatty acids (UFAs). In
some instances a functional deletion is described as a knock-out
mutation.
[0033] One of ordinary skill in the art will appreciate that there
are many methods of attenuating a enzyme activity. For example,
attenuation can be accomplished by introducing amino acid sequence
changes via altering the nucleic acid sequence, placing the gene
under the control of a less active promoter, expressing interfering
RNA, ribozymes or antisense sequences that targeting the gene of
interest, or through any other technique known in the art.
[0034] Carbon source: Generally refers to a substrate or compound
suitable to be used as a source of carbon for prokaryotic or simple
eukaryotic cell growth. Carbon sources can be in various forms,
including, but not limited to polymers, carbohydrates, acids,
alcohols, aldehydes, ketones, amino acids, peptides, etc. These
include, for example, various monosaccharides such as glucose,
oligosaccharides, polysaccharides, cellulosic material, xylose, and
arabinose, disaccharides, such sucrose, saturated or unsaturated
fatty acids, succinate, lactate, acetate, ethanol, etc., or
mixtures thereof. The carbon source can additionally be a product
of photosynthesis, including, but not limited to glucose.
[0035] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and regulatory sequences which
determine transcription. cDNA can be synthesized by reverse
transcription from messenger RNA extracted from cells.
[0036] Deletion: The removal of one or more nucleotides from a
nucleic acid molecule or one or more amino acids from a protein,
the regions on either side being joined together.
[0037] Detectable: Capable of having an existence or presence
ascertained. For example, production of a product from a reactant,
for example, the production of C18 fatty acids, is detectable using
the method provided in Example 11 below.
[0038] DNA: Deoxyribonucleic acid. DNA is a long chain polymer
which includes the genetic material of most living organisms (some
viruses have genes including ribonucleic acid, RNA). The repeating
units in DNA polymers are four different nucleotides, each of which
includes one of the four bases, adenine, guanine, cytosine and
thymine bound to a deoxyribose sugar to which a phosphate group is
attached. Triplets of nucleotides, referred to as codons, in DNA
molecules code for amino acid in a peptide. The term codon is also
used for the corresponding (and complementary) sequences of three
nucleotides in the mRNA into which the DNA sequence is
transcribed.
[0039] Endogenous: As used herein with reference to a nucleic acid
molecule and a particular cell or microorganism refers to a nucleic
acid sequence or peptide that is in the cell and was not introduced
into the cell using recombinant engineering techniques. For
example, a gene that was present in the cell when the cell was
originally isolated from nature. A gene is still considered
endogenous if the control sequences, such as a promoter or enhancer
sequences that activate transcription or translation have been
altered through recombinant techniques.
[0040] Exogenous: As used herein with reference to a nucleic acid
molecule and a particular cell refers to any nucleic acid molecule
that does not originate from that particular cell as found in
nature. Thus, a non-naturally-occurring nucleic acid molecule is
considered to be exogenous to a cell once introduced into the cell.
A nucleic acid molecule that is naturally-occurring also can be
exogenous to a particular cell. For example, an entire coding
sequence isolated from cell X is an exogenous nucleic acid with
respect to cell Y once that coding sequence is introduced into cell
Y, even if X and Y are the same cell type.
[0041] Expression: The process by which a gene's coded information
is converted into the structures and functions of a cell, such as a
protein, transfer RNA, or ribosomal RNA. Expressed genes include
those that are transcribed into mRNA and then translated into
protein and those that are transcribed into RNA but not translated
into protein (for example, transfer and ribosomal RNAs).
[0042] Fatty ester: Includes any ester made from a fatty acid. The
carbon chains in fatty acids can contain any combination of the
modifications described herein. For example, the carbon chain can
contain one or more points of unsaturation, one or more points of
branching, including cyclic branching, and can be engineered to be
short or long. Any alcohol can be used to form fatty acid esters,
for example alcohols derived from the fatty acid biosynthetic
pathway, alcohols produced by the production host through non-fatty
acid biosynthetic pathways, and alcohols that are supplied in the
fermentation broth.
[0043] Fatty acid derivative: Includes products made in part from
the fatty acid biosynthetic pathway of the host organism. The fatty
acid biosynthetic pathway includes fatty acid synthase enzymes
which can be engineered as described herein to produce fatty acid
derivatives, and in some examples can be expressed with additional
enzymes to produce fatty acid derivatives having desired carbon
chain characteristics. Exemplary fatty acid derivatives include for
example, short and long chain alcohols, hydrocarbons, and fatty
acid esters including waxes.
[0044] Fermentation Broth: Includes any medium which supports
microorganism life (i.e. a microorganism that is actively
metabolizing carbon). A fermentation medium usually contains a
carbon source. The carbon source can be anything that can be
utilized, with or without additional enzymes, by the microorganism
for energy.
[0045] Hydrocarbon: includes chemical compounds that containing the
elements carbon (C) and hydrogen (H). All hydrocarbons consist of a
carbon backbone and atoms of hydrogen attached to that backbone.
Sometimes, the term is used as a shortened form of the term
"aliphatic hydrocarbon." There are essentially three types of
hydrocarbons: (1) aromatic hydrocarbons, which have at least one
aromatic ring; (2) saturated hydrocarbons, also known as alkanes,
which lack double, triple or aromatic bonds; and (3) unsaturated
hydrocarbons, which have one or more double or triple bonds between
carbon atoms, are divided into: alkenes, alkynes, and dienes.
Liquid geologically-extracted hydrocarbons are referred to as
petroleum (literally "rock oil") or mineral oil, while gaseous
geologic hydrocarbons are referred to as natural gas. All are
significant sources of fuel and raw materials as a feedstock for
the production of organic chemicals and are commonly found in the
Earth's subsurface using the tools of petroleum geology. Oil
reserves in sedimentary rocks are the principal source of
hydrocarbons for the energy and chemicals industries. Hydrocarbons
are of prime economic importance because they encompass the
constituents of the major fossil fuels (coal, petroleum, natural
gas, etc.) and biofuels, as well as plastics, waxes, solvents and
oils.
[0046] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein, or cell) has been substantially
separated or purified away from other biological components in
which the component naturally occurs, such as other chromosomal and
extrachromosomal DNA and RNA, and proteins. Nucleic acid molecules
and proteins that have been "isolated" include nucleic acid
molecules and proteins purified by standard purification methods.
The term also embraces nucleic acid molecules and proteins prepared
by recombinant expression in a host cell as well as chemically
synthesized nucleic acid molecules and proteins.
[0047] In one example, isolated refers to a naturally-occurring
nucleic acid molecule that is not immediately contiguous with both
of the sequences with which it is immediately contiguous (one on
the 5' end and one on the 3' end) in the naturally-occurring genome
of the organism from which it is derived.
[0048] Microorganism: Includes prokaryotic and eukaryotic microbial
species from the Domains Archaea, Bacteria and Eucarya, the latter
including yeast and filamentous fungi, protozoa, algae, or higher
Protista. The terms "microbial cells" and "microbes" are used
interchangeably with the term microorganism.
[0049] Nucleic Acid Molecule: Encompasses both RNA and DNA
molecules including, without limitation, cDNA, genomic DNA, and
mRNA. Includes synthetic nucleic acid molecules, such as those that
are chemically synthesized or recombinantly produced. The nucleic
acid molecule can be double-stranded or single-stranded. Where
single-stranded, the nucleic acid molecule can be the sense strand
or the antisense strand. In addition, nucleic acid molecule can be
circular or linear.
[0050] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein coding regions, in the same reading frame.
Configurations of separate genes that are transcribed in tandem as
a single messenger RNA are denoted as operons. Thus placing genes
in close proximity, for example in a plasmid vector, under the
transcriptional regulation of a single promoter, constitutes a
synthetic operon.
[0051] ORF (open reading frame): A series of nucleotide triplets
(codons) coding for amino acids without any termination codons.
These sequences are usually translatable into a peptide.
[0052] Over-expressed: When a gene is caused to be transcribed at
an elevated rate compared to the endogenous transcription rate for
that gene. In some examples, over-expression additionally includes
an elevated rate of translation of the gene compared to the
endogenous translation rate for that gene. Methods of testing for
over-expression are well known in the art, for example transcribed
RNA levels can be assessed using rtPCR and protein levels can be
assessed using SDS page gel analysis.
[0053] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified fatty acid derivative preparation, such as a
wax, or a fatty acid ester preparation, is one in which the product
is more concentrated than the product is in its environment within
a cell. For example, a purified wax is one that is substantially
separated from cellular components (nucleic acids, lipids,
carbohydrates, and other peptides) that can accompany it. In
another example, a purified wax preparation is one in which the wax
is substantially-free from contaminants, such as those that might
be present following fermentation.
[0054] In one example, a fatty acid ester is purified when at least
about 50% by weight of a sample is composed of the fatty acid
ester, for example when at least about 60%, 70%, 80%, 85%, 90%,
92%, 95%, 98%, or 99% or more of a sample is composed of the fatty
acid ester. Examples of methods that can be used to purify a waxes,
fatty alcohols, and fatty acid esters, include the methods
described in Example 11 below.
[0055] Recombinant: A recombinant nucleic acid molecule or protein
is one that has a sequence that is not naturally occurring, has a
sequence that is made by an artificial combination of two otherwise
separated segments of sequence, or both. This artificial
combination can be achieved, for example, by chemical synthesis or
by the artificial manipulation of isolated segments of nucleic acid
molecules or proteins, such as genetic engineering techniques.
Recombinant is also used to describe nucleic acid molecules that
have been artificially manipulated, but contain the same regulatory
sequences and coding regions that are found in the organism from
which the nucleic acid was isolated. A recombinant cell or
microorganism is one that contains an exogenous nucleic acid
molecule, such as a recombinant nucleic acid molecule.
[0056] Release: The movement of a compound from inside a cell
(intracellular) to outside a cell (extracellular). The movement can
be active or passive. When release is active it can be facilitated
by one or more transporter peptides and in some examples it can
consume energy. When release is passive, it can be through
diffusion through the membrane and can be facilitated by
continually collecting the desired compound from the extracellular
environment, thus promoting further diffusion. Release of a
compound can also be accomplished by lysing a cell.
[0057] Surfactants: 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 be either ionic or
nonionic, and the hydrocarbon chain is hydrophobic. Surfactants are
used in a variety of products, including detergents and cleaners,
and are also used as auxiliaries for textiles, leather and paper,
in chemical processes, in cosmetics and pharmaceuticals, in the
food industry and in agriculture. In addition, they can be used to
aid in the extraction and isolation of crude oils which are found
hard to access environments or as water emulsions.
[0058] There are four types of surfactants characterized by varying
uses. Anionic surfactants have detergent-like activity and are
generally used for cleaning applications. Cationic surfactants
contain long chain hydrocarbons and are often used to treat
proteins and synthetic polymers or are components of fabric
softeners and hair conditioners. Amphoteric surfactants also
contain long chain hydrocarbons and are typically used in shampoos.
Non-ionic surfactants are generally used in cleaning products.
[0059] Transformed or recombinant cell: A cell into which a nucleic
acid molecule has been introduced, such as an acyl-CoA synthase
encoding nucleic acid molecule, for example by molecular biology
techniques. Transformation encompasses all techniques by which a
nucleic acid molecule can be introduced into such a cell,
including, but not limited to, transfection with viral vectors,
conjugation, transformation with plasmid vectors, and introduction
of naked DNA by electroporation, lipofection, and particle gun
acceleration.
[0060] Under conditions that permit product production: Any
fermentation conditions that allow a microorganism to produce a
desired product, such as fatty acids, hydrocarbons, fatty alcohols,
waxes, or fatty acid esters. Fermentation conditions usually
include temperature ranges, levels of aeration, and media
selection, which when combined allow the microorganism to grow.
Exemplary mediums include broths or gels. Generally, the medium
includes a carbon source such as glucose, fructose, cellulose, or
the like that can be metabolized by the microorganism directly, or
enzymes can be used in the medium to facilitate metabolizing the
carbon source. To determine if culture conditions permit product
production, the microorganism can be cultured for 24, 36, or 48
hours and a sample can be obtained and analyzed. For example, the
cells in the sample or the medium in which the cells were grown can
be tested for the presence of the desired product. When testing for
the presence of a product assays, such as those provided in the
Examples below, can be used.
[0061] Vector: A nucleic acid molecule as introduced into a cell,
thereby producing a transformed cell. A vector can include nucleic
acid sequences that permit it to replicate in the cell, such as an
origin of replication. A vector can also include one or more
selectable marker genes and other genetic elements known in the
art.
[0062] Wax: A variety of fatty acid esters which form solids or
pliable substances under an identified set of physical conditions.
Fatty acid esters that are termed waxes generally have longer
carbon chains than fatty acid esters that are not waxes. For
example, a wax generally forms a pliable substance at room
temperature.
DETAILED DESCRIPTION
I. Production of Fatty Acid Derivatives
[0063] The host organism that exogenous DNA sequences are
transformed into can be a modified host organism, such as an
organism that has been modified to increase the production of
acyl-ACP or acyl-CoA, reduce the catabolism of fatty acid
derivatives and intermediates, or to reduce feedback inhibition at
specific points in the biosynthetic pathway. 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
acetyl-CoA carboxylase (ACC) can be over expressed. The
modifications to the production host described herein can be
through genomic alterations, extrachromosomal expression systems,
or combinations thereof. An overview of the pathway is provided in
FIGS. 1 and 2.
[0064] A. Acetyl-CoA--Malonyl-CoA to Acyl-ACP
[0065] Fatty acid synthase (FAS) is a group of peptides that
catalyze the initiation and elongation of acyl chains (Marrakchi et
al., Biochemical Society, 30:1050-1055, 2002). The acyl carrier
protein (ACP) along with the enzymes in the FAS pathway control the
length, degree of saturation and branching of the fatty acids
produced. Enzymes that can be included in FAS include AccABCD,
FabD, FabH, FabG, FabA, FabZ, FabI, FabK, FabL, FabM, FabB, and
FabF. Depending upon the desired product one or more of these genes
can be attenuated or over-expressed.
[0066] For example, the fatty acid biosynthetic pathway in the
production host suses the precursors acetyl-CoA and malonyl-CoA
(FIG. 2). E. coli or other host organisms engineered to overproduce
these components can serve as the starting point for subsequent
genetic engineering steps to provide the specific output product
(such as, fatty acid esters, hydrocarbons, fatty alcohols). Several
different modifications can be made, either in combination or
individually, to the host strain to obtain increased acetyl
CoA/malonyl CoA/fatty acid and fatty acid derivative production.
For example, to increase acetyl CoA production, a plasmid with pdh,
panK, aceEF, (encoding the E1p 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 DNA encoding fatty-acyl-CoA
reductases and aldehyde decarbonylases, all under the control of a
constitutive, or otherwise controllable promoter, can be
constructed. Exemplary Genbank accession numbers for these genes
are: pdh (BAB34380, AAC73227, AAC73226), panK (also known as coaA,
AAC76952), aceEF (AAC73227, AAC73226), fabH(AAC74175), fabD
(AAC74176), fabG (AAC74177), acpP (AAC74178), fabF (AAC74179).
[0067] Additionally, fadE, gpsA, ldhA, pflb, adhE, pta, poxB, ackA,
and/or ackB can be knocked-out, or their expression levels can be
reduced, in the engineered microorganism by transformation with
conditionally replicative or non-replicative plasmids containing
null or deletion mutations of the corresponding genes, or by
substituting promoter or enhancer sequences. Exemplary Genbank
accession numbers for these genes are; fadE (AAC73325), gspA
(AAC76632), ldhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta
(AAC75357), poxB (AAC73958), ackA (AAC75356), and ackB
(BAB81430).
[0068] The resulting engineered microorganisms can be grown in a
desired environment, for example one with limited glycerol (less
than 1% w/v in the culture medium). As such, these microorganisms
will have increased acetyl-CoA production levels. Malonyl-CoA
overproduction can be effected by engineering the microorganism as
described above, with DNA encoding accABCD (acetyl CoA carboxylase,
for example accession number AAC73296, EC 6.4.1.2) included in the
plasmid synthesized de novo. Fatty acid overproduction can be
achieved by further including DNA encoding lipase (for example
Accessions numbers CAA89087, CAA98876) in the plasmid synthesized
de novo.
[0069] In some examples, acetyl-CoA carboxylase (ACC) is
over-expressed to increase the intracellular concentration thereof
by at least 2-fold, such as at least 5-fold, or at least 10-fold,
for example relative to native expression levels.
[0070] In addition, the plsB (for example Accession number
AAC77011) D311E mutation can be used to remove limitations on the
pool of acyl-CoA.
[0071] In addition, over-expression of an sfa gene (suppressor of
FabA, for example Accession number AAN79592) can be included in the
production host to increase production of monounsaturated fatty
acids (Rock et al., J. Bacteriology 178:5382-5387, 1996).
[0072] B. Acyl-ACP to Fatty Acid
[0073] To engineer a production host for the production of a
homogeneous population of fatty acid derivatives, one or more
endogenous genes can be attenuated or functionally deleted and one
or more thioesterases can be expressed. For example, C10 fatty acid
derivatives can be produced by attenuating thioesterase C18 (for
example accession numbers AAC73596 and P0ADA1), which uses
C18:1-ACP and expressing thioesterase C10 (for example accession
number Q39513), which uses C10-ACP. Thus, resulting in a relatively
homogeneous population of fatty acid derivatives 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 thioesterase
accession number Q39473 (which uses C14-ACP). In yet another
example, C12 fatty acid derivatives can be produced by expressing
thioesterases that use C12-ACP (for example accession number
Q41635) and attenuating thioesterases that produce non-C12 fatty
acids. 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.
TABLE-US-00001 TABLE 1 Thioesterases Preferential Accession product
Number Source Organism Gene produced AAC73596 E. coli tesA without
leader C18:1 sequence Q41635 Umbellularia fatB C12:0 california
Q39513; Cuphea hookeriana fatB2 C8:0-C10:0 AAC49269 Cuphea
hookeriana fatB3 C14:0-C16:0 Q39473 Cinnamonum fatB C14:0 camphorum
CAA85388 Arabidopsis thaliana fatB[M141T]* C16:1 NP 189147;
Arabidopsis thaliana fatA C18:1 NP 193041 CAC39106 Bradyrhiizobium
fatA C18:1 japonicum AAC72883 Cuphea hookeriana fatA C18:1 *Mayer
et al., BMC Plant Biology 7: 1-11, 2007.
[0074] C. Fatty Acid to Acyl-CoA
[0075] Production hosts can be engineered using known peptides to
produce fatty acids of various lengths. One method of making fatty
acids involves increasing the expression of, or expressing more
active forms of, one or more acyl-CoA synthase peptides (EC
2.3.1.86).
[0076] As used herein, acyl-CoA synthase includes peptides in
enzyme classification number EC 2.3.1.86, as well as any other
peptide capable of catalyzing the conversion of a fatty acid to
acyl-CoA. Additionally, one of ordinary skill in the art will
appreciate that some acyl-CoA synthase peptides will catalyze other
reactions as well, for example some acyl-CoA synthase peptides will
accept other substrates in addition to fatty acids. Such
non-specific acyl-CoA synthase peptides are, therefore, also
included. Acyl-CoA synthase peptide sequences are publicly
available. Exemplary GenBank Accession Numbers are provided in FIG.
10.
[0077] D. Acyl-CoA to Fatty Alcohol
[0078] Production hosts can be engineered using known polypeptides
to produce fatty alcohols from acyl-CoA. One method of making fatty
alcohols involves increasing the expression of or expressing more
active forms of fatty alcohol forming acyl-CoA reductase (FAR, EC
1.1.1.*), or acyl-CoA reductases (EC 1.2.1.50) and alcohol
dehydrogenase (EC 1.1.1.1). Hereinafter fatty alcohol forming
acyl-CoA reductase (FAR, EC 1.1.1.*), acyl-CoA reductases (EC
1.2.1.50) and alcohol dehydrogenase (EC 1.1.1.1) are collectively
referred to as fatty alcohol forming peptides. In some examples all
three of the fatty alcohol forming genes can be over expressed in a
production host, and in yet other examples one or more of the fatty
alcohol forming genes can be over-expressed.
[0079] As used herein, fatty alcohol forming peptides include
peptides in enzyme classification numbers EC 1.1.1.*, 1.2.1.50, and
1.1.1.1, as well as any other peptide capable of catalyzing the
conversion of acyl-CoA to fatty alcohol. Additionally, one of
ordinary skill in the art will appreciate that some fatty alcohol
forming peptides will catalyze other reactions as well, for example
some acyl-CoA reductase peptides will accept other substrates in
addition to fatty acids. Such non-specific peptides are, therefore,
also included. Fatty alcohol forming peptides sequences are
publicly available. Exemplary GenBank Accession Numbers are
provided in FIG. 10.
[0080] Fatty alcohols can also be described as hydrocarbon-based
surfactants. For surfactant production the microorganism is
modified so that it produces a surfactant from a renewable carbon
source. Such a microorganism includes a first exogenous DNA
sequence encoding a protein capable of converting a fatty acid to a
fatty aldehyde and a second exogenous DNA sequence encoding a
protein capable of converting a fatty aldehyde to an alcohol. In
some examples, the first exogenous DNA sequence encodes a fatty
acid reductase. In one embodiment, the second exogenous DNA
sequence encodes mammalian microsomal aldehyde reductase or
long-chain aldehyde dehydrogenase. In a further example, the first
and second exogenous DNA sequences are from a multienzyme complex
from Arthrobacter AK 19, Rhodotorula glutinins, Acinobacter sp
strain M-1, or Candida lipolytica. In one embodiment, the first and
second heterologous DNA sequences are from a multienzyme complex
from Acinobacter sp strain M-1 or Candida lipolytica.
[0081] Additional sources of heterologous DNA sequences encoding
fatty acid to long chain alcohol converting proteins that can be
used in surfactant production include, but are not limited to,
Mortierella alpina (ATCC 32222), Crytococcus curvatus, (also
referred to as Apiotricum curvatum), Alcanivorax jadensis (T9T=DSM
12718=ATCC 700854), Acinetobacter sp. HO1-N, (ATCC 14987) and
Rhodococcus opacus (PD630 DSMZ 44193).
[0082] In one example, the fatty acid derivative is a saturated or
unsaturated surfactant product having a carbon atom content limited
to between 6 and 36 carbon atoms. In another example, the
surfactant product has a carbon atom content limited to between 24
and 32 carbon atoms.
[0083] Appropriate hosts for producing surfactants can be either
eukaryotic or prokaryotic microorganisms. Exemplary hosts include
Arthrobacter AK 19, Rhodotorula glutinins, Acinobacter sp strain
M-1, Arabidopsis thalania, or Candida lipolytica, Saccharomyces
cerevisiae, and E. coli engineered to express acetyl CoA
carboxylase. Hosts which demonstrate an innate ability to
synthesize high levels of surfactant precursors in the form of
lipids and oils, such as Rhodococcus opacus, Arthrobacter AK 19,
Rhodotorula glutinins E. coli engineered to express acetyl CoA
carboxylase, and other oleaginous bacteria, yeast, and fungi can
also be used.
[0084] E. Fatty Alcohols to Fatty Esters
[0085] Production hosts can be engineered using known polypeptides
to produce fatty esters of various lengths. One method of making
fatty esters includes increasing the expression of, or expressing
more active forms of, one or more alcohol O-acetyltransferase
peptides (EC 2.3.1.84). These peptides catalyze the reaction of
acetyl-CoA and an alcohol to form CoA and an acetic ester. In some
examples the alcohol O-acetyltransferase peptides can be expressed
in conjunction with selected thioesterase peptides, FAS peptides
and fatty alcohol forming peptides, thus, allowing the carbon chain
length, saturation and degree of branching to be controlled. In
some cases the bkd operon can be coexpressed to enable branched
fatty acid precursors to be produced.
[0086] As used herein, alcohol O-acetyltransferase peptides include
peptides in enzyme classification number EC 2.3.1.84, as well as
any other peptide capable of catalyzing the conversion of
acetyl-CoA and an alcohol to form CoA and an acetic ester.
Additionally, one of ordinary skill in the art will appreciate that
alcohol O-acetyltransferase peptides will catalyze other reactions
as well, for example some alcohol O-acetyltransferase peptides will
accept other substrates in addition to fatty alcohols or acetyl-CoA
thiosester i.e such as other alcohols and other acyl-CoA
thioesters. Such non-specific or divergent specificity alcohol
O-acetyltransferase peptides are, therefore, also included. Alcohol
O-acetyltransferase peptide sequences are publicly available.
Exemplary GenBank Accession Numbers are provided in FIG. 10. Assays
for characterizing the activity of a particular alcohol
O-acetyltransferase peptides are well known in the art. Engineered
O-acetyltransferases and O-acyltransferases can be also created
that have new activities and specificities for the donor acyl group
or acceptor alcohol moiety. Engineered enzymes could be generated
through rational and evolutionary approaches well documented in the
art.
[0087] F. Acyl-CoA to Fatty Esters (Bbiodiesels and Waxes)
[0088] Production hosts can be engineered using known peptides to
produce fatty acid esters from acyl-CoA and alcohols. In some
examples the alcohols are provided in the fermentation media and in
other examples the production host can provide the alcohol as
described herein. One of ordinary skill in the art will appreciate
that structurally, fatty acid esters have an A and a B side. As
described herein, the A side of the ester is used to describe the
carbon chain contributed by the alcohol, and the B side of the
ester is used to describe the carbon chain contributed by the
acyl-CoA. Either chain can be saturated or unsaturated, branched or
unbranched. The production host can be engineered to produce fatty
alcohols or short chain alcohols. The production host can also be
engineered to produce specific acyl-CoA molecules. As used herein
fatty acid esters are esters derived from a fatty acyl-thioester
and an alcohol, wherein the A side and the B side of the ester can
vary in length independently. Generally, the A side of the ester is
at least 1, 2, 3, 4, 5, 6, 7, or 8 carbons in length, while the B
side of the ester is 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26
carbons in length. The A side and the B side can be straight chain
or branched, saturated or unsaturated.
[0089] The production of fatty esters, including waxes from
acyl-CoA and alcohols can be engineered using known polypeptides.
As used herein waxes are long chain fatty acid esters, wherein the
A side and the B side of the ester can vary in length
independently. Generally, the A side of the ester is at least 8,
10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. Similarly
the B side of the ester is at least 8, 10, 12, 14, 16, 18, 20, 22,
24, or 26 carbons in length. The A side and the B side can be
mono-, di-, tri-unsaturated. The production of fatty esters,
including waxes from acyl-CoA and alcohols can be engineered using
known polypeptides. One method of making fatty esters includes
increasing the expression of or expressing more active forms of one
or more wax synthases (EC 2.3.1.75).
[0090] As used herein, wax synthases includes peptides in enzyme
classification number EC 2.3.1.75, as well as any other peptide
capable of catalyzing the conversion of an acyl-thioester to fatty
esters. Additionally, one of ordinary skill in the art will
appreciate that some wax synthase peptides will catalyze other
reactions as well, for example some wax synthase peptides will
accept short chain acyl-CoAs and short chain alcohols to produce
fatty esters. Such non-specific wax synthases are, therefore, also
included. Wax synthase peptide sequences are publicly available.
Exemplary GenBank Accession Numbers are provided in FIG. 10.
Methods to identify wax synthase activity are provided in U.S. Pat.
No. 7,118,896, which is herein incorporated by reference.
[0091] In particular examples, if the desired product is a fatty
ester based biofuel, the microorganism is modified so that it
produces a fatty ester generated from a renewable energy source.
Such a microorganism includes an exongenous DNA sequence encoding a
wax ester synthase that is expressed so as to confer upon said
microorganism the ability to synthesize a saturated, unsaturated,
or branched fatty ester from a renewable energy source. 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 ADP 1 (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.
[0092] Additional sources of heterologous DNA encoding wax
synthesis proteins useful in fatty ester production include, but
are not limited to, Mortierella alpina (for example ATCC 32222),
Crytococcus curvatus, (also referred to as Apiotricum curvatum),
Alcanivorax jadensis (for example T9T=DSM 12718=ATCC 700854),
Acinetobacter sp. HO1-N, (for example ATCC 14987) and Rhodococcus
opacus (for example PD630, DSMZ 44193).
[0093] The methods of described herein permit production of fatty
esters of varied length. In one example, the fatty ester product is
a saturated or unsaturated fatty ester product having a carbon atom
content between 24 and 46 carbon atoms. In one embodiment, the
fatty ester product has a carbon atom content between 24 and 32
carbon atoms. In another embodiment the fatty ester product has a
carbon content of 14 and 20 carbons. In another embodiment the
fatty ester is the methyl ester of C18:1. In another embodiment the
fatty acid ester is the ethyl ester of C16:1. In another embodiment
the fatty ester is the methyl ester of C16:1. In another embodiment
the fatty acid ester is octadecyl ester of octanol.
[0094] Useful hosts for producing fatty esters can be either
eukaryotic or prokaryotic microorganisms. In some embodiments such
hosts include, but are not limited to, Saccharomyces cerevisiae,
Candida lipolytica, E. coli Arthrobacter AK 19, Rhodotorula
glutinins, Acinobacter sp strain M-1, Candida lipolytica and other
oleaginous microorganisms.
[0095] In one example the wax ester synthase from Acinetobacter sp.
ADP1 at locus AAO17391 (described in Kalscheuer and Steinbuchel, J.
Biol. Chem. 278:8075-8082, 2003, herein incorporated by reference)
is used. In another example the wax ester synthase from Simmondsia
chinensis, at locus AAD38041 is used.
[0096] Optionally a wax ester exporter such as a member of the FATP
family can be used to facilitate the release of waxes or esters
into the extracellular environment. One example of a wax ester
exporter that can be used is fatty acid (long chain) transport
protein CG7400-PA, isoform A from Drosophila melanogaster, at locus
NP.sub.--524723.
[0097] G. Acyl-ACP, Acyl-CoA to Hydrocarbon
[0098] A diversity of microorganisms are known to produce
hydrocarbons, such as alkanes, olefins, and isoprenoids. Many of
these hydrocarbons are derived from fatty acid biosynthesis. The
production of these hydrocarbons can be controlled by controlling
the genes associated with fatty acid biosynthesis in the native
hosts. For example, hydrocarbon biosynthesis in the algae
Botryococcus braunii occurs through the decarbonylation of fatty
aldehydes. The fatty aldehydes are produced by the reduction of
fatty acyl--thioesters by fatty acyl-CoA reductase. Thus, the
structure of the final alkanes can be controlled by engineering B.
braunii to express specific genes, such as thioesterases, which
control the chain length of the fatty acids being channeled into
alkane biosynthesis. Expressing the enzymes that result in branched
chain fatty acid biosynthesis in B. braunii will result in the
production of branched chain alkanes. Introduction of genes
effecting the production of desaturation of fatty acids will result
in the production of olefins. Further combinations of these genes
can provide further control over the final structure of the
hydrocarbons produced. To produce higher levels of the native or
engineered hydrocarbons, the genes involved in the biosynthesis of
fatty acids and their precursors or the degradation to other
products can be expressed, overexpressed, or attenuated. Each of
these approaches can be applied to the production of alkanes in
Vibrio furnissi M1 and its functional homologues, which produces
alkanes through the reduction of fatty alcohols (see above for the
biosynthesis and engineering of fatty alcohol production). Each of
these approaches can also be applied to the production of the
olefins produced by many strains of Micrococcus leuteus,
Stenotrophomonas maltophilia, Jeogalicoccus sp. (ATCC8456), and
related microorganisms. These microorganisms produce long chain
internal olefins that are derived from the head to head
condensation of fatty acid precursors. Controlling the structure
and level of the fatty acid precursors using the methods described
herein will result in formation of olefins of different chain
length, branching, and level of saturation.
[0099] Hydrocarbons can also be produced using evolved
oxido/reductases for the reduction of primary alcohols. Primary
fatty alcohols are known to be used to produce alkanes in
microorganisms such as Vibrio furnissii M1 (Myong-Ok, J.
Bacterial., 187:1426-1429, 2005). An NAD(P)H dependent
oxido/reductase is the responsible catalyst. Synthetic NAD(P)H
dependent oxidoreductases can be produced through the use of
evolutionary engineering and be expressed in production hosts to
produce fatty acid derivatives. One of ordinary skill in the art
will appreciate that the process of "evolving" a fatty alcohol
reductase to have the desired activity is well known (Kolkman and
Stemmer Nat Biotechnol. 19:423-8, 2001, Ness et al., Adv Protein
Chem. 55:261-92, 2000, Minshull and Stemmer Curr Opin Chem Biol.
3:284-90, 1999, Huisman and Gray Curr Opin Biotechnol. August;
13:352-8, 2002, and see U.S. patent application 2006/0195947). A
library of NAD(P)H dependent oxidoreductases is generated by
standard methods, such as error prone PCR, site-specific random
mutagenesis, site specific saturation mutagenesis, or site directed
specific mutagenesis. Additionally, a library can be created
through the "shuffling" of naturally occurring NAD(P)H dependent
oxidoreductase encoding sequences. The library is expressed in a
suitable host, such as E. coli. Individual colonies expressing a
different member of the oxido/reductase library is then analyzed
for its expression of an oxido/reductase that can catalyze the
reduction of a fatty alcohol. For example, each cell can be assayed
as a whole cell bioconversion, a cell extract, a permeabilized
cell, or a purified enzyme. Fatty alcohol reductases are identified
by the monitoring the fatty alcohol dependent oxidation of NAD(P)H
spectrophotometrically or fluorometrically. Production of alkanes
is monitored by GC/MS, TLC, or other methods. An oxido/reductase
identified in this manner is used to produce alkanes, alkenes, and
related branched hydrocarbons. This is achieved either in vitro or
in vivo. The later is achieved by expressing the evolved fatty
alcohol reductase gene in an organism that produces fatty alcohols,
such as those described herein. The fatty alcohols act as
substrates for the alcohol reductase which would produce alkanes.
Other oxidoreductases can be also engineered to catalyze this
reaction, such as those that use molecular hydrogen, glutathione,
FADH, or other reductive coenzymes.
II. Genetic Engineering of Production Strain to increase Fatty Acid
Derivative Production
[0100] Heterologous DNA sequences involved in a biosynthetic
pathway for the production of fatty acid derivatives can be
introduced stably or transiently into a host cell using techniques
well known in the art for example electroporation, calcium
phosphate precipitation, DEAE-dextran mediated transfection,
liposome-mediated transfection, conjugation, transduction, and the
like. For stable transformation, a DNA sequence can further include
a selectable marker, such as, antibiotic resistance, for example
resistance to neomycin, tetracycline, chloramphenicol, kanamycin,
genes that complement auxotrophic deficiencies, and the like.
[0101] Various embodiments of this disclosure utilize an expression
vector that includes a heterologous DNA sequence encoding a protein
involved in a metabolic or biosynthetic pathway. Suitable
expression vectors include, but are not limited to, viral vectors,
such as baculovirus vectors, phage vectors, such as bacteriophage
vectors, plasmids, phagemids, cosmids, fosmids, bacterial
artificial chromosomes, viral vectors (e.g. viral vectors based on
vaccinia virus, poliovirus, adenovirus, adeno-associated virus,
SV40, herpes simplex virus, and the like), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as E.
coli, Pseudomonas pisum and Saccharomyces cerevisiae).
[0102] Useful expression vectors can include one or more selectable
marker genes to provide a phenotypic trait for selection of
transformed host cells. The selectable marker gene encodes a
protein necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selectable marker gene
will not survive in the culture medium. Typical selection genes
encode proteins that (a) confer resistance to antibiotics or other
toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline,
(b) complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. In alternative embodiments, the
selectable marker gene is one that encodes dihydrofolate reductase
or confers neomycin resistance (for use in eukaryotic cell
culture), or one that confers tetracycline or ampicillin resistance
(for use in a prokaryotic host cell, such as E. coli).
[0103] The biosynthetic pathway gene product-encoding DNA sequence
in the expression vector is operably linked to an appropriate
expression control sequence, (promoters, enhancers, and the like)
to direct synthesis of the encoded gene product. Such promoters can
be derived from microbial or viral sources, including CMV and SV40.
Depending on the host/vector system utilized, any of a number of
suitable transcription and translation control elements, including
constitutive and inducible promoters, transcription enhancer
elements, transcription terminators, etc. can be used in the
expression vector (see e.g., Bitter et al., Methods in Enzymology,
153:516-544, 1987).
[0104] Suitable promoters for use in prokaryotic host cells
include, but are not limited to, promoters capable of recognizing
the T4, T3, Sp6 and T7 polymerases, the P.sub.R and P.sub.L
promoters of bacteriophage lambda, the trp, recA, heat shock, and
lacZ promoters of E. coli, the alpha-amylase and the sigma-specific
promoters of B. subtilis, the promoters of the bacteriophages of
Bacillus, Streptomyces promoters, the int promoter of bacteriophage
lambda, the bla promoter of the beta-lactamase gene of pBR322, and
the CAT promoter of the chloramphenicol acetyl transferase gene.
Prokaryotic promoters are reviewed by Glick, J. Ind. Microbiol.
1:277, 1987; Watson et al., MOLECULAR BIOLOGY OF THE GENE, 4th Ed.,
Benjamin Cummins (1987); and Sambrook et al., supra.
[0105] Non-limiting examples of suitable eukaryotic promoters for
use within a eukaryotic host are viral in origin and include the
promoter of the mouse metallothionein I gene (Hamer et al., J. Mol.
Appl. Gen. 1:273, 1982); the TK promoter of Herpes virus (McKnight,
Cell 31:355, 1982); the SV40 early promoter (Benoist et al., Nature
(London) 290:304, 1981); the Rous sarcoma virus promoter; the
cytomegalovirus promoter (Foecking et al., Gene 45:101, 1980); the
yeast gal4 gene promoter (Johnston, et al., PNAS (USA) 79:6971,
1982; Silver, et al., PNAS (USA) 81:5951, 1984); and the IgG
promoter (Orlandi et al., PNAS (USA) 86:3833, 1989).
[0106] The microbial host cell can be genetically modified with a
heterologous DNA sequence encoding a biosynthetic pathway gene
product that is operably linked to an inducible promoter. Inducible
promoters are well known in the art. Suitable inducible promoters
include, but are not limited to promoters that are affected by
proteins, metabolites, or chemicals. These include: a bovine
leukemia virus promoter, a metallothionein promoter, a
dexamethasone-inducible MMTV promoter, a SV40 promoter, a MRP
polIII promoter, a tetracycline-inducible CMV promoter (such as the
human immediate-early CMV promoter) as well as those from the trp
and lac operons.
[0107] In some examples a genetically modified host cell is
genetically modified with a heterologous DNA sequence encoding a
biosynthetic pathway gene product that is operably linked to a
constitutive promoter. Suitable constitutive promoters are known in
the art and include, constitutive adenovirus major late promoter, a
constitutive MPSV promoter, and a constitutive CMV promoter.
[0108] In some examples a modified host cell is one that is
genetically modified with an exongenous DNA sequence encoding a
single protein involved in a biosynthesis pathway. In other
embodiments, a modified host cell is one that is genetically
modified with exongenous DNA sequences encoding two or more
proteins involved in a biosynthesis pathway--for example, the first
and second enzymes in a biosynthetic pathway.
[0109] Where the host cell is genetically modified to express two
or more proteins involved in a biosynthetic pathway, those DNA
sequences can each be contained in a single or in separate
expression vectors. When those DNA sequences are contained in a
single expression vector, in some embodiments, the nucleotide
sequences will be operably linked to a common control element
(e.g., a promoter), e.g., the common control element controls
expression of all of the biosynthetic pathway protein-encoding DNA
sequences in the single expression vector.
[0110] When a modified host cell is genetically modified with
heterologous DNA sequences encoding two or more proteins involved
in a biosynthesis pathway, one of the DNA sequences can be operably
linked to an inducible promoter, and one or more of the DNA
sequences can be operably linked to a constitutive promoter.
[0111] In some embodiments, the intracellular concentration (e.g.,
the concentration of the intermediate in the genetically modified
host cell) of the biosynthetic pathway intermediate can be
increased to further boost the yield of the final product. The
intracellular concentration of the intermediate can be increased in
a number of ways, including, but not limited to, increasing the
concentration in the culture medium of a substrate for a
biosynthetic pathway; increasing the catalytic activity of an
enzyme that is active in the biosynthetic pathway; increasing the
intracellular amount of a substrate (e.g., a primary substrate) for
an enzyme that is active in the biosynthetic pathway; and the
like.
[0112] In some examples the fatty acid derivative 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).
III. Carbon Chain Characteristics
[0113] Using the teachings provided herein a range of products can
be produced. These products include hydrocarbons, fatty alcohols,
fatty acid esters, and waxes. Some of these products are useful as
biofuels and specialty chemicals. These products can be designed
and produced in microorganisms. The products can be produced such
that they contain branch points, levels of saturation, and carbon
chain length, thus, making these products desirable starting
materials for use in many applications (FIG. 10 provides a
description of the various enzymes that can be used alone or in
combination to make various fatty acid derivatives).
[0114] In other examples, the expression of exongenous FAS genes
originating from different species or engineered variants can be
introduced into the host cell to result in the biosynthesis of
fatty acid metabolites structurally different (in length,
branching, degree of unsaturation, etc.) as that of the native
host. These heterologous gene products can be also chosen or
engineered so that they are unaffected by the natural complex
regulatory mechanisms in the host cell and, therefore, function in
a manner that is more controllable for the production of the
desired commercial product. For example the FAS enzymes from
Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces spp,
Ralstonia, Rhodococcus, Corynebacteria, Brevibacteria,
Mycobacteria, oleaginous yeast, and the like can be expressed in
the production host.
[0115] One of ordinary skill in the art will appreciate that when a
production host is engineered to produce a fatty acid from the
fatty acid biosynthetic pathway that contains a specific level of
unsaturation, branching, or carbon chain length the resulting
engineered fatty acid can be used in the production of the fatty
acid derivatives. Hence, fatty acid derivatives generated from the
production host can display the characteristics of the engineered
fatty acid. For example, a production host can be engineered to
make branched, short chain fatty acids, and then using the
teachings provided herein relating to fatty alcohol production
(i.e. including alcohol forming enzymes such as FAR) the production
host produce branched, short chain fatty alcohols. Similarly, a
hydrocarbon can be produced by engineering a production host to
produce a fatty acid having a defined level of branching,
unsaturation, and/or carbon chain length, thus, producing a
homogenous hydrocarbon population. Moreover, when an unsaturated
alcohol, fatty acid ester, or hydrocarbon is desired the fatty acid
biosynthetic pathway can be engineered to produce low levels of
saturated fatty acids and an additional desaturase can be expressed
to lessen the saturated product production.
[0116] A. Saturation
[0117] Production hosts can be engineered to produce unsaturated
fatty acids by engineering the production host to over-express
fabB, or by growing the production 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 resulted in the
production of a significant percentage of unsaturated fatty acids
(de Mendoza et al, J. Biol. Chem., 258:2098-101, 1983). These
unsaturated fatty acids can then be used as intermediates in
production hosts that are engineered to produce fatty acid
derivatives, such as fatty alcohols, esters, waxes, olefins,
alkanes, and the like. One of ordinary skill in the art will
appreciate that by attenuating fabA, or over-expressing FabB and
expressing specific thioesterases (described below), unsaturated
fatty acid derivatives having a desired carbon chain length can be
produced. Alternatively, the repressor of fatty acid biosynthesis,
FabR (Genbank accession NP.sub.--418398), can be deleted, which
will also result in increased unsaturated fatty acid production in
E. coli (Zhang et al., J. Biol. Chem. 277:pp. 15558, 2002.).
Further increase in unsaturated fatty acids may be achieved by
over-expression of FabM (trans-2, cis-3-decenoyl-ACP isomerase,
Genbank accession DAA05501) and controlled expression of FabK
(trans-2-enoyl-ACP reductase II, Genbank accession NP.sub.--357969)
from Streptococcus pneumoniae (Marrakchi et al., J. Biol. Chem.
277: 44809, 2002), while deleting E. coli Fab I ((trans-2-enoyl-ACP
reductase, Genbank accession NP.sub.--415804). Additionally, to
increase the percentage of unsaturated fatty acid esters, the
microorganism can also have fabB (encoding .gamma.-ketoacyl-ACP
synthase I, Accessions: BAA16180, EC:2.3.1.41), Sfa (encoding a
suppressor of fabA, Accession: AAC44390) and gnsA and gnsB (both
encoding secG null mutant suppressors, a.k.a. cold shock proteins,
Accession: ABD18647.1, AAC74076.1) over-expressed.
[0118] In some examples, the endogenous fabF gene can be
attenuated, thus, increasing the percentage of palmitoleate (C
16:1) produced.
[0119] B. Branching Including Cyclic Moieties
[0120] Fatty acid derivatives can be produced that contain branch
points, cyclic moieties, and combinations thereof, using the
teachings provided herein.
[0121] Microorganisms that naturally produce straight fatty acids
(sFAs) can be engineered to produce branched chain fatty acids
(brFAs) by expressing one or more exogenous nucleic acid sequences.
For example, E. coli naturally produces straight fatty acids
(sFAs). To engineer E. coli to produce brFAs, several genes can be
introduced and expressed that provide branched precursors (bkd
operon) and allow initiation of fatty acid biosynthesis from
branched precursors (fabH). Additionally, the organism can express
genes for the elongation of brFAs (e.g. ACP, FabF) and/or deleting
the corresponding E. coli genes that normally lead to sFAs and
would compete with the introduced genes (e.g. FabH, FabF).
[0122] The branched acyl-CoAs 2-methyl-buturyl-CoA, isovaleryl-CoA
and isobuturyl-CoA are the precursors of brFA. In most
brFA-containing microorganisms they are synthesized in two steps
(described in detail below) from branched amino acids (isoleucine,
leucine and valine) (Kadena, Microbiol. Rev. 55: pp. 288, 1991). To
engineer a microorganism to produce brFAs, or to overproduce brFAs,
expression or over-expression of one or more of the enzymes in
these two steps can be engineered. For example, in some instances
the production host may have an endogenous enzyme that can
accomplish one step and therefore, only enzymes involved in the
second step need to be expressed recombinantly.
[0123] The first step in forming branched fatty acids is the
production of the corresponding .alpha.-keto acids by a
branched-chain amino acid aminotransferase. E. coli has such an
enzyme, IlvE (EC 2.6.1.42; Genbank accession YP.sub.--026247). In
some examples, a heterologous branched-chain amino acid
aminotransferase may not be expressed. However, E. coli IlvE or any
other branched-chain amino acid aminotransferase, e.g. ilvE from
Lactococcus lactis (Genbank accession AAF34406), ilvE from
Pseudomonas putida (Genbank accession NP.sub.--745648) or ilvE from
Streptomyces coelicolor (Genbank accession NP.sub.--629657) can be
over-expressed in a host microorganism, should the aminotransferase
reaction turn out to be rate limiting in brFA biosynthesis in the
host organism chosen for fatty acid derivative production.
[0124] The second step, the oxidative decarboxylation of the
.alpha.-ketoacids to the corresponding branched-chain acyl-CoA, is
catalyzed by a branched-chain .alpha.-keto acid dehydrogenase
complexes (bkd; EC 1.2.4.4.) (Denoya et al. J. Bacteria 177:pp.
3504, 1995), which consist of E1.alpha./.beta. (decarboxylase), E2
(dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase)
subunits and are similar to pyruvate and .alpha.-ketoglutarate
dehydrogenase complexes. Table 2 shows potential bkd genes from
several microorganisms, that can be expressed in a production host
to provide branched-chain acyl-CoA precursors. Basically, every
microorganism that possesses brFAs and/or grows on branched-chain
amino acids can be used as a source to isolate bkd genes for
expression in production hosts such as, for example, E. coli.
Furthermore, E. coli has the E3 component (as part of its pyruvate
dehydrogenase complex; 1pd, EC 1.8.1.4, Genbank accession
NP.sub.--414658), it can therefore, be sufficient to only express
the E1 /.beta. and E2 bkd genes.
TABLE-US-00002 TABLE 2 Bkd genes from selected microorganisms
Organism Gene Genbank Accession # Streptomyces coelicolor bkdA1
(E1.alpha.) NP_628006 bkdB1 (E1.beta.) NP_628005 bkdC1 (E2)
NP_638004 Streptomyces coelicolor bkdA2 (E1.alpha.) NP_733618 bkdB2
(E1.beta.) NP_628019 bkdC2 (E2) NP_628018 Streptomyces avermitilis
bkdA (E1a) BAC72074 bkdB (E1b) BAC72075 bkdC (E2) BAC72076
Streptomyces avermitilis bkdF (E1.alpha.) BAC72088 bkdG (E1.beta.)
BAC72089 bkdH (E2) BAC72090 Bacillus subtilis bkdAA (E1.alpha.)
NP_390288 bkdAB (E1.beta.) NP_390288 bkdB (E2) NP_390288
Pseudomonas putida bkdA1 (E1.alpha.) AAA65614 bkdA2 (E1.beta.)
AAA65615 bkdC (E2) AAA65617
[0125] In another example, isobuturyl-CoA can be made in a
production host, for example in E. coli through the coexpression of
a crotonyl-CoA reductase (Ccr, EC 1.1.1.9) and isobuturyl-CoA
mutase (large subunit IcmA, EC 5.4.99.2; small subunit IcmB, EC
5.4.99.13) (Han and Reynolds J. Bacteriol. 179:pp. 5157, 1997).
Crotonyl-CoA is an intermediate in fatty acid biosynthesis in E.
coli and other microorganisms. Examples for ccr and icm genes from
selected microorganisms are given in Table 3.
TABLE-US-00003 TABLE 3 Ccr and icm genes from selected
microorganisms Organism Gene Genbank Accession # Streptomyces
coelicolor ccr NP_630556 icmA NP_629554 icmB NP_630904 Streptomyces
cinnamonensis ccr AAD53915 icmA AAC08713 icmB AJ246005
[0126] In addition to expression of the bkd genes (see above), the
initiation of brFA biosynthesis utilizes
.beta.-ketoacyl-acyl-carrier-protein synthase III (FabH, EC
2.3.1.41) with specificity for branched chain acyl CoAs (Li et al.
J. Bacteriol. 187:pp. 3795, 2005). Examples of such FabHs are
listed in Table 4. FabH genes that are involved in fatty acid
biosynthesis of any brFA-containing microorganism can be expressed
in a production host. The Bkd and FabH enzymes from production
hosts that do not naturally make brFA may not support brFA
production and therefore, Bkd and FabH can be expressed
recombinantly. Similarly, the endogenous level of Bkd and FabH
production may not be sufficient to produce brFA, therefore, they
can be over-expressed. Additionally, other components of fatty acid
biosynthesis machinery can be expressed such as acyl carrier
proteins (ACPs) and .beta.-ketoacyl-acyl-carrier-protein synthase
II candidates are acyl carrier proteins (ACPs) and
.beta.-ketoacyl-acyl-carrier-protein synthase II (fabF, EC
2.3.1.41) (candidates are listed in Table 4). In addition to
expressing these genes, some genes in the endogenous fatty acid
biosynthesis pathway may be attenuated in the production host. For
example, in E. coli the most likely candidates to interfere with
brFA biosynthesis are fabH (Genbank accession #NP.sub.--415609)
and/or fabF genes (Genbank accession #NP.sub.--415613).
[0127] 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 Acr1 from Acinetobacter baylyi ADP1 is
coexpressed with a bkd operon, E. coli can synthesize isopentanol,
isobutanol or 2-methyl butanol. Similarly, when Acr1 is coexpressed
with ccr/icm genes, E. coli can synthesize isobutanol.
[0128] In order to convert a production host such as E. coli into
an organism capable of synthesizing .omega.-cyclic fatty acids
(cyFAs), several genes need to be introduced and expressed that
provide the cyclic precursor cyclohexylcarbonyl-CoA (Cropp et al.
Nature Biotech. 18:pp. 980, 2000). The genes listed in Table 4
(fabH, ACP and fabF) can then be expressed to allow initiation and
elongation of .omega.-cyclic fatty acids. Alternatively, the
homologous genes can be isolated from microorganisms that make
cyFAs and expressed in E. coli.
TABLE-US-00004 TABLE 4 FabH, ACP and fabF genes from selected
microorganisms with brFAs Genbank Organism Gene Accession #
Streptomyces coelicolor fabH1 NP_626634 ACP NP_626635 fabF
NP_626636 Streptomyces avermitilis fabH3 NP_823466 fabC3 (ACP)
NP_823467 fabF NP_823468 Bacillus subtilis fabH_A NP_389015 fabH_B
NP_388898 ACP NP_389474 fabF NP_389016 Stenotrophomonas maltophilia
SmalDRAFT_0818 ZP_01643059 (FabH) SmalDRAFT_0821 ZP_01643063 (ACP)
SmalDRAFT_0822 ZP_01643064 (FabF) Legionella pneumophila FabH
YP_123672 ACP YP_123675 fabF YP_123676
[0129] Expression of the following genes are sufficient to provide
cyclohexylcarbonyl-CoA in E. coli: ansJ, ansK, ansL, chcA and ansM
from the ansatrienin gene cluster of Streptomyces collinus (Chen et
al., Eur. J. Biochem. 261:pp. 1999, 1999) or plmJ, plmK, plmL, chcA
and plmM from the phoslactomycin B gene cluster of Streptomyces sp.
HK803 (Palaniappan et al., J. Biol. Chem. 278:pp. 35552, 2003)
together with the chcB gene (Patton et al. Biochem., 39:pp. 7595,
2000) from S. collinus, S. avermitilis or S. coelicolor (see Table
5 for Genbank accession numbers).
TABLE-US-00005 TABLE 5 Genes for the synthesis of
cyclohexylcarbonyl-CoA Organism Gene Genbank Accession #
Streptomyces collinus ansJK U72144* ansL chcA ansL chcB AF268489
Streptomyces sp. HK803 pmlJK AAQ84158 pmlL AAQ84159 chcA AAQ84160
pmlM AAQ84161 Streptomyces coelicolor chcB/caiD NP_629292
Streptomyces avermitilis chcB/caiD NP_629292 Only chcA is annotated
in Genbank entry U72144, ansJKLM are according to Chen et al. (Eur.
J. Biochem. 261: pp. 1999, 1999)
[0130] The genes listed in Table 4 (fabH, ACP and fabF) are
sufficient to allow initiation and elongation of .omega.-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 pm1JKLM/chcAB genes from Table 5 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. Table 6 lists selected
microorganisms that contain .omega.-cyclic fatty acids.
TABLE-US-00006 TABLE 6 Examples of microorganisms that contain
.omega.-cyclic fatty acids Organism Reference Curtobacterium
pusillum ATCC19096 Alicyclobacillus acidoterrestris ATCC49025
Alicyclobacillus acidocaldarius ATCC27009 Alicyclobacillus
cycloheptanicum* Moore, J. Org. Chem. 62: pp. 2173, 1997. *uses
cycloheptylcarbonyl-CoA and not cyclohexylcarbonyl-CoA as precursor
for cyFA biosynthesis
[0131] C. Ester Characteristics
[0132] One of ordinary skill in the art will appreciate that an
ester includes an A side and a B side. As described herein, the B
side is contributed by a fatty acid produced from de novo synthesis
in the host organism. In some instances where the host is
additionally engineered to make alcohols, including fatty alcohols,
the A side is also produced by the host organism. In yet other
examples the A side can be provided in the medium. As described
herein, by selecting the desired thioesterase genes the B side, and
when fatty alcohols are being made the A side, can be designed to
be have certain carbon chain characteristics. These characteristics
include points of unsaturation, branching, and desired carbon chain
lengths. Exemplary methods of making long chain fatty acid esters,
wherein the A and B side are produced by the production host are
provided in Example 6, below. Similarly, Example 5 provides methods
of making medium chain fatty acid esters. When both the A and B
side are contributed by the production host and they are produced
using fatty acid biosynthetic pathway intermediates they will have
similar carbon chain characteristics. For example, at least 50%,
60%, 70%, or 80% of the fatty acid esters produced will have A
sides and B sides that vary by 6, 4, or 2 carbons in length. The A
side and the B side will also display similar branching and
saturation levels.
[0133] In addition to producing fatty alcohols for contribution to
the A side, the host can produce other short chain alcohols such as
ethanol, propanol, isopropanol, isobutanol, and butanol for
incorporation on the A side using techniques well known in the art.
For example, butanol can be made by the host organism. To create
butanol producing cells, the LS9001 strain (described in Example 1,
below) can be further engineered to express atoB (acetyl-CoA
acetyltransferase) from Escherichia 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-alchol dehydrogenase of Clostridium acetobutylicum in the
pBAD24 expression vector under the prpBCDE promoter system.
Similarly, ethanol can be produced in a production host using the
methods taught by Kalscheuer et al., Microbiology 152:2529-2536,
2006, which is herein incorporated by reference.
IV. Fermentation
[0134] The production and isolation of fatty acid derivatives can
be enhanced by employing specific fermentation techniques. One
method for maximizing production while reducing costs is increasing
the percentage of the carbon source that is converted to
hydrocarbon products. 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 microorganisms 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
(reviewed in Camilli and Bassler Science 311:1113, 2006; Venturi
FEMS Microbio Rev 30:274-291, 2006; and Reading and Sperandio FEMS
Microbiol Lett 254:1-11, 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 et al., J. of Bact. 182:1127,
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
product producing genes would be activated, thus minimizing the
need for replication and maintenance pathways to be used while the
fatty acid derivative is being made.
[0135] The percentage of input carbons converted to hydrocarbon
products is a cost driver. The more efficient (i.e. the higher the
percentage), the less expensive the process. For oxygen-containing
carbon sources (i.e. glucose and other carbohydrate based sources),
the oxygen must be released in the form of carbon dioxide. For
every 2 oxygen atoms released, a carbon atom is also released
leading to a maximal theoretical metabolic efficiency of .about.34%
(w/w) (for fatty acid derived products). This figure, however,
changes for other hydrocarbon products and carbon sources. Typical
efficiencies in the literature are .about.<5%. Engineered
microorganisms which produce hydrocarbon products can have greater
than 1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one example
microorganisms will exhibit an efficiency of about 10% to about
25%. In other examples, such microorganisms will exhibit an
efficiency of about 25% to about 30%, and in other examples such
microorganisms will exhibit >30% efficiency.
[0136] In some examples where the final product is released from
the cell, a continuous process can be employed. In this approach, a
reactor with organisms producing fatty acid derivatives can be
assembled in multiple ways. In one example, a portion of the media
is removed and let to sit. Fatty acid derivatives are separated
from the aqueous layer, which will in turn, be returned to the
fermentation chamber.
[0137] In one example, the fermentation chamber will enclose a
fermentation that is undergoing a continuous reduction. In this
instance, a stable reductive environment would be created. The
electron balance would be maintained by the release of carbon
dioxide (in gaseous form). Efforts to augment the NAD/H and NADP/H
balance can also facilitate in stabilizing the electron
balance.
[0138] The availability of intracellular NADPH can be also enhanced
by engineering the production host to express an NADH:NADPH
transhydrogenase. The expression of one or more NADH:NADPH
transhydrogenase converts the NADH produced in glycolysis to NADPH
which enhances the production of fatty acid derivatives.
[0139] Disclosed herein is a system for continuously producing and
exporting fatty acid derivatives 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
derivatives. Thus, in some embodiments an exogenous DNA sequence
encoding an ABC transporter will be functionally expressed by the
recombinant host microorganism, so that the microorganism exports
the fatty acid derivative into the culture medium. In one example,
the ABC transporter is an ABC transporter from Caenorhabditis
elegans, Arabidopsis thalania, Alkaligenes eutrophus or Rhodococcus
erythropolis (locus AAN73268). In another example, the ABC
transporter is an ABC transporter chosen from CER5 (locuses
At1g51500 or AY734542), AtMRP5, AmiS2 and AtPGP1. In some examples,
the ABC transporter is CER5. In yet another example, the CER5 gene
is from Arabidopsis (locuses At1g51500, AY734542, At3g21090 and
At1g51460).
[0140] The transport protein, for example, can also be an efflux
protein selected from: AcrAB, TolC and AcrEF from E. coli, or
tll1618, tll1619 and tll0139 from Thermosynechococcus elongatus
BP-1.
[0141] 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 or any one of the mammalian FATP's. 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, could be
inverted while maintaining the same codons for each particular
amino acid. The identification of these regions is well known in
the art.
[0142] Production hosts can also be chosen for their endogenous
ability to release fatty acid derivatives. The efficiency of
product production and release into the fermentation broth can be
expressed as a ratio intracellular product to extracellular
product. In some examples the ratio can be 5:1, 4:1, 3:1, 2:1, 1:1,
1:2, 1:3, 1:4, or 1:5.
[0143] The production host can be additionally engineered to
express recombinant cellulosomes, such as those described in PCT
application number PCT/US2007/003736, which will allow the
production host to use cellulosic material as a carbon source. For
example, the production host can be additionally engineered to
express invertases (EC 3.2.1.26) so that sucrose can be used as a
carbon source.
[0144] Similarly, the production host can be engineered using the
teachings described in U.S. Pat. Nos. 5,000,000, 5,028,539,
5,424,202, 5,482,846, and 5,602,030 to Ingram et al. so that the
production host can assimilate carbon efficiently and use
cellulosic materials as carbons sources.
IV. Post Production Processing
[0145] The fatty acid derivatives produced during fermentation can
be separated from the fermentation media. Any technique known for
separating fatty acid derivatives from aqueous media can be used.
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 a fatty acid derivative, allowing the
derivative to collect in an organic phase and separating the
organic phase from the aqueous fermentation broth. This method can
be practiced in both a batch and continuous fermentation
setting.
[0146] Bi-phasic separation uses the relative immisiciblity of
fatty acid derivatives to facilitate separation. Immiscible refers
to the relative inability of a compound to dissolve in water and is
defined by the compounds partition coefficient. The partition
coefficient, P, is defined as the equilibrium concentration of
compound in an organic phase (in a bi-phasic system the organic
phase is usually the phase formed by the fatty acid derivative
during the production process, however, in some examples an organic
phase can be provided (such as a layer of octane to facilitate
product separation) divided by the concentration at equilibrium in
an aqueous phase (i.e. fermentation broth). When describing a two
phase system the P is usually discussed in terms of logP. A
compound with a logP of 10 would partition 10:1 to the organic
phase, while a compound of logP of 0.1 would partition 10:1 to the
aqueous phase. One or ordinary skill in the art will appreciate
that by choosing a fermentation broth and the organic phase such
that the fatty acid derivative being produced has a high logP
value, the fatty acid derivative will separate into the organic
phase, even at very low concentrations in the fermentation
vessel.
[0147] The fatty acid derivatives produced by the methods described
herein will be relatively immiscible in the fermentation broth, as
well as in the cytoplasm. Therefore, the fatty acid derivative will
collect in an organic phase either intracellularly or
extracellularly. The collection of the products in an organic phase
will lessen the impact of the fatty acid derivative on cellular
function and will allow the production host to produce more
product. Stated another way, the concentration of the fatty acid
derivative will not have as significant of an impact on the host
cell.
[0148] The fatty alcohols, fatty acid esters, waxes, and
hydrocarbons produced as described herein allow for the production
of homogeneous compounds wherein at least 60%, 70%, 80%, 90%, or
95% of the fatty alcohols, fatty acid esters, and waxes produced
will 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, for example at
least 60%, 70%, 80%, 90%, or 95% of the fatty alcohols, fatty acid
esters, hydrocarbons and waxes will be mono-, di-, or
tri-unsaturated. These compounds can be used directly as fuels,
personal care additives, nutritional supplements. These compounds
can also be used as feedstock for subsequent reactions for example
transesterification, hydrogenation, catalytic cracking via either
hydrogenation, pyrolisis, or both or epoxidations reactions to make
other products.
V. Fuel Compositions
[0149] The fatty acid derivatives described herein can be used as
fuel. One of ordinary skill in the art will appreciate that
depending upon the intended purpose of the fuel different fatty
acid derivatives can be produced and used. For example, for
automobile fuel that is intended to be used in cold climates a
branched fatty acid derivative may be desirable and using the
teachings provided herein, branched hydrocarbons, fatty acid
esters, and alcohols can be made. Using the methods described
herein fuels comprising relatively homogeneous fatty acid
derivatives that have desired fuel qualities can be produced. Such
fuels can be characterized by carbon fingerprinting, their lack of
impurities when compared to petroleum derived fuels or bio-diesel
derived from triglycerides and, moreover, the fatty acid derivative
based fuels can be combined with other fuels or fuel additives to
produce fuels having desired properties.
[0150] A. Carbon Fingerprinting
[0151] Biologically produced fatty acid derivatives represent a new
feedstock for fuels, such as alcohols, diesel and gasoline. Some
biofuels made using fatty acid derivatives have not been produced
from renewable sources and as such, are new compositions of matter.
These new fuels can be distinguished from fuels derived form
petrochemical carbon on the basis of dual carbon-isotopic
fingerprinting. Additionally, the specific source of biosourced
carbon (e.g. glucose vs. glycerol) can be determined by dual
carbon-isotopic fingerprinting (see, U.S. Pat. No. 7,169,588, which
is herein incorporated by reference).
[0152] This method usefully distinguishes chemically-identical
materials, and apportions carbon in products by source (and
possibly year) of growth of the biospheric (plant) component. The
isotopes, .sup.14C and .sup.13C, bring complementary information to
this problem. The radiocarbon dating isotope (.sup.14C), with its
nuclear half life of 5730 years, clearly allows one to apportion
specimen carbon between fossil ("dead") and biospheric ("alive")
feedstocks [Currie, L. A. "Source Apportionment of Atmospheric
Particles," Characterization of Environmental Particles, J. Buffle
and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental
Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3 74].
The basic assumption in radiocarbon dating is that the constancy of
.sup.14C concentration in the atmosphere leads to the constancy of
.sup.14C in living organisms. When dealing with an isolated sample,
the age of a sample can be deduced approximately by the
relationship t=(-5730/0.693)ln(A/A.sub.O) (Equation 5) where t=age,
5730 years is the half-life of radiocarbon, and A and A.sub.O are
the specific .sup.14C activity of the sample and of the modem
standard, respectively [Hsieh, Y., Soil Sci. Soc. Am J., 56, 460,
(1992)]. However, because of atmospheric nuclear testing since 1950
and the burning of fossil fuel since 1850, .sup.14C has acquired a
second, geochemical time characteristic. Its concentration in
atmospheric CO2--and hence in the living biosphere--approximately
doubled at the peak of nuclear testing, in the mid-1960s. It has
since been gradually returning to the steady-state cosmogenic
(atmospheric) baseline isotope rate (.sup.14C /.sup.12C) of ca.
1.2.times.10.sup.12, with an approximate relaxation "half-life" of
7-10 years. (This latter half-life must not be taken literally;
rather, one must use the detailed atmospheric nuclear input/decay
function to trace the variation of atmospheric and biospheric
.sup.14C since the onset of the nuclear age.) It is this latter
biospheric .sup.14C time characteristic that holds out the promise
of annual dating of recent biospheric carbon. .sup.14C can be
measured by accelerator mass spectrometry (AMS), with results given
in units of "fraction of modern carbon" (f.sub.M). f.sub.M is
defined by National Institute of Standards and Technology (NIST)
Standard Reference Materials (SRMs) 4990B and 4990C, known as
oxalic acids standards HOxI and HOxII, respectively. The
fundamental definition relates to 0.95 times the .sup.14C/.sup.12C
isotope ratio HOxI (referenced to AD 1950). This is roughly
equivalent to decay-corrected pre-Industrial Revolution wood. For
the current living biosphere (plant material), f.sub.M approx
1.1.
[0153] The stable carbon isotope ratio (.sup.13C/.sup.12C) provides
a complementary route to source discrimination and apportionment.
The .sup.13C/.sup.12C ratio in a given biosourced material is a
consequence of the .sup.13C/.sup.12C ratio in atmospheric carbon
dioxide at the time the carbon dioxide is fixed and also reflects
the precise metabolic pathway. Regional variations also occur.
Petroleum, C3 plants (the broadleaf), C.sub.4 plants (the grasses),
and marine carbonates all show significant differences in
.sup.13C/.sup.12C and the corresponding delta.sup.13C values.
Furthermore, lipid matter of C3 and C4 plants analyze differently
than materials derived from the carbohydrate components of the same
plants as a consequence of the metabolic pathway. Within the
precision of measurement, .sup.13C shows large variations due to
isotopic fractionation effects, the most significant of which for
the instant invention is the photosynthetic mechanism. The major
cause of differences in the carbon isotope ratio in plants is
closely associated with differences in the pathway of
photosynthetic carbon metabolism in the plants, particularly the
reaction occurring during the primary carboxylation, i.e., the
initial fixation of atmospheric CO.sub.2. Two large classes of
vegetation are those that incorporate the "C3" (or Calvin-Benson)
photosynthetic cycle and those that incorporate the "C4" (or
Hatch-Slack) photosynthetic cycle. C3 plants, such as hardwoods and
conifers, are dominant in the temperate climate zones. In C3
plants, the primary CO.sub.2 fixation or carboxylation reaction
involves the enzyme ribulose-1,5-diphosphate carboxylase and the
first stable product is a 3-carbon compound. C4 plants, on the
other hand, include such plants as tropical grasses, corn and sugar
cane. In C4 plants, an additional carboxylation reaction involving
another enzyme, phosphoenol-pyruvate carboxylase, is the primary
carboxylation reaction. The first stable carbon compound is a
4-carbon acid which is subsequently decarboxylated. The CO.sub.2
thus released is refixed by the C3 cycle.
[0154] Both C4 and C3 plants exhibit a range of .sup.13C/.sup.12C
isotopic ratios, but typical values are ca. -10 to -14 per mil (C4)
and -21 to -26 per mil (C3) [Weber et al., J. Agric. Food Chem.,
45, 2942 (1997)]. Coal and petroleum fall generally in this latter
range. The .sup.13C measurement scale was originally defined by a
zero set by pee dee belemnite (PDB) limestone, where values are
given in parts per thousand deviations from this material. The
".DELTA..sup.13C", values are in parts per thousand (per mil),
abbreviated %, and are calculated as follows:
.delta. 13 C .ident. ( 13 C / 12 C ) sample - ( 13 C / 12 C )
standard ( 13 C / 12 C ) standard .times. 100 % ( Equation 6 )
##EQU00001##
Since the PDB reference material (RM) has been exhausted, a series
of alternative RMs have been developed in cooperation with the
IAEA, USGS, NIST, and other selected international isotope
laboratories. Notations for the per mil deviations from PDB is
.DELTA..sup.13C. Measurements are made on CO.sub.2 by high
precision stable ratio mass spectrometry (IRMS) on molecular ions
of masses 44, 45 and 46.
[0155] The fatty acid derivatives and the associated biofuels,
chemicals, and mixtures may be completely distinguished from their
petrochemical derived counterparts on the basis of .sup.14C (fM)
and dual carbon-isotopic fingerprinting, indicating new
compositions of matter.
[0156] The fatty acid derivatives described herein have utility in
the production of biofuels and chemicals. The new fatty acid
derivative based product compositions provided by the instant
invention additionally may be distinguished on the basis of dual
carbon-isotopic fingerprinting from those materials derived solely
from petrochemical sources. The ability to distinguish these
products is beneficial in tracking these materials in commerce. For
example, fuels or chemicals comprising both "new" and "old" carbon
isotope profiles may be distinguished from fuels and chemicals made
only of "old" materials. Hence, the instant materials may be
followed in commerce on the basis of their unique profile and for
the purposes of defining competition, and for determining shelf
life.
[0157] In some examples a biofuel composition is made that includes
a fatty acid derivative having .delta..sup.13C of from about -10.9
to about -15.4, wherein the fatty acid derivative accounts for at
least about 85% of biosourced material (derived from a renewable
resource such as cellulosic materials and sugars) in the
composition. In other examples, the biofuel composition includes a
fatty acid derivative having the formula
X--(CH(R)).sub.nCH.sub.3 [0158] wherein X represents CH.sub.3,
--CH.sub.2OR.sup.1; --C(O)OR.sup.2; or --C(O)NR.sup.3R.sup.4;
[0159] R is, for each n, independently absent, H or lower
aliphatic; [0160] n is an integer from 8 to 34, such as from 10 to
24; and [0161] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 independently
are selected from H and lower alkyl. Typically, when R is lower
aliphatic, R represents a branched, unbranched or cyclic lower
alkyl or lower alkenyl moiety. Exemplary R groups include, without
limitation, methyl, isopropyl, isobutyl, sec-butyl, cyclopentenyl
and the like. The fatty acid derivative is additionally
characterized as having a .delta..sup.13C of from about -10.9 to
about -15.4; and the fatty acid derivative accounts for at least
about 85% of biosourced material in the composition. In some
examples the fatty acid derivative in the biofuel composition is
characterized by having a fraction of modern carbon
(f.sub.M.sup.14C) of at least about 1.003, 1.010, or 1.5.
[0162] B. Fatty Acid Derivatives
[0163] The centane number (CN), viscosity, melting point, and heat
of combustion for various fatty acid esters have been characterized
in for example, Knothe, Fuel Processing Technology 86:1059-1070,
2005, which is herein incorporated by reference. Using the
teachings provided herein a production host can be engineered to
produce anyone of the fatty acid esters described in the Knothe,
Fuel Processing Technology 86:1059-1070, 2005.
[0164] Alcohols (short chain, long chain, branched or unsaturated)
can be produced by the production hosts described herein. Such
alcohols can be used as fuels directly or they can be used to
create an ester, i.e. the A side of an ester as described above.
Such ester alone or in combination with the other fatty acid
derivatives described herein are useful a fuels.
[0165] Similarly, hydrocarbons produced from the microorganisms
described herein can be used as biofuels. Such hydrocarbon based
fuels can be designed to contain branch points, defined degrees of
saturation, and specific carbon lengths. When used as biofuels
alone or in combination with other fatty acid derivatives the
hydrocarbons can be additionally combined with additives or other
traditional fuels (alcohols, diesel derived from triglycerides, and
petroleum based fuels).
[0166] C. Impurities
[0167] The fatty acid derivatives described herein are useful for
making bio-fuels. These fatty acid derivatives are made directly
from fatty acids and not from the chemical processing of
triglycerides. Accordingly, fuels comprising the disclosed fatty
acid derivatives will contain less of the impurities than are
normally associated with bio-fuels derived from triglycerides, such
as fuels derived from vegetable oils and fats.
[0168] The crude fatty acid derivative bio-fuels described herein
(prior to mixing the fatty acid derivative with other fuels such as
traditional fuels) will contain less transesterification catalyst
than petrochemical diesel or bio-diesel. For example, the fatty
acid derivative can contain less than about 2%, 1.5%, 1.0%, 0.5%,
0.3%, 0.1%, 0.05%, or 0% of a transesterification catalyst or an
impurity resulting from a transesterification catalyst.
Transesterification catalysts include for example, hydroxide
catalysts such as NaOH, KOH, LiOH, and acidic catalysts, such as
mineral acid catalysts and Lewis acid catalysts. Catalysts and
impurities resulting from transesterification catalysts include,
without limitation, tin, lead, mercury, cadmium, zinc, titanium,
zirconium, hafnium, boron, aluminum, phosphorus, arsenic, antimony,
bismuth, calcium, magnesium, strontium, uranium, potassium, sodium,
lithium, and combinations thereof.
[0169] Similarly, the crude fatty acid derivative bio-fuels
described herein (prior to mixing the fatty acid derivative with
other fuels such as petrochemical diesel or bio-diesel) will
contain less glycerol (or glycerin) than bio-fuels made from
triglycerides. For example, the fatty acid derivative can contain
less than about 2%, 1.5%, 1.0%, .5%, .3%, .1%, .05%, or 0%
glycerol.
[0170] The crude biofuel derived from fatty acid derivatives will
also contain less free alcohol (i.e. alcohol that is used to create
the ester) than bio-diesel made from triglycerides. This is in-part
due to the efficiency of utilization of the alcohol by the
production host. For example, the fatty acid derivative will
contain less than about 2%, 1.5%, 1.0%, 0.5%, 0.3%, 0.1%, 0.05%, or
0% free alcohol.
[0171] Biofuel derived from the disclosed fatty acid derivatives
can be additionally characterized by its low concentration of
sulfur compared to petroleum derived diesel. For example, biofuel
derived from fatty acid derivatives can have less than about 2%,
1.5%, 1.0%, 0.5%, 0.3%, 0.1%, 0.05%, or 0% sulfur.
[0172] D. Additives
[0173] Fuel additives are used to enhance the performance of a fuel
or engine. For example, fuel additives can be used to alter the
freezing/gelling point, cloud point, lubricity, viscosity,
oxidative stability, ignition quality, octane level, and flash
point. In the United States, all fuel additives must be registered
with Environmental Protection Agency and companies that sell the
fuel additive and the name of the fuel additive are publicly
available on the agency website and also by contacting the agency.
One of ordinary skill in the art will appreciate that the fatty
acid derivatives described herein can be mixed with one or more
such additives to impart a desired quality.
[0174] One of ordinary skill in the art will also appreciate that
the fatty acid derivatives described herein are can be mixed with
other fuels such as bio-diesel derived from triglycerides, various
alcohols such as ethanol and butanol, and petroleum derived
products such as gasoline. In some examples, a fatty acid
derivative, such as C16:1 ethyl ester or C18:1 ethyl ester, is
produced which has a low gel point. This low gel point fatty acid
derivative is mixed with bio-diesel made from triglycerides to
lessen the overall gelling point of the fuel. Similarly, a fatty
acid derivative such as C16:1 ethyl ester or C18:1 ethyl ester can
be mixed with petroleum derived diesel to provide a mixture that is
at least and often greater than 5% biodiesel. In some examples, the
mixture includes at least 20% or greater of the fatty acid
derivative.
[0175] For example, a biofuel composition can be made that includes
at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95%
of a fatty acid derivative that includes a carbon chain that is
8:0, 10:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3,
20:0, 20:1, 20:2, 20:3, 22:0, 22:1 or 22:3. Such biofuel
compositions can additionally include at least one additive
selected from a cloud point lowering additive that can lower the
cloud point to less than about 5.degree. C., or 0.degree. C., a
surfactant, or a microemulsion, at least about 5%, 10%, 15%, 20%,
30%, 40%, 50%, 60%, 70% or 80%, 85%, 90%, or 95% diesel fuel from
triglycerides, petroleum derived gasoline or diesel fuel from
petroleum.
Examples
[0176] FIG. 1 is a diagram of the FAS pathway showing the enzymes
directly involved in the synthesis of acyl-ACP. To increase the
production of waxes/fatty acid esters, and fatty alcohols one or
more of the enzymes can be over expressed or mutated to reduce
feedback inhibition. Additionally, enzymes that metabolize the
intermediates to make non-fatty acid based products (side
reactions) can be functionally deleted or attenuated to increase
the flux of carbon through the fatty acid biosynthetic pathway.
Examples 1, 2, and 8 below provide exemplary production hosts that
have been modified to increase fatty acid production.
[0177] FIGS. 2, 3 and 4 show biosynthetic pathways that can be
engineered to make fatty alcohols and wax/fatty acid esters,
respectively. As illustrated in FIG. 2 the conversion of each
substrate (acetyl-CoA, malonyl-CoA, acyl-ACP, fatty acid, and
acyl-CoA) to each product (acetyl-CoA, malonyl-CoA, acyl-ACP, fatty
acid, and acyl-CoA) can be accomplished using several different
polypeptides that are members of the enzyme classes indicated. The
Examples below describe microorganisms that have been engineered or
can be engineered to produce specific fatty alcohols and
waxes/fatty acid esters and hydrocarbons.
Example 1
Production Host Construction
[0178] An exemplary production host is LS9001. LS9001 was produced
by modifying C41(DE3) from Overexpress.com (Saint Beausine, France)
to functionally deleting the fadE gene (acyl-CoA
dehydrogenase).
[0179] Briefly, the fadE knock-out strain of E. coli was made using
primers YafV_NotI and Ivry_Ol to amplify about 830 by upstream of
fadE and primers Lpcaf_ol and LpcaR_Bam to amplify about 960 by
downstream of fadE. Overlap PCR was used to create a construct for
in frame deletion of the complete fadE gene. The fadE deletion
construct was cloned into the temperature sensitive plasmid pKOV3,
which contained a SacB gene for counterselection, and a chromosomal
deletion of fadE was made according to the method of Link et al.,
J. Bact. 179:6228-6237, 1997. The resulting strain was not capable
of degrading fatty acids and fatty acyl-CoAs (this functional
deletion is herein designated as .DELTA.fadE)
[0180] Additional modifications that can be included in a
production host include introducing a plasmid carrying the four
genes which are responsible for acetyl-CoA carboxylase activity in
E. coli (accA, B, C, and D, Accessions: NP.sub.--414727,
NP.sub.--417721, NP.sub.--417722, NP.sub.--416819, EC 6.4.1.2). The
accABCD genes were cloned in two steps as bicistronic operons into
the NcoI/HindIII and NdeI/AvrII sites of pACYCDuet-1 (Novagen,
Madison, Wis.) the resulting plasmid was termed pAS004.126.
[0181] Additional modifications that can be included in a
production host include the following: over-expression of aceEF
(encoding the E1p dehydrogase component and the E2p
dihydrolipoamide acyltransferase component of the pyruvate and
2-oxoglutarate dehydrogenase complexes); and
fabH/fabD/fabG/acpP/fabF (encoding FAS) from any organism known in
the art to encode such proteins, including for example E. coli,
Nitrosomonas europaea (ATCC 19718), Bacillus subtilis,
Saccharomyces cerevisiae, Streptomyces spp, Ralstonia, Rhodococcus,
Corynebacteria, Brevibacteria, Mycobacteria, oleaginous yeast, and
the like can be expressed in the production host. Similarly,
production hosts can be engineered to express accABCD (encoding
acetyl co-A carboxylase) from Pisum savitum instead of, or in
addition to, the E. coli homologues. However, when the production
host is also producing butanol it is less desirable to express the
Pisum savitum homologue.
[0182] In some exemplary production hosts, genes can be knocked out
or attenuated using the method of Link, et al., J. Bacteriol.
179:6228-6237, 1997. For example, genes that can be knocked out or
attenuated include gpsA (encoding biosynthetic sn-glycerol
3-phosphate dehydrogenase, accession NP.sub.--418065, EC:
1.1.1.94); ldhA (encoding lactate dehydrogenase, accession
NP.sub.--415898, EC: 1.1.1.28); pflb (encoding formate
acetyltransferase 1, accessions: P09373, EC: 2.3.1.54); adhE
(encoding alcohol dehydrogenase, accessions: CAA47743, EC: 1.1.1.1,
1.2.1.10); pta (encoding phosphotransacetylase, accessions:
NP.sub.--416800, EC: 2.3.1.8); poxB (encoding pyruvate oxidase,
accessions: NP.sub.--415392, EC: 1.2.2.2); ackA (encoding acetate
kinase, accessions: NP.sub.--416799, EC: 2.7.2.1) and combinations
thereof.
[0183] Similarly, the PlsB[D311E] mutation can be introduced into
LS9001 to attenuate PlsB using the method described above for the
fadE deletion. Once introduced, this mutation will decrease the
amount of carbon being diverted to phospholipid production (see,
FIG. 1). Briefly, an allele encoding PlsB[D311E] is made by
replacing the GAC codon for aspartate 311 with a GAA codon for
glutamate. The altered allele is made by gene synthesis and the
chromosomal plsB wildtype allele is exchanged for the mutant
plsB[D311E] allele using the method of Link et al. (see above).
Example 2
Production Host Modifications
[0184] The following plasmids were constructed for the expression
of various proteins that are used in the synthesis of fatty acid
derivatives. The constructs were made using standard molecular
biology methods and all the cloned genes were put under the control
of IPTG-inducible promoters (T7, tac or lac promoters).
[0185] The 'tesA gene (thioesterase A gene accession
NP.sub.--415027 without leader sequence (Cho and Cronan, The J. of
Biol. Chem., 270:4216-9, 1995, EC: 3.1.1.5, 3.1.2.-) of E. coli was
cloned into NdeI/AvrII digested pETDuet-1 (pETDuet-1 described
herein is available from Novagen, Madison, Wis.). Genes encoding
for FatB-type plant thioesterases (TEs) from Umbellularia
California, Cuphea hookeriana and Cinnamonum camphorum (accessions:
UcFatB1=AAA34215, ChFatB2=AAC49269, ChFatB3=AAC72881,
CcFatB=AAC49151 were individually cloned into three different
vectors: (i) NdeI/AvrII digested pETDuet-1, (ii)XhoI/HindIII
digested pBluescript KS+ (Stratagene, La Jolla, Calif.)(used to
create N-terminal lacZ::TE fusion proteins) and (iii)XbaI/HindIII
digested pMAL-c2X (New England Lab, Ipswich, Mass.) (used to create
n-terminal MalE::TE fusions). The fadD gene (encoding acyl-CoA
synthetase) from E. coli was cloned into a NcoI/HindIII digested
pCDFDuet-1 derivative, which contained the acr1 gene (acyl-CoA
reductase) from Acinetobacter baylyi ADP1 within its NdeI/AvrII
sites. Table 7 provides a summary of the plasmids generated to make
several exemplary production strains, one of ordinary skill in the
art will appreciate that different plasmids and genomic
modifications can be used to achieve similar strains.
TABLE-US-00007 TABLE 7 Summary of Plasmids used in Production hosts
Source Organism Plasmid Gene Product Accession No., EC number
pETDuet-1-tesA E. coli Accessions: NP_415027, TesA EC: 3.1.1.5,
3.1.2.- pETDuet-1-TEuc Umbellularia Q41635 pBluescript-TEuc
California pMAL-c2X-TEuc UcFatB1 AAA34215 pETDuet-1-TEch Cuphea
hookeriana ABB71581 pBluescript-TEch ChFatB2 AAC49269 pMAL-c2X-TEch
ChFatB3 AAC72881 pETDuet-1-TEcc Cinnamonun pBluescript-TEcc
camphorum AAC49151 TEci CcFatB pCDFDuet-1- E. coli fadD: Accessions
NP_416319, fadD-acr1 EC 6.2.1.3 acr1: Accessions YP_047869
[0186] The chosen expression plasmids contain compatible replicons
and antibiotic resistance markers, so that a four-plasmid
expression system can be established. Therefore, LS9001 can be
co-transformed with (i) any of the TE-expressing plasmids, (ii) the
FadD-expressing plasmid, which also expresses acr1 and (iii) wax
synthase expression plasmid. When induced with IPTG, the resulting
strain will produce increased concentrations of fatty-alcohols from
carbon sources such as glucose. The carbon chain length and degree
of saturation of the fatty alcohol produced is dependent on the
thioesterase gene that is expressed.
Example 3
Production of Fatty Alcohol in the Recombinant E. coli Strain
[0187] Fatty alcohols were produced by expressing a thioesterase
gene and an acyl-CoA reductase gene (FAR) exogenously in a
production host. More specifically, plasmids pCDFDuet-1-fadD-acr1
(acyl-CoA reductase) and pETDuet-1-'tesA (thioesterase) were
transformed into E. coli strain LS9001 (described in Example 1) and
corresponding transformants were selected in LB plate supplemented
with 100 mg/L of spectinomycin and 50 mg/L of carbenicillin. Four
transformants of LS9001/pCDFDuet-1-fadD-acr1 were independently
inoculated into 3 mL of M9 medium supplemented with 50 mg/L of
carbenicillin and 100 mg/L of spectinomycin). The samples
containing the transformants were grown in at 25.degree. C. in a
shaker (250 rpm) until they reached 0.5 OD.sub.600. 1.5 mL of each
sample was transferred into a 250 mL flask containing 30 mL of the
medium described above. The resulting culture was grown at
25.degree. C. in a shaker until the culture reached between 0.5-1.0
OD.sub.600. IPTG was then added to a final concentration of 1 mM.
and growth continued for 40 hours.
[0188] The cells were then spun down at 4000 rpm and the cell
pellets were suspended in 1.0 mL of methanol. 3 mL of ethyl acetate
was then mixed with the suspended cells. 3 mL of H.sub.2O were then
added to the mixture and the mixture was sonicated for 20 minutes.
The resulting sample was centrifuged at 4000 rpm for 5 minutes and
the organic phase (the upper phase) which contained fatty alcohol
and was subjected to GC/MS analysis. Total alcohol (including
tetradecanol, hexadecanol, hexadecenol and octadecenol) yield was
about 1-10 mg/L. When an E. coli strain carrying only empty vectors
was cultured in the same way, only 0.2-0.5 mg/L of fatty alcohols
were found in the ethyl acetate extract.
Example 4
Production and Release of Fatty Alcohol from Production Host
[0189] Acr1 (acyl-CoA reductase) was expressed in E. coli grown on
glucose as the sole carbon and energy source. The E. coli produced
small amounts of fatty alcohols such as dodecanol (C12:0-OH),
tetradecanol (C14:0-OH) and hexadecanol (C16:0-OH). In other
samples, FadD (acyl-CoA synthetase) was expressed together with
acr1 in E. coli and a five-fold increase in fatty alcohol
production was observed.
[0190] In other samples, acr1, fadD, accABCD (acetyl-CoA
Carboxylase) (plasmid carrying accABCD constructed as described in
Example 1) were expressed along with various individual
thioesterases (TEs) in wildtype E. coliC41(DE3) and an E. coli
C41(DE3 .DELTA.fadE, a strain lacking acyl-CoA dehydrogenase. This
resulted in additional increases in fatty alcohol production and
modulating the profiles of fatty alcohols (see FIG. 5). For
example, over-expression of E. coli 'tesA (pETDuet-1-'tesA) in this
system achieved approximately a 60-fold increase in C12:0-OH,
C14:0-OH and C16:0-OH with C14:0-OH being the major fatty alcohol.
A very similar result was obtained when the ChFatB3 enzyme (FatB3
from Cuphea hookeriana in pMAL-c2X-TEcu) was expressed. When the
UcFatB1 enzyme (FatB1 from Umbellularia californicain in
pMAL-c2X-TEuc) was expressed, fatty alcohol production increased
approximately 20-fold and C12:0-OH was the predominant fatty
alcohol.
[0191] Expression of ChFatB3 and UcFatB1 also led to the production
of significant amounts of the unsaturated fatty alcohols C16:1-OH
and C14:1-OH, respectively. The presence of fatty alcohols was also
found in the supernatant of samples generated from the expression
of tesA (FIG. 6). At 37.degree. C. approximately equal amounts of
fatty alcohols were found in the supernatant and in the cell
pellet, whereas at 25.degree. C. approximately 25% of the fatty
alcohols were found in the supernatant.
Example 5
Medium Chain Fatty Acid Esters
[0192] Alcohol acetyl transferases (AATs, EC 2.3.1.84), which is
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 makes them
good candidates for use as biofuels.
[0193] In this example, a SAAT gene was co-expressed in a
production host C41(DE3, .DELTA.fadE) with fadD from E. coli and
acr1 (alcohol reductase from A. baylyi ADP1) and octanoic acid was
provided in the fermentation broth. This resulted in the production
of octyl octanoate. Similarly, when the wax synthase gene from A.
baylyi ADP1was expressed in the production host instead of the SAAT
gene octyl octanoate was produced.
[0194] A recombinant SAAT gene was synthesized using DNA 2.0 (Menlo
Park, Calif. 94025). The synthesized DNA was based on the published
gene sequence (accession number AF193789) and modified to eliminate
the NcoI site. The synthesized SAAT gene (as a BamHI-HindIII
fragment) was cloned in pRSET B (Invitrogen, Calsbad, Calif.),
linearized with BamHI and HindIII. The resulted plasmid, pHZ1.63A
was cotransformed into an E. coli production host with pAS004.114B,
which carries a fadD gene from E. coli and acr1 gene from A. baylyi
ADP1. The transformants were grown in 3 mL of M9 medium with 2% of
glucose. After IPTG induction and the addition of 0.02% of octanoic
acid, the culture was continued at 25.degree. C. from 40 hours.
After that, 3 mL of acetyl acetate was added to the whole culture
and mixed several times with mixer. The acetyl acetate phase was
analyzed by GC/MS.
[0195] Surprising, in the acetyl acetate extract, there is no acyl
acetate found. However, a new compound was found and the compound
was octyl octanoate. Whereas the control strain without the SAAT
gene [C41(DE3, .DELTA.fadE)/pRSET B+pAS004.114B] did not produce
octyl octanoate. Also the strain [C41(DE3, .DELTA.fadE)/pHZ1.43
B+pAS004.114B], in which the wax synthase gene from A. baylyi ADP1
was carried by pHZ1.43 produced octyl octanoate (see FIG. 7B).
[0196] The finding that SAAT activity produces octyl octanoate has
not reported before and makes it possible to produce medium chain
waxes such as octyl octanoate, octyl decanoate, which have low
melting point and are good candidates to be use for biofuel to
replace triglyceride based biodiesel.
Example 6
Production of Wax Ester in E. coli Strain LS9001
[0197] Wax esters were produced by engineering an E. coli
production host to express a fatty alcohol forming acyl-CoA
reductase, thioesterase, and a wax synthase. Thus, the production
host produced both the A and the B side of the ester and the
structure of both sides was influenced by the expression of the
thioesterase gene.
[0198] More specifically, wax synthase from A. baylyi ADP1 (termed
WSadp1, accessions AA017391, EC: 2.3.175) was amplified with the
following primers using genomic DNA from A. baylyi ADP1 as the
template. The primers were (1) WSadp1_NdeI,
5'-TCATATGCGCCCATTACATCCG-3' and (2) WSadp1_Avr,
5'-TCCTAGGAGGGCTAATTTAGCCCTTTAGTT-3'. The PCR product was digested
with NdeI and AvrII and cloned into pCOALDeut-1 to give pHZ 1.43.
The plasmid carrying WSadp1 was then co-transformed into E. coli
strain LS9001 with both pETDuet-1'tesA and pCDFDuet-1-fadD-acr1 and
transformants were selected in LB plates supplemented with 50 mg/L
of kanamycin, 50 mg/L of carbenicillin and 100 mg/L of
spectinomycin. Three transformants were inoculated in 3 mL of LBKCS
(LB broth supplement with 50 mg/L of kanamycin, 50 mg/L of
carbenicillin, 100 mg/L of spectinomycin and 10 g/L of glucose) and
cultured at 37.degree. C. shaker (250 rpm). When the cultures
reached 0.5 OD.sub.600, 1.5 mL of each culture was transferred into
250 mL flasks containing 50 mL of LBKCS and the flasks were grown
in a shaker (250 rpm) at 37.degree. C. until the culture reached
0.5-1.0 OD.sub.600. IPTG was then added to a final concentration of
1 mM. The induced cultures were grown at 37.degree. C. shaker for
another 40-48 hours.
[0199] The culture was then placed into 50 mL conical tubes and the
cells were spun down at 3500.times.g for 10 minutes. The cell
pellet was then mixed with 5 mL of ethyl acetate. The ethyl acetate
extract was analyzed with GC/MS. The intracellular yield of waxes
(including C16C16, C14:1C16, C18:1C18:1,C2C14, C2C16, C2C16:1,
C16C16:1 and C2C18:1) was about 10 mg/L. When an E. coli strain
only carrying empty vectors was cultured in the same way, only 0.2
mg/L of wax was found in the ethyl acetate extract.
Example 7
Production and Release of Fatty-Ethyl Ester from Production
Host
[0200] The LS9001 strain was modified by transforming it with the
plasmids carrying a wax synthase gene from A. baylyi (plasmid
pHZ1.43), a thioesterase gene from Cuphea hookeriana (plasmid
pMAL-c2X-TEcu) and a fadD gene from E. coli (plasmid
pCDFDuet-1-fadD). This recombinant strain was grown at 25.degree.
C. in 3 mL of M9 medium with 50 mg/L of kanamycin, 100 mg/L of
carbenicillin and 100 mg/L of spectinomycin. After IPTG induction,
the media was adjusted to a final concentration of 1% ethanol and
2% glucose. The culture was allowed to grow for 40 hours after IPTG
induction. The cells were separated from the spent medium by
centrifugation at 3500.times.g for 10 minutes). The cell pellet was
re-suspended with 3 mL of M9 medium. The cell suspension and the
spent medium were then extracted with 1 volume of ethyl acetate.
The resulting ethyl acetate phases from the cells suspension and
the supernatant were subjected to GC-MS analysis. The results
showed that the C16 ethyl ester was the most prominent ester
species (as expected for this thioesterase, see Table 1), and that
20% of the fatty acid ester produced was released from the cell
(see FIG. 8). A control E. coli strain C41(DE3, .DELTA.fadE)
containing pCOLADuet-1 (empty vector for the wax synthase gene),
pMAL-c2X-TEuc (containing fatB from U. california) and
pCDFDuet-1-fadD (fadD gene from E. coli) failed to produce
detectable amounts of fatty ethyl esters. The fatty acid esters
were quantified using commercial palmitic acid ethyl ester as the
reference. Fatty acid esters were also made using the methods
described herein except that methanol, or isopropanol was added to
the fermentation broth and the expected fatty acid esters were
produced.
Example 8
The Influence of Various Thioesterases on the Composition of
Fatty-Ethyl Esters Produced in Recombinant E. coli Strains
[0201] The thioesterases FatB3 (C. hookeriana), TesA (E. coli), and
FatB (U. california) were expressed simultaneously with wax
synthase (A. baylyi). A plasmid termed pHZ1.61 was constructed by
replacing the NotI-AvrII fragment (carrying the acr1 gene) with the
NotI-AvrII fragment from pHZ1.43 so that fadD and the ADP1 wax
synthase were in one plasmid and both coding sequences were under
the control of separate T7 promoter. The construction of pHZ1.61
made it possible to use a two plasmid system instead of the three
plasmid system as described in Example 6. pHZ1.61 was then
co-transformed into E. coli C41(DE3, .DELTA.fadE) with one of the
various plasmids carrying the different thioesterase genes stated
above.
[0202] The total fatty acid ethyl esters (supernatant and
intracellular fatty acid ethyl esters) produced by these
transformants were evaluated using the technique described herein.
The yields and the composition of fatty acid ethyl esters are
summarized in Table 8.
TABLE-US-00008 TABLE 8 The yields (mg/L) and the composition of
fatty acid ethyl esters by recombinant E. coli C41(DE3,
.DELTA.fadE)/pHZ1.61 and plasmids carrying various thioesterase
genes. Thioesterases C2C10 C2C12:1 C2C12 C2C14:1 C2C14 C2C16:1
C2C16 C2C18:1 Total `TesA 0.0 0.0 6.5 0.0 17.5 6.9 21.6 18.1 70.5
ChFatB3 0.0 0.0 0.0 0.0 10.8 12.5 11.7 13.8 48.8 ucFatB 6.4 8.5
25.3 14.7 0.0 4.5 3.7 6.7 69.8 pMAL 0.0 0.0 0.0 0.0 5.6 0.0 12.8
7.6 26.0 Note: `TesA, pETDuet-1-`tesA; ChFatB3, pMAL-c2X-TEcu;
ucFatB, pMAL-c2X-TEuc; pMAL, pMAL-c2X, the empty vector for
thioesterase genes used in the study.
Example 9
Production Host Construction
[0203] The genes that control fatty acid production are conserved
between microorganisms. For example, Table 9 identifies the
homologues of many of the genes described herein which are known to
be expressed in microorganisms that produce hydrocarbons. To
increase fatty acid production and, therefore, hydrocarbon
production in microorganisms such as those identified in Table 9,
heterologous genes, such as those from E. coil can be expressed.
One of ordinary skill in the art will also appreciate that genes
that are endogenous to the micoorganisms provided in Table 9 can
also be over-expressed, or attenuated using the methods described
herein. Moreover, genes that are described in FIG. 10 can be
expressed or attenuated in microorganisms that endogenously produce
hydrocarbons to allow for the production of specific hydrocarbons
with defined carbon chain length, saturation points, and branch
points.
[0204] For example, exogenous nucleic acid sequences encoding
acetyl-CoA carboxylase are introduced into K. radiotolerans. The
following genes comprise the acetyl-CoA carboxylase protein product
in K. radiotolerans; acetyl CoA carboxylase, alpha subunit
(accA/ZP.sub.--00618306), acetyl-CoA carboxylase, biotin carboxyl
carrier protein (accB/ZP.sup.--00618387), acetyl-CoA carboxylase,
biotin carboxylase subunit (accC/ZP.sub.--00618040), and acetyl-CoA
carboxylase, beta (carboxyltranferase) subunit
(accD/ZP.sub.--00618306). These genes are cloned into a plasmid
such that they make a synthetic acetyl-CoA carboxylase operon
(accABCD) under the control of a K. radiotolerans expression system
such as the expression system disclosed in Ruyter et al., Appl
Environ Microbiol. 62:3662-3667, 1996. Transformation of the
plasmid into K. radiotolerans will enhance fatty acid production.
The hydrocarbon producing strain of K. radiotolerans can also be
engineered to make branched, unsaturated hydrocarbons having
specific carbon chain lengths using the methods disclosed
herein.
TABLE-US-00009 TABLE 9 Hydrocarbon production hosts Organism Gene
Name Accession No./Seq ID/Loci EC No. Desulfovibrio desulfuricans
G20 accA YP_388034 6.4.1.2 Desulfovibrio desulfuricans G22 accC
YP_388573/YP_388033 6.3.4.14, 6.4.1.2 Desulfovibrio desulfuricans
G23 accD YP_388034 6.4.1.2 Desulfovibrio desulfuricans G28 fabH
YP_388920 2.3.1.180 Desulfovibrio desulfuricans G29 fabD YP_388786
2.3.1.39 Desulfovibrio desulfuricans G30 fabG YP_388921 1.1.1.100
Desulfovibrio desulfuricans G31 acpP YP_388922/YP_389150 3.1.26.3,
1.6.5.3, 1.6.99.3 Desulfovibrio desulfuricans G32 fabF YP_388923
2.3.1.179 Desulfovibrio desulfuricans G33 gpsA YP_389667 1.1.1.94
Desulfovibrio desulfuricans G34 ldhA YP_388173/YP_390177 1.1.1.27,
1.1.1.28 Erwinia (micrococcus) amylovora accA 942060-943016 6.4.1.2
Erwinia (micrococcus) amylovora accB 3440869-3441336 6.4.1.2
Erwinia (micrococcus) amylovora accC 3441351-3442697 6.3.4.14,
6.4.1.2 Erwinia (micrococcus) amylovora accD 2517571-2516696
6.4.1.2 Erwinia (micrococcus) amylovora fadE 1003232-1000791
1.3.99.- Erwinia (micrococcus) amylovora plsB(D311E) 333843-331423
2.3.1.15 Erwinia (micrococcus) amylovora aceE 840558-843218 1.2.4.1
Erwinia (micrococcus) amylovora aceF 843248-844828 2.3.1.12 Erwinia
(micrococcus) amylovora fabH 1579839-1580789 2.3.1.180 Erwinia
(micrococcus) amylovora fabD 1580826-1581749 2.3.1.39 Erwinia
(micrococcus) amylovora fabG CAA74944 1.1.1.100 Erwinia
(micrococcus) amylovora acpP 1582658-1582891 3.1.26.3, 1.6.5.3,
1.6.99.3 Erwinia (micrococcus) amylovora fabF 1582983-1584221
2.3.1.179 Erwinia (micrococcus) amylovora gpsA 124800-125810
1.1.1.94 Erwinia (micrococcus) amylovora ldhA 1956806-1957789
1.1.1.27, 1.1.1.28 Kineococcus radiotolerans accA ZP_00618306
6.4.1.2 SRS30216 Kineococcus radiotolerans accB ZP_00618387 6.4.1.2
SRS30216 Kineococcus radiotolerans accC ZP_00618040/ 6.3.4.14,
6.4.1.2 SRS30216 ZP_00618387 Kineococcus radiotolerans accD
ZP_00618306 6.4.1.2 SRS30216 Kineococcus radiotolerans fadE
ZP_00617773 1.3.99.- SRS30216 Kineococcus radiotolerans plsB(D311E)
ZP_00617279 2.3.1.15 SRS30216 Kineococcus radiotolerans aceE
ZP_00617600 1.2.4.1 SRS30216 Kineococcus radiotolerans aceF
ZP_00619307 2.3.1.12 SRS30216 Kineococcus radiotolerans fabH
ZP_00618003 2.3.1.180 SRS30216 Kineococcus radiotolerans fabD
ZP_00617602 2.3.1.39 SRS30216 Kineococcus radiotolerans fabG
ZP_00615651 1.1.1.100 SRS30216 Kineococcus radiotolerans acpP
ZP_00617604 3.1.26.3, SRS30216 1.6.5.3, 1.6.99.3 Kineococcus
radiotolerans fabF ZP_00617605 2.3.1.179 SRS30216 Kineococcus
radiotolerans gpsA ZP_00618825 1.1.1.94 SRS30216 Kineococcus
radiotolerans ldhA ZP_00618879 1.1.1.27, SRS30216 1.1.1.28
Rhodospirillum rubrum accA YP_425310 6.4.1.2 Rhodospirillum rubrum
accB YP_427521 6.4.1.2 Rhodospirillum rubrum accC
YP_427522/YP_425144/YP_427028/ 6.3.4.14, 6.4.1.2 YP_426209/
YP_427404 Rhodospirillum rubrum accD YP_428511 6.4.1.2
Rhodospirillum rubrum fadE YP_427035 1.3.99.- Rhodospirillum rubrum
aceE YP_427492 1.2.4.1 Rhodospirillum rubrum aceF YP_426966
2.3.1.12 Rhodospirillum rubrum fabH YP_426754 2.3.1.180
Rhodospirillum rubrum fabD YP_425507 2.3.1.39 Rhodospirillum rubrum
fabG YP_425508/YP_425365 1.1.1.100 Rhodospirillum rubrum acpP
YP_425509 3.1.26.3, 1.6.5.3, 1.6.99.3 Rhodospirillum rubrum fabF
YP_425510/YP_425510/ 2.3.1.179 YP_425285 Rhodospirillum rubrum gpsA
YP_428652 1.1.1.94 Rhodospirillum rubrum ldhA YP_426902/YP_428871
1.1.1.27, 1.1.1.28 Vibrio furnissii accA 1, 16 6.4.1.2 Vibrio
furnissii accB 2, 17 6.4.1.2 Vibrio furnissii accC 3, 18 6.3.4.14,
6.4.1.2 Vibrio furnissii accD 4, 19 6.4.1.2 Vibrio furnissii fadE
5, 20 1.3.99.- Vibrio furnissii plsB(D311E) 6, 21 2.3.1.15 Vibrio
furnissii aceE 7, 22 1.2.4.1 Vibrio furnissii aceF 8, 23 2.3.1.12
Vibrio furnissii fabH 9, 24 2.3.1.180 Vibrio furnissii fabD 10, 25
2.3.1.39 Vibrio furnissii fabG 11, 26 1.1.1.100 Vibrio furnissii
acpP 12, 27 3.1.26.3, 1.6.5.3, 1.6.99.3 Vibrio furnissii fabF 13,
28 2.3.1.179 Vibrio furnissii gpsA 14, 29 1.1.1.94 Vibrio furnissii
ldhA 15, 30 1.1.1.27, 1.1.1.28 Stenotrophomonas maltophilia accA
ZP_01643799 6.4.1.2 R551-3 Stenotrophomonas maltophilia accB
ZP_01644036 6.4.1.2 R551-3 Stenotrophomonas maltophilia accC
ZP_01644037 6.3.4.14, 6.4.1.2 R551-3 Stenotrophomonas maltophilia
accD ZP_01644801 6.4.1.2 R551-3 Stenotrophomonas maltophilia fadE
ZP_01645823 1.3.99.- R551-3 Stenotrophomonas maltophilia
plsB(D311E) ZP_01644152 2.3.1.15 R551-3 Stenotrophomonas
maltophilia aceE ZP_01644724 1.2.4.1 R551-3 Stenotrophomonas
maltophilia aceF ZP_01645795 2.3.1.12 R551-3 Stenotrophomonas
maltophilia fabH ZP_01643247 2.3.1.180 R551-3 Stenotrophomonas
maltophilia fabD ZP_01643535 2.3.1.39 R551-3 Stenotrophomonas
maltophilia fabG ZP_01643062 1.1.1.100 R551-3 Stenotrophomonas
maltophilia acpP ZP_01643063 3.1.26.3, R551-3 1.6.5.3, 1.6.99.3
Stenotrophomonas maltophilia fabF ZP_01643064 2.3.1.179 R551-3
Stenotrophomonas maltophilia gpsA ZP_01643216 1.1.1.94 R551-3
Stenotrophomonas maltophilia ldhA ZP_01645395 1.1.1.27, R551-3
1.1.1.28 For Table 9, Accession Numbers are from GenBank, Release
159.0 as of Apr. 15, 2007, EC Numbers are from KEGG, Release 42.0
as of April 2007 (plus daily updates up to and including May 09,
2007), results for Erwinia amylovora strain Ea273 are taken from
the Sanger sequencing center, completed shotgun sequence as of May
09, 2007, positions for Erwinia represent locations on the Sanger
psuedo-chromosome, sequences from Vibrio furnisii M1 are from the
LS9 VFM1 pseudochromosome, v2 build, as of Sep. 28, 2006, and
include the entire gene, and may also include flanking sequence
Example 10
Additional Exemplary Production Strains
[0205] Table 10, below provides additional exemplary production
strains. Two example biosynthetic pathways are described for
producing fatty acids, fatty alcohols, and wax esters. A
genetically engineered host can be produced by cloning the
expression of the accABCD genes from E. coli, the 'tesA gene from
E. coli, and fadD gene from E. coli into a host cell. Host cells
can be selected from E. coli, yeast, add to the list. These genes
can also be transformed into a host cell that is modified to
contain one or more of the genetic manipulations described in
Examples 1 and 2, above. As provided in Table 10, additional
production hosts can be created using the indicated exogenous
genes.
TABLE-US-00010 TABLE 10 Combination of genes useful for making
genetically engineered production strains Sources of Fatty acids
Fatty alcohols wax/fatty esters Peptide genes Genes example 1
example 2 example 1 example 2 example 1 example 2 acetyl-CoA E.
coli accABCD X X X X X X carboxylase thio- E. coli tesA X X X X
esterase Cinnamomum ccFatB camphora Umbellularia umFatB X X
californica Cuphea chFatB2 hookeriana Cuphea chFatB3 hookeriana
Cuphea chFatA hookerian Arabidopsis AtFatA1 thaliana Arabidopsis
AtFatB1[M141T] thalian acyl-CoA E. coli fadD X X X X X X synthase
acyl-CoA Bombyx mori bFAR reductase Acinetobacter acr 1 X X baylyi
L ADP1 Simmondsia jjFAR X X chinensis Triticum aestivum Mus mFAR1
musculus Mus mFAR2 musculus Acinetpbacter acr M1 sp M1 Homo hFAR
sapiens wax Fundibacter WST9 synthase/ jadensis DSM alcohol 12178
acyltransferase Acinetobacter WSHN X sp. HO1-N Acinetobacter WSadp1
X baylyi ADP1 Mus mWS musculus Homo hWS sapiens Fragaria x SAAT
ananassa Malus x MpAAT domestica Simmondsia JjWS chinensis
(AAD38041) Decarbonylase Arabidopsis cer1 thaliana Oryza sativa
cer1 Transporter Acinetobacter X X sp. HO1-N Arabidopsis Cer5
thaliana
Example 11
Fermentation
[0206] Host microorganisms can be also engineered to express umuC
and umuD from E. coli in pBAD24 under the prpBCDE promoter system
through de novo synthesis of this gene with the appropriate
end-product production genes. For small scale hydrocarbon product
production, E. coli BL21(DE3) cells harbouring pBAD24 (with
ampicillin resistance and the end-product synthesis pathway) as
well as pUMVC1 (with kanamycin resistance and the acetyl
CoA/malonyl CoA over-expression system) are incubated overnight at
at 37.degree. C. shaken at >200 rpm 2L flasks in 500 ml LB
medium supplemented with 75 .mu.g/mL ampicillin and 50 .mu.g/ml
kanamycin until cultures reached an OD.sub.600 of >0.8. Upon
achieving an OD.sub.600 of >0.8, cells are supplemented with 25
mM sodium proprionate (pH 8.0) to activate the engineered gene
systems for production as well as to stop cellular proliferation
(through activation of umuC and umuD proteins). Induction is
performed for 6 hours at 30.degree. C. After incubation, media is
examined for product using GC-MS (as described below).
[0207] For large scale product production, the engineered
microorganisms are grown in 10 L, 100 L or larger batches,
fermented and induced to express desired products based on the
specific genes encoded in plasmids as appropriate. E. coli
BL21(DE3) cells harbouring pBAD24 (with ampicillin resistance and
the end-product synthesis pathway) as well as pUMVC1 (with
kanamycin resistance and the acetyl-CoA/malonyl-CoA over-expression
system) are incubated from a 500 mL seed culture for 10 L
fermentations (5 L for 100 L fermentations) in LB media (glycerol
free) at 37.degree. C. shaken at >200 rpm until cultures reached
an OD.sub.600 of >0.8 (typically 16 hours) incubated with 50
.mu.g/mL kanamycin and 75 .mu.g/mL ampicillin. Media is treated
with continuously supplemented to maintain a 25 mM sodium
proprionate (pH 8.0) to activate the engineered in gene systems for
production as well as to stop cellular proliferation (through
activation of umuC and umuD proteins). Media is continuously
supplemented with glucose to maintain a concentration 90 g/100 mL.
After the first hour of induction, aliquots of no more than 10% of
the total cell volume are removed each hour and allowed to sit
unaggitated so as to allow the hydrocarbon product to rise to the
surface and undergo a spontaneous phase separation. The hydrocarbon
component is then collected and the aqueous phase returned to the
reaction chamber. The reaction chamber is operated continuously.
When the OD.sub.600 drops below 0.6, the cells are replaced with a
new batch grown from a seed culture.
[0208] For wax ester production, subsequent to isolation, the wax
esters are washed briefly in 1 M HCl to split the ester bond, and
returned to pH 7 with extensive washing with distilled water.
Example 12
Product Characterization
[0209] To characterize and quantify the fatty alcohols and fatty
acid esters, gas chromatography (GC) coupled with electron impact
mass spectra (MS) detection was used. Fatty alcohol samples were
first derivatized with an excess of N-trimethylsilyl (TMS)
imidazole to increase detection sensitivity. Fatty acid esters did
not required derivatization. Both fatty alcohol-TMS derivatives and
fatty acid esters were dissolved in an appropriate volatile
solvent, like ethyl acetate. The samples were analyzed on a 30 m
DP-5 capillary column using the following method. After a 1 .mu.L
splitless injection onto the GC/MS column, the oven is held at
100.degree. C. for 3 minutes. The temperature was ramped up to
320.degree. C. at a rate of 20.degree. C./minute. The oven was held
at 320.degree. C. for an additional 5 minutes. The flow rate of the
carrier gas helium was 1.3 mL/minute. The MS quadrapole scans from
50 to 550 m/z. Retention times and fragmentation patterns of
product peaks were compared with authentic references to confirm
peak identity.
[0210] For example, hexadeconic acid ethyl ester eluted at 10.18
minutes (FIGS. 9A and 9B). The parent ion of 284 mass units was
readily observed. More abundent were the daughter ions produced
during mass fragmentation. This included the most prevalent
daughter ion of 80 mass units. The derivatized fatty alcohol
hexadecanol-TMS eluted at 10.29 minutes and the parent ion of 313
could be observed. The most prevalent ion was the M-14 ion of 299
mass units.
[0211] Quantification was carried out by injecting various
concentrations of the appropriate authentic references using the
GC/MS method described above. This information was used to generate
a standard curve with response (total integrated ion count) versus
concentration.
Equivalents
[0212] While specific examples of the subject inventions are
explicitly disclosed herein, the above specification and examples
herein are illustrative and not restrictive. Many variations of the
inventions will become apparent to those skilled in the art upon
review of this specification including the examples. The full scope
of the inventions should be determined by reference to the
examples, along with their full scope of equivalents, and the
specification, along with such variations.
* * * * *