U.S. patent application number 15/385257 was filed with the patent office on 2017-06-08 for methods of using natural and engineered organisms to produce small molecules for industrial application.
This patent application is currently assigned to Kiverdi, Inc.. The applicant listed for this patent is Kiverdi, Inc.. Invention is credited to Lisa Dyson, Henrik Fyrst, Itzhak Kurek, John S. Reed.
Application Number | 20170159087 15/385257 |
Document ID | / |
Family ID | 51528789 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170159087 |
Kind Code |
A1 |
Reed; John S. ; et
al. |
June 8, 2017 |
Methods of Using Natural and Engineered Organisms to Produce Small
Molecules for Industrial Application
Abstract
Aspects of the invention relate to methods of producing small
molecules for industrial application using natural organisms and
engineered organisms.
Inventors: |
Reed; John S.; (Emeryville,
CA) ; Kurek; Itzhak; (San Francisco, CA) ;
Fyrst; Henrik; (Oakland, CA) ; Dyson; Lisa;
(Berkeley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kiverdi, Inc. |
Hayward |
CA |
US |
|
|
Assignee: |
Kiverdi, Inc.
Hayward
CA
|
Family ID: |
51528789 |
Appl. No.: |
15/385257 |
Filed: |
December 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14214784 |
Mar 15, 2014 |
9556462 |
|
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15385257 |
|
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61791456 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 5/005 20130101;
C12P 13/225 20130101; C12P 13/08 20130101; C12P 17/10 20130101;
C12P 13/001 20130101; C12P 13/222 20130101; C12P 7/625 20130101;
C12P 7/6409 20130101; C12P 7/18 20130101 |
International
Class: |
C12P 13/22 20060101
C12P013/22; C12P 13/00 20060101 C12P013/00; C12P 7/62 20060101
C12P007/62; C12P 5/00 20060101 C12P005/00; C12P 7/18 20060101
C12P007/18; C12P 7/64 20060101 C12P007/64; C12P 13/08 20060101
C12P013/08; C12P 17/10 20060101 C12P017/10 |
Claims
1.-8. (canceled)
9. A method for harvesting amino acids, comprising: culturing in
growth medium comprising a carbon-containing gas a bacterial cell
that can grow in the presence of a carbon-containing gas and that
secretes amino acids into the growth medium; and separating the
secreted amino acids from the growth medium.
10. The method of claim 9, wherein the amino acid is lysine,
tyrosine or phenylalanine.
11. The method of claim 9, wherein the bacterial cell is
recombinant.
12. The method of claim 11, wherein the bacterial cell exhibits
increased expression or activity relative to a wild type cell of an
enzyme involved in lysine biosynthesis or in lysine secretion.
13. The method of claim 12, wherein the enzyme involved in lysine
biosynthesis is an aspartate kinase or a dihydrodipicolinate
synthase.
14. The method of claim 12, wherein the enzyme involved in lysine
secretion is a lysine exporter.
15. The method of claim 11, wherein the bacterial cell exhibits
decreased expression or activity relative to a wild type cell of
one or more enzymes in the citric acid cycle.
16. The method of claim 15, wherein the enzyme in the citric acid
cycle is succinyl-CoA synthase.
17. The method of claim 11, wherein the bacterial cell exhibits
increased expression or activity relative to a wild type cell of an
enzyme involved in the Shikimate pathway.
18. The method of claim 17, wherein the enzyme involved in the
Shikimate pathway is chorismate synthase.
19. The method of claim 9, wherein the bacterial cell is of the
genus Ralstonia or of the genus Rhodococcus.
20. The method of claim 19, wherein the bacterial cell is a
Ralstonia eutropha cell or a Rhodococcus opacus cell.
21. A method for producing putrescine, comprising: culturing in
growth medium comprising a carbon-containing gas a bacterial cell
that can grow in the presence of a carbon-containing gas and that
secretes putrescine into the growth medium; and separating the
putrescine from the growth medium.
22. The method of claim 21, wherein the bacterial cell is
recombinant.
23. The method of claim 22, wherein the bacterial cell exhibits
increased expression or activity relative to a wild type cell of an
arginine decarboxylase and/or an ornithine decarboxylase.
24. A method for producing caprolactam, comprising: culturing in
growth medium comprising a carbon-containing gas a recombinant
bacterial cell that can grow in the presence of a carbon-containing
gas and that secretes caprolactam into the growth medium; and
separating the caprolactam from the growth medium.
25. The method of claim 24, wherein the bacterial cell exhibits
increased expression or activity relative to a wild type cell of an
enzyme selected from a carbon nitrogen lyase, an
.alpha.-.beta.-enoate reductase, an amidohydrolase and a tyrosine
phenol lyase.
26. A method for producing styrene, comprising: culturing in growth
medium comprising a carbon-containing gas a recombinant bacterial
cell that can grow in the presence of a carbon-containing gas and
that secretes styrene into the growth medium; and separating the
styrene from the growth medium.
27. The method of claim 26, wherein the bacterial cell exhibits
increased expression or activity relative to a wild type cell of a
phenylalanine ammonium lyase enzyme and/or an oxylate decarboxylase
enzyme.
28. A method for producing 1,3-butanediol, comprising: culturing in
growth medium comprising a carbon-containing gas a bacterial cell
that can grow in the presence of a carbon-containing gas and that
secretes 1,3-butanediol into the growth medium; and separating the
1,3-butanediol from the growth medium.
29. The method of claim 28, wherein the bacterial cell exhibits
increased expression or activity relative to a wild type cell of an
oleate hydratase enzyme.
30. A recombinant cell that exhibits increased expression or
activity of: (i) one or more of an aspartate kinase, a
dihydrodipicolinate synthase or a lysine exporter, wherein the
recombinant cell can produce a cell culture that contains lysine;
(ii) a chorismate synthase enzyme, wherein the recombinant cell can
produce a cell culture that contains lysine or phenylalanine; (iii)
an arginine decarboxylase and/or an ornithine decarboxylase,
wherein the recombinant cell can produce a cell culture that
contains putrescine; (iv) an enzyme selected from a carbon nitrogen
lyase, an .alpha.-.beta.-enoate reductase, an amidohydrolase and a
tyrosine phenol lyase, wherein the recombinant cell can produce a
cell culture that contains caprolactam; (v) a phenylalanine
ammonium lyase enzyme and/or an oxylate decarboxylase enzyme,
wherein the recombinant cell can produce a cell culture that
contains styrene; or (vi) an oleate hydratase enzyme, wherein the
recombinant cell can produce a cell culture that contains
1,3-butadiene.
31. The recombinant cell of claim 30, wherein the recombinant cell
is of the genus Ralstonia or of the genus Rhodococcus.
32. The recombinant cell of claim 31, wherein the recombinant cell
is a Ralstonia eutropha cell or a Rhodococcus opacus cell.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. 119(e)
of U.S. Provisional Application Ser. No. 61/791,456, entitled
"Methods of using natural and engineered organisms to produce small
molecules for industrial application," filed on Mar. 15, 2013, the
entire disclosure of which is incorporated by reference herein in
its entirety.
FIELD OF THE INVENTION
[0002] The disclosure relates to methods of producing small
molecules for industrial application using natural organisms and
engineered organisms.
BACKGROUND OF THE INVENTION
[0003] Microorganisms have been used for a variety of industrial
applications. Genetic engineering of microorganisms has increased
their potential, such as by manipulating enzymatic pathways, or
creating new enzymatic pathways, within cells (Adrio et al. (2010)
Bioeng. Bugs. 1(2):116-131).
SUMMARY OF INVENTION
[0004] The instant invention provides methods of using engineered
organisms and natural organisms to produce small molecules for
industrial application.
[0005] Aspects of the invention relate to methods for the
production of omega-7 fatty acids from feedstock comprising syngas
and containing at least one of CO or a mixture of CO.sub.2 and
H.sub.2, the process comprising passing syngas to a bioreactor for
contact therein with Rhodococcus microorganisms.
[0006] In some embodiments, methods further comprise a separation
step wherein a cell mass is separated from a supernatant by
centrifugation to create a biomass pellet. In some embodiments, the
biomass pellet contains omega-7 fatty acids. In some embodiments,
the omega-7 fatty acids comprise palmitoleic acid (C16:1) and
vaccenic acid (C18:1).
[0007] In some embodiments, methods further comprise an extraction
step wherein the supernatant is discarded and the biomass pellet is
applied to a Silica-60 column. In some embodiments, the extraction
step further includes eluting lipids with an organic solvent. In
some embodiments, the organic solvent is selected from a group
comprising: hexane, chloroform, isopropanol, methanol, and acetone.
In some embodiments, the microorganism is a Rhodococcus opacus
strain.
[0008] The details of one or more embodiments of the invention are
set forth in the description below. Other features or advantages of
the present invention will be apparent from the following drawings
and detailed description of several embodiments, and also from the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The accompanying drawings are not intended to be drawn to
scale. The figures are illustrative only and are not required for
enablement of the disclosure. For purposes of clarity, not every
component may be labeled in every drawing. In the drawings:
[0010] FIGS. 1A and 1B show gas chromatography-mass spectrometry
analysis of secreted bacterial fermentation products from C.
necator.
[0011] FIG. 2 shows samples of polymer extracted from C. necator
DSM 531.
[0012] FIG. 3A-3D show gas chromatography-mass spectrometry
analysis of monomers derived from extracted polymers from C.
necator cultures.
[0013] FIG. 4 presents results from gas chromatography-mass
spectrometry analysis of fatty acid methyl esters produced by
Rhodococcus opacus.
[0014] FIG. 5 shows enhanced production of Lysine in C. necator DSM
541 cultures.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In some embodiments, the instant invention provides a method
of using a natural strain microorganism that grows on
carbon-containing gas such as syngas, producer gas, CO.sub.2,
carbon monoxide and mixtures of the same containing hydrogen gas;
and/or C1 compounds, gaseous or liquid, including but not limited
to methanol or methane, to produce and/or secrete amino containing
compounds including amino acids by combining the natural
microorganism and said carbon-containing gas in a bioreactor or
solution. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531 or DSM 541 (Table 2). In some
non-limiting embodiments the microorganism is Ralstonia eutropha
N-1, DSM 13513.
[0016] In some embodiments, the amino containing compounds secreted
and/or produced include glutamic acid, sarcosine, serine, glycine,
alanine, threonine, valine, isoleucine, ornithine, histidine,
arginine, phenylalanine, lysine, tyrosine, cytosine, asparatic
acid, glutamine, proline, leucine, tryptophan, methionine,
.beta.-alanine, S-adenosylmethionine, S-adenosylhomocysteine,
methionine sulfoxide and putrescine (Table 2).
[0017] In some embodiments, the instant invention provides for an
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
amino containing compounds and encodes one or more genes useful for
the production of amino containing compounds.
[0018] In some embodiments, the instant invention provides for a
method of producing amino containing compounds by combining, in a
bioreactor or solution, a carbon-containing gas and an engineered
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into amino
containing compounds and encodes one or more genes useful for the
production of amino containing compounds. (Table 2).
[0019] In some embodiments, the instant invention provides for a
method of producing styrene by combining, in a bioreactor or
solution, one or more enzymes useful for the production of amino
containing compounds, a carbon-containing gas, and an engineered or
natural microorganism that converts a carbon-containing gas such as
syngas, producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into the
precursors utilized by the enzymes to produce amino containing
compounds. (Table 2).
[0020] In some embodiments, the instant invention provides for a
natural or engineered microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO2, carbon
monoxide and mixtures of the same containing hydrogen gas; and/or
C1 compounds, gaseous or liquid, including but not limited to
methanol or methane, into putrescine and encodes one or more genes
including but not limited to the enzyme ornithine decarboxylase. In
some non-limiting embodiments the microorganism is Cupriavidus
necator DSM 531 or DSM 541 (Table 2). In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0021] In some embodiments, the instant invention provides for a
method of producing putrescine by combining, in a bioreactor or
solution, a carbon-containing gas and a natural or engineered
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds liquid or gaseous
including but not limited to methanol or methane, into putrescine
and encodes one or more genes including but not limited to the
enzyme ornithine decarboxylase. In some non-limiting embodiments
the microorganism is Cupriavidus necator DSM 531 or DSM 541. In
some non-limiting embodiments the microorganism is Ralstonia
eutropha N-1, DSM 13513. In some embodiments, the instant invention
further provides for the additional step of adding adipic acid to
the putrescine to produce nylon-4,6.
[0022] In some embodiments, the instant invention provides for a
method of producing putrescine by combining, in a bioreactor or
solution, a carbon-containing gas and a natural strain
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds liquid or gaseous
including but not limited to methanol or methane, into putrescine.
In some non-limiting embodiments the microorganism is Cupriavidus
necator DSM 531 or DSM 541 (Table 2). In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM 13513.
In some embodiments, the instant invention further provides for the
additional step of adding adipic acid to the putrescine to produce
nylon-4,6.
[0023] In some embodiments, the instant invention provides for a
method of producing putrescine by combining, in a bioreactor or
solution, ornithine decarboxylase, a carbon-containing gas, and an
engineered or natural microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO.sub.2,
carbon monoxide and mixtures of the same containing hydrogen gas;
and/or C1 compounds liquid or gaseous including but not limited to
methanol or methane, into ornithine. In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541 (Table 2). In some non-limiting embodiments the microorganism
is Ralstonia eutropha N-1, DSM 13513. In some embodiments, the
instant invention further provides for the additional step of
converting ornithine into putrescine through the catalytic action
of the enzyme ornithine decarboxylase. In some embodiments, the
instant invention further provides for the additional step of
adding adipic acid to the putrescine to produce nylon-4,6.
[0024] In one embodiment, the instant invention provides a
composition containing an engineered or natural microorganism that
converts a carbon-containing gas such as syngas, producer gas, CO2,
carbon monoxide and mixtures of the same containing hydrogen gas;
and/or C1 compounds, gaseous or liquid, including but not limited
to methanol or methane, into lysine. In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541 (Table 2). In some non-limiting embodiments the microorganism
is Ralstonia eutropha N-1, DSM 13513.
[0025] In some embodiments, the instant invention further provides
for the engineered microorganism that converts a carbon-containing
gas such as syngas, producer gas, CO2, carbon monoxide and mixtures
of the same containing hydrogen gas; and/or C1 compounds, gaseous
or liquid, including but not limited to methanol or methane, into
lysine and encodes one or more genes including but not limited to,
a carbon nitrogen lyase, an oxidoreductase, .alpha.-.beta.-enoate
reductase (EC 1.3.1.-) and/or an amidohydrolase (EC 3.5.2.-).
[0026] In some embodiments, the instant invention further provides
for the engineered microorganism that converts a carbon-containing
gas such as syngas, producer gas, CO.sub.2, carbon monoxide and
mixtures of the same containing hydrogen gas; and/or C1 compounds,
gaseous or liquid, including but not limited to methanol or
methane, into tyrosine and encodes one or more genes including but
not limited to, tyrosine phenol lyase (EC 4.1.99.2). In some
embodiments the reaction of tyrosine catalyzed by tyrosine phenol
lyase results in the production of phenol.
[0027] In some embodiments, the instant invention provides for a
method of producing phenol by combining, in a bioreactor or
solution, one or more enzymes including but not limited to tyrosine
ammonium lyase and, a carbon-containing gas, and an engineered or
natural microorganism that converts a carbon-containing gas such as
syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into a
tyrosine. (Table 2). In some non-limiting embodiments the
microorganism is Cupriavidus necator DSM 531 or DSM 541. In some
non-limiting embodiments the microorganism is Ralstonia eutropha
N-1, DSM 13513.
[0028] In some embodiments, the instant invention further provides
for a method of producing caprolactam by combining the engineered
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO.sub.2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into lysine, with
said carbon-containing gas and an enzyme encoded into the
microorganism or supplied to the organism including but not limited
to .alpha.-.beta.-enoate reductase and/or amidohydrolase in a
bioreactor or solution. In some embodiments the caprolactam is used
to produce nylon-6 using chemical conversion processes known to one
well versed in the art and science.
[0029] In some embodiments, the instant invention further provides
for a method of producing caprolactam by combining the engineered
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO.sub.2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into tyrosine
(Table 2), with said carbon-containing gas and an enzyme encoded
into the microorganism or supplied to the organism including but
not limited to tyrosine phenol lyase (EC 4.1.99.2) in a bioreactor
or solution. In some embodiments the action of tyrosine phenol
lyase on tyrosine produces phenol. In some embodiments the phenol
is separated from the aqueous broth using methods known to one well
versed in the art and science. In some embodiments commercial
technologies that have been developed for the recovery of phenol
from an aqueous process stream are utilized for the separation of
phenol from the aqueous broth. In some non-limiting embodiments a
pervaporation process is used to separate phenol from the aqueous
broth. In some non-limiting embodiments pervaporation is followed
by liquid-liquid phase separation to produce a higher purity phenol
output. In some embodiments the phenol product resulting from
pervaporation followed by liquid-liquid phase separation is over 72
wt % phenol. In some embodiments the aqueous residual from
pervaporation followed by liquid-liquid phase separation is 7-8 wt
% phenol, which is recycled to the feed stream entering the
pervaporation step. In some embodiments the phenol-rich liquid
produced through pervaporation is further purified by distillation
using methods known to one well versed in the art and science. In
some other non-limiting embodiments solvent extraction is used to
separate phenol from the aqueous broth. In some non-limiting
embodiments the cells are filtered from the aqueous broth using
methods known to one well versed in the art and science and are
recycled into the bioreactor prior to the aqueous broth entering
solvent. In some embodiments Methylisobutylketone (MIBK), is used
as an extracting agent to extract phenol from the aqueous broth in
an extraction column. In some embodiments the phenol is further
purified and the MIBK recovered for further extractions using a
distillation column. In some embodiments the stream of aqueous
broth after having phenol removed by solvent extraction flowing out
of the solvent extraction unit may carry traces of the extracting
agent. In some embodiments the traces of extracting agent are
recovered in a stripper column. In some embodiments the MIBK/water
azeotrope accumulates at the head of the stripper column, where it
is separated in the separator into the light MIBK phase and the
heavy water phase. In some embodiments the recovered MIBK is used
for additional solvent extraction. In some embodiments the
recovered phenol is converted to cyclohexanone using methods known
to one well versed in the art and science. In some embodiments
phenol is converted to cyclohexanone using a commercial process for
the hydrogenation of phenol to cyclohexanone. In some non-limiting
embodiments the hydrogenation of phenol occurs in the vapor phase.
In some non-limiting embodiments the hydrogenation of phenol occurs
in the liquid phase. In some non-limiting embodiments the
hydrogenation of phenol to cyclohexanone utilizes a palladium based
catalyst.
[0030] In some non-limiting embodiments vapor phase phenol
hydrogenation is conducted at temperatures from 140-170.degree. C.
and a pressure slightly above atmospheric. In some embodiments the
conversion of phenol to cyclohexanone occurs in a single reactor.
In some embodiments over a 90% yield in cyclohexanone is achieved
in a single reactor. In some non-limiting embodiments a nickel-type
catalyst is used instead of a palladium based catalyst. In some
non-limiting embodiments the conversion of phenol to cyclohexanone
has two distinct reaction steps, 1) full hydrogenation to
cyclohexanol followed by 2) dehydrogenation to cyclohexanone. In
some non-limiting embodiments a liquid-phase hydrogenation of
phenol to cyclohexanone is performed at temperatures below the
atmospheric boiling point. In some non-limiting embodiments a
liquid-phase hydrogenation of phenol to cyclohexanone is performed
at temperatures from 140-150.degree. C. In some non-limiting
embodiments over 99% yield of cyclohexanone is achieved at greater
than or equal to 90% conversion. In some embodiments the
cyclohexanone produced is converted to caprolactam using chemical
conversion processes known to one well versed in the art and
science. In some embodiments the cyclohexanone is converted to the
oxime by reaction with hydroxylamine which in turn rearranges to
form caprolactam in the presence of a sulfuric acid catalyst. In
some embodiments the caprolactam is used to produce nylon-6 using
chemical conversion processes known to one well versed in the art
and science. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531 or DSM 541. In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0031] In some embodiments, the instant invention further provides
for a method of producing caprolactam by combining the engineered
or natural microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of
the same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
lysine (Table 2), recovering lysine from the microbial broth using
methods known in the art and science of microbial lysine
production, and using high temperatures and alcohol as described in
and specifically incorporated by reference from U.S. Pat. No.
8,283,466 (U.S. patent application Ser. No. 12/527,848) to convert
the lysine into caprolactam. In some embodiments the caprolactam is
used to produce nylon-6 using chemical conversion processes known
to one well versed in the art and science. In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541. In some non-limiting embodiments the microorganism is
Ralstonia eutropha N-1, DSM 13513.
[0032] In some embodiments, the instant invention further provides
for a method of producing caprolactam by combining the engineered
or natural microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
lysine (Table 2), with said carbon-containing gas and a chemical
catalyst as described in and specifically incorporated by reference
from U.S. Pat. No. 7,399,855 used to convert the lysine into
caprolactam. In some embodiments the caprolactam is used to produce
nylon-6 using chemical conversion processes known to one well
versed in the art and science. In some non-limiting embodiments the
microorganism is Cupriavidus necator DSM 531 or DSM 541. In some
non-limiting embodiments the microorganism is Ralstonia eutropha
N-1, DSM 13513.
[0033] In an alternative embodiment, the instant invention provides
for an engineered or natural microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO.sub.2,
carbon monoxide and mixtures of the same containing hydrogen gas;
and/or C1 compounds, gaseous or liquid, including but not limited
to methanol or methane, into phenylalanine (Table 2). In some
embodiments the engineered or natural microorganism encodes one or
more genes including but not limited to enzymes in the Shikimate
pathway including but not limited to chorismate synthase. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0034] In some embodiments, the instant invention provides for a
method of producing phenylalanine by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered or natural
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into
phenylalanine (Table 2), and in some embodiments it encodes one or
more genes including but not limited to enzymes in the Shikimate
pathway including but not limited to chorismate synthase. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0035] In some embodiments, the instant invention provides for a
method of producing phenylalanine by combining, in a bioreactor or
solution, one or more enzymes including but not limited to enzymes
in the Shikimate pathway including but not limited to chorismate
synthase, a carbon-containing gas, and an engineered or natural
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into a precursor
to phenylalanine that the enzymes use. (Table 2). In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0036] In some embodiments, the instant invention provides for an
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of
the same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
cinnamic acid and encodes one or more genes including but not
limited to the enzyme phenylalanine ammonium lyase (EC
4.3.1.24).
[0037] In some embodiments, the instant invention provides for a
method of producing cinnamic acid by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO.sub.2, carbon monoxide and mixtures of the same containing
hydrogen gas; and/or C1 compounds, gaseous or liquid, including but
not limited to methanol or methane, into cinnamic acid and encodes
one or more genes including but not limited to the enzyme
phenylalanine ammonium lyase. In some non-limiting embodiments the
phenylalanine ammonium lyase gene is taken from Streptomyces
maritimus (Piel et al., 2000).
[0038] In some embodiments, the instant invention provides for a
method of producing cinnamic acid by combining, in a bioreactor or
solution, one or more enzymes including but not limited to
phenylalanine ammonium lyase, a carbon-containing gas, and an
engineered or natural microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO2, carbon
monoxide and mixtures of the same containing hydrogen gas; and/or
C1 compounds, gaseous or liquid, including but not limited to
methanol or methane, into a phenylalanine. (Table 2). In some
embodiments the phenylalanine is converted to cinnamic acid. In
some non-limiting embodiments the phenylalanine ammonium lyase
enzyme is taken from Streptomyces maritimus (Piel et al., 2000). In
some non-limiting embodiments the microorganism is Cupriavidus
necator DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0039] In some embodiments, the instant invention provides for a
method of producing cinnamic acid by combining, in a bioreactor or
solution, one or more enzymes including but not limited to tyrosine
ammonium lyase and, a carbon-containing gas, and an engineered or
natural microorganism that converts a carbon-containing gas such as
syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into a
tyrosine. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531 or DSM 541. In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0040] In some embodiments, the instant invention provides for an
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of
the same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
styrene and encodes one or more genes including but not limited to
the enzyme cinnamic acid decarboxylase. In some embodiments two or
more genes are encoded including phenylalanine ammonium lyase and
cinnamic acid decarboxylase. In some embodiments phenylalanine is
converted to cinnamic acid. In some embodiments cinnamic acid is
converted to styrene. In some embodiments phenylalanine ammonium
lyase (EC 4.3.1.24) activity converts phenylalanine into cinnamic
acid. In some embodiments cinnamic acid is further converted into
styrene through a decarboxylation step catalyzed by enzymes
belonging to a family of oxalate decarboxylases (EC 4.1.1.2). In
some non-limiting embodiments the genes that encode phenylalanine
ammonium lyase (EC 4.3.1.24) are taken from Streptomyces maritimus
[Piel et al., 2000]. In some non-limiting embodiments the genes
that encode oxylate decarboxylase are taken from Rhodococcus jostii
RHA1 (McLeod et al., 2006). In some non-limiting embodiments the
microorganism is Cupriavidus necator DSM 531 or DSM 541. In some
non-limiting embodiments the microorganism is Ralstonia eutropha
N-1, DSM 13513.
[0041] In some embodiments, the instant invention provides for a
method of producing styrene by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO.sub.2, carbon monoxide and mixtures of the same containing
hydrogen gas; and/or C1 compounds, gaseous or liquid, including but
not limited to methanol or methane, into styrene and encodes one or
more genes including but not limited to the enzyme cinnamic acid
decarboxylase. In some embodiments two or more genes are encoded
including phenylalanine ammonium lyase and cinnamic acid
decarboxylase. In some embodiments phenylalanine is converted to
cinnamic acid. In some embodiments cinnamic acid is converted to
styrene. In some embodiments phenylalanine ammonium lyase (EC
4.3.1.24) activity converts phenylalanine into cinnamic acid. In
some embodiments cinnamic acid is further converted into styrene
through a decarboxylation step catalyzed by enzymes belonging to a
family of oxalate decarboxylases (EC 4.1.1.2). In some non-limiting
embodiments the genes that encode phenylalanine ammonium lyase (EC
4.3.1.24) are taken from Streptomyces maritimus (Piel et al.,
2000). In some non-limiting embodiments the genes that encode
oxylate decarboxylase are taken from Rhodococcus jostii RHA1
(McLeod et al., 2006). In some non-limiting embodiments the
microorganism is Cupriavidus necator DSM 531 or DSM 541. In some
non-limiting embodiments the microorganism is Ralstonia eutropha
N-1, DSM 13513.
[0042] In some embodiments, the instant invention provides for a
method of producing styrene by combining, in a bioreactor or
solution, one or more enzymes including but not limited to cinnamic
acid decarboxylase, a carbon-containing gas, and an engineered or
natural microorganism that converts a carbon-containing gas such as
syngas, producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into a cinnamic
acid. In some embodiments the cinnamic acid is converted to
styrene. In some embodiments cinnamic acid is converted into
styrene through a decarboxylation step catalyzed by enzymes
belonging to a family of oxalate decarboxylases (EC 4.1.1.2). In
some non-limiting embodiments the oxalate decarboxylase enzyme that
decarboxylates the cinnamic acid to produce styrene is encode by a
genes taken from Rhodococcus jostii RHA1 (McLeod et al., 2006). In
some non-limiting embodiments the microorganism is Cupriavidus
necator DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0043] In some embodiments, the instant invention provides for an
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
tyrosine and encodes one or more genes including but not limited to
the enzyme phenylalanine hydroxylase. In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541. In some non-limiting embodiments the microorganism is
Ralstonia eutropha N-1, DSM 13513.
[0044] In some embodiments, the instant invention provides for a
method of producing tyrosine by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered or natural
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into tyrosine and
encodes one or more genes including but not limited to the enzyme
phenylalanine hydroxylase (Table 2). In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541. In some non-limiting embodiments the microorganism is
Ralstonia eutropha N-1, DSM 13513.
[0045] In some embodiments, the instant invention provides for a
method of producing tyrosine by combining, in a bioreactor or
solution, one or more enzymes including but not limited to
phenylalanine hydroxylase, a carbon-containing gas, and an
engineered or natural microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO2, carbon
monoxide and mixtures of the same containing hydrogen gas; and/or
C1 compounds, gaseous or liquid, including but not limited to
methanol or methane, into a phenylalanine (Table 2). In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0046] In some embodiments, the instant invention provides for an
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane into
phenol and encodes one or more genes including but not limited to
the enzyme tyrosine phenol lyase. In some embodiments tyrosine is
converted into phenol. In some non-limiting embodiments the
microorganism is Cupriavidus necator DSM 531 or DSM 541. In some
non-limiting embodiments the microorganism is Ralstonia eutropha
N-1, DSM 13513.
[0047] In some embodiments, the instant invention provides for a
method of producing phenol by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO2, carbon monoxide and mixtures of the same containing hydrogen
gas; and/or C1 compounds, gaseous or liquid, including but not
limited to methanol or methane, into phenol and encodes one or more
genes including but not limited to the enzyme tyrosine phenol
lyase. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531 or DSM 541. In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0048] In some embodiments, the instant invention provides for a
method of producing phenol by combining, in a bioreactor or
solution, one or more enzymes including but not limited to tyrosine
phenol lyase, a carbon-containing gas, and an engineered or natural
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into a tyrosine.
In some embodiments tyrosine is converted to phenol. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0049] In some embodiments, the instant invention provides for a
method of producing benzene by first producing phenol by combining,
in a bioreactor or solution, a carbon-containing gas and an
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
phenol and encodes one or more genes including but not limited to
the enzyme tyrosine phenol lyase; then by converting phenol into
benzene chemically by one or more methods known to one well versed
in the art and science including but not limited to combining heat,
zinc metal and phenol to produce benzene according to well
established and known protocols. In some embodiments, the instant
invention further provides for a method of converting the benzene
produced by said method into caprolactam known to one well versed
in the art and science through commercial chemical processes for
the hydrogenation of benzene into cyclohexane, then oxidizing
cyclohexane into cyclohexanol which is dehydrogenated into
cyclohexanone which in some embodiments is converted into
caprolactam through the Beckman rearrangement. In an alternative
embodiment, the instant invention further provides for a method of
converting said benzene into teraphthalic acid through commercial
chemical processes well known to one well versed in the art and
science including but not limited to by converting benzene through
dehydrocyclodimerization into xylene, which is then converted by
oxidation into teraphthalic acid. In some non-limiting embodiments
the microorganism is Cupriavidus necator DSM 531 or DSM 541. In
some non-limiting embodiments the microorganism is Ralstonia
eutropha N-1, DSM 13513.
[0050] In some embodiments, the instant invention provides for a
method of producing cyclohexanone by combining, in a bioreactor or
solution, a carbon-containing gas, and an engineered or natural
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO.sub.2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into a phenol,
then converting the phenol into cyclohexanone chemically through
hydrogenation, a commercial chemical process well known to one well
versed in the art and science. In some embodiments the
cyclohexanone is converted into caprolactam through, a commercial
chemical process well known to one well versed in the art and
science known as the Beckman rearrangement. In some embodiments the
caprolactam is a precursor for nylon-6. In some embodiments it
encodes one or more genes including but not limited to the enzyme
tyrosine phenol lyase. In some embodiments tyrosine is converted to
phenol. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531 or DSM 541. In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0051] In some embodiments, the instant invention provides for an
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of
the same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including, but not limited to methanol or methane, into
caprolactam and encodes one or more genes including but not limited
to the enzyme carbon nitrogen lyase, .alpha.-.beta.-enoate
reductase, amidohydrolase. In some embodiments the caprolactam is
used as a precursor for the production of nylon-6. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0052] In some embodiments, the instant invention provides for a
method of producing caprolactam by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO.sub.2, carbon monoxide and mixtures of the same containing
hydrogen gas; and/or C1 compounds, gaseous or liquid, including but
not limited to methanol or methane, into caprolactam and encodes
one or more genes including but not limited to the enzyme carbon
nitrogen lyase, .alpha.-.beta.-enoate reductase, amidohydrolase. In
some embodiments the caprolactam is used as a precursor for
nylon-6. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531 or DSM 541. In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0053] In some embodiments, the instant invention provides for a
method of producing caprolactam by combining, in a bioreactor or
solution, one or more enzymes including but not limited to Carbon
nitrogen lyase, .alpha.-.beta.-enoate reductase, amidohydrolase, a
carbon-containing gas, and an engineered or natural microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO.sub.2, carbon monoxide and mixtures of the same containing
hydrogen gas; and/or C1 compounds, gaseous or liquid, including but
not limited to methanol or methane, into a lysine (Table 2). In
some non-limiting embodiments the microorganism is Cupriavidus
necator DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0054] In some embodiments, the instant invention provides for a
method of producing caprolactam by combining, in a bioreactor or
solution, a carbon-containing gas, and an engineered or natural
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO.sub.2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into a
cyclohexanone, then performing the acid induced Beckman
rearrangement on the cyclohexanone produced. In some embodiments
the cyclohexanone is converted to caprolactam. In some embodiments
the caprolactam is converted to nylon-6. In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541. In some non-limiting embodiments the microorganism is
Ralstonia eutropha N-1, DSM 13513.
[0055] In some embodiments, the instant invention provides for a
method of producing polyhydroxybutyrate (PHB) by combining, in a
bioreactor or solution, a carbon-containing gas and a natural
strain microorganism that converts a carbon-containing gas such as
syngas, producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into
polyhydroxybutyrate. (FIGS. 2, 3A and 3B). In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531.
[0056] In some embodiments, the instant invention provides for a
method of producing polyhydroxybutyrate by combining, in a
bioreactor or solution, one or more enzymes used to convert a
carbon containing gas into polyhydroxybutyrate including but not
limited to HMG-CoA lyase, 3-hydroxybutyrate dehydrogenase, and/or
3-hydroxybuterate polymerase, a carbon-containing gas, and a
natural microorganism that converts a carbon-containing gas such as
syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into a
precursor compound that can be enzymatically converted to
polyhydroxybutyrate including but not limited to acetyl-CoA,
pyruvate, acetoacetate, and 3-hydroxybuterate. (FIG. 1). In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531.
[0057] In some embodiments, the instant invention further provides
for a method of producing 3-hydroxybuterate by combining, in a
bioreactor or solution, a carbon-containing gas and a natural or
engineered strain microorganism that converts a carbon-containing
gas such as syngas, producer gas, CO2, carbon monoxide and mixtures
of the same containing hydrogen gas; and/or C1 compounds, gaseous
or liquid, including but not limited to methanol or methane, into
3-hydroxybuterate. (FIG. 1). In some non-limiting embodiments the
microorganism is Cupriavidus necator DSM 531.
[0058] In some embodiments, the instant invention further provides
for a method of producing 3-hydoxybuterate by combining, in a
bioreactor or solution, one or more enzymes including but not
limited to PHB depolymerase, a carbon-containing gas, and a natural
or engineered microorganism that converts a carbon-containing gas
such as syngas, producer gas, CO2, carbon monoxide and mixtures of
the same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into a
polyhydroxybutyrate. (FIGS. 2, 3A, 3B). In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531. In
some non-limiting embodiments the microorganism is Ralstonia
eutropha N-1, DSM 13513.
[0059] In some embodiments, the instant invention further provides
for a method of producing 1,3-butanediol by combining, in a
bioreactor or solution, a carbon-containing gas and a natural or
engineered microorganism that converts a carbon-containing gas such
as syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of
the same containing hydrogen gas; and/or C1 compounds, gaseous or
liquid, including but not limited to methanol or methane, into
1,3-butanediol. (FIG. 1). In some embodiments the 1,3-butanediol
produced in certain embodiments of the present invention is
recovered from the aqueous broth using methods known to one well
versed in the art and science. In some embodiments 1,3-butanediol
is recovered from the aqueous broth by solvent extraction. In some
embodiments 1,3-butanediol is recovered from the aqueous broth
using in situ solvent extraction. In some embodiments a
biocompatible but not bioavailable solvent is used for in situ
solvent extraction of 1,3-BDO. In some non-limiting embodiments the
biocompatible but not bioavailable solvent used to extract 1,3-BDO
from the aqueous broth is cis-9-octadecen-1-ol. In some embodiments
one or more of the following separation steps are used to recover
1,3-BDO from the aqueous broth: steam stripping, pervaporation,
reverse osmosis, and/or solvent extraction. In some embodiments one
or more of the following solvents are used in liquid-liquid
extraction of the 1,3-BDO from the aqueous broth: solvent
extractants, e.g., ethyl acetate, tributylphosphate, diethyl ether,
n-butanol, dodecanol, and/or oleyl alcohol. In some embodiments
prior to exposure to solvent, the aqueous broth is dewatered by
evaporation or both microfiltration and reverse osmosis. In some
embodiments reactive extraction is used to recover 1,3-butanediol
from the aqueous broth whereby 1,3-butanediol is reacted with
formaldehyde to form a formal under catalysis of acid (Senkus
1946), with the 1,3-butanediol formal collected in the top oil
phase and allowed to react with acid methanol to form
1,3-butanediol and methylal, and the methylal can be hydrolyzed to
methanol and formaldehyde, with each of the three reaction steps
using acids as catalyst. In some embodiments pervaporation or
vacuum membrane distillation is used for the concentration of
1,3-butanediol from the aqueous broth (Qureshi et al. 1994). In
some embodiments an integrated process for fed-batch fermentation
of 1,3-butanediol combined with recovery of 1,3-butanediol by
vacuum membrane distillation is used. In some non-limiting
embodiments a microporous polytetrafluoroethylene (PTFE) membrane
is used for the recovery by pervaporation or vacuum membrane
distillation. In some non-limiting embodiments a silicone membrane
is used for the pervaporative recovery of 1,3-butanediol from the
aqueous broth. In some embodiments the recovered 1,3-butanediol is
converted to butadiene by dehydration using methods known to one
well versed in the art and science. In some embodiments a
commercially used process for the conversion of 1,3-butanediol to
butadiene by dehydration will be utilized. In some non-limiting
embodiments the 1,3-butanediol is dehydrated in the gas phase at
270.degree. C. using a Na polyphosphate catalyst to produce
1,3-butadiene. In some non-limiting embodiments the selectivity of
the conversion from 1,3-butanediol to butadiene is about 70%. In
some non-limiting embodiments the microorganism is Cupriavidus
necator DSM 531. In some non-limiting embodiments the microorganism
is Ralstonia eutropha N-1, DSM 13513.
[0060] In some embodiments, the instant invention further provides
for a method of producing 1,3-butanediol by combining, in a
bioreactor or solution, one or more enzymes including but not
limited to PHB depolymerase, aldehyde dehydrogenase, and/or alcohol
dehydrogenase, a carbon-containing gas, and a natural or engineered
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO.sub.2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into a
polyhydroxybutyrate. (FIGS. 1, 2, 3A, 3B). In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531. In
some non-limiting embodiments the microorganism is Ralstonia
eutropha N-1, DSM 13513.
[0061] In some embodiments, the instant invention provides for a
natural or engineered microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO.sub.2,
carbon monoxide and mixtures of the same containing hydrogen gas;
and/or C1 compounds, gaseous or liquid, including but not limited
to methanol or methane, into butanediol (BDO) and encodes one or
more genes including but not limited to the enzyme acetolactate
synthase, .alpha.-acetolactate decarboxylase, and/or 2,3-butanediol
dehydrogenase. In some non-limiting embodiments the microorganism
is Cupriavidus necator DSM 531 or DSM 541. In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0062] In some embodiments, the instant invention provides for a
method of producing butanediol by combining, in a bioreactor or
solution, a carbon-containing gas and a natural or engineered
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO.sub.2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into butanediol
and encodes one or more genes including but not limited to the
enzyme acetolactate synthase, .alpha.-acetolactate decarboxylase,
and/or 2,3-butanediol dehydrogenase. In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541. In some non-limiting embodiments the microorganism is
Ralstonia eutropha N-1, DSM 13513.
[0063] In some embodiments, the instant invention provides for a
method of producing butanediol by combining, in a bioreactor or
solution, one or more enzymes including but not limited to
acetolactate synthase, .alpha.-acetolactate decarboxylase, and/or
2,3-butanediol dehydrogenase, a carbon-containing gas, and an
engineered or natural microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO.sub.2,
carbon monoxide and mixtures of the same containing hydrogen gas;
and/or C1 compounds, gaseous or liquid, including but not limited
to methanol or methane, into a pyruvate. In some non-limiting
embodiments the microorganism is Cupriavidus necator DSM 531 or DSM
541. In some non-limiting embodiments the microorganism is
Ralstonia eutropha N-1, DSM 13513.
[0064] In some embodiments, the instant invention provides for a
natural or engineered microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO2, carbon
monoxide and mixtures of the same containing hydrogen gas; and/or
C1 compounds, gaseous or liquid, including but not limited to
methanol or methane, into butadiene and encodes one or more genes
including but not limited to a fatty acid hydrotase including but
not limited to oleate hydratase. In some non-limiting embodiments
the microorganism is Cupriavidus necator DSM 531 or DSM 541. In
some non-limiting embodiments the microorganism is Ralstonia
eutropha N-1, DSM 13513.
[0065] In some embodiments, the instant invention provides for a
method of producing butadiene by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO2, carbon monoxide and mixtures of the same containing hydrogen
gas; and/or C1 compounds, gaseous or liquid, including but not
limited to methanol or methane, into butadiene and encodes one or
more genes including but not limited to the enzyme a fatty acid
hydratase including but not limited to oleate hydratase. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0066] In some embodiments, the instant invention provides for a
method of producing butadiene by combining, in a bioreactor or
solution, one or more enzymes including but not limited to a fatty
acid hydratase including but not limited to oleate hydratase, a
carbon-containing gas, and an engineered or natural microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO2, carbon monoxide and mixtures of the same containing hydrogen
gas; and/or C1 compounds, gaseous or liquid, including but not
limited to methanol or methane, into a 1,3-butanediol. (FIG. 1). In
some embodiments 1,3-butanediol is recovered from the aqueous broth
using methods known to one well versed in the art and science. In
some embodiments 1,3-butanediol is recovered from the aqueous broth
by solvent extraction. In some embodiments 1,3-butanediol is
recovered from the aqueous broth using in situ solvent extraction.
In some embodiments a biocompatible but not bioavailable solvent is
used for in situ solvent extraction of 1,3-BDO. In some
non-limiting embodiments the biocompatible but not bioavailable
solvent used to extract 1,3-BDO from the aqueous broth is
cis-9-octadecen-1-ol. In some embodiments one or more of the
following separation steps are used to recover 1,3-BDO from the
aqueous broth: steam stripping, pervaporation, reverse osmosis,
and/or solvent extraction. In some embodiments one or more of the
following solvents are used in liquid-liquid extraction of the
1,3-BDO from the aqueous broth: solvent extractants, e.g., ethyl
acetate, tributylphosphate, diethyl ether, n-butanol, dodecanol,
and/or oleyl alcohol. In some embodiments prior to exposure to
solvent, the aqueous broth is dewatered by evaporation or both
microfiltration and reverse osmosis. In some embodiments reactive
extraction is used to recover 1,3-butanediol from the aqueous broth
whereby 1,3-butanediol is reacted with formaldehyde to form a
formal under catalysis of acid (Senkus 1946), with the
1,3-butanediol formal collected in the top oil phase and allowed to
react with acid methanol to form 1,3-butanediol and methylal, and
the methylal can be hydrolyzed to methanol and formaldehyde, with
each of the three reaction steps using acids as catalyst. In some
embodiments pervaporation or vacuum membrane distillation is used
for the concentration of 1,3-butanediol from the aqueous broth
(Qureshi et al. 1994). In some embodiments an integrated process
for fed-batch fermentation of 1,3-butanediol combined with recovery
of 1,3-butanediol by vacuum membrane distillation is used. In some
non-limiting embodiments a microporous polytetrafluoroethylene
(PTFE) membrane is used for the recovery by pervaporation or vacuum
membrane distillation. In some non-limiting embodiments a silicone
membrane is used for the pervaporative recovery of 1,3-butanediol
from the aqueous broth. In some embodiments the recovered
1,3-butanediol is converted to butadiene by dehydration using
methods known to one well versed in the art and science. In some
embodiments a commercially used process for the conversion of
1,3-butanediol to butadiene by dehydration will be utilized. In
some non-limiting embodiments the 1,3-butanediol is dehydrated in
the gas phase at 270.degree. C. using a Na polyphosphate catalyst
to produce 1,3-butadiene. In some non-limiting embodiments the
selectivity of the conversion from 1,3-butanediol to butadiene is
about 70%. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0067] In some embodiments, the instant invention provides for a
method of producing butadiene by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO.sub.2, carbon monoxide and mixtures of the same containing
hydrogen gas; and/or C1 compounds, gaseous or liquid, including but
not limited to methanol or methane, into 1,3-butanediol and encodes
one or more genes including but not limited to the enzyme a fatty
acid hydratase including but not limited to oleate hydratase. In
some non-limiting embodiments the microorganism is Cupriavidus
necator DSM 531. In some non-limiting embodiments the microorganism
is Ralstonia eutropha N-1, DSM 13513.
[0068] In some embodiments, the instant invention provides for a
method of producing butadiene by combining, in a bioreactor or
solution, one or more enzymes including but not limited to a fatty
acid hydratase including but not limited to oleate hydratase, a
carbon-containing gas, and an engineered or natural microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO2, carbon monoxide and mixtures of the same containing hydrogen
gas; and/or C1 compounds, gaseous or liquid, including, but not
limited to methanol or methane, into a 2,3-butanediol. In some
embodiments the 2,3-butandiol is converted to butadiene by
dehydration. In some non-limiting embodiments the microorganism is
Cupriavidus necator DSM 531 or DSM 541. In some non-limiting
embodiments the microorganism is Ralstonia eutropha N-1, DSM
13513.
[0069] In some embodiments, the instant invention provides for a
method of producing butadiene by combining, in a bioreactor or
solution, a carbon-containing gas and an engineered microorganism
that converts a carbon-containing gas such as syngas, producer gas,
CO2, carbon monoxide and mixtures of the same containing hydrogen
gas; and/or C1 compounds, gaseous or liquid, including but not
limited to methanol or methane, into 2,3-butanediol and encodes one
or more genes including but not limited to the enzyme a fatty acid
hydratase including but not limited to oleate hydratase. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0070] In some embodiments, the instant invention provides for a
method of producing butadiene by first producing butanediol by
combining, in a bioreactor or solution, a carbon-containing gas and
a natural or engineered microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO.sub.2,
carbon monoxide and mixtures of the same containing hydrogen gas;
and/or C1 compounds, gaseous or liquid, including but not limited
to methanol or methane into butanediol. In some embodiments the
butanediol is recovered from the aqueous broth using methods known
to one well versed in the art and science. In some embodiments
butanediol is recovered from the aqueous broth by solvent
extraction. In some embodiments butanediol is recovered from the
aqueous broth using in situ solvent extraction. In some embodiments
a biocompatible but not bioavailable solvent is used for in situ
solvent extraction of BDO. In some non-limiting embodiments the
biocompatible but not bioavailable solvent used to extract BDO from
the aqueous broth is cis-9-octadecen-1-ol. In some embodiments one
or more of the following separation steps are used to recover BDO
from the aqueous broth: steam stripping, pervaporation, reverse
osmosis, and/or solvent extraction. In some embodiments one or more
of the following solvents are used in liquid-liquid extraction of
the BDO from the aqueous broth: solvent extractants, e.g., ethyl
acetate, tributylphosphate, diethyl ether, n-butanol, dodecanol,
and/or oleyl alcohol. In some embodiments prior to exposure to
solvent, the aqueous broth dewatered by evaporation or both
microfiltration and reverse osmosis. In some embodiments reactive
extraction is used to recover butanediol from the aqueous broth
whereby butanediol is reacted with formaldehyde to form a formal
under catalysis of acid (Senkus 1946), with the butanediol formal
collected in the top oil phase and allowed to react with acid
methanol to form butanediol and methylal, and the methylal can be
hydrolyzed to methanol and formaldehyde, with each of the three
reaction steps using acids as catalyst. In some embodiments
pervaporation or vacuum membrane distillation is used for the
concentration of butanediol from the aqueous broth (Qureshi et al.
1994). In some embodiments an integrated process for fed-batch
fermentation of butanediol combined with recovery of butanediol by
vacuum membrane distillation is used. In some non-limiting
embodiments a microporous polytetrafluoroethylene (PTFE) membrane
is used for the recovery by pervaporation or vacuum membrane
distillation. In some non-limiting embodiments a silicone membrane
is used for the pervaporative recovery of butanediol from the
aqueous broth. In some embodiments the butanediol recovered from
the aqueous broth is then by converting into butadiene chemically
by dehydrating butanediol over a catalyst such as thorium oxide
using methods known to one well versed in the art and science. In
some embodiments the isomer of butanediol produced in the
bioreactor and recovered from the broth is 1,3-butanediol. In some
embodiments a commercially used process for the conversion of
1,3-butanediol to butadiene by dehydration will be utilized. In
some non-limiting embodiments the 1,3-butanediol is dehydrated in
the gas phase at 270.degree. C. using a Na polyphosphate catalyst
to produce 1,3-butadiene. In some non-limiting embodiments the
selectivity of the conversion from 1,3-butanediol to butadiene is
about 70%. In some embodiments, 1,3-butadiene is further converted
into styrene-butadiene rubber by mixing butadiene with styrene
using methods known to one well versed in the art and science of
synthetic rubber manufacturing. In some embodiments the butadiene
is further converted to caprolactam using methods known to one well
versed in the art and science by first carbonylating butadiene into
methyl 3-pentenoate, then isomerizing methyl 3-pentenoate into
methyl 4-pentenoate, then hydroformylating methyl 4-pentenoate into
methyl 5-formylvalerate, which is mixed with hydrogen and ammonia
into methyl 6-aminocaproate, which is mixed in a multitubular
reactor with xylene to produce caprolactam and methanol. In some
embodiments, butadiene is further converted into adiponitrile using
methods known to one well versed in the art and science by a
nickel-catalyzed hydrocynation of butadiene, involving butadiene
monohydocynated into isomers of pentenenitriles, and 2- and
3-methylbutenentriles, the unsaturated nitriles are isomerized into
3- and 4-pentenenitriles, which are hydrocynated to produce
adiponitrile. In some embodiments, using methods known to one well
versed in the art and science adiponitrile is further hydrogenated
into 1,6-diaminohexane, which is then mixed with adipic acid for
the production of nylon 6,6. In some embodiments using methods
known to one well versed in the art and science the butadiene is
used to produce synthetic rubber through polymerization. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0071] In some embodiments, the instant invention provides for a
method of producing omega-7 fatty acids including but not limited
to palmitoleic acid also known as 7-hexadecenoic acid, by
combining, in a bioreactor or solution, a carbon-containing gas,
and a natural strain microorganism that converts a
carbon-containing gas such as syngas, producer gas, CO2, carbon
monoxide and mixtures of the same containing hydrogen gas; and/or
C1 compounds, gaseous or liquid, including but not limited to
methanol or methane, into omega-7 fatty acids, including but not
limited to palmitoleic acid (FIG. 4). In some non-limiting
embodiments the microorganism is Rhodococcus opacus DSM 43205. In
some non-limiting embodiments the microorganism is Rhodococcus sp.
DSM 3346.
[0072] In some embodiments, the instant invention provides for a
method of producing polyunsaturated fatty acids including but not
limited to alpha-linoleic acid, by combining, in a bioreactor or
solution, a carbon-containing gas, and a natural strain
microorganism that converts a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into a
polyunsaturated fatty acids, including but not limited to
alpha-linoleic acid (FIG. 4). In some non-limiting embodiments the
microorganism is Rhodococcus opacus DSM 43205. In some non-limiting
embodiments the microorganism is Rhodococcus sp. DSM 3346.
[0073] In some embodiments, the instant invention provides for a
method of producing carotenoids including but not limited to
beta-carotene, by combining, in a bioreactor or solution, a
carbon-containing gas, and a natural strain microorganism that
converts a carbon-containing gas such as syngas, producer gas,
CO.sub.2, carbon monoxide and mixtures of the same containing
hydrogen gas; and/or C1 compounds, gaseous or liquid, including but
not limited to methanol or methane, into a carotenoid, including
but not limited to beta-carotene.
[0074] In some embodiments, the instant invention provides for a
method of producing long chain alkanes including but not restricted
to eicosane, by combining, in a bioreactor or solution, a
carbon-containing gas, and a natural strain microorganism that
converts a carbon-containing gas such as syngas, producer gas,
CO.sub.2, carbon monoxide and mixtures of the same containing
hydrogen gas; and/or C1 compounds, gaseous or liquid, including but
not limited to methanol or methane, into long chain alkanes
including but not restricted to eicosane (FIG. 4). In some
non-limiting embodiments the microorganism is Rhodococcus opacus
DSM 43205. In some non-limiting embodiments the microorganism is
Rhodococcus sp. DSM 3346.
[0075] In some embodiments, the instant invention provides for an
engineered or natural microorganism that converts carbon monoxide
and water (H.sub.2O) into hydrogen gas (H.sub.2) and carbon dioxide
and encodes one or more enzyme genes including but not limited to
hydrogenase, and/or carbon monoxide dehydrogenase.
[0076] In some embodiments, the instant invention provides for a
method of producing hydrogen gas and carbon dioxide by combining,
in a bioreactor or solution, carbon monoxide, water (H.sub.2O) and
an engineered or natural microorganism that converts carbon
monoxide and water (H.sub.2O) into hydrogen gas (H.sub.2) and
carbon dioxide and encodes one or more genes including but not
limited to the enzyme hydrogenase, and/or carbon monoxide
dehydrogenase.
[0077] In some embodiments, the instant invention provides for a
method of producing hydrogen gas (H.sub.2) and carbon dioxide by
combining, in a bioreactor or solution, one or more enzymes
including but not limited to hydrogenase and/or carbon monoxide
dehydrogenase, carbon monoxide, and water (H.sub.2O).
[0078] In some embodiments, the instant invention provides for a
method of producing hydrogen gas and carbon dioxide by combining,
in a bioreactor or solution, carbon monoxide, water (H.sub.2O) and
a natural strain or engineered microorganism that converts carbon
monoxide and water (H.sub.2O) into hydrogen gas (H.sub.2) and
carbon dioxide.
[0079] In some embodiments the natural or engineered strain
includes but is not limited to carbon monoxide utilizing microbes
including but not limited to Rhodospirillum rubrum or
Rhodopseudomonas sp. In some embodiments the natural or engineered
strain includes but is not limited to hydrogen utilizing microbes
including but not limited to the genera Rhodococcus or Gordonia,
Ralstonia or Cupriavidus. In some embodiments the natural or
engineered strain includes but is not limited to Corynebacterium
autotrophicum. In some embodiments the natural or engineered strain
includes but is not limited to Corynebacterium glutamicum. In some
embodiments the natural or engineered strain includes but is not
limited to the chemoautotrophic microorganisms from the group
consisting of one or more of the following genera: Acetoanaerobium
sp.; Acetobacterium sp.; Acetogenium sp.; Achromobacter sp.;
Acidianus sp.; Acinetobacter sp.; Actinomadura sp.; Aeromonas sp.;
Alcaligenes sp.; Alcaliqenes sp.; Arcobacter sp.; Aureobacterium
sp.; Bacillus sp.; Beggiatoa sp.; Butyribacterium sp.;
Carboxydothermus sp.; Clostridium sp.; Comamonas sp.; Dehalobacter
sp.; Dehalococcoide sp.; Dehalospirillum sp.; Desulfobacterium sp.;
Desulfomonile sp.; Desulfotomaculum sp.; Desulfovibrio sp.;
Desulfurosarcina sp.; Ectothiorhodospira sp.; Enterobacter sp.;
Eubacterium sp.; Ferroplasma sp.; Halothibacillus sp.;
Hydrogenobacter sp.; Hydrogenomonas sp.; Leptospirillum sp.;
Metallosphaera sp.; Methanobacterium sp.; Methanobrevibacter sp.;
Methanococcus sp.; Methanosarcina sp.; Micrococcus sp.; Nitrobacter
sp.; Nitrosococcus sp.; Nitrosolobus sp.; Nitrosomonas sp.;
Nitrosospira sp.; Nitrosovibrio sp.; Nitrospina sp.; Oleomonas sp.;
Paracoccus sp.; Peptostreptococcus sp.; Planctomycetes sp.;
Pseudomonas sp.; Ralstonia sp.; Rhodobacter sp.; Rhodococcus sp.;
Rhodocyclus sp.; Rhodomicrobium sp.; Rhodopseudomonas sp.;
Rhodospirillum sp.; Shewanella sp.; Streptomyces sp.; Sulfobacillus
sp.; Sulfolobus sp.; Thiobacillus sp.; Thiomicrospira sp.;
Thioploca sp.; Thiosphaera sp.; Thiothrix sp. In some embodiments
the natural or engineered strain includes but is not limited to the
following genera: Rhizobium sp.; Thiocapsa sp.; Nocardia sp.;
Hydrogenovibrio sp.; Helicobacter sp.; Xanthobacter sp.;
Bradyrhizobium sp.; Gordonia sp.; Mycobacteria sp.; Variovorax sp.;
Acidovorax sp.; Anabaena sp.; Scenedesmus sp.; Chlamydomonas sp.,
Ankistrodesmus sp., and Rhaphidium sp.
[0080] In some embodiments, a natural strain or engineered
microorganism converts a carbon-containing gas such as syngas,
producer gas, CO.sub.2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or C1 compounds, gaseous or liquid,
including but not limited to methanol or methane, into organic
compounds including but not limited to one or more of the
following: Methyl undecanoate (11:0), Cyclopentasiloxane,
decamethyl-, Cyclotetrasiloxane, octamethyl-,
7H-Dibenzo[b,g]carbazole, 7-methyl-, Cyclohexasiloxane,
dodecamethyl-, Cyclopentasiloxane, decamethyl-, Cyclopentasiloxane,
decamethyl-, Benzeneacetic acid, .alpha.,3,4-tris[(t, Malonic acid,
2-(2,3-dihydro-benzo[b]th, .beta.-D-Fructofuranose, 2,3,4,6-tetrak,
Benzoic acid, 2,5-bis(trimethylsiloxy)-,
2-Acetyl-1,3,3,4,4-pentamethylcyclopent, Cycloheptasiloxane,
tetradecamethyl-, Cyclohexasiloxane, dodecamethyl-,
7-Chloro-2,3-dihydro-3-(4-N,N-dimethyla, Cyclohexasiloxane,
dodecamethyl-, Benzothiophene, 5-chloro-3-methyl-2-(2-,
7-Chloro-2,3-dihydro-3-(4-N,N-dimethyla, Ethyl
alpha-hydroxy-O-nitrocinnamate,
2-Methyl-4-ethoxycarbonyl-3H-imidazo[1,Heptasiloxane,
1,1,3,3,5,5,7,7,9,9,11,1, 2-Acetyl-1,3,3,4,4-pentamethylcyclopent,
Silane, [[4-[1,2-bis[(trimethylsilyl)ox, TETRADECANOIC ACID, METHYL
ESTER (14:0), Thiocyanic acid carbazol-3,6-diyl ester,
Cycloheptasiloxane, tetradecamethyl-, Thiophene,
2-(trimethylsilyl)-5-[(trime,
2-Acetyl-1,3,3,4,4-pentamethylcyclopent, ISOPENTADECANOIC ACID,
METHYL ESTER (IS, ANTEISO PENTADECANOIC ACID (A15:0),
3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5, Cyclononasiloxane,
octadecamethyl-, ISO HEXADECANOIC ACID, METHYL ESTER,
7-HEXADECENOIC ACID, METHYL ESTER (16:1, 7-HEXADECENOIC ACID,
METHYL ESTER (16:1, HEXADECANOIC ACID, METHYL ESTER (16:0),
HEXADECANOIC ACID, METHYL ESTER (16:0), HEXADECANOIC ACID, METHYL
ESTER (16:0), Cyclohexane, (1,2-dimethylbutyl)-,
cis-13-Octadecenoic acid, Cyclopentane, 1-pentyl-2-propyl-,
Cyclohexanone, 2-ethyl-2-propyl-, 4-Methyl-dodec-3-en-1-ol,
n-Hexadecanoic acid, Cyclopentane, 1-pentyl-2-propyl-, Cyclohexane,
1,2,4-trimethyl-, 7,8-Epoxide-Octadecanoic Acid, Methyl E,
CYCLOPROPYLHEPTADECANOIC ACID, METHYL E, CYCLOPROPYLHEPTADECANOIC
ACID, METHYL E, 3-HYDROXY DODECANOIC ACID, METHYL ESTER,
Cyclononasiloxane, octadecamethyl-, 2-HYDROXY HEXADECANOIC ACID,
METHYL EST, Cycloheptane, 3-HYDROXY DODECANOIC ACID, METHYL ESTER,
2,4-Difluoroaniline, 9-OCTADECENOIC ACID, METHYL ESTER (18:1,
Succinic acid, 2-hexyl pentyl ester, 7-OCTADECENOIC ACID, METHYL
ESTER (18:1, OCTADECANOIC ACID, METHYL ESTER (18:0),
2,2-Dimethylpropanoic acid, undecyl est, Nonanoic acid,
trimethylsilyl ester, Carbamic acid, 1-naphthyl-, propyl este,
2-(5-Nitro-2-furyl)benzimidazole monohy, 10-METHYL OCTADECANOIC
ACID (10ME18:0), Decanoic acid, methyl ester,
Cyclopentaneundecanoic acid, methyl est, 12-NONADECENOIC ACID,
METHYL ESTER (19: Cyclopropyl Nonadecanoic Acid (Cyc 19:0,
13-Tetradecynoic acid, methyl ester, Propenoic acid,
3-(5-ethoxycarbonyl-2,4, Octadecanoic acid, 11-methoxy-, methyl,
Succinic acid, di(2-(2-methoxyethyl)hep, 6-Aza-2-thiothymine,
Octadecanoic acid, 11-methoxy-, methyl,
3-Methoxymethoxy-2,3-dimethylundec-1-en, 10-Hydroxydecanoic acid,
methyl ester, 1,4-Benzenedicarboxylic acid, methyl tr,
Cyclohexanebutanoic acid, .alpha.,4-dim, Di-n-octyl phthalate,
3,6-Dioxa-2,4,5,7-tetrasilaoctane, 2,2, Picein, Methyl undecanoate
(11:0), TETRADECANOIC ACID, METHYL ESTER (14:0), ISOPENTADECANOIC
ACID, METHYL ESTER (IS, 7-HEXADECENOIC ACID, METHYL ESTER (16:1,
7-HEXADECENOIC ACID, METHYL ESTER (16:1, HEXADECANOIC ACID, METHYL
ESTER (16:0), Oleyl alcohol, trifluoroacetate, 4-Octene,
2,6-dimethyl-, [S-(E)]-, Cyclooctane, 1,2-dimethyl-, Cyclopentane,
pentyl-, Cyclohexane, 1,2,4-trimethyl-, CYCLOPROPYLHEPTADECANOIC
ACID, METHYL E, cis-10-Heptadecenoic acid, methyl ester,
CYCLOPROPYLHEPTADECANOIC ACID, METHYL E, Octadecanoic acid,
9,10,12-trimethoxy-,7-OCTADECENOIC ACID, METHYL ESTER (18:1,
OCTADECANOIC ACID, METHYL ESTER (18:0), Nonanoic acid,
trimethylsilyl ester, Dipyrido[3,2-b; 2,3-d]pyrrole, 1,9-dioxi,
Adipic acid, di(trans-2-methylcyclohexy, Nonanoic acid, 9-oxo-,
methyl ester, Cyclopropyl Nonadecanoic Acid (Cyc 19:0,
TETRADECANOIC ACID, METHYL ESTER (14:0), ISOPENTADECANOIC ACID,
METHYL ESTER (ISO 15:0), ANTEISO PENTADECANOIC ACID (A15:0), ISO
HEXADECANOIC ACID, METHYL ESTER, 7-HEXADECENOIC ACID, METHYL ESTER
(16:1W7TRANS), 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7CIS),
HEXADECANOIC ACID, METHYL ESTER (16:0), CYCLOPROPYLHEPTADECANOIC
ACID, METHYL ESTER (CYC17:0), n-Hexadecanoic acid, Cyclopentane,
undecyl-, 1-Nonene, 11-Methyl Hexadecanoic Acid, Methyl Ester
(11-Me 16:0), 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS),
7-HEXADECENOIC ACID, METHYL ESTER (16:1W7CIS), HEPTADECANOIC ACID,
METHYL ESTER (17:0), 3-HYDROXY TETRADECANOIC ACID, METHYL ESTER
(3-OH 14:0), 3-HYDROXY TETRADECANOIC ACID, METHYL ESTER (3-OH
14:0), Methane, diethoxy-, Heptanedioic acid, 4-methyl-, dimethyl
ester, 1,2,4-Triazine-6-carboxylic acid,
2,3,4,5-tetrahydro-5-oxo-3-thioxo-, ethyl ester, OCTADECANOIC ACID,
METHYL ESTER (18:0), Cyclopentanone, 2-methyl-3-(1-methylethyl)-,
Carbamic acid, 1-naphthyl-, propyl ester,
1-Methoxycarbonylethyl-5-methoxycarbonylpentyl ether, Decanoic
acid, methyl ester,
1,6-Anhydro-3,4-O-isopropylidene-2-tosyl-D-galactose,
12-NONADECENOIC ACID, METHYL ESTER (19:1W12 CIS), Cyclopropyl
Nonadecanoic Acid (Cyc 19:0), 9-Hydroxypentadecanoic acid, methyl
ester, Methyl 5,9-dimethyldecanoate, Octadecanoic acid,
11-methoxy-, methyl ester, 9-OCTADECENOIC ACID, METHYL ESTER
(18:1W9 TRANS), 1,1-Dipropyl-3-[2-thiazolyl]-2-thiourea,
Octadecanoic acid, 11-methoxy-, methyl ester, (.+/-.)-,
Nordextromethorphan, Dodecanoic acid, 2-methyl-, Cyclohexyl
undecanoic acid, methyl ester (Cyclohexyl 17:0), Methyl
5,9-dimethyldecanoate, Cyclotrisiloxane, hexamethyl-,
Iso-Tridecanoic Acid, Methyl Ester (Iso 13:0), ISOTETRADECANOIC
ACID, METHYL ESTER (ISO 14:0), TETRADECANOIC ACID, METHYL ESTER
(14:0), ISOPENTADECANOIC ACID, METHYL ESTER (ISO 15:0), ANTEISO
PENTADECANOIC ACID (A15:0), ISOPENTADECANOIC ACID, METHYL ESTER
(ISO 15:0), HEXADECANOIC ACID, METHYL ESTER (16:0), 7-HEXADECENOIC
ACID, METHYL ESTER (16:1W7TRANS), HEXADECANOIC ACID, METHYL ESTER
(16:0), 7-Hexadecenoic acid, methyl ester, (Z)-, n-Hexadecanoic
acid, 2-Propenoic acid, 2-methyl-, dodecyl ester, Cyclooctane,
methyl-, ISO HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), ANTEISO
HEPTADECANOIC ACID, METHYL ESTER, 7-HEXADECENOIC ACID, METHYL ESTER
(16:1W7TRANS), 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7CIS), ISO
HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), 3-HYDROXY TETRADECANOIC
ACID, METHYL ESTER (3-OH 14:0), 2-HYDROXY HEXADECANOIC ACID, METHYL
ESTER (2-OH 16:0), 3-HYDROXY TETRADECANOIC ACID, METHYL ESTER (3-OH
14:0), 2-Octanol, 2-methyl-6-methylene-, Octadecanoic acid, methyl
ester, 1,2,4-Triazine-6-carboxylic acid,
2,3,4,5-tetrahydro-5-oxo-3-thioxo-, ethyl ester, 7-OCTADECENOIC
ACID, METHYL ESTER (18:1W7 CIS), OCTADECANOIC ACID, METHYL ESTER
(18:0), Cyclohexane, 1,2,3-trimethyl-,
(1.alpha.,2.alpha.,3.beta.)-, Octadecanoic acid, 3-hydroxy-, methyl
ester, Cyclopropaneoctanoic acid, 2-octyl-, methyl ester, trans-,
Octadecanoic acid, 11-methoxy-, methyl ester, Methyl
trans-9-(2-butylcyclopentyl)nonanoate, 1,2-Dodecanediol,
NONADECENOIC ACID, METHYL ESTER (19:1W12 TRANS), 12-NONADECENOIC
ACID, METHYL ESTER (19:1W12 CIS), Cyclopropyl Nonadecanoic Acid
(Cyc 19:0), Decanoic acid, methyl ester, Undecanoic acid,
11-bromo-, methyl ester, Cyclobutane, 1-butyl-2-ethyl-, Ethyl
tetradecyl ether, Octadecanoic acid, 11-methoxy-, methyl ester,
Methyl 8-methyl-nonanoate, 1,1-Dipropyl-3-[2-thiazolyl]-2-thiourea,
3,8-Dinitrocarbazole, Dodecanoic acid, 2-methyl-,
Cyclopropaneoctanoic acid, 2-octyl-, methyl ester, Piperidine,
1-(1-oxo-3-phenyl-2-propynyl)-, Tridecanoic acid, methyl ester,
Cyclopenteno[4.3-b]tetrahydrofuran,
3-[(4-methyl-5-oxo-3-phenylthio)tetrahydrofuran-2-yloxymethylene]-,
TETRADECANOIC ACID, METHYL ESTER (14:0), ISOPENTADECANOIC ACID,
METHYL ESTER (IS, ANTEISO PENTADECANOIC ACID (A15:0), ISO
HEXADECANOIC ACID, METHYL ESTER, 7-HEXADECENOIC ACID, METHYL ESTER
(16:1, 7-HEXADECENOIC ACID, METHYL ESTER (16:1, 7-HEXADECENOIC
ACID, METHYL ESTER (16:1, HEXADECANOIC ACID, METHYL ESTER (16:0),
2(1H)-Naphthalenone, octahydro-1,4a-dim,
3,4,4-Trimethyl-cyclohex-2-en-1-ol, Cyclopentane,
1-pentyl-2-propyl-, Cyclohexane, 1,2,3-trimethyl-, (1.alpha,
Triallylsilane, Cyclopentane, pentyl-, Cyclohexane,
1,2,3-trimethyl-, (1.alpha, 7-HEXADECENOIC ACID, METHYL ESTER
(16:1, CYCLOPROPYLHEPTADECANOIC ACID, METHYL E, Octadecanoic acid,
9,10,12-trimethoxy-,7-OCTADECENOIC ACID, METHYL ESTER (18:1,
4-Hydroxy-3-methylpent-2-enoic acid, me, 9-OCTADECENOIC ACID,
METHYL ESTER (18:1, OCTADECANOIC ACID, METHYL ESTER (18:0),
2-Octene, 4-ethyl-, 1,2-Benzenediol, o-isonicotinoyl-o'-val,
Carbamic acid, 1-naphthyl-, propyl este, Methyl
(13S)-(E)-13-trimethylsilyloxy-9, Octadecanoic acid, 11-methoxy-,
methyl, Decanoic acid, methyl ester, 5-Decanol, Methyl
tetradecanoate, 12-NONADECENOIC ACID, METHYL ESTER (19, Cyclopropyl
Nonadecanoic Acid (Cyc 19:0, Methyl 5,9-dimethyldecanoate,
1-Methoxycarbonylethyl-5-methoxycarbony, TETRADECANOIC ACID, METHYL
ESTER (14:0), ISOPENTADECANOIC ACID, METHYL ESTER (IS, ANTEISO
PENTADECANOIC ACID (A15:0), ISO HEXADECANOIC ACID, METHYL ESTER,
7-HEXADECENOIC ACID, METHYL ESTER (16:1, 7-HEXADECENOIC ACID,
METHYL ESTER (16:1, 7-HEXADECENOIC ACID, METHYL ESTER (16:1,
HEXADECANOIC ACID, METHYL ESTER (16:0), Oxacyclodecan-2-one,
1,2-Decanediol, Cyclohexane, 1,2,3-trimethyl-, (1.alpha,
2-Hexanone, 5-methyl-3-methylene-, Cyclopentane, pentyl-,
Cyclohexanone, 3,5-dimethyl-, cis-, 7-HEXADECENOIC ACID, METHYL
ESTER (16:1, CYCLOPROPYLHEPTADECANOIC ACID, METHYL E, Octadecanoic
acid, 9,10,12-trimethoxy-,7-OCTADECENOIC ACID, METHYL ESTER (18:1,
Succinic acid, 2,2-dichloroethyl nonyl, OCTADECANOIC ACID, METHYL
ESTER (18:0), Cyclopentanone, 2-methyl-3-(1-methyleth,
1,2-Benzenediol, o-isonicotinoyl-o'-val, Carbamic acid,
1-naphthyl-, propyl este, 1H-1-Benzazepine-2,5-dione, 1,4-dimethy,
4-Acetyl-2,3-O-acetone-d-mannosan, Nonanoic acid, 9-oxo-, methyl
ester, 1-Azabicyclo[2.2.2]octane-2-carboxylic, Cyclopropyl
Nonadecanoic Acid (Cyc 19:0, Nonanoic acid, 9-hydroxy-, methyl
ester, 1-Methoxycarbonylethyl-5-methoxycarbony ISOTETRADECANOIC
ACID, METHYL ESTER (ISO 14:0), TETRADECANOIC ACID, METHYL ESTER
(14:0), ISOPENTADECANOIC ACID, METHYL ESTER (ISO 15:0), ANTEISO
PENTADECANOIC ACID (A15:0), PENTADECANOIC ACID, METHYL ACID (15:0),
ISO HEXADECANOIC ACID, METHYL ESTER, 7-HEXADECENOIC ACID, METHYL
ESTER (16:1W7TRANS), 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7CIS),
HEXADECANOIC ACID, METHYL ESTER (16:0), Methyl 8-heptadecenoate,
Cyclooctane, methyl-, n-Hexadecanoic acid, Cyclooctane, methyl-,
ISO HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), ANTEISO
HEPTADECANOIC ACID, METHYL ESTER, 7-HEXADECENOIC ACID, METHYL ESTER
(16:1W7TRANS), HEPTADECANOIC ACID, METHYL ESTER (17:0), Methyl
2-hydroxy-hexadecanoate, 3-HYDROXY TETRADECANOIC ACID, METHYL ESTER
(3-OH 14:0), Ethyl tetradecyl ether, Heptanedioic acid, 4-methyl-,
dimethyl ester, 1,2,4-Triazine-6-carboxylic acid,
2,3,4,5-tetrahydro-5-oxo-3-thioxo-, ethyl ester,
Dichloromethyldimethylsilyloxycyclobutane, OCTADECANOIC ACID,
METHYL ESTER (18:0), Oxirane, 2-decyl-3-(5-methylhexyl)-, cis-,
2,4,5-Trifluorobenzonitrile, Octadecanoic acid, 11-methoxy-, methyl
ester, Nonanoic acid, 9-oxo-, methyl ester, cis-Vaccenic acid,
1-Nitrododecane, 12-NONADECENOIC ACID, METHYL ESTER (19:1W12 CIS),
Cyclopropyl Nonadecanoic Acid (Cyc 19:0), Decanoic acid, 2-methyl-,
Heptanoic acid, methyl ester, Octadecanoic acid, 11-methoxy-,
methyl ester, Phosphonous acid, phenyl-,
bis[5-methyl-2-(1-methylethyl)cyclohexyl] ester,
[1R-[1.alpha.(1R*,2S*,5R*),2.beta.,5.alpha.]]-, Octadecanoic acid,
11-methoxy-, methyl ester, Dodecanoic acid, methyl ester,
10-Nonadecenoic acid, methyl ester, 1,2-Bis(trimethylsilyl)benzene
ISOTETRADECANOIC ACID, METHYL ESTER (ISO 14:0), TETRADECANOIC ACID,
METHYL ESTER (14:0), ISOPENTADECANOIC ACID, METHYL ESTER (ISO
15:0), ANTEISO PENTADECANOIC ACID (A15:0), ISOPENTADECANOIC ACID,
METHYL ESTER (ISO 15:0), ISO HEXADECANOIC ACID, METHYL ESTER,
7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS), 7-HEXADECENOIC
ACID, METHYL ESTER (16:1W7CIS), 7-HEXADECENOIC ACID, METHYL ESTER
(16:1W7CIS), HEXADECANOIC ACID, METHYL ESTER (16:0),
cis-10-Heptadecenoic acid, methyl ester, n-Hexadecanoic acid,
Heptafluorobutyric acid, n-octadecyl ester, Cyclooctane, methyl-,
ISO HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), ISO HEPTADECANOIC
ACID, METHYL ESTER (ISO17:0), CYCLOPROPYLHEPTADECANOIC ACID, METHYL
ESTER (CYC17:0), HEPTADECANOIC ACID, METHYL ESTER (17:0), 2-HYDROXY
HEXADECANOIC ACID, METHYL ESTER (2-OH 16:0), 3-HYDROXY DODECANOIC
ACID, METHYL ESTER (3-OH 12:0), 2-Octanol, 2-methyl-6-methylene-,
Octadecanoic acid, methyl ester, 1,2,4-Triazine-6-carboxylic acid,
2,3,4,5-tetrahydro-5-oxo-3-thioxo-, ethyl ester, OCTADECANOIC ACID,
METHYL ESTER (18:0), Cyclohexane, 1,2,3-trimethyl-,
(1.alpha.,2.alpha.,3.beta.)-, 1,2-Benzenediamine,
4-(4-aminophenoxy)-, Octadecanoic acid, 11-methoxy-, methyl ester,
9-Hydroxypentadecanoic acid, methyl ester, Decanoic acid, methyl
ester, 12-NONADECENOIC ACID, METHYL ESTER (19:1W12 CIS),
Cyclopropyl Nonadecanoic Acid (Cyc 19:0), Octadecanoic acid,
9,10-dichloro-, methyl ester, Octadecanoic acid, 11-methoxy-,
methyl ester, 1-Methyl-1-(2-pentadecyl)oxy-1-silacyclopentane,
Octadecanoic acid, 11-methoxy-, methyl ester, Undecanoic acid,
10-methyl-, methyl ester, METHYL DODECANOATE (12:0),
12-NONADECENOIC ACID, METHYL ESTER (19:1W12 CIS), Cyclotrisiloxane,
hexamethyl-, TETRADECANOIC ACID (14:0), ISO/ANTEISO-PENTADECANOIC
ACID (C14:0-Me), ISO HEXADECANOIC ACID, HEXADECANOIC ACID (16:0),
7-HEXADECENOIC ACID (16:1), HEXADECANOIC ACID, 11-Methyl,
ISO/ANTEISO-HEPTADECANOIC ACID (C16:0-ME), CYCLOPROPYLHEPTADECANOIC
ACID (Cyc 17:0)/10-HEPTADECENOIC ACID (17:1), HEPTADECANOIC ACID
(C17:0), OCTADECANOIC ACID (18:0), 7-OCTADECENOIC ACID (18:1),
9-OCTADECENOIC ACID (18:1), OCTADECANOIC ACID, 11-METHOXY,
OCTADECANOIC ACID, 9,10,12-TRIMETHOXY, CYCLOPROPYL NONADECANOIC
ACID (Cyc 19:0)/12-NONADECENOIC ACID (19:1), TETRADECANOIC ACID
(C14:0), ISO/ANTEISO-PENTADECANOIC ACID (C14:0-Me),
ISO-HEXADECANOIC ACID (C15:0-Me), HEXADECANOIC ACID (16:0),
7-HEXADECENOIC ACID (16:1), ISO/ANTEISO-HEPTADECANOIC ACID
(C16:0-Me), CYCLOPROPYLHEPTADECANOIC ACID (Cyc 17:0), HEPTADECANOIC
ACID (C17:0), OCTADECANOIC ACID (18:0), 7-OCTADECENOIC ACID (18:1),
9-OCTADECENOIC ACID (18:1), OCTADECANOIC ACID, 11-METHOXY,
OCTADECANOIC ACID, 9,10,12-TRIMETHOXY, CYCLOPROPYL NONADECANOIC
ACID (Cyc 19:0)/12-NONADECENOIC ACID (19:1), Methyl undecanoate
(11:0), Cyclohexasiloxane, dodecamethyl-,
7-Chloro-2,3-dihydro-3-(4-N,N-dimethyla, Hexasiloxane,
tetradecamethyl-, Cycloheptasiloxane, tetradecamethyl-,
Cycloheptasiloxane, tetradecamethyl-, Cycloheptasiloxane,
tetradecamethyl-, 3,5-Dioxa-4-phospha-2-silaheptan-7-oic,
2,4-Dimethyldodecane, Pentadecane, 3-methyl-, 5-Tetradecene, (E)-,
1-Undecene, 8-methyl-, Cyclotetradecane, Heptasiloxane,
hexadecamethyl-, Hexasiloxane, tetradecamethyl-, Hexasiloxane,
tetradecamethyl-, 2-Methyl-E-7-hexadecene, Benzoic acid,
2,4-bis[(trimethylsilyl)o, Cyclohexanecarboxylic acid, 6-chlorohex,
Succinic acid, hexyl 2-pentyl ester, 5,5-Dibutylnonane, 2-Heptenoic
acid, methyl ester, Isopropylmalic acid, O-(tert-butyldimet,
3-Dodecanol, 3,7,11-trimethyl-, Dodecane, 2,6,11-trimethyl-,
Eicosane, 2,4-dimethyl-, Hexadecane, 3-methyl-, Piperidine,
1-(5-trifluoromethyl-2-pyri, 9-Octadecene, (E)-, Hexadecanoic acid,
2-hydroxy-, methyl e, 5-Octadecene, (E)-, Trifluoroacetoxy
hexadecane, 9-Octadecene, (E)-, 5-Octadecene, (E)-, Ethanol,
2-(octadecyloxy)-, Cyclononasiloxane, octadecamethyl-,
Cyclononasiloxane, octadecamethyl-, Cyclononasiloxane,
octadecamethyl-, Adipic acid, cyclohexyl isobutyl ester, Succinic
acid, butyl tetradecyl ester, 2-Amino-2-oxo-acetic acid,
N-[3,4-dimet, Nonadecane, 9-methyl-, cis-10-Heptadecenoic acid,
methyl ester, Hexasiloxane, tetradecamethyl-, Cyclononasiloxane,
octadecamethyl-, Cyclodecasiloxane, eicosamethyl-, Piperidine,
1-(5-trifluoromethyl-2-pyri, Cyclononasiloxane, octadecamethyl-,
Cyclodecasiloxane, eicosamethyl-, Piperidine,
1-(5-trifluoromethyl-2-pyri, n-Nonadecanoic acid,
pentamethyldisilyl, Piperidine, 1-(5-trifluoromethyl-2-pyri,
Piperidine, 1-(5-trifluoromethyl-2-pyri, n-Nonadecanoic acid,
pentamethyldisilyl, Hexasiloxane, tetradecamethyl-,
1,2-Benzenedicarboxylic acid, mono(2-et,
Piperidine, 1-(5-trifluoromethyl-2-pyri, Silane,
[[4-[1,2-bis[(trimethylsilyl)ox, Hexasiloxane, tetradecamethyl-,
Heptasiloxane, hexadecamethyl-, Hexasiloxane, tetradecamethyl-,
Cyclononasiloxane, octadecamethyl-,
2,6,10,14,18,22-Tetracosahexaene, 2,6,1, 2-Amino-2-oxo-acetic acid,
N-[3,4-dimet, Cyclononasiloxane, octadecamethyl-, Heptasiloxane,
hexadecamethyl-, 1,2-Benzisothiazole-3-acetic acid, meth,
2-Amino-2-oxo-acetic acid, N-[3,4-dimet, Hexasiloxane,
tetradecamethyl-, Heptasiloxane, hexadecamethyl-,
Cyclononasiloxane, octadecamethyl-, Heptasiloxane, hexadecamethyl-,
Cyclononasiloxane, octadecamethyl-, DL-Leucine, N-acetyl-, methyl
ester, Pentadecanal-, ISOTETRADECANOIC ACID, METHYL ESTER (ISO
14:0), ISO PENTADECENOIC ACID, METHYL ESTER (ISO 15:1),
AnteISOPENTADECANOIC ACID, METHYL ESTER (a 15:0), ANTEISO
PENTADECANOIC ACID (A15:0), Tetradecanal, ISOPENTADECANOIC ACID,
METHYL ESTER (ISO 15:0), HEXADECANOIC ACID, METHYL ESTER (16:0),
7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS), Oxirane,
tridecyl-, HEXADECANOIC ACID, METHYL ESTER (16:0), ISO
HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), ANTEISO HEPTADECANOIC
ACID, METHYL ESTER, Hexadecanal, HEPTADECANOIC ACID, METHYL ESTER
(17:0), 6,9-OCTADECADIENOIC ACID, METHYL ESTER (18:2W6,9 ALL CIS),
9-OCTADECENOIC ACID, METHYL ESTER (18:1W9TRANS), 7-OCTADECENOIC
ACID, METHYL ESTER (18:1W7 CIS), Oxirane, heptadecyl-, OCTADECANOIC
ACID, METHYL ESTER (18:0), Octadecanoic acid, 3-hydroxy-, methyl
ester, 10-METHYL OCTADECANOIC ACID (10ME18:0),
Bicyclo[10.8.0]eicosane, cis-, EICOSANOIC ACID, METHYL ESTER
(20:0), 3-Methoxymethoxy-2,3-dimethylundec-1-ene, Octadecanoic
acid, 3-hydroxy-, methyl ester, Octadecanoic acid, 9,10-dichloro-,
methyl ester, 9-DOCOSENOIC ACID, METHYL ESTER (22:1W9CIS),
1-Methoxycarbonylethyl-5-methoxycarbonylpentyl ether, DOCOSANOIC
ACID, METHYL ESTER (22:0), trans-13-Octadecenoic acid, methyl
ester, 2,6,10,14,18,22-Tetracosahexaene,
2,6,10,15,19,23-hexamethyl-, (all-E)-, Cyclotrisiloxane,
hexamethyl-, 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS), ISO
HEXADECANOIC ACID, METHYL ESTER, ANTEISO HEPTADECANOIC ACID, METHYL
ESTER, HEPTADECANOIC ACID, METHYL ESTER (17:0), OCTADECATRIENOIC
ACID, METHYL ESTER (18:3W6), Methyl 10-oxohexadecanoate,
6,9-OCTADECADIENOIC ACID, METHYL ESTER (18:2W6,9 ALL CIS),
9-OCTADECENOIC ACID, METHYL ESTER (18:1W9 CIS),
4-Hydroxy-2-methylthio-5-pyrimidinehydroxamic acid, OCTADECANOIC
ACID, METHYL ESTER (18:0), 10-METHYL OCTADECANOIC ACID (10ME18:0),
Cyclopropyl Nonadecanoic Acid (Cyc 19:0),
3-Methoxymethoxy-2,3-dimethylundec-1-ene, Thiazole,
2-ethyl-4,5-dimethyl-, n-Decylsuccinic anhydride,
Cyclopropanecarboxylic acid, heptadecyl ester, Octadecanoic acid,
11-methoxy-, methyl ester, 4-Bromo-2-methoxybut-2-enoic acid,
methyl ester, Octadecanoic acid, 9,10-dichloro-, methyl ester,
Adipic acid, di(trans-2-methylcyclohexyl) ester, 11-Octadecenoic
acid, methyl ester, Cyclotrisiloxane, hexamethyl-,
Cyclopentasiloxane, decamethyl-, 4-(Nonafluoro-tert-butyl)
nitrobenzene, Cyclohexasiloxane, dodecamethyl-, Methyl undecanoate
(11:0), Cycloheptasiloxane, tetradecamethyl-, Silane,
[[4-[1,2-bis[(trimethylsilyl)oxy]ethyl]-1,2-phenylene]bis(oxy)]bis[trimet-
hyl-, TETRADECANOIC ACID, METHYL ESTER (14:0),
1,2-Bis(trimethylsilyl)benzene, ISOPENTADECANOIC ACID, METHYL ESTER
(ISO 15:0), ANTEISO PENTADECANOIC ACID (A15:0), ISOPENTADECANOIC
ACID, METHYL ESTER (ISO 15:0), 3-HYDROXY TETRADECANOIC ACID, METHYL
ESTER (3-OH 14:0), Cyclononasiloxane, octadecamethyl-, Pentanamide,
N-(4-methoxyphenyl)-, ISO HEXADECANOIC ACID, METHYL ESTER,
7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS), 7-HEXADECENOIC
ACID, METHYL ESTER (16:1W7TRANS), Cyclododecanol, HEXADECANOIC
ACID, METHYL ESTER (16:0), HEXADECANOIC ACID, METHYL ESTER (16:0),
Eicosane, n-Hexadecanoic acid, 10-Methyl Hexadecanoic Acid, Methyl
Ester (10-Me 16:0), Cyclobarbital, 2,4-Cyclohexadien-1-one,
3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 9,12-Octadecadienoic acid
(Z,Z)-, cis-10-Heptadecenoic acid, methyl ester, Butanoic acid,
undec-2-enyl ester, HEPTADECANOIC ACID, METHYL ESTER (17:0),
Cyclodecasiloxane, eicosamethyl-, OCTADECATRIENOIC ACID, METHYL
ESTER (18:3W6), 9,12-Octadecadienoic acid, methyl ester, (E,E)-,
1-(2-Isopropyl-5-methylcyclopentyl)ethanone, 6,9-OCTADECADIENOIC
ACID, METHYL ESTER (18:2W6,9 ALL CIS), 7-OCTADECENOIC ACID, METHYL
ESTER (18:1W7 CIS), 7-OCTADECENOIC ACID, METHYL ESTER (18:1W7 CIS),
9-OCTADECENOIC ACID, METHYL ESTER (18:1W9 CIS),
Z-11(13-Methyl)tetradecen-1-ol acetate, OCTADECANOIC ACID, METHYL
ESTER (18:0), Octadecanoic acid, 10-METHYL OCTADECANOIC ACID
(10ME18:0), Methyl 10-trans,12-cis-octadecadienoate, i-Propyl
tricosanoate, 2H-Pyran, 2-(8-dodecynyloxy)tetrahydro-,
12-NONADECENOIC ACID, METHYL ESTER (19:1W12 CIS), 3-Pentane
isothiocyanate, Cyclotrisiloxane, hexamethyl-, Methyl
8,11,14-eicosatrienoate, .alpha.-D-Ribopyranose, 5-thio-, cyclic
1,2:3,4-bis(ethylboronate), Cyclononasiloxane, octadecamethyl-,
Adipic acid, di(trans-2-methylcyclohexyl) ester, Octadecanoic acid,
9,10-dichloro-, methyl ester, Cyclononasiloxane, octadecamethyl-,
Cyclononasiloxane, octadecamethyl-, Octasiloxane,
1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl-,
Trimethyl(4-tert.-butylphenoxy)silane,
Trimethyl[4-(1,1,3,3,-tetramethylbutyl)phenoxy]silane,
ISOTETRADECANOIC ACID, METHYL ESTER (ISO 14:0), ISOPENTADECANOIC
ACID, METHYL ESTER (ISO 15:0), AnteISOPENTADECANOIC ACID, METHYL
ESTER (a 15:0), PENTADECANOIC ACID, METHYL ACID (15:0), ISO
HEXADECANOIC ACID, METHYL ESTER, 7-HEXADECENOIC ACID, METHYL ESTER
(16:1W7CIS), 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS),
HEXADECANOIC ACID, METHYL ESTER (16:0), 10-Methyl Hexadecanoic
Acid, Methyl Ester (10-Me 16:0), ISO HEPTADECANOIC ACID, METHYL
ESTER (ISO17:0), Methyl 9,10-methylene-hexadecanoate, ISO
HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), 9-OCTADECENOIC ACID,
METHYL ESTER (18:1W9TRANS), 7-OCTADECENOIC ACID, METHYL ESTER
(18:1W7 CIS), OCTADECANOIC ACID, METHYL ESTER (18:0), 10-METHYL
OCTADECANOIC ACID (10ME18:0), Cyclopropaneoctanoic acid, 2-octyl-,
methyl ester, 2-Bromo-4,6-di-tert-butylphenol, TETRADECENOIC ACID,
METHYL ESTER (14:1W5), TETRADECANOIC ACID, METHYL ESTER (14:0),
Heneicosane, 1-Iodo-2-methylundecane, PENTADECANOIC ACID, METHYL
ACID (15:0), ANTEISO PENTADECANOIC ACID (A15:0), 9-Octadecenoic
acid (Z)-, methyl ester, PENTADECANOIC ACID, METHYL ACID (15:0),
ISO HEXADECANOIC ACID, METHYL ESTER, 7-HEXADECENOIC ACID, METHYL
ESTER (16:1W7TRANS), 7-HEXADECENOIC ACID, METHYL ESTER
(16:1W7TRANS), 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS),
HEXADECANOIC ACID, METHYL ESTER (16:0), Heptadecane, 9-octyl-,
10-Methyl Hexadecanoic Acid, Methyl Ester (10-Me 16:0), ISO
HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), Eicosane,
cis-10-Heptadecenoic acid, methyl ester, Methyl
9,10-methylene-hexadecanoate, HEPTADECANOIC ACID, METHYL ESTER
(17:0), Heptacosane, 10-Methyl Heptadecanoic Acid, Methyl Ester
(10-Me 17:0), 2-Furanethanol, .beta.-ethoxy-, 6,9-OCTADECADIENOIC
ACID, METHYL ESTER (18:2W6,9 ALL CIS), 9-OCTADECENOIC ACID, METHYL
ESTER (18:1W9 CIS), 7-OCTADECENOIC ACID, METHYL ESTER (18:1W7 CIS),
OCTADECANOIC ACID, METHYL ESTER (18:0), Hexadecanenitrile,
10-METHYL OCTADECANOIC ACID (10ME18:0), Methyl
10-trans,12-cis-octadecadienoate, Octadecanoic acid, 10-oxo-,
methyl ester, 12-NONADECENOIC ACID, METHYL ESTER (19:1W12 CIS),
NONADECENOIC ACID, METHYL ESTER (19:1W12CIS), Heptadecane,
9-octyl-, Cyclopropyl Nonadecanoic Acid (Cyc 19:0), 12-Methyl
Octadecanoic Acid, Methyl Ester (12-Me 18:0),
3-Methoxymethoxy-2,3-dimethylundec-1-ene, Naphthalene, 1,1
'-(1,2-ethanediyl)bis-, Heneicosane, 1,2-Benzisothiazole,
3-(hexahydro-1H-azepin-1-yl)-, 1,1-dioxide, 9-EICOSENOIC ACID,
METHYL ESTER (20:1W9TRANS), 9-EICOSENOIC ACID, METHYL ESTER
(20:1W9TRANS), Cyclopropanecarboxylic acid, heptadecyl ester,
EICOSANOIC ACID, METHYL ESTER (20:0), Hexadecane, 2-methyl-,
Undecanoic acid, methyl ester, 6-Octadecenoic acid, (Z)-,
Heptacosane, Pyrimidine, 2,4-diamino-5-(3-pyridylmethyl)-, Adipic
acid, di(trans-2-methylcyclohexyl) ester, Octadecanoic acid,
9,10-dichloro-, methyl ester, 9-DOCOSENOIC ACID, METHYL ESTER
(22:1W9TRANS), 4-Acetyl-2,3-O-acetone-d-mannosan, DOCOSANOIC ACID,
METHYL ESTER (22:0), 10-Undecynoic acid, methyl ester, Heneicosane,
Heptacosane, TETRACOSENOIC ACID, METHYL ESTER (24:1), Eicosane,
Cyclotrisiloxane, hexamethyl-, Heneicosane, 3-methyl-,
ISOPENTADECANOIC ACID, METHYL ESTER (ISO 15:0), 7-HEXADECENOIC
ACID, METHYL ESTER (16:1W7TRANS), ISO HEXADECANOIC ACID, METHYL
ESTER, 9,12-Octadecadienoic acid (Z,Z)-, cis-10-Heptadecenoic acid,
methyl ester, HEPTADECANOIC ACID, METHYL ESTER (17:0),
OCTADECATRIENOIC ACID, METHYL ESTER (18:3W6), 6,9-OCTADECADIENOIC
ACID, METHYL ESTER (18:2W6,9 ALL CIS), 3,6,9-OCTADECATRIENOIC ACID,
METHYL ESTER (18:3W3), 7-OCTADECENOIC ACID, METHYL ESTER (18:1W7
CIS), 7-OCTADECENOIC ACID, METHYL ESTER (18:1W7 CIS), 11-Dodecenoic
acid, 10-hydroxy-, methyl ester, OCTADECANOIC ACID, METHYL ESTER
(18:0), 10-METHYL OCTADECANOIC ACID (10ME18:0), Cyclopropyl
Nonadecanoic Acid (Cyc 19:0), NONADECANOIC ACID, METHYL ESTER
(19:0), 4-Amino-5-methyl-2(1H)-pyrimidinethione, Methyl
8,11,14-eicosatrienoate, 1,2-Benzisothiazole,
3-(hexahydro-1H-azepin-1-yl)-, 1,1-dioxide, Dodecanoic acid,
11-hydroxy-, methyl ester, N-1-Naphthyl-N'-4-[N-aziridyl]butylurea,
Succinic acid, 1-cyclopentylethyl ethyl ester, Octadecanoic acid,
9,10-dichloro-, methyl ester,
1-Methoxycarbonylethyl-5-methoxycarbonylpentyl ether,
16-Hexadecanoyl hydrazide, L-Leucine, N-acetyl-, methyl ester,
Methyl undecanoate (11:0), Decanedioic acid, dimethyl ester,
Pentadecanal-, TETRADECANOIC ACID, METHYL ESTER (14:0),
Undecanedioic acid, dimethyl ester, ISOPENTADECANOIC ACID, METHYL
ESTER (ISO 15:0), ANTEISO PENTADECANOIC ACID (A15:0), Hexadecanal,
PENTADECANOIC ACID, METHYL ACID (15:0), HEXADECANOIC ACID, METHYL
ESTER (16:0), 7-HEXADECENOIC ACID, METHYL ESTER (16:1W7TRANS),
Methyl hexadec-9-enoate, Oxirane, hexadecyl-, HEXADECANOIC ACID,
METHYL ESTER (16:0), HEPTADECANOIC ACID, METHYL ESTER (17:0),
ANTEISO HEPTADECANOIC ACID, METHYL ESTER, Oxirane, heptadecyl-,
HEPTADECANOIC ACID, METHYL ESTER (17:0), 3,6,9-OCTADECATRIENOIC
ACID, METHYL ESTER (18:3W3), 6,9-OCTADECADIENOIC ACID, METHYL ESTER
(18:2W6,9 ALL CIS), 7-OCTADECENOIC ACID, METHYL ESTER (18:1W7 CIS),
7-OCTADECENOIC ACID, METHYL ESTER (18:1W7 CIS), Oxirane,
hexadecyl-, OCTADECANOIC ACID, METHYL ESTER (18:0), Octadecanoic
acid, 3-hydroxy-, methyl ester, 10-METHYL OCTADECANOIC ACID
(10ME18:0), 6,9-TETRACOSADIENOIC ACID, METHYL ESTER (24:2W6),
Z,E-2,13-Octadecadien-1-ol, Cyclopropyl Nonadecanoic Acid (Cyc
19:0), Bicyclo[10.8.0]eicosane, cis-, NONADECANOIC ACID, METHYL
ESTER (19:0), Oxirane, tetradecyl-, Octadecanoic acid, 3-hydroxy-,
methyl ester, Methyl 12-oxo-octadecanoate, 9-EICOSENOIC ACID,
METHYL ESTER (20:1W9TRANS), 9-EICOSENOIC ACID, METHYL ESTER
(20:1W9TRANS), Cyclopropanecarboxylic acid, heptadecyl ester,
Oxirane, tetradecyl-, EICOSANOIC ACID, METHYL ESTER (20:0),
Heptane, 1,1-diethoxy-, 1-Nonadecene, 1-Nonadecene, Oxirane,
tetradecyl-, Docosanoic acid, 4,4-dimethyl-, methyl ester,
Octadecanoic acid, 3-hydroxy-, methyl ester, Octadecanoic acid,
9,10-dichloro-, methyl ester, 9-DOCOSENOIC ACID, METHYL ESTER
(22:1W9TRANS), 9-DOCOSENOIC ACID, METHYL ESTER (22:1W9CIS),
3-Methoxymethoxy-2,3-dimethylundec-1-ene, Phthalic acid,
2-ethylhexyl tridecyl ester, DOCOSANOIC ACID, METHYL ESTER (22:0),
10-Undecynoic acid, methyl ester, TRICOSANOIC ACID, METHYL ESTER
(23:0), 2,2-Dimethylpropanoic acid, heptadecyl ester, TETRACOSENOIC
ACID, METHYL ESTER (24:1), TETRACOSENOIC ACID, METHYL ESTER (24:1),
TETRACOSENOIC ACID, METHYL ESTER (24:1), TETRACOSANOIC ACID, METHYL
ESTER (24:0), 2,6,10,14,18,22-Tetracosahexaene,
2,6,10,15,19,23-hexamethyl-, (all-E)-, Cyclotrisiloxane,
hexamethyl-, Cyclotrisiloxane, hexamethyl-, Cyclotrisiloxane,
hexamethyl-, 1,2,8,9-Dibenzpentacene, TETRADECANOIC ACID, METHYL
ESTER (14:0), PENTADECANOIC ACID, METHYL ACID (15:0), ANTEISO
PENTADECANOIC ACID (A15:0), PENTADECANOIC ACID, METHYL ACID (15:0),
HEXADECANOIC ACID, METHYL ESTER (16:0), 7-HEXADECENOIC ACID, METHYL
ESTER (16:1W7TRANS), 7-HEXADECENOIC ACID, METHYL ESTER
(16:1W7TRANS), HEXADECANOIC ACID, METHYL ESTER (16:0), ISO
HEPTADECANOIC ACID, METHYL ESTER (ISO17:0), ISO HEPTADECANOIC ACID,
METHYL ESTER (15017:0), 9-Hexadecenoic acid, methyl ester, (Z)-,
HEPTADECANOIC ACID, METHYL ESTER (17:0), Iso-Octadecanoic Acid,
Methyl Ester (Iso 18:0), 9-OCTADECENOIC ACID, METHYL ESTER
(18:1W9CIS), 7-OCTADECENOIC ACID, METHYL ESTER (18:1W7 CIS),
OCTADECANOIC ACID, METHYL ESTER (18:0), 10-METHYL OCTADECANOIC ACID
(10ME18:0), Cyclopropaneoctanoic acid, 2-octyl-, methyl ester,
Methyl 13-eicosenoate. In some non-limiting embodiments the
microorganism is Rhodococcus opacus DSM 43205. In some non-limiting
embodiments the microorganism is Rhodococcus sp. DSM 3346. In some
non-limiting embodiments the microorganism is Cupriavidus necator
DSM 531 or DSM 541. In some non-limiting embodiments the
microorganism is Ralstonia eutropha N-1, DSM 13513.
[0081] Aspects of the invention relate to engineered organisms for
use in the production of molecules for industrial application. As
used herein, "engineered organisms" refer to organisms that
recombinantly express nucleic acids. In some embodiments, such
nucleic acids encode enzymes as discussed herein. Homologs and
alleles of genes associated with the invention can be identified by
conventional techniques. Also encompassed by the invention are
nucleic acids, referred to as "primers" or "primer sets," that
hybridize under stringent conditions to the genes described herein.
The term "stringent conditions" as used herein refers to parameters
with which the art is familiar. Nucleic acid hybridization
parameters may be found in references which compile such methods,
e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al.,
eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 2012, or Current Protocols in Molecular
Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc.,
New York.
[0082] It should be appreciated that the genes associated with
invention can be obtained from a variety of sources. It should be
further appreciated that any of the nucleic acids and/or
polypeptides described herein can be codon-optimized and expressed
recombinantly in a codon-optimized form.
[0083] As one of ordinary skill in the art would be aware,
homologous genes for enzymes described herein could be obtained
from other species and could be identified by homology searches,
for example through a protein BLAST search, available at the
National Center for Biotechnology Information (NCBI) internet site
(ncbi.nlm.nih.gov). Genes associated with the invention can be PCR
amplified from DNA from any source of DNA which contains the given
gene. In some embodiments, genes associated with the invention are
synthetic. Any means of obtaining a gene encoding the enzymes
associated with the invention are compatible with the instant
invention.
[0084] In general, homologs and alleles typically will share at
least 75% nucleotide identity and/or at least 80% amino acid
identity to the sequences of nucleic acids and polypeptides,
respectively, in some instances will share at least 90% nucleotide
identity and/or at least 90% amino acid identity and in still other
instances will share at least 95% nucleotide identity and/or at
least 95% or 99% amino acid identity. The homology can be
calculated using various, publicly available software tools
developed by NCBI (Bethesda, Md.) that can be obtained through the
NCBI internet site. Exemplary tools include the BLAST software,
also available at the NCBI internet site (www.ncbi.nlm.nih.gov).
Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well
as Kyte-Doolittle hydropathic analysis can be obtained using the
MacVector sequence analysis software (Oxford Molecular Group).
Watson-Crick complements of the foregoing nucleic acids also are
embraced by the invention.
[0085] The invention also includes degenerate nucleic acids which
include alternative codons to those present in the native
materials. For example, serine residues are encoded by the codons
TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is
equivalent for the purposes of encoding a serine residue. Thus, it
will be apparent to one of ordinary skill in the art that any of
the serine-encoding nucleotide triplets may be employed to direct
the protein synthesis apparatus, in vitro or in vivo, to
incorporate a serine residue into an elongating polypeptide.
Similarly, nucleotide sequence triplets which encode other amino
acid residues include, but are not limited to: CCA, CCC, CCG and
CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine
codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT
(asparagine codons); and ATA, ATC and ATT (isoleucine codons).
Other amino acid residues may be encoded similarly by multiple
nucleotide sequences. Thus, the invention embraces degenerate
nucleic acids that differ from the biologically isolated nucleic
acids in codon sequence due to the degeneracy of the genetic code.
The invention also embraces codon optimization to suit optimal
codon usage of a host cell.
[0086] The invention also provides modified nucleic acid molecules
which include additions, substitutions and deletions of one or more
nucleotides. In preferred embodiments, these modified nucleic acid
molecules and/or the polypeptides they encode retain at least one
activity or function of the unmodified nucleic acid molecule and/or
the polypeptides, such as enzymatic activity. In certain
embodiments, the modified nucleic acid molecules encode modified
polypeptides, preferably polypeptides having conservative amino
acid substitutions as are described elsewhere herein. The modified
nucleic acid molecules are structurally related to the unmodified
nucleic acid molecules and in preferred embodiments are
sufficiently structurally related to the unmodified nucleic acid
molecules so that the modified and unmodified nucleic acid
molecules hybridize under stringent conditions known to one of
skill in the art.
[0087] For example, modified nucleic acid molecules which encode
polypeptides having single amino acid changes can be prepared. Each
of these nucleic acid molecules can have one, two or three
nucleotide substitutions exclusive of nucleotide changes
corresponding to the degeneracy of the genetic code as described
herein. Likewise, modified nucleic acid molecules which encode
polypeptides having two amino acid changes can be prepared which
have, e.g., 2-6 nucleotide changes. Numerous modified nucleic acid
molecules like these will be readily envisioned by one of skill in
the art, including for example, substitutions of nucleotides in
codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and
so on. In the foregoing example, each combination of two amino
acids is included in the set of modified nucleic acid molecules, as
well as all nucleotide substitutions which code for the amino acid
substitutions. Additional nucleic acid molecules that encode
polypeptides having additional substitutions (i.e., 3 or more),
additions or deletions (e.g., by introduction of a stop codon or a
splice site(s)) also can be prepared and are embraced by the
invention as readily envisioned by one of ordinary skill in the
art. Any of the foregoing nucleic acids or polypeptides can be
tested by routine experimentation for retention of structural
relation or activity to the nucleic acids and/or polypeptides
disclosed herein.
[0088] The invention embraces variants of polypeptides. As used
herein, a "variant" of a polypeptide is a polypeptide which
contains one or more modifications to the primary amino acid
sequence of the polypeptide. Modifications which create a variant
can be made to a polypeptide, for example to: 1) reduce or
eliminate an activity of a polypeptide; 2) enhance a property of a
polypeptide; 3) provide a novel activity or property to a
polypeptide, such as addition of an antigenic epitope or addition
of a detectable moiety; or 4) provide equivalent or better binding
between molecules (e.g., an enzymatic substrate). Modifications to
a polypeptide are typically made to the nucleic acid which encodes
the polypeptide, and can include deletions, point mutations,
truncations, amino acid substitutions and additions of amino acids
or non-amino acid moieties. Alternatively, modifications can be
made directly to the polypeptide, such as by cleavage, addition of
a linker molecule, addition of a detectable moiety, such as biotin,
addition of a fatty acid, and the like. Modifications also embrace
fusion proteins comprising all or part of the amino acid sequence.
One of skill in the art will be familiar with methods for
predicting the effect on protein conformation of a change in
protein sequence, and can thus "design" a variant of a polypeptide
according to known methods. One example of such a method is
described by Dahiyat and Mayo in Science 278:82 87, 1997, whereby
proteins can be designed de novo. The method can be applied to a
known protein to vary only a portion of the polypeptide sequence.
By applying the computational methods of Dahiyat and Mayo, specific
variants of a polypeptide can be proposed and tested to determine
whether the variant retains a desired conformation.
[0089] In general, variants include polypeptides which are modified
specifically to alter a feature of the polypeptide unrelated to its
desired physiological activity. For example, cysteine residues can
be substituted or deleted to prevent unwanted disulfide linkages.
Similarly, certain amino acids can be changed to enhance expression
of a polypeptide by eliminating proteolysis by proteases in an
expression system (e.g., dibasic amino acid residues in yeast
expression systems in which KEX2 protease activity is present).
[0090] Mutations of a nucleic acid which encode a polypeptide
preferably preserve the amino acid reading frame of the coding
sequence, and preferably do not create regions in the nucleic acid
which are likely to hybridize to form secondary structures, such a
hairpins or loops, which can be deleterious to expression of the
variant polypeptide.
[0091] Mutations can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid which encodes the polypeptide. Variant polypeptides
are then expressed and tested for one or more activities to
determine which mutation provides a variant polypeptide with the
desired properties. Further mutations can be made to variants (or
to non-variant polypeptides) which are silent as to the amino acid
sequence of the polypeptide, but which provide preferred codons for
translation in a particular host. The preferred codons for
translation of a nucleic acid in, e.g., E. coli, are well known to
those of ordinary skill in the art. Still other mutations can be
made to the noncoding sequences of a gene or cDNA clone to enhance
expression of the polypeptide. The activity of variant polypeptides
can be tested by cloning the gene encoding the variant polypeptide
into a bacterial or mammalian expression vector, introducing the
vector into an appropriate host cell, expressing the variant
polypeptide, and testing for a functional capability of the
polypeptides as disclosed herein.
[0092] The skilled artisan will also realize that conservative
amino acid substitutions may be made in polypeptides to provide
functionally equivalent variants of the foregoing polypeptides,
i.e., the variants retain the functional capabilities of the
polypeptides. As used herein, a "conservative amino acid
substitution" refers to an amino acid substitution which does not
alter the relative charge or size characteristics of the protein in
which the amino acid substitution is made. Variants can be prepared
according to methods for altering polypeptide sequence known to one
of ordinary skill in the art such as are found in references which
compile such methods, e.g. Molecular Cloning: A Laboratory Manual,
J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2012, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. Exemplary functionally equivalent
variants of polypeptides include conservative amino acid
substitutions in the amino acid sequences of proteins disclosed
herein. Conservative substitutions of amino acids include
substitutions made amongst amino acids within the following groups:
(a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f)
Q, N; and (g) E, D.
[0093] In general, it is preferred that fewer than all of the amino
acids are changed when preparing variant polypeptides. Where
particular amino acid residues are known to confer function, such
amino acids will in some embodiments not be replaced, or
alternatively, will in some embodiments be replaced by conservative
amino acid substitutions. In some embodiments, preferably, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
residues can be changed when preparing variant polypeptides. In
some embodiments, it is generally preferred that the fewest number
of substitutions is made. Thus, one method for generating variant
polypeptides is to substitute all other amino acids for a
particular single amino acid, then assay activity of the variant,
then repeat the process with one or more of the polypeptides having
the best activity.
[0094] Conservative amino-acid substitutions in the amino acid
sequence of a polypeptide to produce functionally equivalent
variants of the polypeptide typically are made by alteration of a
nucleic acid encoding the polypeptide. Such substitutions can be
made by a variety of methods known to one of ordinary skill in the
art. For example, amino acid substitutions may be made by
PCR-directed mutation, site-directed mutagenesis according to the
method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,
1985), or by chemical synthesis of a gene encoding a
polypeptide.
[0095] It should be appreciated that some cells compatible with the
invention may express an endogenous copy of one or more of the
genes associated with the invention as well as a recombinant copy.
In some embodiments if a cell has an endogenous copy of one or more
of the genes associated with the invention then the methods will
not necessarily require adding a recombinant copy of the gene(s)
that are endogenously expressed. In some embodiments the cell may
endogenously express one or more enzymes from the pathways
described herein.
[0096] In some embodiments, one or more of the genes associated
with the invention is expressed in a recombinant expression vector.
As used herein, a "vector" may be any of a number of nucleic acids
into which a desired sequence or sequences may be inserted by
restriction and ligation for transport between different genetic
environments or for expression in a host cell. Vectors are
typically composed of DNA although RNA vectors are also available.
Vectors include, but are not limited to: plasmids, fosmids,
phagemids, virus genomes and artificial chromosomes.
[0097] A cloning vector is one which is able to replicate
autonomously or integrated in the genome in a host cell, and which
is further characterized by one or more endonuclease restriction
sites at which the vector may be cut in a determinable fashion and
into which a desired DNA sequence may be ligated such that the new
recombinant vector retains its ability to replicate in the host
cell. In the case of plasmids, replication of the desired sequence
may occur many times as the plasmid increases in copy number within
the host cell such as a host bacterium or just a single time per
host before the host reproduces by mitosis. In the case of phage,
replication may occur actively during a lytic phase or passively
during a lysogenic phase.
[0098] An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. Vectors may further contain one or more marker
sequences suitable for use in the identification of cells which
have or have not been transformed or transfected with the vector.
Markers include, for example, genes encoding proteins which
increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art
(e.g., .beta.-galactosidase, luciferase or alkaline phosphatase),
and genes which visibly affect the phenotype of transformed or
transfected cells, hosts, colonies or plaques (e.g., green
fluorescent protein). Preferred vectors are those capable of
autonomous replication and expression of the structural gene
products present in the DNA segments to which they are operably
joined.
[0099] As used herein, a coding sequence and regulatory sequences
are said to be "operably" joined when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
sequences. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operably joined if induction of a promoter in the 5' regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript can be translated into the desired protein or
polypeptide.
[0100] When a nucleic acid molecule encoding an enzyme associated
with aspects of the invention is expressed in a cell, a variety of
transcription control sequences (e.g., promoter/enhancer sequences)
can be used to direct its expression. The promoter can be a native
promoter, i.e., the promoter of the gene in its endogenous context,
which provides normal regulation of expression of the gene. In some
embodiments the promoter can be constitutive, i.e., the promoter is
unregulated allowing for continual transcription of its associated
gene. A variety of conditional promoters also can be used, such as
promoters controlled by the presence or absence of a molecule.
[0101] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CAAT sequence, and the like. In particular, such
5' non-transcribed regulatory sequences will include a promoter
region which includes a promoter sequence for transcriptional
control of the operably joined gene. Regulatory sequences may also
include enhancer sequences or upstream activator sequences as
desired. The vectors of the invention may optionally include 5'
leader or signal sequences. The choice and design of an appropriate
vector is within the ability and discretion of one of ordinary
skill in the art.
[0102] Expression vectors containing elements for expression are
commercially available and known to those skilled in the art. See,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,
Fourth Edition, Cold Spring Harbor Laboratory Press, 2012. Cells
can be genetically engineered by the introduction into the cells of
heterologous nucleic acids, such as DNA or RNA. As used herein a
"recombinant cell" refers to a cell that expresses heterologous
nucleic acids. That heterologous nucleic acid, such as DNA or RNA
can be placed under operable control of transcriptional elements to
permit the expression of the heterologous DNA in the host cell.
Heterologous expression of genes associated with the invention, is
described in non-limiting examples of bacterial cells. As one of
ordinary skill in the art would appreciate, methods disclosed
herein can include other bacterial cells, and in some embodiments
could include archaeal cells, fungi (including yeast cells),
mammalian cells, plant cells, etc.
[0103] A nucleic acid molecule that encodes an enzyme associated
with aspects of the invention can be introduced into a cell or
cells using methods and techniques that are standard in the art.
For example, nucleic acid molecules can be introduced by standard
protocols such as transformation including chemical transformation
and electroporation, transduction, particle bombardment, etc.
Expressing a nucleic acid molecule encoding an enzymes associated
with aspects of the invention also may be accomplished by
integrating the nucleic acid molecule into the genome.
[0104] Aspects of the invention relate to expression of bacterial
cells. Bacterial cells associated with the invention can be
cultured in some embodiments in media of any type (rich or
minimal), including fermentation medium, and any composition. As
would be understood by one of ordinary skill in the art, routine
optimization would allow for use of a variety of types of media.
The selected medium can be supplemented with various additional
components. Some non-limiting examples of supplemental components
include glucose, antibiotics, IPTG for gene induction and ATCC
Trace Mineral Supplement. Similarly, other aspects of the medium
and growth conditions of the cells of the invention may be
optimized through routine experimentation. For example, pH and
temperature are non-limiting examples of factors which can be
optimized. In some embodiments, factors such as choice of media,
media supplements, and temperature can influence production levels
of a desired molecule. In some embodiments the concentration and
amount of a supplemental component may be optimized. In some
embodiments, how often the media is supplemented with one or more
supplemental components, and the amount of time that the media is
cultured before harvesting the desired molecule is optimized.
[0105] The liquid cultures used to grow cells associated with the
invention can be housed in any of the culture vessels known and
used in the art. In some embodiments large scale production in a
bioreactor vessel can be used to produce large quantities of a
desired molecule.
[0106] Unless otherwise defined herein, scientific and technical
terms used in connection with the present disclosure shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. The methods and techniques of the present disclosure are
generally performed according to conventional methods well-known in
the art. Generally, nomenclatures used in connection with, and
techniques of biochemistry, enzymology, molecular and cellular
biology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well-known
and commonly used in the art. The methods and techniques of the
present disclosure are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification unless otherwise
indicated.
[0107] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
[0108] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by reference, in
particular for the teaching that is referenced hereinabove.
However, the citation of any reference is not intended to be an
admission that the reference is prior art.
EXAMPLES
Example 1: Production of Amino Acids from Feedstock Consisting of a
Syngas, or Components Thereof
[0109] Cupriavidus necator (also known as Ralstonia eutropha)
strains DSM 531 and DSM541 were cultured using a
H.sub.2/CO.sub.2/O.sub.2 gas mixture and mineral salt fermentation
medium. The culture was grown for 96 hrs in 20 ml MSM medium (1 L
Medium A: 9 g Na2HPO412H2O, 1.5 g H2PO4, 1.0 g NH4Cl and 0.2 g
MgSO47H2O per 1 L; 10 ml Medium B: 50 mg Ferric ammonium citrate
and 100 mg CaCl2 per 100 ml; 10 ml Medium C: 5 g NaHCO3 per 100 ml;
and 1 ml Trace Mineral Solution: 100 mg ZnSO47H2O, 30 mg MnCl24H2O,
300 mg H3BO3, 200 mg COCl26H2O, 10 mg CuCl22H2O, 20 mg NiCl26H2O
and 30 mg Na2MoO42H2O per 1 L) in a serum bottle supplemented with
66.7% H2, 9.5% CO2, 5% 02 and 18.8% N2 at 30.degree. C. and 200
rpm.
[0110] For lysine detection in the growth media, 1 ml of the cells
(O.D=0.1) were separated by centrifugation (10,000 rpm, 5 min at
room temperature) and the supernatant (200 .mu.l) was further
filtrated (0.22.mu.). Samples of the supernatants were collected
and analyzed for secretion of amino-containing compounds, such as
amino acids including lysine, tyrosine, and phenylalanine, as shown
in Table 2. It was observed that C. necator strain DSM541 secreted
higher concentrations of lysine, tyrosine, and phenylalanine into
the medium compared to C. necator strain DSM531. The analyses were
performed on 200 .mu.l of sterile filtered fermentation medium.
Compounds were isolated and derivatized using a clean-up and
derivatization kit (e.g. EZ-FaaST (Phenomenex) followed by liquid
chromatography-mass spectrometry to separate and identify compounds
that had been secreted by the bacterial strains into the medium
(Table 2). The levels of lysine found in the media from DSM 541
were 125 fold higher than DSM 531. (FIG. 5).
Example 2: Production of Amino Acids from Feedstock Consisting of a
Syngas, or Components Thereof, Using an Engineered
Microorganism
[0111] Bacterial strains can be genetically modified to increase
production of amino-containing molecules such as lysine. Ralstonia
eutropha N-1 strain DSM 13513 can be engineered to express
exogenous genes or up-regulate native genes coding for enzymes
involved in lysine biosynthesis such as aspartate kinase (EC
2.7.2.4, see, for example, SEQ ID NO:1) (Kalinowski et al., 1991),
dihydrodipicolinate synthase (EC 4.2.1.52, see, for example, SEQ ID
NO:2), (Patek et al., 1997) or in lysine secretion such as lysine
exporter (Vrljic et al., 1996) (see, for example, SEQ ID NO:3). In
some embodiments, increased lysine production is obtained by
down-regulation of enzymes in the citric acid cycle, such as
succinyl-CoA synthase (EC 6.2.1.4, see, for example, alpha subunit
SEQ ID NO:4 and beta subunit SEQ ID NO:5) (Johnson et al.,
1998).
[0112] Aromatic amino acids are synthesized through the Shikimate
pathway. Bacterial strains are engineered to produce increased
quantifies of the aromatic amino acids phenylalanine and tyrosine
by over-expressing enzymes in this pathway, such as chorismate
synthase (EC 4.2.3.5, SEQ ID NO:6).
[0113] Rhodococcus opacus PD630 produces a chorismate synthase for
the production of aromatic amino acids (Holder et al., 2011). Basic
Local Alignment Search Tool (BLAST) of the protein sequence of the
chorismate synthase from Rhodococcus opacus PD360 against the NCBI
nr database (All non-redundant GenBank CDS
translations+PDB+SwissProt+PIR+PRF excluding environmental samples
from WGS projects) reveals multiple protein sequences with high
homology. As described herein, homologous proteins can be used for
practice of the invention.
[0114] To generate a bacterial strain that produces increased
quantities of amino acids, expression vectors are constructed. The
aspartate kinase, dihydrodipicolinate synthase, and chorismate
synthase genes from the Ralstonia eutropha are amplified from the
R. eutropha genome using the primer pairs provided by SEQ ID NO:11
and 12, 13 and 14, and 19 and 20, respectively. The genes can also
be synthesized. The cloned genes are inserted into a pBBR1
MCS-derived broad host-range vector. In some embodiments, all genes
are regulated by a single promoter to yield a single, polycistronic
mRNA transcript. Gene expression can be modulated by placing the
over-expressed genes under control of the heterologous araBAD
promoter or the native rbc promoter (RuBisCO carboxylase/oxygenase
promoter). Plasmid manipulation is performed in E. coli.
[0115] Plasmids prepared as described above are transformed into
Ralstonia eutropha using electroporation (11.5 kV/cm, 25 .mu.F, 5
ms). For plasmid containing an inducible promoter, the inducer
molecule will be added to the culture at mid-log phase. In
embodiments in which the plasmid contains the rbc promoter, the
genes are expressed constitutively.
[0116] The genetically modified Ralstonia eutropha is grown to high
cell density using a gas-based feedstock such as syngas. Bacterial
cells and fermentation supernatant are separated by centrifugation
at 10,000 rpm for 10 minutes. To lyse the bacterial cells, pellets
are washed twice with 10 mM Tris pH 8.0 containing 10 mM EDTA (TE
buffer) and resuspended in TE buffer containing 10% SDS followed by
tip sonication (3.times.30 sec, 30% duty cycle, power level 7).
Supernatant is filtered through a 0.2 micron filter. The
identification and quantity of free amino acids in the cell extract
and in the supernatant (secreted) are determined after purification
and derivatization using a clean-up and derivatization kit (e.g.,
EZ-faast kit (Phenomenex, Torrance, Calif.) followed by High
Performance Liquid Chromatography coupled to Mass Spectrometry
(HPLC-MS).
[0117] Total bacterial protein is isolated by salt precipitation
using high concentrations of (NH.sub.4).sub.2SO.sub.4 followed by
dialysis. The isolated bacterial protein is hydrolyzed by treatment
with HO, and the amino acid composition is determined following
separation and quantitation by Ion-Exchange Chromatography. Results
of these experiments are expected to show production of amino acids
from feedstock consisting of a syngas or components thereof using
overexpression in a chemoautotrophic production strain that can be
grown to high cell density using a gas-based feedstock.
2.1 Enhanced Production of Lysine
[0118] Cupriavidus necator wild type (DSM 531) and the mutant
strain that does not form poly-.beta.-hydroxy-butyrate (DSM 541),
were grown under chemoautotrophic conditions for 96 hrs in 20 ml
MSM medium (1 L Medium A: 9 g Na.sub.2HPO.sub.412H.sub.2O, 1.5 g
H.sub.2PO.sub.4, 1.0 g NH.sub.4Cl and 0.2 g MgSO.sub.47H.sub.2O per
1 L; 10 ml Medium B: 50 mg Ferric ammonium citrate and 100 mg
CaCl.sub.2 per 100 ml; 10 ml Medium C: 5 g NaHCO.sub.3 per 100 ml;
and 1 ml Trace Mineral Solution: 100 mg ZnSO.sub.47H.sub.2O, 30 mg
MnCl.sub.24H.sub.2O, 300 mg H.sub.3BO.sub.3, 200 mg
COCl.sub.26H.sub.2O, 10 mg CuCl.sub.22H.sub.2O, 20 mg
NiCl.sub.26H.sub.2O and 30 mg Na.sub.2MoO.sub.42H.sub.2O per 1 L)
in a serum bottle supplemented with 66.7% H.sub.2, 9.5% CO.sub.2,
5% O.sub.2 and 18.8% N.sub.2 at 30.degree. C. and 200 rpm.
[0119] For lysine detection in the growth media, 1 ml of the cells
(O.D=0.1) were separated by centrifugation (10,000 rpm, 5 min at
room temperature) and the supernatant (200.mu.1) was further
filtrated (0.22.mu.). The compounds were isolated from the
supernatant and derivatized using EZ-FaaST and analyzed by LC-MS.
The levels of lysine found in the media from DSM 541 were 125 fold
higher than DSM 531. (FIG. 5)
Example 3: Production of Putrescine from Feedstock Consisting of a
Syngas, or Components Thereof
[0120] Cupriavidus Necator (also known as Ralstonia eutropha)
strains DSM 531 and DSM541 were grown on an
H.sub.2/CO.sub.2/O.sub.2 gas mixture and mineral salt fermentation
medium (1 L Medium A: 9 g Na2HPO412H2O, 1.5 g H2PO4, 1.0 g NH4Cl
and 0.2 g MgSO47H2O per 1 L; 10 ml Medium B: 50 mg Ferric ammonium
citrate and 100 mg CaCl2 per 100 ml; 10 ml Medium C: 5 g NaHCO3 per
100 ml; and 1 ml Trace Mineral Solution: 100 mg ZnSO47H2O, 30 mg
MnCl24H2O, 300 mg H3BO3, 200 mg COCl26H2O, 10 mg CuCl22H2O, 20 mg
NiCl26H2O and 30 mg Na2MoO42H2O per 1 L) for 96 hours in 20 ml
medium in a serum bottle supplemented with 66.7% H2, 9.5% CO2, 5%
02 and 18.8% N2 at 30.degree. C. and 200 rpm.
[0121] For putresine detection in the growth media, 1 ml of the
cells (O.D=0.1) were separated by centrifugation (10,000 rpm, 5 min
at room temperature) and the supernatant was further filtrated
(0.22.mu.). Samples of the supernatant were collected and analyzed
for secretion of putrescine into the aqueous broth, as shown in
Table 2. It was observed that C. necator strain DSM531 secreted
higher concentrations of putrescine into the medium compared to C.
necator strain DSM541. The analyses were performed on 200 .mu.l of
sterile filtered fermentation broth. Compounds were isolated and
derivatized using a clean-up and derivatization kit (e.g., EZ-FaaST
(Phenomenex) followed by liquid chromatography-mass spectrometry to
separate and identify the compounds that had been secreted by the
bacterial strains into the medium (Table 2).
Example 4: Production of Putrescine from Feedstock Consisting of a
Syngas, or Components Thereof, Using an Engineered
Microorganism
[0122] Bacterial strains such as Ralstonia eutropha N-1 strain DSM
13513 can be genetically engineered to increase production of
putrescine. Biochemical synthesis of putrescine is achieved by
enzymatic degradation of arginine by arginase (EC 3.5.3.1, SEQ ID
NO:7), followed by enzymatic decarboxylation of ornithine in a
reaction catalyzed by ornithine decarboxylase (EC 4.1.1.17, SEQ ID
NO:8). Corynebacterium glutamicum produces an exemplary ornithine
decarboxylase that can be used for the production of putrescine
(Schneider and Wendisch, 2010).
[0123] To generate a bacterial strain that produces increased
quantities of putrescine, expression vectors are constructed. The
arginase and ornithine decarboxylase genes from Ralstonia eutropha
are amplified from the R. eutropha genome using the cloning primer
pairs provided by SEQ ID NO 17 and 18, and 21 and 22, respectively.
Alternatively, the arginase and ornithine genes can be synthesized.
The genes are inserted into a pBBR1 MCS-derived broad host-range
vector. In some embodiments, all genes are regulated by a single
promoter to yield a single, polycistronic mRNA transcript. Gene
expression can be modulated by placing the over-expressed genes
under control of the heterologous araBAD promoter or the native rbc
promoter (RuBisCO carboxylase/oxygenase promoter). Plasmid
manipulation is performed in E. coli.
[0124] Plasmids prepared as described above are transformed into
Ralstonia eutropha using electroporation (11.5 kV/cm, 25 .mu.F, 5
ms). For plasmids containing an inducible promoter, the inducer
molecule is added to the culture at mid-log phase. In embodiments
in which the plasmid contains the rbc promoter, the genes are
expressed constitutively.
[0125] The genetically modified Ralstonia eutropha is grown to high
cell densities using a gas-based feedstock such as syngas.
Bacterial cells and fermentation supernatant are separated by
centrifugation at 10; 000 rpm for 10 minutes. The recovered
supernatant is filtered through a 0.2 micron filter. Production and
secretion of putrescine are evaluated. The supernatant is also
acidified to pH 2 to precipitate soluble proteins; the precipitate
is removed by centrifugation.
[0126] The pellet of bacterial cells is washed with 0.1 M HCl in
0.25/0.75 water/methanol to recover co-precipitated putrescine
hydrochloride. Putrescine purity is assessed by NMR and quantified
by pH titration.
Example 5: Production of Caprolactam from Feedstock Consisting of a
Syngas, or Components Thereof, Using an Engineered
Microorganism
[0127] Bacterial strains such as Ralstonia eutropha N-1 strain DSM
13513 can be genetically engineered for the production of
caprolactam by expressing genes involved in the biosynthetic
pathway. Biochemical synthesis of caprolactam is achieved by
enzymatic conversion of lysine through the metabolic pathway:
lysine.fwdarw.6-aminohex-2-enoic acid.fwdarw.6-aminocaproic
acid.fwdarw.caprolactam. Carbon nitrogen lyase (EC 4.3.1.-)
activity converts lysine into 6-aminohex-2-enoic acid
(Raemarkers-Franken et al., 2011), which can then be converted into
6-aminocaproic acid using enzymes with .alpha.-.beta.-enoate
reductase (EC 1.3.1.-) activity, for example enzymes from species
such as Acremonium, Clostridium, Moorella and Ochrobactrum
(Raemarkers-Franken et al., 2009). An enzyme with amidohydrolase
(EC 3.5.2.-) activity (Burgard et al., 2010) catalyzes the
conversion of 6-aminocaproic acid to caprolactam.
[0128] In some embodiments, an enzyme with tyrosine phenol lyase
activity (EC 4.1.99.2, SEQ ID NO:9) can convert tyrosine into
phenol, which can then be chemically converted into benzene and
caprolactam. Erwinia herbicola produces an exemplary tyrosine
phenol lyase that can be used for the production of phenol (Mikami
et al., 2000).
[0129] Alternatively, phenol can be chemically converted to
cyclohexanone which can then be converted to caprolactam.
[0130] As an additional alternative, purified lysine can be used
for the chemical synthesis of caprolactam (see, for example U.S.
Pat. No. 7,399,855 B2 and U.S. Patent Publication US 2010/0145003
A1; Frost 2008; Frost 2010)].
[0131] To generate a bacterial strain that produces increased
quantities of caprolactam, expression vectors are constructed.
Genes for the biosynthesis of caprolactam can be amplified from the
genomes of appropriate microorganisms or be synthesized. The genes
can be inserted into a pBBR1 MCS-derived broad host-range vector.
In some embodiments, all genes are regulated by a single promoter
to yield a single, polycistronic mRNA transcript. Gene expression
can be modulated by placing the over-expressed genes under control
of the heterologous araBAD promoter or the native rbc promoter
(RuBisCO carboxylase/oxygenase promoter). Plasmid manipulation is
performed in E. coli.
[0132] Plasmids prepared as described above can be transformed into
Ralstonia eutropha using electroporation (11.5 kV/cm, 25 .mu.F, 5
ms). For plasmids containing an inducible promoter, the inducer
molecule can be added to the culture at mid-log phase. In
embodiments in which the plasmid contains the rbc promoter, the
genes are expressed constitutively.
[0133] The genetically modified Ralstonia eutropha is grown to a
high cell density using a gas-based feedstock such as syngas.
Bacterial cells and fermentation supernatant are separated by
centrifugation at 10; 000 rpm for 10 minutes. The recovered
supernatant is filtered through a 0.2 micron filter. Production and
secretion of lysine, benzene, and caprolactam are evaluated by
high-performance liquid chromatography-mass spectrometry or by gas
chromatography coupled to mass spectrometry.
[0134] Secreted lysine can be purified by running the supernatant
through a commercially available ion-exchange resin (e.g., DOWEX
(Dow, Pittsburgh, Calif.)).
Example 6: Production of Styrene from Feedstock Consisting of a
Syngas, or Components Thereof, Using an Engineered
Microorganism
[0135] Bacterial strains such as the Cupriavidus necator strain
that produces amino acids described in Example 1 can be genetically
engineered for the production of styrene.
[0136] Styrene can be produced from the aromatic amino acid
phenylalanine through the following metabolic pathway:
phenylalanine cinnamic acid styrene. Phenylalanine ammonium lyase
(EC 4.3.1.24) activity catalyzes the conversion of phenylalanine
into cinnamic acid, which can be further converted into styrene
through a decarboxylation step. Streptomyces maritimus produces an
exemplary phenylalanine ammonium lyase that can be used for the
production of cinnamic acid (Piel et al., 2000). Exemplary enzymes
that are compatible with the decarboxylation step include those
belonging to a family of oxalate decarboxylases (EC 4.1.1.2), for
example an oxalate decarboxylase from Rhodococcus jostii RHA1 that
can produce styrene ([McLeod et al., 2006)S. In some embodiments,
in order to further increase production of styrene, the endogenous
styrene degradation pathway can be minimized by down-regulation of
the styrene monooxygenase complex (Mooney et al., 2006).
[0137] To generate a bacterial strain that produces increased
quantities of styrene, expression vectors are constructed. Genes
for the biosynthesis of styrene can be amplified from the genomes
of appropriate microorganisms or be synthesized. The genes can be
inserted into a pBBR1 MCS-derived broad host-range vector. In some
embodiments, all genes can be regulated by a single promoter to
yield a single, polycistronic mRNA transcript. Gene expression can
in some embodiments be modulated by placing the over-expressed
genes under control of the heterologous araBAD promoter or the
native rbc promoter (RuBisCO carboxylase/oxygenase promoter).
Plasmid manipulation is performed in E. coli.
[0138] Plasmids prepared as described above can be transformed into
Ralstonia eutropha using electroporation (11.5 kV/cm, 25 .mu.F, 5
ms). For plasmids containing an inducible promoter, the inducer
molecule can be added to the culture at mid-log phase. In
embodiments in which the plasmid contains the rbc promoter, the
genes are expressed constitutively.
[0139] The genetically modified Ralstonia eutropha is grown to a
high cell density using a gas-based feedstock such as syngas.
Bacterial cells and fermentation supernatant are separated by
centrifugation at 10,000 rpm for 10 minutes. The recovered
supernatant is filtered through a 0.2 micron filter. Production and
secretion of styrene are evaluated.
Example 7: Production of 1,3-Butanediol from Feedstock Consisting
of a Syngas, or Components Thereof
[0140] Cupriavidus necator (also known as Ralstonia eutropha)
strain DSM541 was cultured using a H.sub.2/CO.sub.2/O.sub.2 gas
mixture and mineral salt fermentation medium (1 L Medium A: 9 g
Na2HPO412H2O, 1.5 g H2PO4, 1.0 g NH4Cl and 0.2 g MgSO47H2O per 1 L;
10 ml Medium B: 50 mg Ferric ammonium citrate and 100 mg CaCl2 per
100 ml; 10 ml Medium C: 5 g NaHCO3 per 100 ml; and 1 ml Trace
Mineral Solution: 100 mg ZnSO47H2O, 30 mg MnCl24H2O, 300 mg H3BO3,
200 mg COCl26H2O, 10 mg CuCl22H2O, 20 mg NiCl26H2O and 30 mg
Na2MoO42H2O per 1 L). Samples of the supernatant were collected and
analyzed for the secretion of 1,3-butanediol by gas
chromatography-mass spectrometry (GC-MS), as shown in FIGS. 1A and
1B.
GC-MS
[0141] For butanediol analysis, samples were injected manually
using a SPME syringe (SPME fiber assembly Polydimethylsiloxane,
Sigma Aldrich). Reactor broth samples were placed in 2 mL GC vials
with a septum top. The SPME was injected into the samples for 30
seconds and then placed into the GC injector for 5 seconds.
Compounds were detected on an Agilent 6890N GC/MS (Agilent, Santa
Clara, Calif.) on a HP1 60 m column.times.0.25 mm ID. The injector
temperature was 250.degree. C. and was run in split mote (8:1) with
an initial GC temperature of 60.degree. C., ramp at 5.degree.
C./min to a final temp of 100.degree. C. which was held for 5
minutes. Peak ID was accomplished through a NIST08 library search
and comparison with 1,4-Butanediol standard material. Butanediol
standards were prepared by adding known quantities of standard to
fresh media and injecting with the same method as the broth
samples.
Example 8: Production of 1,3-Butadiene from Feedstock Consisting of
a Syngas, or Components Thereof, Using an Engineered
Microorganism
[0142] Cupriavidus necator strain DSM541 can grow on a
carbon-containing gas such as syngas, producer gas, CO2, carbon
monoxide and mixtures of the same containing hydrogen gas, to
produce polymers of 3-hydroxybutyrate (poly-3-hydroxybutyrate,
P3HB). During fermentative growth, P3HB can be degraded, and
3-hydroxybutyrate and 1,3-butanediol are produced. C. necator
strains can be genetically engineered to produce increased
quantities of butadiene by introducing an exogenous oleate
hydratase enzyme (EC 4.2.1.53, SEQ ID NO:10).
[0143] To generate a bacterial strain that produces increased
quantities of 1,3-butadiene, expression vectors are constructed. A
gene encoding an oleate hydratase can be amplified from the genomes
of an appropriate microorganism or be synthesized. The gene can be
inserted into a pBBR1 MCS-derived broad host-range vector. Gene
expression can in some embodiments be modulated by placing the
over-expressed genes under control of the heterologous araBAD
promoter or the native rbc promoter (RuBisCO carboxylase/oxygenase
promoter). Plasmid manipulation is performed in E. coli.
[0144] Plasmids prepared as described above can be transformed into
Ralstonia eutropha using electroporation (11.5 kV/cm, 25 .mu.F, 5
ms). For plasmids containing an inducible promoter, the inducer
molecule can be added to the culture at mid-log phase. In
embodiments in which the plasmid contains the rbc promoter, the
genes are expressed constitutively.
[0145] The genetically modified Ralstonia eutropha is grown to a
high cell density using a gas-based feedstock such as syngas.
Bacterial cells and fermentation supernatant are separated by
centrifugation at 10; 000 rpm for 10 minutes. The recovered
supernatant is filtered through a 0.2 micron filter. Production and
secretion of butadiene are evaluated.
8.1 PHB Purification and Extraction
[0146] PHB is purified and extracted according to the following
methods:
Polymer Analysis
Isolation/Extraction of Polymers:
[0147] 1: Add chloroform to lyophilized bacteria (6 ml of
chloroform/g bacteria). Incubate at 60.degree. C. for 4 hours. 2:
Recover chloroform extract and dry down to approximately 1/5 of
volume under nitrogen at 40.degree. C. 3: Add concentrated
chloroform extract to ice-cold methanol (at least a 1:4 ratio of
chloroform to methanol). 4: Isolate precipitated polymers. 5: Wash
by re-dissolving polymers in chloroform and precipitate with
methanol (1:4 ratio). Repeat washing a couple of times.
Production of Monomers (Methanolysis):
[0148] 1: Add 1 ml of chloroform and 1 ml of methanol containing
2.8 M sulfuric acid. 2: Incubate for 2 hours at 100.degree. C. 3:
Cool and add 0.5 ml of distilled water. 4: Collect organic phase
containing methyl-esters.
8.2: GC-MS
[0149] For polymer analysis, compounds were detected on an Agilent
6890N GC/MS (Agilent, Santa Clara, Calif.) on a HP1 60 m
column.times.0.25 mm ID. Samples were placed in GC vial inserts
with a final volume in hexane of 50 uL. Samples were injected using
an automatic injector, injector temperature was 250.degree. C. and
was run in split mote (8:1) with an initial GC temperature of
40.degree. C., ramp at 4.degree. C./min to a final temp of
100.degree. C., then a ramp of 10.degree. C./min to 225.degree. C.,
finally a 20.degree. C./min ramp to 312 C. Peak ID was accomplished
through a NIST08 library.
Example 9: Production of Omega-7 Fatty Acids from Feedstock
Consisting of a Syngas, or Components Thereof
[0150] Rhodococcus opacus strain DSM 43205 was cultured using a
H.sub.2/CO.sub.2/O.sub.2 gas mixture and mineral salt fermentation
medium. The cell mass was separated from the supernatant of the
culture by centrifugation. The supernatant was discarded and a
chloroform/methanol (C/M) extraction was performed on the biomass
pellet. Lipids were applied to Silica-60 columns, and different
lipid groups were separated and eluted from the column with organic
solvents including hexane, chloroform, isopropanol, methanol and
acetone. Mild alkaline methylation was performed to methylate
non-fatty acid lipids and acid methylation was performed to
methylate fatty acids. Fatty acid methyl esters (FAMEs) were
analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS
analysis revealed that Rhodococcus opacus strain DSM 43205 cultured
with the gas mixture produced triacylglycerols, which contained
high amounts of omega-7 fatty acids, including palmitoleic acid
(C16:1, also known as 9-hexadecenoic acid) and vaccenic acid
(C18:1, also known as 11-octadecenoic acid) as shown in FIG. 4.
Analysis of the biomass showed 40% lipid content; further analysis
of the lipid content showed 13% C16:1 omega 7 fatty acid
(palmitoleic acid) and 21% C18:1 omega 7 fatty acid (vaccenic
acid). Table 1 provides the lipid content and growth conditions for
the samples presented in FIG. 4.
GC-MS
[0151] For FAME analysis, compounds were detected on an Agilent
6890N GC/MS (Agilent, Santa Clara, Calif.) on a HP1 60 m
column.times.0.25 mm ID. Samples were placed in GC vial inserts
with a final volume in hexane of 50 uL. Samples were injected using
an automatic injector, the injector temperature was 250.degree. C.
and was run in split mote (8:1) with an initial GC temperature of
100.degree. C., ramp at 10.degree. C./min to a final temp of
150.degree. C., then a ramp of 3.degree. C./min to 250.degree. C.,
finally a 10.degree. C./min ramp to 312.degree. C. which is held
for 7 min. Peak ID was accomplished through a NIST08 library and
comparison to known standards (Supelco 37 Component FAME Mix).
Quantification was accomplished through an external standard added
to each sample prior to injection (methyl undecanoate) and
extraction efficiency was quantified by an internal standard
(1,2-dinonadecanoyl-sn-glycero-3-phosphocholine).
TABLE-US-00001 TABLE 1 Lipid content of Rhodococcus opacus
cultures. Total Lipid Total Lipid Growth in C/M extract soluble in
hexane Strain Conditions Sample (% of dry wt) (1% of dry wt) R.
opacus Gas A 42 14 R. opacus Gas B 27 3
Example 10: Production of Omega-7 Fatty Acids from Feedstock
Consisting of a Syngas, or Components Thereof
[0152] As demonstrated in Example 9 and FIG. 4, Rhodococcus opacus
strain DSM 43205 can grow on a carbon-containing gas such as
syngas, producer gas, CO.sub.2, carbon monoxide and mixtures of the
same containing hydrogen gas; and/or C1 compounds, to produce and
accumulate lipids, including triacylglycerols, which contain high
amounts of omega-7 fatty acids, including palmitoleic acid (C16:1)
and vaccenic acid (C18:1).
[0153] Production of omega-7 fatty acids is further enhanced by
culturing Rhodococcus opacus strain DSM 43205 to a high cell
density using a gas feedstock, turning off the input of nitrogen
nutrient (ammonium) and/or phosphorous nutrient (phosphate) to
facilitate the accumulation of lipids including triacylglycerols.
The cells are harvested by spinning at 10000 rpm for 10 minutes and
the wet mass is applied to a lyophilizer overnight to obtain dry
cell material. The lipids are extracted by adding methanol to the
dry cell material and incubating at 60.degree. C. for 30 minutes
followed by the addition of 2 volumes of chloroform and shaking at
40.degree. C. for 30 minutes. The extract is spun at 1500 rpm for 5
minutes and supernatant containing the lipids is recovered. The
lipids are applied to Silica-60 columns and the different lipid
groups are separated and eluted from the column with organic
solvents including hexane, chloroform, isopropanol, methanol and
acetone. The different lipid fractions are analyzed by gas
chromatography-mass spectrometry.
TABLE-US-00002 TABLE 2 Analysis of secreted amino-compounds. DSMZ
531: DSMZ 541: C. necator C. necator Compound Blank umol/L umol/L
fold difference Glu Glutamic acid 0.1952 11.556 40.614 3.5 Sar
Sarcosine 1.7232 2.5708 36.4692 14.2 Ser Serine 1.7688 7.9428
35.8164 4.5 Gly Glycine 9.4757 10.3272 35.0351 3.4 Ala Alanine
0.6504 5.996 32.3436 5.4 Thr Threonine 0.216 5.4152 22.9456 4.2 Val
Valine 0.0984 4.182 21.5904 5.2 Ile Isoleucine 0.0272 2.1476
14.0068 6.5 Orn Ornithine 0.9324 10.4876 13.056 1.2 His Histidine
0.99 2.3816 12.0852 5.1 Arg Arginine 0.2988 0.4112 9.3428 22.7 Phe
Phenylalanine 0.1 3.4216 8.6652 2.5 Lys Lysine 0.1012 0.063 7.9088
125.5 Tyr Tyrosine 0.386 2.9448 7.3972 2.5 Cit Citosine 0.3332
0.6572 6.8248 10.4 Asp Asparatic acid 2.1964 3.2776 4.6132 1.4 Gln
Glutamine 0.1412 1.2548 4.2944 3.4 Pro Proline 0.0477 1.2567 4.1107
3.3 Leu Leucine 0.054 2.5558 3.7205 1.5 Trp Tryptophan 0.0352
0.9464 2.7072 2.9 Met Methionine 0.0156 1.3944 1.614 1.2 Tpr Tpr
0.034 0.5208 0.8052 1.5 B-Ala B-Alanine 0 2.0904 0.6688 0.3 SAM
S-Adenosyl- 0 0 0.5604 methionine SAH S-Adenosylhomo- 1.194 2.3232
0.2812 0.1 cysteine MetSo Methionine 0.0128 0.3696 0.2528 0.7
Sulfoxide Hcy-PCA Hcy-PCA 0.024 0.1944 0.2344 1.2 a-AAA a-AAA
0.0096 0.2008 0.1492 0.7 APA APA 0 0.0248 0.134 5.4 Put Putracine
0.1912 15.0568 0.128 0.0 Cys-PCA Cys-PCA 0.0392 0.7148 0.1272 0.2
GSH-PCA GSH-PCA 0.0056 0.0052 0.0468 9.0 Spd Spd 0.0652 0.0728
0.0444 0.6 3-His 3-His 0.0264 0.0384 0.0276 0.7 Cy2 Cy2 0.0364
0.0628 0.0128 0.2 Cth Cth 0.0072 0.0072 0.0124 1.7 CysGly-
CysGly-PCA 0.002 0.01 0.0112 1.1 PCA Erg Erg 0.0076 0.0512 0.0084
0.2 Hcy2 Hcy2 0.0116 0.008 0.0048 0.6
Example 11: Production of Polyhydroxybutyrate (PHB) from Feedstock
Consisting of a Syngas or Components Thereof
[0154] Cupriavidus Necator (also known as Ralstonia eutropha)
strain DSM531 was cultured using a H2/CO2/O2 gas mixture and
mineral salt fermentation medium (1 L Medium A: 9 g Na2HPO412H2O,
1.5 g H2PO4, 1.0 g NH4Cl and 0.2 g MgSO47H2O per 1 L; 10 ml Medium
B: 50 mg Ferric ammonium citrate and 100 mg CaCl2 per 100 ml; 10 ml
Medium C: 5 g NaHCO3 per 100 ml; and 1 ml Trace Mineral Solution:
100 mg ZnSO47H2O, 30 mg MnCl24H2O, 300 mg H3BO3, 200 mg COCl26H2O,
10 mg CuCl22H2O, 20 mg NiCl26H2O and 30 mg Na2MoO42H2O per 1 L) and
found to produce and accumulate biopolymers including PHBs.
[0155] Strain DSM531 was grown to high cell density using a gas
feedstock (up to optical density between 60 to 130 at 650 nm).
Cells were harvested by spinning at 10000 rpm for 10 minutes. Wet
mass was applied to a lyophilizer overnight to obtain dry cell
material. Biopolymers were extracted by adding chloroform to the
dry cell material and incubating at 60 degrees Celsius for 4 hours.
Extract was spun at 1500 rpm for 5 minutes and supernatant
containing the extracted biopolymers was recovered. Supernatant was
concentrated under nitrogen and biopolymers was precipitated and
recovered by adding concentrated supernatant to 4 volumes of
ice-cold methanol and spinning at 1500 rpm for 5 minutes (FIG. 2).
Recovered biopolymers were re-dissolved in chloroform. Monomer
production was facilitated by adding methanol and sulfuric acid and
incubation at 100 degrees Celsius for 2 hours. Following the
addition of water to create a 2-phase extraction system, the
monomeric methyl-ester was isolated in the organic phase and
analyzed by GC/MS (FIG. 3A-3D).
Polymer Analysis
Isolation/Extraction of Polymers:
[0156] 1: Added chloroform to lyophilized bacteria (6 ml of
chloroform/g bacteria). Incubated at 60.degree. C. for 4 hours. 2:
Recovered chloroform extract and dried down to approximately 1/5 of
volume under nitrogen at 40.degree. C. 3: Added concentrated
chloroform extract to ice-cold methanol (at least a 1:4 ratio of
chloroform to methanol). 4: Isolated precipitated polymers. 5:
Washed by re-dissolving polymers in chloroform and precipitate with
methanol (1:4 ratio). Repeated washing a couple of times.
Production of Monomers (Methanolysis):
[0157] 1: Added 1 ml of chloroform and 1 ml of methanol containing
2.8 M sulfuric acid. 2: Incubated for 2 hours at 100.degree. C. 3:
Cooled and added 0.5 ml of distilled water. 4: Collected organic
phase containing methyl-esters.
GC-MS
[0158] For polymer analysis, compounds were detected on an Agilent
6890N GC/MS (Agilent, Santa Clara, Calif.) on a HP1 60 m
column.times.0.25 mm ID. Samples were placed in GC vial inserts
with a final volume in hexane of 50 uL. Samples were injected using
an automatic injector, injector temperature was 250.degree. C. and
was run in split mote (8:1) with an initial GC temperature of
40.degree. C., ramp at 4.degree. C./min to a final temp of
100.degree. C., then a ramp of 10.degree. C./min to 225.degree. C.,
finally a 20.degree. C./min ramp to 312 C. Peak ID was accomplished
through a NIST08 library.
Example 12: Increasing Production of Polyhydroxybutyrate (PHB) from
Feedstock Consisting of a Syngas or Components Thereof
[0159] Cupriavidus Necator (also known as Ralstonia eutropha)
strain DSM531) can grow on a carbon-containing gas such as syngas,
producer gas, CO2, carbon monoxide and mixtures of the same
containing hydrogen gas; and/or Cl compounds to produce and
accumulate biopolymers including PHBs.
[0160] Strain DSM531 can be grown to high cell density using a gas
feedstock (over optical density at 650 nm). The input of nitrogen
nutrient (ammonium) and/or phosphorous nutrient (phosphate) is
turned off to facilitate the accumulation of biopolymers including
PHBs. Cells are harvested by spinning at 10000 rpm for 10 minutes.
Wet mass is applied to a lyophilizer overnight to obtain dry cell
material. Biopolymers are extracted by adding chloroform to the dry
cell material and incubating at 60 degrees Celsius for 4 hours.
Extract is spun at 1500 rpm for 5 minutes and supernatant
containing the extracted biopolymers is recovered. Supernatant is
concentrated under nitrogen and biopolymers are precipitated and
recovered by adding concentrated supernatant to 4 volumes of
ice-cold methanol and spinning at 1500 rpm for 5 minutes. Recovered
biopolymers are re-dissolved in chloroform. Monomer production is
facilitated by adding methanol and sulfuric acid and incubation at
100 degrees Celsius for 2 hours. Following the addition of water to
create a 2-phase extraction system, the monomeric methyl-ester is
isolated in the organic phase and analyzed by GC/MS.
Polymer Analysis
Isolation/Extraction of Polymers:
[0161] 1: Add chloroform to lyophilized bacteria (6 ml of
chloroform/g bacteria). Incubate at 60.degree. C. for 4 hours. 2:
Recover chloroform extract and dry down to approximately 1/5 of
volume under nitrogen at 40.degree. C. 3: Add concentrated
chloroform extract to ice-cold methanol (at least a 1:4 ratio of
chloroform to methanol). 4: Isolate precipitated polymers. 5: Wash
by re-dissolving polymers in chloroform and precipitate with
methanol (1:4 ratio). Repeat washing a couple of times.
Production of Monomers (Methanolysis):
[0162] 1: Add 1 ml of chloroform and 1 ml of methanol containing
2.8 M sulfuric acid. 2: Incubate for 2 hours at 100.degree. C. 3:
Cool and add 0.5 ml of distilled water. 4: Collect organic phase
containing methyl-esters.
GC-MS
[0163] For polymer analysis, compounds are detected on an Agilent
6890N GC/MS (Agilent, Santa Clara, Calif.) on a HP1 60 m
column.times.0.25 mm ID. Samples are placed in GC vial inserts with
a final volume in hexane of 50 uL. Samples are injected using an
automatic injector, injector temperature is 250.degree. C. and is
run in split mote (8:1) with an initial GC temperature of
40.degree. C., ramp at 4.degree. C./min to a final temp of
100.degree. C., then a ramp of 10.degree. C./min to 225.degree. C.,
finally a 20.degree. C./min ramp to 312 C. Peak ID is accomplished
through a NIST08 library.
REFERENCES
[0164] Burgard et al., 2010. Microorganisms for the production of
adipic acid and other compounds. U.S. Pat. No. 7,799,545 B2. [0165]
Frost 2008. Synthesis of caprolactam from lysine. U.S. Pat. No.
7,399,855 B2. [0166] Frost 2010. Catalytic deamination for
caprolactam production. Patent # US 2010/0145003 A1. [0167] Holder
et al., 2011. Comparative and functional genomics of Rhodococcus
opacus PD630 for biofuel development. PLoS Genet. (9) e-pub:
E1002219. [0168] Johnson et al., 1998. Genetic evidence for the
expression of ATP- and GTP-specific succinyl-coA synthetases in
multicellular eukaryotes. J. Biol. Chem. (273) 27580-6. [0169]
Kalinowski et al., 1991. Genetic and biochemical analysis of the
aspartokinase from Corynebacterium glutamicum. Mol. Microbiol. (5)
1197-1204. [0170] Mikami et al., 2000. Tyrosine phenol-lyase from
Erwinia herbicola. Protein databank entry ID #1C7G. [0171] Patek et
al., 1997. Identification and transcriptional analysis of the
dapB-orf2-dapA-orf4 operon of Corynebacterium glutamicum, encoding
two enzymes involved in 1-lysine synthesis. Biotech. Letters 19
(11) 1113-17. [0172] Piel et al., 2000. Cloning, sequencing and
analysis of the enterocin biosynthesis gene cluster from the marine
isolate `Streptomyces maritimus`: evidence for the derailment of an
aromatic polyketide synthase. Chem. Biol. (7) 943-55. [0173]
Raemakers-Franken et al., 2009. Biochemical synthesis of
6-aminocaproic acid. U.S. Pat. No. 7,491,520 B2. [0174]
Raemakers-Franken et al., 2011. Methods of finding a biocatalyst
having ammonia lyase activity. Patent App # WO/2011/078667. [0175]
Schneider and Wendisch, 2010. Putrescine production by engineered
Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. (88)
859-68. [0176] Vrljic et al., 1996. A new type of transporter with
a new type of cellular function: I-lysine export from
Corynebacterium glutamicum. Mol. Microbiol. (22) 815-26.
Sequence CWU 1
1
221416PRTCupriavidus necator 1Met Ala Leu Ile Val His Lys Tyr Gly
Gly Thr Ser Met Gly Ser Thr 1 5 10 15 Glu Arg Ile Lys Asn Val Ala
Lys Arg Val Ala Lys Trp His Arg Ala 20 25 30 Gly His Arg Val Val
Val Val Pro Ser Ala Met Ser Gly Glu Thr Asn 35 40 45 Arg Leu Leu
Gly Leu Ala Lys Glu Ile Ser Pro Gln Pro Asn Pro Arg 50 55 60 Glu
Leu Asp Met Leu Ala Ser Thr Gly Glu Gln Ala Ser Val Ala Leu 65 70
75 80 Leu Ala Ile Ala Leu His Gly Glu Asp Ile Asp Ala Val Ser Tyr
Thr 85 90 95 Gly Trp Gln Val Pro Val Lys Thr Asp Ser Ala Tyr Thr
Lys Ala Arg 100 105 110 Ile Glu Ser Ile Asp Asp Glu Arg Ile Leu Ala
Asp Leu Asp Ala Gly 115 120 125 Arg Val Val Val Ile Thr Gly Phe Gln
Gly Ile Asp Asp Asp Gly Asn 130 135 140 Ile Thr Thr Leu Gly Arg Gly
Gly Ser Asp Thr Ser Ala Val Ala Ile 145 150 155 160 Ala Ala Ala Ile
Glu Ala Asp Glu Cys Leu Ile Tyr Thr Asp Val Asp 165 170 175 Gly Val
Tyr Thr Thr Asp Pro Arg Val Val Glu Asp Ala Arg Arg Leu 180 185 190
Asp Gln Ile Thr Phe Glu Glu Met Leu Glu Met Ala Ser Leu Gly Ser 195
200 205 Lys Val Leu Gln Ile Arg Ser Val Glu Phe Ala Gly Lys Tyr Arg
Val 210 215 220 Lys Thr Arg Val Leu Ser Ser Leu Thr Asp Pro Leu Met
Pro Leu Glu 225 230 235 240 Gln Glu Met His Ser Gly Thr Leu Ile Thr
Phe Glu Glu Asp Ser Thr 245 250 255 Met Glu Ala Ala Val Ile Ser Gly
Ile Ala Phe Ala Arg Asp Glu Ala 260 265 270 Lys Ile Thr Val Leu Gly
Val Pro Asp Lys Pro Gly Ile Ala Tyr Gln 275 280 285 Ile Leu Gly Pro
Val Ala Asp Ala Asn Ile Asp Val Asp Met Ile Ile 290 295 300 Gln Asn
Gln Ser Val Asp Gly Lys Thr Asp Phe Thr Phe Thr Val Pro 305 310 315
320 Arg Gly Glu Tyr Gln Arg Ala Leu Ala Ile Leu Asn Asp Gly Val Lys
325 330 335 Ser His Ile Gly Ala Gly Ser Val Ser Gly Asp Pro Lys Val
Ser Lys 340 345 350 Val Ser Val Val Gly Val Gly Met Arg Ser His Val
Gly Ile Ala Ser 355 360 365 Lys Met Phe Arg Thr Leu Ser Glu Glu Gly
Ile Asn Ile Gln Met Ile 370 375 380 Ser Thr Ser Glu Ile Lys Ile Ser
Val Leu Ile Asp Glu Lys Tyr Met 385 390 395 400 Glu Leu Ala Val Arg
Ala Leu His Lys Ala Phe Glu Leu Glu Gln Ala 405 410 415
2304PRTCupriavidus necator 2Met Asn Asn Lys Leu Thr Ala Ala Asp Leu
Arg Gly Ile Phe Pro Ala 1 5 10 15 Ile Pro Thr Pro Val Thr Ala Asp
Asp Arg Ile Asp Gln Asp Ala Thr 20 25 30 Arg Lys Leu Met Ala Tyr
Leu Leu Ala Asn Gly Val Ser Gly Val Val 35 40 45 Pro Leu Gly Gly
Thr Gly Glu Tyr Gly Ala Leu Ala Arg Glu Glu Arg 50 55 60 Val Arg
Met Ala Ala Leu Cys Val Glu Ala Ala Ala Gly Gln Val Pro 65 70 75 80
Val Ile Pro Gly Val Leu Asp Pro Gly Phe His Asp Ala Leu Asp Ala 85
90 95 Gly Lys Ala Phe Ala Gly Val Gly Ala Ser Ala Leu Met Val Leu
Thr 100 105 110 Pro Tyr Tyr Thr Ser Pro Thr Gln Gln Gly Ile Arg Asp
Tyr Phe Leu 115 120 125 Arg Tyr Ala Asp Ala Ser Pro Val Pro Val Met
Ile Tyr Glu Ile Pro 130 135 140 Tyr Arg Thr Arg Ile Ala Ile Ala Pro
Glu Val Leu His Glu Leu Ser 145 150 155 160 Arg His Glu Asn Ile Ile
Gly Met Lys Ala Cys Asn Thr Asp Met Tyr 165 170 175 His Phe Leu Lys
Val Val Ala Gly Val Asp Asp Ser Phe Ser Val Phe 180 185 190 Ser Gly
Glu Asp Ser Leu Phe Pro Leu His Met Ala Gly Gly Ala Arg 195 200 205
Gly Gly Val Val Val Thr Ala Ser Val Leu Pro Arg Thr Trp Arg Ala 210
215 220 Ile Tyr Glu Leu Gly Val Ala Gly Asn Thr Ala Gln Ala Val Arg
Leu 225 230 235 240 His Arg Glu Leu Ile Pro Leu Leu Asp Leu Ala Phe
Ser Glu Thr Asn 245 250 255 Pro Gly Pro Leu Lys Ser Val Leu Asp Leu
Val Gly Val Thr Ala Pro 260 265 270 Lys Val Leu Ala Pro Leu Val Ala
Pro Ala Pro Gly Leu Gln Ala Gln 275 280 285 Leu Arg Ala Glu Leu Thr
Gln Arg Leu Gln Ala Glu Ala Ala Leu Ala 290 295 300
3216PRTRalstonia eutropha 3Met Asn Ser Leu Pro Asp Thr Ala Ser Leu
Ala Leu Ser Ile Ala Leu 1 5 10 15 Gln Gly Leu Ala Leu Ser Leu Gly
Leu Ile Val Ala Ile Gly Ala Gln 20 25 30 Asn Ala Phe Val Leu Arg
Gln Gly Leu Arg Arg Gln His Val Gly Ser 35 40 45 Val Val Leu Phe
Cys Ala Ala Ala Asp Ala Leu Leu Ile Ala Ala Gly 50 55 60 Val Met
Gly Met Ala Gln Ala Leu Gly Asp Arg Pro Gly Leu Ala Arg 65 70 75 80
Ala Leu Ala Val Ala Gly Ser Val Phe Leu Ala Ile Tyr Gly Trp Gln 85
90 95 Ala Leu Gln Arg Ala Arg Gln Ser His Gln Leu Lys Ala Ala Asp
Gly 100 105 110 Val Asp Gly Leu Gly Arg Gly Ala Val Leu Ala Gln Ala
Ala Ala Phe 115 120 125 Thr Leu Leu Asn Pro His Val Tyr Leu Asp Thr
Val Leu Leu Val Gly 130 135 140 Ser Ile Gly Ala Gln Gln Pro Ala Ala
Leu Arg Gly Trp Phe Val Ala 145 150 155 160 Gly Ala Ser Ala Ala Ser
Leu Phe Trp Phe Gly Leu Leu Gly Phe Gly 165 170 175 Ala Arg Trp Leu
Ala Pro Trp Phe Ala Arg Pro Lys Ala Trp Arg Val 180 185 190 Leu Asp
Gly Val Ile Ala Met Thr Met Phe Val Leu Ser Ala Leu Leu 195 200 205
Val Arg His Val Phe Asn Ala Val 210 215 4293PRTRalstonia eutropha
4Met Ser Ile Leu Ile Asn Lys Asp Thr Lys Val Ile Thr Gln Gly Ile 1
5 10 15 Thr Gly Lys Thr Gly Gln Phe His Thr Arg Gly Cys Arg Asp Tyr
Ala 20 25 30 Asn Gly Lys Asn Cys Phe Val Ala Gly Val Asn Pro Lys
Lys Ala Gly 35 40 45 Glu Asp Phe Glu Gly Ile Pro Ile Tyr Ala Ser
Val Lys Asp Ala Lys 50 55 60 Ala Gln Thr Gly Ala Thr Val Ser Val
Ile Tyr Val Pro Pro Ala Gly 65 70 75 80 Ala Ala Ala Ala Ile Trp Glu
Ala Val Asp Ala Asp Leu Asp Leu Val 85 90 95 Val Cys Ile Thr Glu
Gly Ile Pro Val Arg Asp Met Met Glu Val Lys 100 105 110 Asp Arg Met
Arg Arg Glu Asn Lys Lys Thr Leu Leu Leu Gly Pro Asn 115 120 125 Cys
Pro Gly Leu Ile Thr Pro Asp Glu Ile Lys Ile Gly Ile Met Pro 130 135
140 Gly His Ile His Arg Lys Gly Arg Ile Gly Val Val Ser Arg Ser Gly
145 150 155 160 Thr Leu Thr Tyr Glu Ala Val Gly Gln Leu Thr Ala Leu
Gly Leu Gly 165 170 175 Gln Ser Ser Ala Val Gly Ile Gly Gly Asp Pro
Ile Asn Gly Leu Lys 180 185 190 His Ile Asp Val Met Lys Met Phe Asn
Asp Asp Pro Glu Thr Asp Ala 195 200 205 Val Val Met Ile Gly Glu Ile
Gly Gly Pro Asp Glu Ala Asn Ala Ala 210 215 220 Tyr Trp Ile Lys Asp
Asn Met Lys Lys Pro Val Val Gly Phe Ile Ala 225 230 235 240 Gly Val
Thr Ala Pro Pro Gly Lys Arg Met Gly His Ala Gly Ala Leu 245 250 255
Ile Ser Gly Gly Ala Asp Thr Ala Gln Ala Lys Leu Glu Ile Met Glu 260
265 270 Ala Cys Gly Ile Thr Val Thr Lys Asn Pro Ser Glu Met Ala Arg
Leu 275 280 285 Leu Lys Ala Lys Leu 290 5389PRTRalstonia eutropha
5Met Asn Ile His Glu Tyr Gln Gly Lys Glu Ile Leu Arg Lys Tyr Asn 1
5 10 15 Val Pro Val Pro Arg Gly Ile Pro Ala Phe Ser Val Ala Glu Ala
Leu 20 25 30 Lys Ala Ala Glu Glu Leu Gly Gly Pro Val Trp Val Val
Lys Ala Gln 35 40 45 Ile His Ala Gly Gly Arg Gly Lys Gly Gly Gly
Val Lys Val Ala Lys 50 55 60 Ser Ile Asp Asp Val Lys Thr Tyr Ala
Thr Asn Ile Leu Gly Met Gln 65 70 75 80 Leu Val Thr His Gln Thr Gly
Pro Glu Gly Lys Lys Val Asn Arg Leu 85 90 95 Leu Ile Glu Glu Gly
Ala Asp Ile Lys Lys Glu Leu Tyr Val Ser Leu 100 105 110 Val Val Asp
Arg Val Ser Gln Lys Ile Ala Leu Met Ala Ser Ser Glu 115 120 125 Gly
Gly Met Asp Ile Glu Glu Val Ala Ala His Thr Pro Glu Lys Ile 130 135
140 His Thr Leu Ile Ile Glu Pro Ser Thr Gly Leu Thr Asp Ala Asp Ala
145 150 155 160 Asp Asp Ile Ala Arg Lys Ile Gly Val Pro Asp Ala Ser
Val Ala Gln 165 170 175 Ala Arg Gln Ala Leu Gln Gly Leu Tyr Lys Ala
Phe Tyr Asp Thr Asp 180 185 190 Ala Ser Leu Ala Glu Ile Asn Pro Leu
Ile Leu Thr Gly Glu Gly Lys 195 200 205 Val Ile Ala Leu Asp Ala Lys
Phe Asn Phe Asp Ser Asn Ala Leu Phe 210 215 220 Arg His Pro Glu Ile
Val Ala Tyr Arg Asp Leu Asp Glu Glu Asp Ala 225 230 235 240 Asn Glu
Ile Glu Ala Ser Lys Phe Asp Leu Ala Tyr Ile Ser Leu Asp 245 250 255
Gly Asn Ile Gly Cys Leu Val Asn Gly Ala Gly Leu Ala Met Ala Thr 260
265 270 Met Asp Thr Ile Lys Leu Phe Gly Gly Glu Pro Ala Asn Phe Leu
Asp 275 280 285 Val Gly Gly Gly Ala Thr Thr Glu Lys Val Thr Glu Ala
Phe Lys Leu 290 295 300 Met Leu Lys Asn Pro Asn Val Glu Ala Ile Leu
Val Asn Ile Phe Gly 305 310 315 320 Gly Ile Met Arg Cys Asp Val Ile
Ala Glu Gly Val Ile Ser Ala Ser 325 330 335 Lys Ala Val Asn Leu Thr
Val Pro Leu Val Val Arg Met Lys Gly Thr 340 345 350 Asn Glu Asp Leu
Gly Lys Lys Met Leu Ala Asp Ser Gly Leu Pro Ile 355 360 365 Ile Ala
Ala Asp Thr Met Glu Glu Ala Ala Gln Lys Val Val Ala Ala 370 375 380
Ala Ala Gly Lys Lys 385 6370PRTRalstonia eutropha 6Met Ser Gly Asn
Thr Leu Gly Leu Leu Phe Thr Val Thr Thr Phe Gly 1 5 10 15 Glu Ser
His Gly Pro Ala Ile Gly Ala Val Val Asp Gly Cys Pro Pro 20 25 30
Gly Met Asp Leu Thr Glu Ala Asp Ile Gln Gly Asp Leu Asp Arg Arg 35
40 45 Lys Pro Gly Thr Ser Arg His Val Thr Gln Arg Lys Glu Pro Asp
Gln 50 55 60 Val Glu Ile Leu Ser Gly Val Phe Glu Gly Lys Thr Thr
Gly Thr Pro 65 70 75 80 Ile Cys Leu Leu Ile Arg Asn Thr Asp Gln Arg
Ser Lys Asp Tyr Gly 85 90 95 Asn Ile Val Glu Thr Phe Arg Pro Gly
His Ala Asp Tyr Thr Tyr Trp 100 105 110 Gln Lys Tyr Gly Ile Arg Asp
Tyr Arg Gly Gly Gly Arg Ser Ser Ala 115 120 125 Arg Leu Thr Ala Pro
Val Val Ala Ala Gly Ala Val Ala Lys Lys Trp 130 135 140 Leu Arg Glu
Gln Phe Gly Thr Glu Ile Arg Gly Tyr Met Ser Lys Leu 145 150 155 160
Gly Glu Ile Glu Val Pro Phe Ser Asp Trp Ser His Val Pro Glu Asn 165
170 175 Pro Phe Phe Ala Ala Asn Ala Asp Ile Val Pro Glu Leu Glu Thr
Tyr 180 185 190 Met Asp Ala Leu Arg Arg Asp Gly Asp Ser Val Gly Ala
Arg Ile Glu 195 200 205 Val Val Ala Ser Asn Val Pro Val Gly Leu Gly
Glu Pro Leu Phe Asp 210 215 220 Arg Leu Asp Ala Asp Ile Ala His Ala
Met Met Gly Leu Asn Ala Val 225 230 235 240 Lys Gly Val Glu Ile Gly
Ala Gly Phe Lys Ser Val Glu Gln Arg Gly 245 250 255 Ser Glu His Gly
Asp Glu Leu Thr Ala Gln Gly Phe Arg Gly Asn Asn 260 265 270 Ala Gly
Gly Ile Leu Gly Gly Ile Ser Thr Gly Gln Asp Ile Thr Val 275 280 285
Ser Leu Ala Ile Lys Pro Thr Ser Ser Ile Arg Thr Pro Arg Glu Ser 290
295 300 Ile Asp Lys Ala Gly Asn Ala Ala Thr Val Glu Thr Phe Gly Arg
His 305 310 315 320 Asp Pro Cys Val Gly Ile Arg Ala Thr Pro Ile Ala
Glu Ala Leu Leu 325 330 335 Ala Leu Val Leu Val Asp His Ala Leu Arg
His Arg Ala Gln Cys Gly 340 345 350 Asp Val Lys Val Asp Thr Pro Arg
Ile Pro Ala Gln Ala Pro Gly Gln 355 360 365 Pro Gly 370
7304PRTRalstonia eutropha 7Met Lys Pro Ile Thr Leu Phe Gly Ala Pro
Thr Asp Val Gly Ala Ser 1 5 10 15 Thr Arg Gly Cys Thr Met Gly Pro
Glu Ala Leu Arg Ile Ala Asp Ile 20 25 30 Val Pro Ala Leu Ala Arg
Met Gly Leu Asp Val Ser Asp Ala Gly Asn 35 40 45 Ile Ala Gly Pro
Val Asn Pro Leu Ala Pro Pro Val Gln Gly Leu Arg 50 55 60 His Leu
Asp Glu Val Val Ala Trp Asn Arg Gly Val Phe Glu Ala Ser 65 70 75 80
Thr Arg Ile Leu Gln Ala Gly Arg Met Pro Val Leu Leu Gly Gly Asp 85
90 95 His Cys Leu Ala Val Gly Ser Val Ser Ala Val Ala Arg His Cys
Arg 100 105 110 Glu Ala Gly Arg Lys Leu Val Val Leu Trp Leu Asp Ala
His Ala Asp 115 120 125 Ala Asn Ile Gly Thr Ser Thr Pro Thr Gly Asn
Met His Gly Met Pro 130 135 140 Val Ala Cys Leu Cys Gly Asp Gly Pro
Thr Pro Leu Thr Thr Leu Gly 145 150 155 160 Gly Gln Pro Pro Ala Val
Arg Pro Glu Glu Ile Arg Gln Val Gly Ile 165 170 175 Arg Ser Val Asp
Ala Gln Glu Lys Leu Arg Leu His Ala Leu Gly Leu 180 185 190 Lys Val
Phe Asp Met Arg Tyr Ile Asp Glu Tyr Gly Met Arg Gln Thr 195 200 205
Met Glu Gln Ala Leu Ala Gly Val Asp Asp Asp Thr His Leu His Val 210
215 220 Ser Phe Asp Val Asp Phe Ile Asp Ser Ala Ile Ala Pro Gly Val
Gly 225 230 235 240 Thr Thr Val Leu Gly Gly Pro Thr Tyr Arg Glu Thr
Gln Leu Cys Met 245 250 255 Glu Met Ile Ala Asp Thr Gly Arg Leu Ser
Ser Leu Asp Val Met Glu 260 265 270 Leu Asn Pro Ala Cys Asp Val Arg
Asn Glu Thr Ala Arg Leu Val Val 275 280 285 Asp Phe Leu Glu
Ser Leu Phe Gly Lys Ser Thr Leu Leu Arg Ala Arg 290 295 300
8756PRTRalstonia eutropha 8Met Lys Phe Arg Phe Pro Val Ile Ile Ile
Asp Glu Asp Phe Arg Ser 1 5 10 15 Glu Asn Ile Ser Gly Ser Gly Ile
Arg Ala Leu Ala Glu Ala Ile Glu 20 25 30 Lys Glu Gly Met Glu Val
Met Gly Leu Thr Ser Tyr Gly Asp Leu Thr 35 40 45 Ser Phe Ala Gln
Gln Ser Ser Arg Ala Ser Thr Phe Ile Val Ser Ile 50 55 60 Asp Asp
Asp Glu Phe Val Thr Ala Asp Asp Gln Pro Glu Ala Ala Ala 65 70 75 80
Ile Glu Lys Leu Arg Ala Phe Val Asn Glu Val Arg Arg Arg Asn Thr 85
90 95 Asp Leu Pro Ile Phe Leu Tyr Gly Glu Thr Arg Thr Ser Arg His
Ile 100 105 110 Pro Asn Asp Ile Leu Arg Glu Leu His Gly Phe Ile His
Met Phe Glu 115 120 125 Asp Thr Pro Glu Phe Val Ala Arg His Ile Ile
Arg Glu Ala Lys Val 130 135 140 Tyr Leu Asp Thr Leu Ala Pro Pro Phe
Phe Lys Ala Leu Ile Asp Tyr 145 150 155 160 Ala Gln Asp Ser Ser Tyr
Ser Trp His Cys Pro Gly His Ser Gly Gly 165 170 175 Val Ala Phe Leu
Lys Ser Pro Val Gly Gln Val Phe His Gln Phe Phe 180 185 190 Gly Glu
Asn Met Leu Arg Ala Asp Val Cys Asn Ala Val Asp Glu Leu 195 200 205
Gly Gln Leu Leu Asp His Thr Gly Pro Val Ala Ala Ser Glu Arg Asn 210
215 220 Ala Ala Arg Ile Phe Asn Ser Asp His Met Phe Phe Val Thr Asn
Gly 225 230 235 240 Thr Ser Thr Ser Asn Lys Met Val Trp His Ala Asn
Val Ala Pro Gly 245 250 255 Asp Ile Val Val Val Asp Arg Asn Cys His
Lys Ser Ile Leu His Ala 260 265 270 Ile Met Met Thr Gly Ala Ile Pro
Val Phe Leu Met Pro Thr Arg Asn 275 280 285 His Tyr Gly Ile Ile Gly
Pro Ile Pro Lys Ser Glu Phe Asp Pro Glu 290 295 300 Thr Ile Arg Arg
Lys Ile Ala Asn His Pro Phe Ala Ser Lys Ala Lys 305 310 315 320 Asn
Gln Lys Pro Arg Ile Leu Thr Ile Thr Gln Gly Thr Tyr Asp Gly 325 330
335 Val Leu Tyr Asn Ala Glu Gln Ile Lys Glu Met Leu Ala Ser Glu Ile
340 345 350 Asp Thr Leu His Phe Asp Glu Ala Trp Leu Pro His Ala Ala
Phe His 355 360 365 Glu Phe Tyr His Asn Met His Ala Ile Gly Arg Asp
Arg Pro Arg Ser 370 375 380 Lys Asp Ala Leu Val Phe Ala Thr Gln Ser
Thr His Lys Leu Leu Ala 385 390 395 400 Gly Leu Ser Gln Ala Ser Gln
Ile Leu Val Gln Asp Ser Glu Thr Arg 405 410 415 Lys Leu Asp Arg Tyr
Arg Phe Asn Glu Ala Tyr Leu Met His Thr Ser 420 425 430 Thr Ser Pro
Gln Tyr Ala Ile Ile Ala Ser Cys Asp Val Ala Ala Ala 435 440 445 Met
Met Glu Ala Pro Gly Gly Pro Ala Leu Val Glu Glu Ser Ile Gln 450 455
460 Glu Ala Leu Asp Phe Arg Arg Ala Met Arg Lys Val Glu Gly Asp Phe
465 470 475 480 Glu Ala Gly Asn Asn Gly Asp Trp Trp Phe Lys Val Trp
Gly Pro Asp 485 490 495 Thr Leu Asn Asp Glu Gly Met Pro Glu Arg Glu
Gln Trp Met Leu Lys 500 505 510 Ala Asn Glu Arg Trp His Gly Phe Gly
Asp Leu Ala Asp Gly Phe Asn 515 520 525 Leu Leu Asp Pro Ile Lys Ala
Thr Ile Ile Thr Pro Gly Leu Asp Val 530 535 540 Asp Gly Glu Phe Ser
Asp Arg Gly Ile Pro Ala Ala Ile Val Thr Lys 545 550 555 560 Tyr Leu
Ala Glu His Gly Ile Ile Ile Glu Lys Thr Gly Leu Tyr Ser 565 570 575
Phe Phe Ile Met Phe Thr Ile Gly Ile Thr Lys Gly Arg Trp Asn Ser 580
585 590 Leu Val Thr Glu Leu Gln Gln Phe Lys Asp Asp Tyr Asp Gln Asn
Gln 595 600 605 Pro Leu Trp Arg Val Leu Pro Glu Phe Val Gly Lys His
Pro Gln Tyr 610 615 620 Glu Arg Met Gly Leu Arg Asp Leu Cys Asp Ala
Val His Ser Val Tyr 625 630 635 640 Lys Ala Asn Asp Val Ala Arg Val
Thr Thr Glu Met Tyr Leu Ser Asp 645 650 655 Met Glu Pro Ala Met Lys
Pro Ser Asp Ala Trp Ser Met Met Ala His 660 665 670 Arg Glu Ile Glu
Arg Val Pro Val Asp Asp Leu Glu Gly Arg Val Thr 675 680 685 Ala Ile
Leu Leu Thr Pro Tyr Pro Pro Gly Ile Pro Leu Leu Ile Pro 690 695 700
Gly Glu Arg Phe Asn Arg Thr Ile Val Gln Tyr Leu Lys Phe Ala Arg 705
710 715 720 Glu Phe Asn Lys Leu Phe Pro Gly Phe Glu Thr Asp Val His
Gly Leu 725 730 735 Val Glu Glu Glu Val Asp Gly Arg Lys Ala Tyr Phe
Val Asp Cys Val 740 745 750 Lys Gln Gly Ser 755 9 454PRTRhodobacter
capsulatus 9Met Lys Thr Ile Ile Glu Pro Phe Arg Ile Lys Ser Val Glu
Pro Ile 1 5 10 15 Arg Leu Thr Ser Arg Pro Glu Arg Glu Arg Leu Ala
Arg Ala Ala Gly 20 25 30 Tyr Asn Leu Phe Gly Leu His Ser Asp Asp
Val Leu Ile Asp Leu Leu 35 40 45 Thr Asp Ser Gly Thr Gly Ala Met
Ser Ser Leu Gln Trp Ala Ala Val 50 55 60 Met Gln Gly Asp Glu Ser
Tyr Ala Gly Ser Pro Ser Phe Phe Arg Phe 65 70 75 80 Glu Ala Ala Val
Gln Asn Leu Met Pro Phe Lys His Ile Ile Pro Thr 85 90 95 His Gln
Gly Arg Ala Ala Glu Ala Ile Leu Phe Ser Ile Phe Gly Gly 100 105 110
Lys Gly Arg Arg Ile Pro Ser Asn Thr His Phe Asp Thr Thr Arg Gly 115
120 125 Asn Ile Glu Ala Ser Gly Ala Thr Gly Asp Asp Leu Val Ile Ala
Glu 130 135 140 Gly Lys Asp Pro Gln Asn Leu His Pro Phe Lys Gly Asn
Met Asp Leu 145 150 155 160 Ala Arg Leu Glu Ala Tyr Leu Glu Ala His
His Ala Glu Val Pro Leu 165 170 175 Val Met Ile Thr Ile Thr Asn Asn
Ala Gly Gly Gly Gln Pro Val Ser 180 185 190 Leu Ala Asn Ile Arg Ala
Val Ala Asp Leu Ala His Arg Tyr Gly Lys 195 200 205 Pro Phe Val Ile
Asp Gly Cys Arg Phe Ala Glu Asn Ala Trp Phe Ile 210 215 220 Lys Thr
Arg Glu Glu Gly Gln Ala Asp Arg Ser Ile Pro Glu Ile Val 225 230 235
240 Arg Asp Cys Phe Ala Val Ala Asp Gly Met Thr Met Ser Ala Lys Lys
245 250 255 Asp Ala Phe Gly Asn Ile Gly Gly Trp Leu Ala Leu Asn Asp
Asp Asp 260 265 270 Leu Ala Glu Glu Ala Arg Gly His Leu Ile Arg Thr
Glu Gly Phe Pro 275 280 285 Thr Tyr Gly Gly Leu Ala Gly Arg Asp Leu
Asp Ala Leu Ala Gln Gly 290 295 300 Leu Val Glu Ile Val Asp Glu Asp
Tyr Leu Arg Tyr Arg Ile Arg Thr 305 310 315 320 His Gln Tyr Ile Val
Glu Arg Leu Asp Ala Met Gly Val Pro Val Val 325 330 335 Lys Pro Ala
Gly Gly His Ala Val Phe Ile Asp Ala Arg Ala Trp Leu 340 345 350 Ser
His Ile Pro Pro Leu Glu Tyr Pro Gly Gln Ala Leu Ala Val Ala 355 360
365 Leu Tyr Glu Ile Ala Gly Val Arg Ser Cys Glu Ile Gly Thr Ala Met
370 375 380 Phe Gly Arg Gln Pro Asp Gly Ser Glu Lys Pro Ala Ala Met
Asp Leu 385 390 395 400 Val Arg Leu Ala Phe Pro Arg Arg Thr Tyr Thr
Gln Ser His Ala Asp 405 410 415 Tyr Ile Val Glu Ala Phe Glu Glu Leu
Ala Ala Thr Lys Asp Ala Leu 420 425 430 Arg Gly Tyr Arg Ile Val Lys
Glu Pro Lys Leu Met Arg His Phe Thr 435 440 445 Cys Arg Phe Glu Lys
Leu 450 10645PRTSulfurospirillum multivorans 10Met Asn Asn Gln Lys
Ser Thr Ile Asp Thr Ser Lys Phe Asp Asn Val 1 5 10 15 Leu Asp Ser
Ser Lys Thr Phe Val Asp His Glu Pro Asp Ser Ser Lys 20 25 30 Glu
Ile Gln Arg Asn Thr Pro Gln Lys Thr Met Pro Phe Ser Asp Gln 35 40
45 Ile Gly Asn Tyr Gln Arg Asn Arg Gly Ile Pro Ala Tyr Ser Tyr Asp
50 55 60 Glu Ser Lys Val Tyr Ile Val Gly Ser Gly Ile Ala Gly Leu
Ser Ala 65 70 75 80 Ala Phe Tyr Leu Ile Arg Asp Gly Arg Ile Pro Ala
Gln Asn Ile Thr 85 90 95 Phe Leu Glu Lys Leu Ser Val Glu Gly Gly
Ser Met Asp Gly Ala Gly 100 105 110 Asp Ala Arg Glu Gly Tyr Ile Ile
Arg Gly Gly Arg Glu Met Asp Met 115 120 125 Thr Tyr Glu Asn Leu Trp
Asp Leu Phe Gln Asp Val Pro Ala Val Glu 130 135 140 Leu Pro Glu Pro
Tyr Ser Val Leu Asp Glu Tyr Arg Leu Leu Asn Asp 145 150 155 160 Asn
Asp Ser Asn Tyr Ser Lys Ala Arg Phe Ile His Asn Lys Gly His 165 170
175 Ile Thr Asp Phe Ser Lys Phe Gly Leu Ser Lys Lys Asp Gln Leu Ala
180 185 190 Ile Ile Lys Leu Leu Leu Lys Lys Lys Glu Asp Leu Asp Asp
Val Thr 195 200 205 Ile Gln Asp Tyr Phe Ser Glu Ser Phe Leu Ala Ser
Asp Phe Trp Thr 210 215 220 Leu Trp Arg Thr Met Phe Ala Phe Glu Asn
Trp His Ser Val Leu Glu 225 230 235 240 Cys Lys Leu Tyr Met His Arg
Phe Leu His Val Leu Asp Gly Met Lys 245 250 255 Asp Leu Ser Ala Leu
Val Phe Pro Lys Tyr Asn Gln Tyr Asp Thr Phe 260 265 270 Ile Ala Pro
Leu Arg Lys Leu Leu Gln Glu Lys Gly Val Gln Phe Gln 275 280 285 Phe
Asp Thr Leu Val Glu Asp Leu Glu Ile Thr Met Thr His Asn Glu 290 295
300 Lys Ile Val Glu Asn Ile Val Thr Ile His Asn Glu Thr Ser Ser Lys
305 310 315 320 Ile Ala Val Gly Arg Asp Asp Tyr Val Ile Val Thr Thr
Gly Ser Met 325 330 335 Thr Glu Asp Thr Phe Tyr Gly Asp Asn His Asn
Ala Pro Ile Ile Ser 340 345 350 Ile Asp Asn Thr Thr Ser Gly Gln Ser
Ser Gly Trp Lys Leu Trp Lys 355 360 365 Asn Leu Ala Lys Lys Ser Glu
Val Phe Gly Lys Pro Glu Lys Phe Cys 370 375 380 Ser Thr Ile Glu His
Ser Ser Trp Glu Ser Ala Thr Leu Thr Cys Lys 385 390 395 400 Pro Ser
Ala Phe Val Glu Lys Leu Lys Lys Leu Ser Val Asn Asp Pro 405 410 415
Tyr Ser Gly Lys Thr Val Thr Gly Gly Ile Ile Thr Ile Thr Asp Ser 420
425 430 Asn Trp Leu Met Ser Phe Thr Cys Asn Arg Gln Pro His Phe Ile
Glu 435 440 445 Gln Pro Asp Asp Ile Leu Val Ile Trp Leu Tyr Ala Leu
Phe Met Asp 450 455 460 Lys Glu Gly Asn Tyr Val Lys Lys Pro Met Pro
Glu Cys Ser Gly Asp 465 470 475 480 Glu Ile Leu Thr Glu Leu Cys Tyr
His Leu Gly Ile Lys Asp Asp Leu 485 490 495 Glu Asn Val Leu Lys Asn
Thr Ile Val Arg Thr Ala Phe Met Pro Tyr 500 505 510 Ile Thr Ser Met
Phe Met Pro Arg Ala Lys Gly Asp Arg Pro Arg Ile 515 520 525 Val Pro
Lys Gly Cys Lys Asn Leu Gly Leu Ile Gly Gln Phe Val Glu 530 535 540
Thr Asn Asn Asp Ile Val Phe Thr Met Glu Ser Ser Ile Arg Thr Ala 545
550 555 560 Arg Ile Ala Val Tyr Thr Leu Leu Asn Leu Asn Lys Gln Val
Pro Asp 565 570 575 Ile Asn Pro Leu Gln Tyr Asp Ile Arg Gln Leu Leu
Lys Ala Val Lys 580 585 590 Ser Leu Asn Asp Asp Gln Pro Phe Ile Gly
Glu Gly Ile Leu Arg Lys 595 600 605 Phe Leu Lys Asp Thr Tyr Tyr Glu
Tyr Ile Leu Pro Pro Met Ser Lys 610 615 620 Glu Ser Glu Gln Glu Ser
Ser Phe Met Glu His Ile Glu Lys Ile Lys 625 630 635 640 Glu Trp Ile
Leu Arg 645 1120DNAartificial sequencesynthetic polynucleotide
11atggctctca tcgttcacaa 201219DNAartificial sequencesynthetic
polynucleotide 12tcacgcctgt tccagttcg 191325DNAartificial
sequencesynthetic polynucleotide 13gttctgggac gaggtgacgc tgcag
251424DNAartificial sequencesynthetic polynucleotide 14gtatgcggcc
aggttgcgtg ccag 241528DNAartificial sequencesynthetic
polynucleotide 15atgaactccc tgcctgacac cgcttccc 281630DNAartificial
sequencesynthetic polynucleotide 16tcacactgcg ttgaatacgt gacgcaccag
301728DNAartificial sequencesynthetic polynucleotide 17tgaagcgccc
aggaagcatt ctggacag 281825DNAartificial sequencesynthetic
polynucleotide 18cgcgcgaaga acccgcccgt ttcac 251926DNAartificial
sequencesynthetic polynucleotide 19cctcgtcgcc tacgtcgaac gcatgc
262025DNAartificial sequencesynthetic polynucleotide 20cagcacgccg
atctgcacgg cgttg 252127DNAartificial sequencesynthetic
polynucleotide 21ccgagtggga aggctatgtg acgctgg 272224DNAartificial
sequencesynthetic polynucleotide 22gtggtagtgg cagccgagcc ggtg
24
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