U.S. patent application number 15/903744 was filed with the patent office on 2018-09-27 for methods, reagents and cells for biosynthesizing compounds.
This patent application is currently assigned to INVISTA NORTH AMERICA S.A.R.L.. The applicant listed for this patent is INVISTA NORTH AMERICA S.A.R.L.. Invention is credited to Adriana Leonora BOTES, Alex Van Eck CONRADIE, Alexander Brett FOSTER, Nadia Fatma KADI, Mariusz Stanislaw KAMIONKA.
Application Number | 20180273987 15/903744 |
Document ID | / |
Family ID | 53487464 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273987 |
Kind Code |
A1 |
KADI; Nadia Fatma ; et
al. |
September 27, 2018 |
METHODS, REAGENTS AND CELLS FOR BIOSYNTHESIZING COMPOUNDS
Abstract
This document describes biochemical pathways for producing
2,4-pentadienoyl-CoA by forming one or two terminal functional
groups, comprised of carboxyl or hydroxyl group, in a C5 backbone
substrate such as glutaryl-CoA, glutaryl-[acp] or glutarate methyl
ester. 2,4-pentadienoyl-CoA can be enzymatically converted to
1,3-butadiene.
Inventors: |
KADI; Nadia Fatma; (Redcar,
GB) ; KAMIONKA; Mariusz Stanislaw; (Redcar, GB)
; FOSTER; Alexander Brett; (Redcar, GB) ;
CONRADIE; Alex Van Eck; (Redcar, GB) ; BOTES; Adriana
Leonora; (Redcar, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INVISTA NORTH AMERICA S.A.R.L. |
WILMINGTON |
DE |
US |
|
|
Assignee: |
INVISTA NORTH AMERICA
S.A.R.L.
WILMINGTON
DE
|
Family ID: |
53487464 |
Appl. No.: |
15/903744 |
Filed: |
February 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14741414 |
Jun 16, 2015 |
9938543 |
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15903744 |
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62012722 |
Jun 16, 2014 |
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62012586 |
Jun 16, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/1096 20130101;
C12N 15/52 20130101; C12Y 201/01197 20130101; C12Y 301/01085
20130101; C12P 7/62 20130101; C12N 9/1007 20130101; C12P 13/001
20130101; C12Y 102/99006 20130101; C12N 9/18 20130101; C12P 7/44
20130101; C12Y 206/01038 20130101; C07C 229/08 20130101; C07C 69/42
20130101; C12N 9/0008 20130101; C12N 9/16 20130101; C12Y 206/01018
20130101; C12P 7/42 20130101 |
International
Class: |
C12P 7/62 20060101
C12P007/62; C12N 9/02 20060101 C12N009/02; C07C 69/42 20060101
C07C069/42; C12N 9/10 20060101 C12N009/10; C12N 9/16 20060101
C12N009/16; C12N 15/52 20060101 C12N015/52; C12P 13/00 20060101
C12P013/00; C12P 7/42 20060101 C12P007/42; C12P 7/44 20060101
C12P007/44; C07C 229/08 20060101 C07C229/08; C12N 9/18 20060101
C12N009/18 |
Claims
1-22. (canceled)
23. A method of making glutarate, said method comprising (i)
enzymatically converting glutaryl-[acp] methyl ester to
glutaryl-[acp] or glutaryl-CoA methyl ester to glutaryl-CoA using a
polypeptide having pimeloyl-[acp] methyl ester methylesterase
activity, and (ii) enzymatically converting glutaryl-[acp] or
glutaryl-CoA to glutarate using at least one polypeptide having
thioesterase activity, reversible CoA-ligase activity, a
CoA-transferase activity, an acylating dehydrogenase activity, an
aldehyde dehydrogenase activity, a glutarate semialdehyde
dehydrogenase activity, or a succinate-semialdehyde dehydrogenase
activity.
24. The method of claim 23, wherein said polypeptide having
pimeloyl-[acp] methyl ester methylesterase activity has at least
70% sequence identity to the amino acid sequence set forth in SEQ
ID NO: 1.
25. The method of claim 23, wherein glutaryl-[acp] or glutaryl-CoA
is enzymatically converted to glutaric acid using a polypeptide
having thioesterase activity.
26. The method of claim 23, wherein glutaryl-[acp] or glutaryl-CoA
is enzymatically converted to glutaric acid using a polypeptide
having reversible CoA-ligase activity or a CoA-transferase
activity.
27. The method of claim 23, wherein glutaryl-[acp] or glutaryl-CoA
is enzymatically converted to glutaric acid using a polypeptide
having an acylating dehydrogenase activity, an aldehyde
dehydrogenase activity, a glutarate semialdehyde dehydrogenase
activity, or a succinate-semialdehyde dehydrogenase activity.
28. The method of claim 23, wherein the method is performed in a
host subjected to a cultivation strategy under aerobic or
micro-aerobic cultivation conditions, a host cultured under
conditions of nutrient limitation either via nitrogen, phosphate or
oxygen limitation, and/or a host retained using a ceramic membrane
to maintain a high cell density during fermentation.
29-30. (canceled)
31. The method of claim 23, wherein a principal carbon source fed
to the fermentation is derived from a biological feedstock which
is, or derives from monosaccharides, disaccharides, lignocellulose,
hemicellulose, cellulose, lignin, levulinic acid and formic acid,
triglycerides, glycerol, fatty acids, agricultural waste, condensed
distillers' solubles, or municipal waste or a non-biological
feedstock which is, or derives from, natural gas, syngas,
CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue
(NVR) or a caustic wash waste stream from cyclohexane oxidation
processes, or terephthalic acid/isophthalic acid mixture waste
streams.
32-34. (canceled)
35. The method of claim 23, wherein the host is method is performed
in a prokaryote selected from the group consisting of Escherichia;
Clostridia; Corynebacteria; Cupriavidus; Pseudomonas; Delftia;
Bacilluss; Lactobacillus; Lactococcus; and Rhodococcus or a
eukaryote is selected from the group consisting of Aspergillus,
Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces,
Arxula, and Kluyveromyces.
36. (canceled)
37. The method of claim 35, wherein the prokaryote is selected from
the group consisting of Escherichia coli, Clostridium ljungdahlii,
Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium
glutamicum, Cupriavidus necator, Cupriavidus metallidurans.
Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas
oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus
delbrueckii, Lactococcus lactis, and Rhodococcus equi.
38-39. (canceled)
40. The method of claim 35, wherein the eukaryote is selected from
the group consisting of Aspergillus niger, Saccharomyces
cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia
orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and
Kluyveromyces lactis.
41. The method of claim 23, wherein the method is performed in a
host exhibiting tolerance to high concentrations of a C5 building
block, and wherein the tolerance to high concentrations of a C5
building block is improved through continuous cultivation in a
selective environment.
42. The method of claim 23, wherein the method is performed in a
host expressing one or more of the following exogenous polypeptides
having an acetyl-CoA synthetase, a 6-phosphogluconate
dehydrogenase; a transketolase; a feedback resistant threonine
deaminase; a puridine nucleotide transhydrogenase; a formate
dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a
glucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; a
propionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamate
dehydrogenase; a L-glutamine synthetase; a lysine transporter; a
dicarboxylate transporter; and/or a multidrug transporter
activity.
43. The method of claim 23, wherein the method is performed in a
host comprises comprising an attenuation of one or more
polypeptides having an activity selected from the group consisting
of: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, an
acetyl-CoA specific .beta.-ketothiolase, an acetoacetyl-CoA
reductase, a phosphotransacetylase forming acetate, an acetate
kinase, a lactate dehydrogenase, a menaquinol-fumarate
oxidoreductase, a 2-oxoacid decarboxylase producing isobutanol, an
alcohol dehydrogenase forming ethanol, a triose phosphate
isomerase, a pyruvate decarboxylase, a glucose-6-phosphate
isomerase, a transhydrogenase dissipating the cofactor imbalance,
aglutamate dehydrogenase specific for the co-factor for which an
imbalance is created, a NADH/NADPH-utilizing glutamate
dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA
dehydrogenase accepting C5 building blocks and central precursors
as substrates; a glutaryl-CoA dehydrogenase; and a pimeloyl-CoA
synthetase.
44. A recombinant host cell comprising at least one exogenous
nucleic acid encoding a polypeptide having malonyl-CoA
O-methyltransferase activity; and a polypeptide having thioesterase
activity, the host producing glutarate methyl ester.
45. The host of claim 44, the host further comprising an exogenous
polypeptide having carboxylate reductase activity, said host
further producing glutarate semialdehyde methyl ester.
46. The host of claim 44, the host further comprising one or more
exogenous polypeptides having an activity selected from the group
consisting of synthase activity, dehydrogenase activity,
dehydratase activity, and reductase activity.
47. The host of claim 44, the host further comprising one or more
exogenous polypeptides having an activity selected from the group
consisting of synthase activity, .beta.-ketothiolase activity,
dehydrogenase activity, hydratase activity, and reductase
activity.
48. The host of claim 44, wherein the polypeptide having
malonyl-CoA O-methyltransferase activity has at least 70% sequence
identity to the amino acid sequence set forth in SEQ ID NO:13.
49. The host of claim 44, wherein the polypeptide having
thioesterase activity has at least 70% sequence identity to any one
of the amino acid sequences set forth in SEQ ID NOs: 14 or 15.
50. The host of claim 45, wherein the polypeptide having reductase
activity has at least 70% sequence identity to any one of the amino
acid sequences set forth in SEQ ID NOs: 11 or 12.
51. The host of claim 44 further comprising an exogenous
polypeptide having esterase activity, the host further producing
glutaric acid or 5-oxopentanoic acid.
52. The host of claim 44 further comprising one or more exogenous
polypeptides having an activity selected from the group consisting
of esterase activity, 6-hydroxyhexanoate dehydrogenase activity,
4-hydroxybutyrate dehydrogenase activity, 5-hydroxypentanoate
dehydrogenase activity, and alcohol dehydrogenase activity, the
host producing 5-hydroxypentanoic acid.
53. The host of claim 52, further comprising one or more exogenous
polypeptides having an activity selected from the group consisting
of CoA-transferase activity, a synthase activity, and dehydratase
activity, the host producing 2,4-pentadienoyl-CoA from
5-hydroxypentanoic acid.
54. The host of claim 53, further comprising one or more exogenous
polypeptides having an activity selected from the group consisting
of hydratase activity, thioesterase activity, decarboxylase
activity, dehydrogenase activity, CoA-transferase activity, and
dehydratase activity, the host producing 1,3-butadiene from
2,4-pentadienoyl-CoA.
55. The host of claim 54, wherein said polypeptide having
thioesterase activity has at least 70% sequence identity to the
amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15
56. The host of claim 45, wherein the polypeptide having
carboxylate reductase activity has at least 70% sequence identity
to any one of the amino acid sequences set forth in any one of SEQ
ID NOs: 2-7.
57. A recombinant host comprising at least one exogenous nucleic
acid encoding a polypeptide having pimeloyl-[acp] methyl ester
methylesterase activity, and at least one polypeptide having an
activity selected from the group consisting of thioesterase
activity, reversible CoA-ligase activity, a CoA-transferase
activity, an acylating dehydrogenase activity, an aldehyde
dehydrogenase activity, a glutarate semialdehyde dehydrogenase
activity, and a succinate-semialdehyde dehydrogenase activity.
58. The recombinant host of claim 57, wherein said polypeptide
having pimeloyl-[acp] methyl ester methylesterase activity has at
least 70% sequence identity to the amino acid sequence set forth in
SEQ ID NO: 1.
59. A bio-derived product, bio-based product or
fermentation-derived product, wherein said product comprises: i. a
composition comprising at least one bio-derived, bio-based or
fermentation-derived compound according to claim 23, or any one of
FIGS. 1-7, or any combination thereof, ii. a bio-derived, bio-based
or fermentation-derived polymer comprising the bio-derived,
bio-based or fermentation-derived composition or compound of i., or
any combination thereof, iii. a bio-derived, bio-based or
fermentation-derived resin comprising the bio-derived, bio-based or
fermentation-derived compound or bio-derived, bio-based or
fermentation-derived composition of i. or any combination thereof
or the bio-derived, bio-based or fermentation-derived polymer of
ii. or any combination thereof, iv. a molded substance obtained by
molding the bio-derived, bio-based or fermentation-derived polymer
of ii. or the bio-derived, bio-based or fermentation-derived resin
of iii., or any combination thereof, v. a bio-derived, bio-based or
fermentation-derived formulation comprising the bio-derived,
bio-based or fermentation-derived composition of i., bio-derived,
bio-based or fermentation-derived compound of i., bio-derived,
bio-based or fermentation-derived polymer of ii., bio-derived,
bio-based or fermentation-derived resin of iii., or bio-derived,
bio-based or fermentation-derived molded substance of iv, or any
combination thereof, or vi. a bio-derived, bio-based or
fermentation-derived semi-solid or a non-semi-solid stream,
comprising the bio-derived, bio-based or fermentation-derived
composition of i., bio-derived, bio-based or fermentation-derived
compound of i., bio-derived, bio-based or fermentation-derived
polymer of ii., bio-derived, bio-based or fermentation-derived
resin of iii., bio-derived, bio-based or fermentation-derived
formulation of v., or bio-derived, bio-based or
fermentation-derived molded substance of iv., or any combination
thereof.
60. A method of increasing the activity of a polypeptide having
carboxylate reductase activity on a substituted or unsubstituted
C.sub.4-C.sub.8 dicarboxylic acid, the method comprising
enzymatically converting said C.sub.4-C.sub.8 dicarboxylic acid to
a HOC(.dbd.O)(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 ester using
a polypeptide having malonyl-CoA methyltransferase activity before
enzymatically converting the HOC(.dbd.O)(C.sub.2-C.sub.6
alkyl)-C(.dbd.O)OCH.sub.3 ester to a HC(.dbd.O)(C.sub.2-C.sub.6
alkyl)-C(.dbd.O)OCH.sub.3 using a polypeptide having carboxylate
reductase activity.
61. The method of claim 60, further comprising converting said
HC(.dbd.O)(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 to a
HOCH.sub.2(C2-C6 alkyl)-C(.dbd.O)OCH.sub.3 using a polypeptide
having dehydrogenase activity.
62. The method of claim 61, further comprising enzymatically
converting the HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3
to a HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OH using a
polypeptide having esterase activity.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
14/741,414 filed Jun. 16, 2015, which claims the benefit of
priority from U.S. Provisional Application Ser. No. 62/012,722,
filed Jun. 16, 2014, and U.S. Provisional Application Ser. No.
62/012,586, filed Jun. 16, 2014, the disclosures of which are
incorporated by reference herein in their entireties.
TECHNICAL FIELD
[0002] This invention relates to a method of increasing the
activity of a polypeptide having carboxylate reductase activity on
a dicarboxylic acid by enzymatically converting the dicarboxylic
acid to a methyl ester using a polypeptide having malonyl-CoA
methyltransferase activity. This invention also relates to methods
for biosynthesizing 2,4-pentadienoyl-CoA (e.g., as a precursor to
the biosynthesis of 1,3-butadiene), and more particularly to
synthesizing 2,4-pentadienoyl-CoA using one or more isolated
enzymes such as one or more of a malonyl-CoA O-methyltransferase,
methyl ester esterase, a carboxylate reductase, or a
5-hydroxyvaleryl-CoA dehydratase, or using recombinant host cells
expressing one or more of such enzymes.
BACKGROUND
[0003] 1,3-butadiene (hereinafter butadiene) is an important
monomer for the production of synthetic rubbers including
styrene-butadiene-rubber (SBR), polybutadiene (PB),
styrene-butadiene latex (SBL), acrylonitrile-butadiene-styrene
resins (ABS), nitrile rubber, and adiponitrile, which is used in
the manufacture of Nylon-66 (White, Chemico-Biological
Interactions, 2007, 166, 10-14).
[0004] Butadiene is typically produced as a co-product from the
steam cracking process, distilled to a crude butadiene stream, and
purified via extractive distillation (White, Chemico-Biological
Interactions, 2007, 166, 10-14).
[0005] On-purpose butadiene has been prepared among other methods
by dehydrogenation of n-butane and n-butene (Houdry process); and
oxidative dehydrogenation of n-butene (Oxo-D or O-X-D process)
(White, Chemico-Biological Interactions, 2007, 166, 10-14).
[0006] Industrially, 95% of global butadiene production is
undertaken via the steam cracking process using petrochemical-based
feedstocks such as naphtha. Production of on-purpose butadiene is
not significant, given the high cost of production and low process
yield (White, Chemico-Biological Interactions, 2007, 166,
10-14).
[0007] Given a reliance on petrochemical feedstocks and, for
on-purpose butadiene, energy intensive catalytic steps;
biotechnology offers an alternative approach via biocatalysis.
Biocatalysis is the use of biological catalysts, such as enzymes,
to perform biochemical transformations of organic compounds.
[0008] Accordingly, against this background, it is clear that there
is a need for sustainable methods for producing intermediates
wherein the methods are biocatalyst based (Jang et al.,
Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).
SUMMARY
[0009] This disclosure is based at least in part on the development
of enzymatic systems and recombinant hosts for biosynthesizing
2,4-pentadienoyl-CoA or precursors thereof, which are useful for
producing, for example, 1,3-butadiene and polymers or copolymers of
1,3-butadiene. In particular, as described herein,
2,4-pentadienoyl-CoA can be biosynthetically produced from
renewable feedstocks without the need for any chemical catalysts
such as metal oxides. For example, in the pathways described
herein, 2,4-pentadienoyl-CoA can be produced from malonyl-CoA or
malonyl-[acp] via various methyl-ester shielded routes. Such
methyl-ester shielded routes include using a methyl ester esterase
such as apimelyl-[acp] methyl ester esterase or esterase to
hydrolyze the methyl ester of glutaryl-[acp] methyl ester,
glutaryl-CoA methyl ester, glutarate methyl ester, glutarate
semialdehyde methyl ester, or 5-hydroxypentanoate methyl ester, and
using a 5-hydroxyvaleryl-CoA dehydratase to introduce the first
terminal vinyl group of 1,3-butadiene. For example, 1,3-butadiene
can be produced from precursors stemming from 2,4-pentadienoyl-CoA
as outlined in FIG. 7.
[0010] In some embodiments, the C5 aliphatic backbone for
conversion to 1,3-butadiene can be formed from malonyl-[acp] or
malonyl-CoA via conversion to glutaryl-[acp] methyl ester or
glutaryl-CoA methyl ester, followed by (i) de-esterification of
glutaryl-[acp] methyl ester or glutaryl-CoA methyl ester to
glutaryl-[acp] or glutaryl-CoA respectively, or (ii) hydrolysis of
glutaryl-[acp] methyl ester or glutaryl-CoA methyl ester to
glutarate methyl ester. See FIG. 1-3.
[0011] In some embodiments, an enzyme in the pathway generating the
C5 aliphatic backbone purposefully contains irreversible enzymatic
steps.
[0012] In some embodiments, the terminal carboxyl groups can be
enzymatically formed using an esterase, a thioesterase, a
reversible CoA-ligase, a CoA-transferase, an aldehyde
dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate
dehydrogenase or a 5-oxopentanoate dehydrogenase. See FIG. 4.
[0013] In some embodiments, the terminal hydroxyl group can be
enzymatically formed using an alcohol dehydrogenase, a
4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate
dehydrogenase and a 6-hydroxyhexanoate dehydrogenase. See FIG.
5.
[0014] In some embodiments, the terminal vinyl group can be
enzymatically formed using a 5-hydroxyvaleryl-CoA dehydratase. See
FIG. 6.
[0015] In one aspect this document features a method of
biosynthesizing glutarate methyl ester in a recombinant host. The
method includes enzymatically converting at least one of
malonyl-[acp] and malonyl-CoA to glutarate methyl ester in the host
using at least one polypeptide having malonyl-CoA
O-methyltransferase activity and at least one polypeptide having
thioesterase activity.
[0016] In some embodiments, the malonyl-[acp] is enzymatically
converted to malonyl-[acp] methyl ester using the at least one
polypeptide having malonyl-CoA O-methyltransferase activity. The
the malonyl-[acp] methyl ester can be enzymatically converted to
glutaryl-[acp] methyl ester using at least one polypeptide having
an activity selected from the group consisting of synthase
activity, dehydrogenase activity, dehydratase activity, and
reductase activity. The glutaryl-[acp] methyl ester can be
enzymatically converted to glutarate methyl ester using the at
least one polypeptide having thioesterase activity.
[0017] In some embodiments, malonyl-CoA is enzymatically converted
to malonyl-CoA methyl ester using the at least one polypeptide
having malonyl-CoA O-methyltransferase activity. The malonyl-CoA
methyl ester can be enzymatically converted to glutaryl-CoA methyl
ester using at least one polypeptide having an activity selected
from the group consisting of synthase activity, .beta.-ketothiolase
activity, dehydrogenase activity, hydratase activity, and reductase
activity. The glutaryl-CoA methyl ester can be enzymatically
converted to glutarate methyl ester using the at least one
polypeptide having thioesterase activity.
[0018] The polypeptide having malonyl-CoA O-methyltransferase
activity can have at least 70% sequence identity (e.g., at least
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%) to the amino acid sequence set forth in SEQ ID NO:13.
[0019] The polypeptide having reductase activity can have at least
70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid
sequence set forth in SEQ ID NO: 11 or 12.
[0020] In some embodiments, the method further includes
enzymatically converting glutarate methyl ester to glutarate
semialdehyde methyl ester in the host using at least one
polypeptide having carboxylate reductase activity. The polypeptide
having carboxylate reductase activity can be used in combination
with a polypeptide having phosphopantetheine transferase enhancer
activity.
[0021] In some embodiments, the method further includes
enzymatically converting glutarate methyl ester to 5-oxopentanoic
acid using at least one polypeptide having an activity selected
from the group consisting of carboxylate reductase activity and
esterase activity. The polypeptide having carboxylate reductase
activity can be used in combination with a polypeptide having
phosphopantetheine transferase enhancer activity.
[0022] In some embodiments, the method further includes
enzymatically converting glutarate semialdehyde methyl ester to
5-hydroxypentanoic acid using at least one polypeptide having
esterase activity. In some embodiments, the method further includes
using at least one polypeptide having dehydrogenase activity to
enzymatically convert glutarate semialdehyde methyl ester to
5-hydroxypentanoic acid.
[0023] The polypeptide having esterase activity can have at least
70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid
sequence set forth in SEQ ID NO:8.
[0024] In some embodiments, the method further includes
enzymatically converting glutarate methyl ester to glutaric acid
using at least one polypeptide having esterase activity. The method
can further include enzymatically converting glutaric acid to
5-hydroxypentanoic acid using at least one polypeptide having
carboxylate reductase activity and at least one polypeptide having
dehydrogenase activity classified under EC 1.1.1.-. The polypeptide
having carboxylate reductase activity can be used in combination
with a polypeptide having phosphopantetheine transferase enhancer
activity.
[0025] The polypeptide having carboxylate reductase activity can
have at least 70% sequence identity (e.g., at least 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any
one of the amino acid sequences set forth in any one of SEQ ID NOs:
2-7.
[0026] The polypeptide having thioesterase activity can have at
least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of
the amino acid sequences set forth in SEQ ID NO: 9, 10, 14 or
15.
[0027] In some embodiments, the method further includes
enzymatically converting 5-hydroxypentanoic acid to
2,4-pentadienoyl-CoA using at least one polypeptide having an
activity selected from the group consisting of CoA-transferase
activity, a synthase activity, and dehydratase activity. A
polypeptide having a CoA-transferase activity or a synthase
activity and a polypeptide having dehydratase activity can
enzymatically convert 5-hydroxypentanoic acid to
2,4-pentadienoyl-CoA. The method can further include enzymatically
converting 2,4-pentadienoyl-CoA into 1,3 butadiene using at least
one polypeptide having an activity selected from the group
consisting of hydratase activity, thioesterase activity,
decarboxylase activity, dehydrogenase activity, CoA-transferase
activity, and dehydratase activity.
[0028] The polypeptide having thioesterase activity can have at
least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of
the amino acid sequences set forth in SEQ ID NOs: 14-15.
[0029] In another aspect, this document features a method of making
glutarate. The method includes (i) enzymatically converting
glutaryl-[acp] methyl ester to glutaryl-[acp] or glutaryl-CoA
methyl ester to glutaryl-CoA using a polypeptide having
pimeloyl-[acp] methyl ester methylesterase activity, and (ii)
enzymatically converting glutaryl-[acp] or glutaryl-CoA to
glutarate using at least one polypeptide having thioesterase
activity, reversible CoA-ligase activity, a CoA-transferase
activity, an acylating dehydrogenase activity, an aldehyde
dehydrogenase activity, a glutarate semialdehyde dehydrogenase
activity, or a succinate-semialdehyde dehydrogenase activity.
[0030] The polypeptide having pimeloyl-[acp] methyl ester
methylesterase activity can have at least 70% sequence identity
(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ
ID NO: 1.
[0031] In some embodiments, glutaryl-[acp] or glutaryl-CoA is
enzymatically converted to glutaric acid using a polypeptide having
thioesterase activity. In some embodiments, glutaryl-[acp] or
glutaryl-CoA is enzymatically converted to glutaric acid using a
polypeptide having reversible CoA-ligase activity or a
CoA-transferase activity. In some embodiments, glutaryl-[acp] or
glutaryl-CoA is enzymatically converted to glutaric acid using a
polypeptide having an acylating dehydrogenase activity, an aldehyde
dehydrogenase activity, a glutarate semialdehyde dehydrogenase
activity, or a succinate-semialdehyde dehydrogenase activity. In
another aspect, this document features a recombinant host cell. The
recombinant host cell includes at least one exogenous nucleic acid
encoding a polypeptide having malonyl-CoA O-methyltransferase
activity and a polypeptide having thioesterase activity, the host
producing glutarate methyl ester.
[0032] The host can further include an exogenous polypeptide having
carboxylate reductase activity, the host further producing
glutarate semialdehyde methyl ester. In some embodiments, the host
furthers include one or more exogenous polypeptides having an
activity selected from the group consisting of synthase activity,
dehydrogenase activity, dehydratase activity, and reductase
activity. In some embodiments, the host further includes one or
more exogenous polypeptides having an activity selected from the
group consisting of synthase activity, .beta.-ketothiolase
activity, dehydrogenase activity, hydratase activity, and reductase
activity.
[0033] The polypeptide having malonyl-CoA O-methyltransferase
activity can have at least 70% sequence identity (e.g., at least
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%) to the amino acid sequence set forth in SEQ ID NO: 13.
[0034] The polypeptide having thioesterase activity can have at
least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of
the amino acid sequences set forth in any one of SEQ ID NOs:
14-15.
[0035] The polypeptide having reductase activity can have at least
70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the amino
acid sequences set forth in SEQ ID NOs: 11 or 12.
[0036] The host can further include an exogenous polypeptide having
esterase activity, the host further producing glutaric acid or
5-oxopentanoic acid.
[0037] In some embodiments, the host includes one or more exogenous
polypeptides having an activity selected from the group consisting
of esterase activity, 6-hydroxyhexanoate dehydrogenase activity,
4-hydroxybutyrate dehydrogenase activity, 5-hydroxypentanoate
dehydrogenase activity, and alcohol dehydrogenase activity, the
host producing 5-hydroxypentanoic acid. The host can further
include one or more exogenous polypeptides having an activity
selected from the group consisting of CoA-transferase activity, a
synthase activity, and dehydratase activity, the host producing
2,4-pentadienoyl-CoA from 5-hydroxypentanoic acid. The host can
further include one or more exogenous polypeptides having an
activity selected from the group consisting of hydratase activity,
thioesterase activity, decarboxylase activity, dehydrogenase
activity, CoA-transferase activity, and dehydratase activity, the
host producing 1,3-butadiene from 2,4-pentadienoyl-CoA.
[0038] The polypeptide having thioesterase activity can at least
70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid
sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15
[0039] The polypeptide having carboxylate reductase activity can
have at least 70% sequence identity (e.g., at least 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any
one of the amino acid sequences set forth in any one of SEQ ID NOs:
2-7.
[0040] In some embodiments, when the host includes an exogenous
polypeptide having carboxylate reductase activity it is used in
combination with an exogenous polypeptide having phosphopantetheine
transferase enhancer activity.
[0041] In another aspect, this document features a recombinant host
including at least one exogenous nucleic acid encoding a
polypeptide having pimeloyl-[acp] methyl ester methylesterase
activity, and at least one polypeptide having an activity selected
from the group consisting of thioesterase activity, reversible
CoA-ligase activity, a CoA-transferase activity, an acylating
dehydrogenase activity, an aldehyde dehydrogenase activity, a
glutarate semialdehyde dehydrogenase activity, and a
succinate-semialdehyde dehydrogenase activity.
[0042] The polypeptide having pimeloyl-[acp] methyl ester
methylesterase activity can have at least 70% sequence identity
(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ
ID NO: 1.
[0043] In some embodiments, when the host includes an exogenous
polypeptide having carboxylate reductase activity it is used in
combination with an exogenous polypeptide having phosphopantetheine
transferase enhancer activity.
[0044] In another aspect, this document features a bio-derived
product, bio-based product or fermentation-derived product, wherein
the product includes (i.) a composition including at least one
bio-derived, bio-based or fermentation-derived compound as
described herein, or any combination thereof; (ii.) a bio-derived,
bio-based or fermentation-derived polymer including the
bio-derived, bio-based or fermentation-derived composition or
compound of (i.), or any combination thereof; (iii.) a bio-derived,
bio-based or fermentation-derived resin including the bio-derived,
bio-based or fermentation-derived compound or bio-derived,
bio-based or fermentation-derived composition of (i.) or any
combination thereof or the bio-derived, bio-based or
fermentation-derived polymer of ii. or any combination thereof;
(iv.) a molded substance obtained by molding the bio-derived,
bio-based or fermentation-derived polymer of (ii.) or the
bio-derived, bio-based or fermentation-derived resin of (iii.), or
any combination thereof; (v.) a bio-derived, bio-based or
fermentation-derived formulation including the bio-derived,
bio-based or fermentation-derived composition of (i.), bio-derived,
bio-based or fermentation-derived compound of (i.), bio-derived,
bio-based or fermentation-derived polymer of (ii.), bio-derived,
bio-based or fermentation-derived resin of (iii.), or bio-derived,
bio-based or fermentation-derived molded substance of (iv.), or any
combination thereof; or (vi.) a bio-derived, bio-based or
fermentation-derived semi-solid or a non-semi-solid stream,
including the bio-derived, bio-based or fermentation-derived
composition of (i.), bio-derived, bio-based or fermentation-derived
compound of (i.), bio-derived, bio-based or fermentation-derived
polymer of (ii.), bio-derived, bio-based or fermentation-derived
resin of (iii.), bio-derived, bio-based or fermentation-derived
formulation of (v.), or bio-derived, bio-based or
fermentation-derived molded substance of (iv.), or any combination
thereof.
[0045] This document also features a method of increasing the
activity of a polypeptide having carboxylate reductase activity on
a substituted or unsubstituted C.sub.4-C.sub.8 dicarboxylic acid
such as glutaric acid or adipic acid. The method includes
enzymatically converting the C.sub.4-C.sub.8 dicarboxylic acid to a
HOC(.dbd.O)(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 ester using a
polypeptide having malonyl-CoA methyltransferase activity before
enzymatically converting the HOC(.dbd.O)(C.sub.2-C.sub.6
alkyl)-C(.dbd.O)OCH.sub.3 ester to a HC(.dbd.O)(C.sub.2-C.sub.6
alkyl)-C(.dbd.O)OCH.sub.3 using a polypeptide having carboxylate
reductase activity. The method further can include enzymatically
converting the HC(.dbd.O)(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3
to HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 using a
polypeptide having dehydrogenase activity. In some embodiments, the
method further includes enzymatically converting the
HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 product to a
HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OH product using a
polypeptide having the activity of an esterase.
[0046] This document also features a recombinant host that includes
at least one exogenous nucleic acid encoding a (i) malonyl-[acp]
O-methyltransferase, (ii) a pimeloyl-[acp] methyl ester
methylesterase and (iii) a thioesterase, and produce glutarate
methyl ester, glutaryl-[acp] or glutaryl-CoA.
[0047] Such a recombinant host producing glutarate methyl ester
further can include an esterase, and further produce glutaric
acid.
[0048] Such a recombinant host producing glutaryl-[acp] further can
include a thioesterase and produce glutaric acid.
[0049] Such a recombinant host producing glutaryl-CoA further can
include one or more of (i) a thioesterase, (ii) a reversible
CoA-ligase, (iii) a CoA-transferase, or (iv) an acylating
dehydrogenase, and (v) an aldehyde dehydrogenase such as such as
7-oxoheptanoate dehydrogenase, 6-oxohexanoate dehydrogenase or
5-oxopentanoate dehydrogenase and further produce glutaric acid or
5-oxopentanoate.
[0050] A recombinant host producing 5-oxopentanoate or glutaric
acid further can include one or more of (i) an alcohol
dehydrogenase or (ii) a carboxylate reductase and further produce
5-hydroxypentanoate.
[0051] A recombinant host producing glutarate methyl ester further
can include one or more of (i) an alcohol dehydrogenase, (ii) an
esterase or (iii) a carboxylate reductase and further produce
5-hydroxypentanoate.
[0052] In another aspect, this document features a method for
producing a bioderived four or five carbon compound. The method
includes culturing or growing a host as described herein under
conditions and for a sufficient period of time to produce the
bioderived four or five carbon compound, wherein, optionally, the
bioderived four or five carbon compound can be selected from the
group consisting of 2,4-pentadienoyl-CoA, glutaryl-[acp] methyl
ester, 5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol,
1,3-butadiene and combinations thereof.
[0053] In another aspect, this document features a composition
including a bioderived for or five carbon compound and a compound
other than the bioderived four or five carbon compound, wherein the
bioderived four or five carbon compound is selected from the group
consisting of 2,4-pentadienoyl-CoA, glutaryl-[acp] methyl ester,
5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol, 1,3-butadiene
and combinations thereof. For example, the bioderived 4-carbon
compound can be a cellular portion of a host cell or an
organism.
[0054] This document also features a biobased polymer including the
bioderived four or five carbon compound including
2,4-pentadienoyl-CoA, glutaryl-[acp] methyl ester,
5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol, 1,3-butadiene
and combinations thereof.
[0055] This document also features biobased resin including the
bioderived four or five carbon compound including
2,4-pentadienoyl-CoA, glutaryl-[acp] methyl ester,
5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol, 1,3-butadiene
and combinations thereof, as well as a molded product obtained by
molding a biobased resin.
[0056] In another aspect this document also features a process for
producing a biobased polymer including chemically reacting the
bioderived four or five carbon compoundwith itself or another
compound in a polymer-producing reaction.
[0057] In another aspect this document features a process for
producing a biobased resin as described herein including chemically
reacting the bioderived four or five carbon compound with itself or
another compound in a resin producing reaction.
[0058] This document also features a biochemical network including
a malonyl-CoA O-methyltransferase, wherein the malonyl-CoA
O-methyltransferase enzymatically converts malonyl-[acp] to
malonyl-[acp] methyl ester. The biochemical network can further
include a synthase, a dehydrogenase, a dehydratase, a reductase,
and a thioesterase, wherein the synthase, the dehydrogenase, the
dehydratase, the reductase, and the thioesterase, enzymatically
convert the malonyl-[acp] methyl ester to glutarate methyl
ester.
[0059] In some embodiments the biochemical network further includes
a carboxylate reductase, wherein the carboxylate reductase
enzymatically converts glutarate methyl ester to glutarate
semialdehyde methyl ester. The biochemical network can further
include an esterase and a dehydrogenase, wherein the esterase and
dehydrogenase enzymatically convert glutarate semialdehyde methyl
ester to 5-hydroxypentanoic acid.
[0060] In some embodiments, the biochemical network can further
include a CoA-transferase and dehydratase, wherein the
CoA-transferase and dehydratase enzymatically convert
5-hydroxypentanoic acid to 2,4-pentadienoyl-CoA. The biochemical
network can further include a hydratase, a thioesterase, a
decarboxylase, a dehydrogenase, a CoA-transferase, and a
dehydratase, wherein the hydratase, the thioesterase, the
decarboxylase, the dehydrogenase, the CoA-transferase, and the
dehydratase enzymatically convert the 2,4-pentadienoyl-CoA to
1,3-butadiene.
[0061] This document also features a biochemical network including
a malonyl-CoA O-methyltransferase, wherein the malonyl-CoA
O-methyltransferase enzymatically converts malonyl-CoA to
malonyl-CoA methyl ester. The biochemical network can further
include a synthase, a .beta.-ketothiolase, a dehydrogenase, a
hydratase, a reductase, and a thioesterase, wherein the synthase,
the .beta.-ketothiolase, the dehydrogenase, the hydratase, the
reductase, and the thioesterase, enzymatically convert the
malonyl-CoA methyl ester to glutarate methyl ester.
[0062] In some embodiments the biochemical network further includes
a carboxylate reductase, wherein the carboxylate reductase
enzymatically converts glutarate methyl ester to glutarate
semialdehyde methyl ester. The biochemical network can further
include an esterase and a dehydrogenase, wherein the esterase and
dehydrogenase enzymatically convert glutarate semialdehyde methyl
ester to 5-hydroxypentanoic acid.
[0063] In some embodiments, the biochemical network can further
include a CoA-transferase and dehydratase, wherein the
CoA-transferase and dehydratase enzymatically convert
5-hydroxypentanoic acid to 2,4-pentadienoyl-CoA. The biochemical
network can further include a hydratase, a thioesterase, a
decarboxylase, a dehydrogenase, a CoA-transferase, and a
dehydratase, wherein the hydratase, the thioesterase, the
decarboxylase, the dehydrogenase, the CoA-transferase, and the
dehydratase enzymatically convert the 2,4-pentadienoyl-CoA to
1,3-butadiene.
[0064] This document also features a method of increasing the
activity of a polypeptide having carboxylate reductase activity on
a substituted or unsubstituted C.sub.4-C.sub.8 dicarboxylic acid
such as glutaric acid or adipic acid. The method includes
enzymatically converting the C.sub.4-C.sub.8 dicarboxylic acid to a
HOC(.dbd.O)(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 ester using a
polypeptide having malonyl-CoA methyltransferase activity before
enzymatically converting the HOC(.dbd.O)(C.sub.2-C.sub.6
alkyl)-C(.dbd.O)OCH.sub.3 ester to a HC(.dbd.O)(C.sub.2-C.sub.6
alkyl)-C(.dbd.O)OCH.sub.3 using a polypeptide having carboxylate
reductase activity. The method further can include enzymatically
converting the HC(.dbd.O)(C.sub.2-C.sub.6alkyl)-C(.dbd.O)OCH.sub.3
to HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 using a
polypeptide having dehydrogenase activity. In some embodiments, the
method further includes enzymatically converting the
HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OCH.sub.3 product to a
HOCH.sub.2(C.sub.2-C.sub.6 alkyl)-C(.dbd.O)OH product using a
polypeptide having the activity of an esterase.
[0065] Any of the methods can be performed in a recombinant host by
fermentation. The host can be subjected to a cultivation strategy
under aerobic, anaerobic, or micro-aerobic cultivation conditions.
The host can be cultured under conditions of nutrient limitation
such as phosphate, oxygen or nitrogen limitation. The host can be
retained using a ceramic membrane to maintain a high cell density
during fermentation.
[0066] In some embodiments, the host is subjected to a cultivation
strategy under aerobic or micro-aerobic cultivation conditions.
[0067] In some embodiments, a biological feedstock can be used as
the principal carbon source for the fermentation. For example, the
biological feedstock can be, or can derive from monosaccharides,
disaccharides, lignocellulose, hemicellulose, cellulose, lignin,
levulinic acid and formic acid, triglycerides, glycerol, fatty
acids, agricultural waste, condensed distillers' solubles, or
municipal waste.
[0068] In some embodiments, a non-biological feedstock can be used
as the principal carbon source for the fermentation. The
non-biological feedstock can be, or can be derived from, natural
gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate,
non-volatile residue (NVR) or a caustic wash waste stream from
cyclohexane oxidation processes, or terephthalic acid/isophthalic
acid mixture waste streams.
[0069] In any of the embodiments described herein, the host can be
a prokaryote. The prokaryote can be selected from the group
consisting of Escherichia; Clostridia; Corynebacteria; Cupriavidus;
Pseudomonas; Delftia; Bacilluss; Lactobacillus; Lactococcus; and
Rhodococcus. For example, the prokaryote can be selected from the
group consisting of Escherichia coli, Clostridium ljungdahlii,
Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium
glutamicum, Cupriavidus necator, Cupriavidus metallidurans.
Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas
oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus
delbrueckii, Lactococcus lactis, and Rhodococcus equi.
[0070] In any of the embodiments described herein, the host can be
a eukaryote. The eukaryote can selected from the group consisting
of Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia,
Debagomyces, Arxula, and Kluyveromyces. For example, the eukaryote
can be selected from the group consisting of Aspergillus niger,
Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica,
Issathenkia orientalis, Debaryomyces hansenii, Arxula
adenoinivorans, and Kluyveromyces lactis.
[0071] In some embodiments, the host exhibits tolerance to high
concentrations of a C5 building block, and wherein the tolerance to
high concentrations of a C5 building block is improved through
continuous cultivation in a selective environment.
[0072] In some embodiments, the host's endogenous biochemical
network is attenuated or augmented to (1) ensure the intracellular
availability of acetyl-CoA, (2) create a cofactor, i.e. NADH or
NADPH, imbalance that may be balanced via the formation of
glutarate methyl ester, 2,4-pentadienoyl-CoA, or 1,3-butadiene, (3)
prevent degradation of central metabolites, central precursors
leading to and including glutarate methyl ester,
2,4-pentadienoyl-CoA, or 1,3-butadiene and (4) ensure efficient
efflux from the cell.
[0073] In some embodiments, the host includes one or more of the
following: the intracellular concentration of oxaloacetate for
biosynthesis of a C5 building block is increased in the host by
overexpressing recombinant genes forming oxaloacetate; wherein an
imbalance in NADPH is generated that can be balanced via the
formation of a C5 building block; wherein an exogenous lysine
biosynthesis pathway synthesizing lysine from 2-oxoglutarate via
2-oxoadipate is introduced in a host using the meso 2,6
diaminopimelate pathway for lysine synthesis; wherein an exogenous
lysine biosynthesis pathway synthesizing lysine from oxaloacetate
to meso 2,6 diaminopimelate is introduced in a host using the
2-oxoadipate pathway for lysine synthesis; wherein endogenous
degradation pathways of central metabolites and central precursors
leading to and including C5 building blocks are attenuated in the
host; or wherein the efflux of a C5 building block across the cell
membrane to the extracellular media is enhanced or amplified by
genetically engineering structural modifications to the cell
membrane or increasing any associated transporter activity for a C5
building block.
[0074] Any of the recombinant hosts described herein further can
include one or more of the following attenuated polypeptides having
attenuated activity of a: polyhydroxyalkanoate synthase, an
acetyl-CoA thioesterase, an acetyl-CoA specific
.beta.-ketothiolase, an acetoacetyl-CoA reductase, a
phosphotransacetylase forming acetate, an acetate kinase, a lactate
dehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoacid
decarboxylase producing isobutanol, an alcohol dehydrogenase
forming ethanol, a triose phosphate isomerase, a pyruvate
decarboxylase, a glucose-6-phosphate isomerase, a transhydrogenase
dissipating the cofactor imbalance, aglutamate dehydrogenase
specific for the co-factor for which an imbalance is created, a
NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA
dehydrogenase; an acyl-CoA dehydrogenase accepting C5 building
blocks and central precursors as substrates; a glutaryl-CoA
dehydrogenase; or a pimeloyl-CoA synthetase.
[0075] Any of the recombinant hosts described herein further can
overexpress one or more genes encoding a polypeptide having: an
acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a
transketolase; a feedback resistant threonine deaminase; a puridine
nucleotide transhydrogenase; aformate dehydrogenase; a
glyceraldehyde-3P-dehydrogenase; a malic enzyme; a
glucose-6-phosphate dehydrogenase; afructose 1,6 diphosphatase; a
propionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamate
dehydrogenase; a L-glutamine synthetase; a lysine transporter; a
dicarboxylate transporter; and/or a multidrug transporter
activity.
[0076] The reactions of the pathways described herein can be
performed in one or more cell (e.g., host cell) strains (a)
naturally expressing one or more relevant enzymes, (b) genetically
engineered to express one or more relevant enzymes, or (c)
naturally expressing one or more relevant enzymes and genetically
engineered to express one or more relevant enzymes. Alternatively,
relevant enzymes can be extracted from of the above types of host
cells and used in a purified or semi-purified form. Extracted
enzymes can optionally be immobilized to the floors and/or walls of
appropriate reaction vessels. Moreover, such extracts include
lysates (e.g. cell lysates) that can be used as sources of relevant
enzymes. In the methods provided by the document, all the steps can
be performed in cells (e.g., host cells), all the steps can be
performed using extracted enzymes, or some of the steps can be
performed in cells and others can be performed using extracted
enzymes.
[0077] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein
including GenBank and NCBI submissions with accession numbers are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0078] One of skill in the art understands that compounds
containing carboxylic acid groups (including, but not limited to,
organic monoacids, hydroxyacids, aminoacids, and dicarboxylic
acids) are formed or converted to their ionic salt form when an
acidic proton present in the parent compound either is replaced by
a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or
an aluminum ion; or coordinates with an organic base. Acceptable
organic bases include, but are not limited to, ethanolamine,
diethanolamine, triethanolamine, tromethamine, N-methylglucamine,
and the like. Acceptable inorganic bases include, but are not
limited to, aluminum hydroxide, calcium hydroxide, potassium
hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt
of the present invention is isolated as a salt or converted to the
free acid by reducing the pH to below the pKa, through addition of
acid or treatment with an acidic ion exchange resin.
[0079] One of skill in the art understands that compounds
containing amine groups (including, but not limited to, organic
amines, aminoacids, and diamines) are formed or converted to their
ionic salt form, for example, by addition of an acidic proton to
the amine to form the ammonium salt, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like; or formed with organic acids
including, but not limited to, acetic acid, propionic acid,
hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic
acid, lactic acid, malonic acid, succinic acid, malic acid, maleic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
2-naphthalenesulfonic acid,
4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid),
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, lauryl sulfuric acid, gluconic acid, glutamic acid,
hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid,
and the like. Acceptable inorganic bases include, but are not
limited to, aluminum hydroxide, calcium hydroxide, potassium
hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt
of the present invention is isolated as a salt or converted to the
free amine by raising the pH to above the pKb through addition of
base or treatment with a basic ion exchange resin.
[0080] One of skill in the art understands that compounds
containing both amine groups and carboxylic acid groups (including,
but not limited to, aminoacids) are formed or converted to their
ionic salt form by either 1) acid addition salts, formed with
inorganic acids including, but not limited to, hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and
the like; or formed with organic acids including, but not limited
to, acetic acid, propionic acid, hexanoic acid,
cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic
acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric
acid, tartaric acid, citric acid, benzoic acid,
3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic
acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
2-naphthalenesulfonic acid,
4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic
acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid),
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, lauryl sulfuric acid, gluconic acid, glutamic acid,
hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid,
and the like. Acceptable inorganic bases include, but are not
limited to, aluminum hydroxide, calcium hydroxide, potassium
hydroxide, sodium carbonate, sodium hydroxide, and the like, or 2)
when an acidic proton present in the parent compound either is
replaced by a metal ion, e.g., an alkali metal ion, an alkaline
earth ion, or an aluminum ion; or coordinates with an organic base.
Acceptable organic bases include, but are not limited to,
ethanolamine, diethanolamine, triethanolamine, tromethamine,
N-methyiglucamine, and the like. Acceptable inorganic bases
include, but are not limited to, aluminum hydroxide, calcium
hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide,
and the like. A salt can of the present invention is isolated as a
salt or converted to the free acid by reducing the pH to below the
pKa through addition of acid or treatment with an acidic ion
exchange resin.
[0081] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and the drawings, and from the
claims. The word "comprising" in the claims may be replaced by
"consisting essentially of or with "consisting of," according to
standard practice in patent law.
DESCRIPTION OF DRAWINGS
[0082] FIG. 1 is a schematic of exemplary biochemical pathways
leading to glutarate methyl ester or glutaryl-[acp] from
malonyl-[acp].
[0083] FIG. 2 is a schematic of exemplary biochemical pathways
leading to glutarate methyl ester or glutaryl-CoA from malonyl-CoA
using NADPH as reducing equivalent.
[0084] FIG. 3 is a schematic of exemplary biochemical pathways
leading to glutarate methyl ester or glutaryl-CoA from malonyl-CoA
using NADH as reducing equivalent.
[0085] FIG. 4 is a schematic of exemplary biochemical pathways
leading to glutarate using glutarate methyl ester, glutaryl-[acp]
or glutaryl-CoA as central precursor.
[0086] FIG. 5 is a schematic of an exemplary biochemical pathway
leading to 5-hydroxypentanoate using glutarate methyl ester or
glutarate as a central precursor.
[0087] FIG. 6 is a schematic of an exemplary biochemical pathway
leading to 2,4-pentadienoyl-CoA using 5-hydroxypentanoate as a
central precursor.
[0088] FIG. 7 is a schematic of an exemplary biochemical pathway
leading to 1,3-butadiene using 2,4-pentadienoyl-CoA as central
precursor.
[0089] FIGS. 8A, 8B, 8C, 8D, 8E, 8F, 8G and 8H contain the amino
acid sequences of an Escherichia coli pimeloyl-[acp] methyl ester
methylesterase (see Genbank Accession No. AAC76437.1, SEQ ID NO:
1), a Mycobacterium marinum carboxylate reductase (see Genbank
Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis
carboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID
NO: 3), a Segniliparus rugosus carboxylate reductase (see Genbank
Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis
carboxylate reductase (see Genbank Accession No. ABK75684.1, SEQ ID
NO: 5), a Mycobacterium massiliense carboxylate reductase (see
Genbank Accession No. EIV11143.1, SEQ ID NO: 6), a Segniliparus
rotundus carboxylate reductase (see Genbank Accession No.
ADG98140.1, SEQ ID NO: 7), an Pseudomonas fluorescens esterase (see
Genbank Accession No. AAC60471.2, SEQ ID NO: 8), a Lactobacillus
brevis acyl-[acp] thioesterase (see Genbank Accession NO:
ABJ63754.1, SEQ ID NO:9), a Lactobacillus plantarum acyl-[acp]
thioesterase (see Genbank Accession Nos. CCC78182.1, SEQ ID NO:
10), a Treponema denticola enoyl-CoA reductase (see, e.g., Genbank
Accession No. AAS11092.1, SEQ ID NO: 11), an Euglena gracilis
enoyl-CoA reductase (see, e.g., Genbank Accession No. AAW66853.1,
SEQ ID NO: 12), a Bacillus cereus malonyl-[acp] O-methyltransferase
(see, e.g., Genbank Accession No. AAP11034.1, SEQ ID NO: 13), an
Escherichia coli thioesterase (see, e.g., Genbank Accession No.
AAB59067.1, SEQ ID NO: 14), and an Escherichia coli thioesterase
(see, e.g., Genbank Accession No. AAA24665.1, SEQ ID NO: 15), a
Bacillus subtilis phosphopantetheinyl transferase (see Genbank
Accession No. CAA44858.1, SEQ ID NO:16), a Nocardia sp. NRRL 5646
phosphopantetheinyl transferase (see Genbank Accession No.
ABI83656.1, SEQ ID NO:17),
[0090] FIG. 9 is a bar graph summarizing the change in absorbance
at 340 nm after 20 minutes, which is a measure of the consumption
of NADPH and activity of five carboxylate reductase preparations in
enzyme only controls (no substrate).
[0091] FIG. 10 is a bar graph of the change in absorbance at 340 nm
after 20 minutes, which is a measure of the consumption of NADPH
and activity of a carboxylate reductase preparation for converting
glutarate methyl ester to glutarate semialdehyde methyl ester
relative to the empty vector control.
[0092] FIG. 11 is a table of conversion after 1 hour of
glutaryl-CoA methyl ester to glutaryl-CoA bypimeloyl-[acp] methyl
ester methylesterase.
DETAILED DESCRIPTION
[0093] This document provides enzymes, non-natural pathways,
cultivation strategies, feedstocks, host microorganisms and
attenuations to the host's biochemical network, which can be used
to synthesize 2,4-pentadienoyl-CoA and, optionally, 1,3-butadiene
(also known as buta-1,3-diene, biethylene, or vinylethylene) from
central precursors or central metabolites. Production of butadiene
thus can proceed through a common intermediate,
2,4-pentadienoyl-CoA, even though there are a number of different
feedstocks and different pathways that can be used to produce
2,4-pentadienoyl-CoA. For example, malonyl-CoA or malonyl-[acp] can
be used to produce 2,4-pentadienoyl-CoA via different methyl-ester
shielded routes. As used herein, the term "central precursor" is
used to denote any metabolite in any metabolic pathway shown herein
leading to the synthesis of 5-hydroxypentanoate,
2,4-pentadienoyl-CoA or butadiene. The term "central metabolite" is
used herein to denote a metabolite that is produced in all
microorganisms to support growth.
[0094] As such, host microorganisms described herein can include
endogenous pathways that can be manipulated such that
2,4-pentadienoyl-CoA can be produced. In an endogenous pathway, the
host microorganism naturally expresses all of the enzymes
catalyzing the reactions within the pathway. A host microorganism
containing an engineered pathway does not naturally express all of
the enzymes catalyzing the reactions within the pathway but has
been engineered such that all of the enzymes within the pathway are
expressed in the host.
[0095] The term "exogenous" as used herein with reference to a
nucleic acid (or a protein) and a host refers to a nucleic acid
that does not occur in (and cannot be obtained from) a cell of that
particular type as it is found in nature or a protein encoded by
such a nucleic acid. Thus, a non-naturally-occurring nucleic acid
is considered to be exogenous to a host once in the host. It is
important to note that non-naturally-occurring nucleic acids can
contain nucleic acid subsequences or fragments of nucleic acid
sequences that are found in nature provided the nucleic acid as a
whole does not exist in nature. For example, a nucleic acid
molecule containing a genomic DNA sequence within an expression
vector is non-naturally-occurring nucleic acid, and thus is
exogenous to a host cell once introduced into the host, since that
nucleic acid molecule as a whole (genomic DNA plus vector DNA) does
not exist in nature. Thus, any vector, autonomously replicating
plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus)
that as a whole does not exist in nature is considered to be
non-naturally-occurring nucleic acid. It follows that genomic DNA
fragments produced by PCR or restriction endonuclease treatment as
well as cDNAs are considered to be non-naturally-occurring nucleic
acid since they exist as separate molecules not found in nature. It
also follows that any nucleic acid containing a promoter sequence
and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an
arrangement not found in nature is non-naturally-occurring nucleic
acid. A nucleic acid that is naturally-occurring can be exogenous
to a particular host microorganism. For example, an entire
chromosome isolated from a cell of yeast x is an exogenous nucleic
acid with respect to a cell of yeast y once that chromosome is
introduced into a cell of yeast y.
[0096] In contrast, the term "endogenous" as used herein with
reference to a nucleic acid (e.g., a gene) (or a protein) and a
host refers to a nucleic acid (or protein) that does occur in (and
can be obtained from) that particular host as it is found in
nature. Moreover, a cell "endogenously expressing" a nucleic acid
(or protein) expresses that nucleic acid (or protein) as does a
host of the same particular type as it is found in nature.
Moreover, a host "endogenously producing" or that "endogenously
produces" a nucleic acid, protein, or other compound produces that
nucleic acid, protein, or compound as does a host of the same
particular type as it is found in nature.
[0097] For example, depending on the host and the compounds
produced by the host, one or more of the following enzymes may be
expressed in the host including a malonyl-[acp]
O-methyltransferase, a pimeloyl-[acp] methyl ester methyl esterase,
an esterase, a reversible CoA-ligase, CoA-transferase, a
4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate
dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, an alcohol
dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate
dehydrogenase, a 7-oxoheptanoate dehydrogenase, an aldehyde
dehydrogenase, or a carboxylate reductase. In recombinant hosts
expressing a carboxylate reductase, a phosphopantetheinyl
transferase also can be expressed as it enhances activity of the
carboxylate reductase.
[0098] This document also features a recombinant host that includes
at least one exogenous nucleic acid encoding (i) a polypeptide
having malonyl-[acp] O-methyltransferase activity, (ii) a
polypeptide having pimeloyl-[acp] methyl ester methylesterase
activity and (iii) a polypeptide having thioesterase activity, and
produce glutarate methyl ester, glutaryl-[acp] or glutaryl-CoA.
[0099] Such a recombinant host producing glutarate methyl ester
further can include a polypeptide having esterase activity, and
further produce glutaric acid.
[0100] Such a recombinant host producing glutaryl-[acp] further can
include a polypeptide having thioesterase activity and produce
glutaric acid.
[0101] Such a recombinant host producing glutaryl-CoA further can
include one or more of (i) a polypeptide having thioesterase
activity, (ii) a polypeptide having reversible CoA-ligase activity,
(iii) a polypeptide having CoA-transferase activity, or (iv) a
polypeptide having acylating dehydrogenase activity, and (v) a
polypeptide having aldehyde dehydrogenase activity such as a
7-oxoheptanoate dehydrogenase, 6-oxohexanoate dehydrogenase or
5-oxopentanoate dehydrogenase activity and further produce glutaric
acid or 5-oxopentanoate.
[0102] A recombinant host producing 5-oxopentanoate or glutaric
acid further can include one or more of (i) a polypeptide having
alcohol dehydrogenase activity or (ii) a polypeptide having
carboxylate reductase activity and further produce
5-hydroxypentanoate.
[0103] A recombinant host producing glutarate methyl ester further
can include one or more of (i) a polypeptide having alcohol
dehydrogenase activity, (ii) a polypeptide having esterase activity
or (iii) a polypeptide having carboxylate reductase activity and
further produce 5-hydroxypentanoate.
[0104] Within an engineered pathway, the enzymes can be from a
single source, i.e., from one species or genus, or can be from
multiple sources, i.e., different species or genera. Nucleic acids
encoding the enzymes described herein have been identified from
various organisms and are readily available in publicly available
databases such as GenBank or EMBL.
[0105] Any of the enzymes described herein that can be used for
production of one or more C5 building blocks can have at least 70%
sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino
acid sequence of the corresponding wild-type enzyme. It will be
appreciated that the sequence identity can be determined on the
basis of the mature enzyme (e.g., with any signal sequence removed)
or on the basis of the immature enzyme (e.g., with any signal
sequence included). It also will be appreciated that the initial
methionine residue may or may not be present on any of the enzyme
sequences described herein.
[0106] For example, a polypeptide having pimeloyl-[acp] methyl
ester methylesterase activity described herein can have at least
70% sequence identity (homology) (e.g., at least 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the
amino acid sequence of an Escherichia coli (see Genbank Accession
Nos. AAC76437.1, SEQ ID NO: 1)pimeloyl-[acp] methyl ester
methylesterase. See FIG. 1-3.
[0107] For example, a polypeptide having carboxylate reductase
activity described herein can have at least 70% sequence identity
(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a
Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID
NO: 2), a Mycobacterium smegmatis (see Genbank Accession No.
ABK71854.1, SEQ ID NO: 3), a Segniliparus rugosus (see Genbank
Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis
(see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), a
Mycobacterium massiliense (see Genbank Accession No. EIV11143.1,
SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession
No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase. See, FIG.
5.
[0108] For example, a polypeptide having phosphopantetheinyl
transferase activity described herein can have at least 70%
sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino
acid sequence of a Bacillus subtilis phosphopantetheinyl
transferase (see Genbank Accession No. CAA44858.1, SEQ ID NO: 16)
or a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see
Genbank Accession No. ABI83656.1, SEQ ID NO: 17). See FIG. 5.
[0109] For example, a polypeptide having esterase activity
described herein can have at least 70% sequence identity (homology)
(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%) to the amino acid sequence of a Pseudomonas
fluorescens esterase (see Genbank Accession Nos. AAC60471.2, SEQ ID
NO: 8). See FIG. 4, 5.
[0110] For example, a polypeptide having thioesterase activity
described herein can have at least 70% sequence identity (homology)
(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%) to the amino acid sequence of a
Lactobacillus brevis acyl-[acp] thioesterase (see Genbank Accession
Nos. ABJ63754.1, SEQ ID NO: 9), a Lactobacillus plantarum
acyl-[acp] thioesterase (see Genbank Accession Nos. CCC78182.1, SEQ
ID NO: 10), or a Escherichia coli thioesterase (see Genbank
Accession Nos. AAB59067.1 or AAA24665.1, SEQ ID NO: 14 or 15). See
FIG. 4.
[0111] For example, a polypeptide having malonyl-[acp]
O-methyltransferase activity described herein can have at least 70%
sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino
acid sequence of a Bacillus cereus (see Genbank Accession Nos.
AAP11034.1, SEQ ID NO: 13) malonyl-[acp] O-methyltransferase. See
FIG. 1-3.
[0112] For example, a polypeptide having enoyl-CoA reductase
activity described herein can have at least 70% sequence identity
(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a
Treponema denticola (see Genbank Accession Nos. AAS11092.1, SEQ ID
NO:11), or a Euglena gracilis (see Genbank Accession Nos.
AAW66853.1, SEQ ID NO:12) enoyl-CoA reductase. See FIGS. 1-3.
[0113] The percent identity (homology) between two amino acid
sequences can be determined as follows. First, the amino acid
sequences are aligned using the BLAST 2 Sequences (Bl2seq) program
from the stand-alone version of BLASTZ containing BLASTP version
2.0.14. This stand-alone version of BLASTZ can be obtained from
Fish & Richardson's web site (e.g., www.fr.com/blast/) or the
U.S. government's National Center for Biotechnology Information web
site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the
Bl2seq program can be found in the readme file accompanying BLASTZ.
Bl2seq performs a comparison between two amino acid sequences using
the BLASTP algorithm. To compare two amino acid sequences, the
options of Bl2seq are set as follows: -i is set to a file
containing the first amino acid sequence to be compared (e.g.,
C:\seq1.txt); -j is set to a file containing the second amino acid
sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp;
-o is set to any desired file name (e.g., C:\output.txt); and all
other options are left at their default setting. For example, the
following command can be used to generate an output file containing
a comparison between two amino acid sequences: C:\Bl2seq -i
c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two
compared sequences share homology (identity), then the designated
output file will present those regions of homology as aligned
sequences. If the two compared sequences do not share homology
(identity), then the designated output file will not present
aligned sequences. Similar procedures can be following for nucleic
acid sequences except that blastn is used.
[0114] Once aligned, the number of matches is determined by
counting the number of positions where an identical amino acid
residue is presented in both sequences. The percent identity
(homology) is determined by dividing the number of matches by the
length of the full-length polypeptide amino acid sequence followed
by multiplying the resulting value by 100. It is noted that the
percent identity (homology) value is rounded to the nearest tenth.
For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to
78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to
78.2. It also is noted that the length value will always be an
integer.
[0115] It will be appreciated that a number of nucleic acids can
encode a polypeptide having a particular amino acid sequence. The
degeneracy of the genetic code is well known to the art; i.e., for
many amino acids, there is more than one nucleotide triplet that
serves as the codon for the amino acid. For example, codons in the
coding sequence for a given enzyme can be modified such that
optimal expression in a particular species (e.g., bacteria or
fungus) is obtained, using appropriate codon bias tables for that
species.
[0116] Functional fragments of any of the enzymes described herein
can also be used in the methods of the document. The term
"functional fragment" as used herein refers to a peptide fragment
of a protein that has at least 25% (e.g., at least: 30%; 40%; 50%;
60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 100%; or even greater
than 100%) of the activity of the corresponding mature,
full-length, wild-type protein. The functional fragment can
generally, but not always, be comprised of a continuous region of
the protein, wherein the region has functional activity.
[0117] This document also provides (i) functional variants of the
enzymes used in the methods of the document and (ii) functional
variants of the functional fragments described above. Functional
variants of the enzymes and functional fragments can contain
additions, deletions, or substitutions relative to the
corresponding wild-type sequences. Enzymes with substitutions will
generally have not more than 50 (e.g., not more than one, two,
three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25,
30, 35, 40, or 50) amino acid substitutions (e.g., conservative
substitutions). This applies to any of the enzymes described herein
and functional fragments. A conservative substitution is a
substitution of one amino acid for another with similar
characteristics. Conservative substitutions include substitutions
within the following groups: valine, alanine and glycine; leucine,
valine, and isoleucine; aspartic acid and glutamic acid; asparagine
and glutamine; serine, cysteine, and threonine; lysine and
arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine and glutamine. The positively charged (basic) amino
acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid. Any substitution of one member of the above-mentioned polar,
basic or acidic groups by another member of the same group can be
deemed a conservative substitution. By contrast, a nonconservative
substitution is a substitution of one amino acid for another with
dissimilar characteristics.
[0118] Deletion variants can lack one, two, three, four, five, six,
seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
amino acid segments (of two or more amino acids) or non-contiguous
single amino acids. Additions (addition variants) include fusion
proteins containing: (a) any of the enzymes described herein or a
fragment thereof; and (b) internal or terminal (C or N) irrelevant
or heterologous amino acid sequences. In the context of such fusion
proteins, the term "heterologous amino acid sequences" refers to an
amino acid sequence other than (a). A heterologous sequence can be,
for example a sequence used for purification of the recombinant
protein (e.g., FLAG, polyhistidine (e.g., hexahistidine),
hemagglutinin (HA), glutathione-S-transferase (GST), or
maltosebinding protein (MBP)). Heterologous sequences also can be
proteins useful as detectable markers, for example, luciferase,
green fluorescent protein (GFP), or chloramphenicol acetyl
transferase (CAT). In some embodiments, the fusion protein contains
a signal sequence from another protein. In certain host cells
(e.g., yeast host cells), expression and/or secretion of the target
protein can be increased through use of a heterologous signal
sequence. In some embodiments, the fusion protein can contain a
carrier (e.g., KLH) useful, e.g., in eliciting an immune response
for antibody generation) or ER or Golgi apparatus retention
signals. Heterologous sequences can be of varying length and in
some cases can be a longer sequences than the full-length target
proteins to which the heterologous sequences are attached.
[0119] Engineered hosts can naturally express none or some (e.g.,
one or more, two or more, three or more, four or more, five or
more, or six or more) of the enzymes of the pathways described
herein. Thus, a pathway within an engineered host can include all
exogenous enzymes, or can include both endogenous and exogenous
enzymes. Endogenous genes of the engineered hosts also can be
disrupted to prevent the formation of undesirable metabolites or
prevent the loss of intermediates in the pathway through other
enzymes acting on such intermediates. Engineered hosts can be
referred to as recombinant hosts or recombinant host cells. As
described herein recombinant hosts can include nucleic acids
encoding one or more polypeptide having the activity of a
reductase, deacetylase, N-acetyltransferase, malonyl-[acp]
O-methyltransferase, esterase, thioesterase, hydratase,
dehydrogenase, CoA-ligase, and/or CoA-transferase as described
herein.
[0120] In addition, the production of one or more C5 building
blocks can be performed in vitro using the isolated enzymes
described herein, using a lysate (e.g., a cell lysate) from a host
microorganism as a source of the enzymes, or using a plurality of
lysates from different host microorganisms as the source of the
enzymes.
Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis
of 2,4-Pentadienoyl-CoA
[0121] As depicted in FIG. 4, a terminal carboxyl group can be
enzymatically formed using (i) a polypeptide having thioesterase
activity, (ii) a polypeptide having reversible CoA-ligase activity,
(iii) a polypeptide having CoA-transferase activity, (iv) a
polypeptide having acylating dehydrogenase activity, or (v) a
polypeptide having aldehyde dehydrogenase activity such as a
7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a
5-oxopentanoate dehydrogenase activity, or (vi) a polypeptide
having esterase activity.
[0122] In some embodiments, a terminal carboxyl group leading to
the synthesis of glutarate is enzymatically formed by a
thioesterase classified under EC 3.1.2.-, such as the gene product
of YciA (SEQ ID NO: 14), tesB (Genbank Accession No. AAA24665.1,
SEQ ID NO: 15) or Acot13 (see, for example, Cantu et al., Protein
Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008,
47(9), 2789-2796; or Naggert et al., J. Biol. Chem., 1991, 266(17),
11044-11050).
[0123] In some embodiments, the second terminal carboxyl group
leading to the synthesis of glutaric acid is enzymatically formed
by a CoA-transferase such as a glutaconate CoA-transferase
classified, for example, under EC 2.8.3.12 such as from
Acidaminococcus fermentans. See, for example, Buckel et al., 1981,
Eur. J. Biochem., 118:315-321. FIG. 4.
[0124] In some embodiments, the second terminal carboxyl group
leading to the synthesis of glutaric acid is enzymatically formed
by a reversible CoA-ligase such as a succinate-CoA ligase
classified, for example, under EC 6.2.1.5 such as from Thermococcus
kodakaraensis. See, for example, Shikata et al., 2007, J. Biol.
Chem., 282(37):26963-26970.
[0125] In some embodiments, the second terminal carboxyl group
leading to the synthesis of glutaric acid is enzymatically formed
by an acyl-[acp] thioesterase classified under EC 3.1.2.-, such as
the acyl-[acp] thioesterase from Lactobacillus brevis (GenBank
Accession No. ABJ63754.1, SEQ ID NO: 9) or from Lactobacillus
plantarum (GenBank Accession No. CCC78182.1, SEQ ID NO: 10). Such
acyl-[acp] thioesterases have C6-C8 chain length specificity (see,
for example, Jing et al., 2011, BMC Biochemistry, 12(44)). See,
e.g., FIG. 4.
[0126] In some embodiments, the second terminal carboxyl group
leading to the synthesis of glutaric acid is enzymatically formed
by an aldehyde dehydrogenase classified, for example, under EC
1.2.1.3 (see, Guerrillot & Vandecasteele, Eur. J. Biochem.,
1977, 81, 185-192). See, FIG. 4.
[0127] In some embodiments, the second terminal carboxyl group
leading to the synthesis of glutaric acid is enzymatically formed
by an aldehyde dehydrogenase classified under EC 1.2.1.- such as a
glutarate semialdehyde dehydrogenase classified, for example, under
EC 1.2.1.20, a succinate-semialdehyde dehydrogenase classified, for
example, under EC 1.2.1.16 or EC 1.2.1.79, or an aldehyde
dehydrogenase classified under EC 1.2.1.3. For example, an aldehyde
dehydrogenase classified under EC 1.2.1.- can be a 5-oxopentanoate
dehydrogenase such as the gene product of CpnE, a 6-oxohexanoate
dehydrogenase (e.g., the gene product of ChnE from Acinetobacter
sp.), or a 7-oxoheptanoate dehydrogenase (e.g., the gene product of
ThnG from Sphingomonas macrogolitabida) (Iwaki et al., Appl.
Environ. Microbiol., 1999, 65(11), 5158-5162; Lopez-Sanchez et al.,
Appl. Environ. Microbiol., 2010, 76(1), 110-118). For example, a
6-oxohexanoate dehydrogenase can be classified under EC 1.2.1.63
such as the gene product of ChnE. For example, a 7-oxoheptanoate
dehydrogenase can be classified under EC 1.2.1.-.
[0128] In some embodiments, the second terminal carboxyl group
leading to the synthesis of glutaric acid is enzymatically formed
by a polypeptide having esterase activity such as an esterase
classified under EC 3.1.1.- such as EC 3.1.1.1 or EC 3.1.1.6.
[0129] Enzymes Generating the Terminal Hydroxyl Groups in the
Biosynthesis of a 2,4-Pentadienoyl-CoA
[0130] As depicted in FIGS. 5, a terminal hydroxyl group can be
enzymatically formed using an alcohol dehydrogenase such as a
6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate
dehydrogenase, or a 4-hydroxybutyrate dehydrogenase.
[0131] For example, a terminal hydroxyl group leading to the
synthesis of 5-hydroxypentanoate can be enzymatically formed by a
dehydrogenase classified, for example, under EC 1.1.1.- such as a
6-hydroxyhexanoate dehydrogenase classified, for example, under EC
1.1.1.258 (e.g., the gene from of ChnD), a 5-hydroxypentanoate
dehydrogenase classified, for example, under EC 1.1.1.- such as the
gene product of CpnD (see, for example, Iwaki et al., 2002, Appl.
Environ. Microbiol., 68(11):5671-5684), a 5-hydroxypentanoate
dehydrogenase from Clostridium viride, or a 4-hydroxybutyrate
dehydrogenase such as gabD (see, for example, Lutke-Eversloh &
Steinbuchel, 1999, FEMS Microbiology Letters, 181(1):63-71). See,
FIG. 5.
Enzymes Generating the Terminal Vinyl Group in the Biosynthesis of
a 2,4-Pentadienoyl-CoA
[0132] As depicted in FIG. 6, a terminal vinyl group can be
enzymatically formed using a dehydratase such as
5-hydroxypentanoyl-CoA dehydratase from Clostridium viride
(Eikmanns and Buckel, 1991, Eur. J. Biochem., 197, 661 -668).
Biochemical Pathways
[0133] Pathway to Glutarate Methyl Ester, Glutaryl-CoA or
Glutaryl-[acp] from Malonyl-[acp] or Malonyl-CoA
[0134] As shown in FIG. 1, glutarate methyl ester can be
synthesized from malonyl-[acp] by conversion of malonyl-[acp] to
malonyl-[acp] methyl ester by a malonyl-CoA O-methyltransferase
classified, for example, under EC 2.1.1.197 such as the gene
product of bioC; followed by conversion to 3-oxoglutaryl-[acp]
methyl ester by condensation with malonyl-[acp] and a
.beta.-ketoacyl-[acp] synthase classified, for example, under EC
2.3.1.- such as EC 2.3.1.41, EC 2.3.1.179 or EC 2.3.1.180 (e.g.,
the gene product of fabB,fabF or fabII); followed by conversion to
3-hydroxy-glutaryl-[acp] methyl ester by a 3-hydroxyacyl-CoA
dehydrogenase classified, for example, under EC 1.1.1.- such as EC
1.1.1.100 (e.g., the gene product of fabG); followed by conversion
to 2,3-dehydroglutaryl-[acp] methyl ester by a 3-hydroxyacyl-[acp]
dehydratase classified, for example, under EC 4.2.1.59 such as the
gene product of fabZ; followed by conversion to glutaryl-[acp]
methyl ester by a trans-2-enoyl-CoA reductase classified, for
example, EC 1.3.1.- such as EC 1.3.1.10 such as the gene product of
fabI; followed by (i) conversion to glutarate methyl ester by a
thioesterase classified, for example, under EC 3.1.2.- such as the
tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a Bacteroides
thetaiotaomicron acyl-ACP thioesterase (GenBank Accession No.
AAO77182) or a Lactobacillus plantarum acyl-CoA thioesterase
(GenBank Accession No. CCC78182.1) or (ii) conversion to
glutaryl-[acp] by a pimeloyl-[acp] methyl ester methylesterase
classified, for example, under EC 3.1.1.85 such as bioH(SEQ ID NO:
1).
[0135] As shown in FIG. 2, glutarate methyl ester can be
synthesized from malonyl-CoA by conversion of malonyl-CoA to
malonyl-CoA methyl ester by a malonyl-CoA O-methyltransferase
classified, for example, under EC 2.1.1.197 such as the gene
product of bioC; followed by conversion to 3-oxoglutaryl-CoA methyl
ester by condensation with acetyl-CoA by a .beta.-ketothiolase
classified, for example, under EC 2.3.1.16 such as the gene product
of bktB or by condensation with malonyl-CoA by a
.beta.-ketoacyl-[acp]synthase classified, for example, under EC
2.3.1.180 such as the gene product of fabH; followed by conversion
to 3-hydroxy-glutaryl-CoA methyl ester by a 3-hydroxyacyl-CoA
dehydrogenase classified, for example, under EC 1.1.1.- such as EC
1.1.1.100 (e.g., the gene product of fabG) or EC 1.1.1.36 (e.g.,
the gene product ofphaB); followed by conversion to
2,3-dehydroglutaryl-CoA methyl ester by an enoyl-CoA hydratase
classified, for example, under EC 4.2.1.119 such as the gene
product of phaJ (Shen et al., Appl. Environ. Microbiol., 2011,
77(9), 2905-2915; Fukui et al., Journal of Bacteriology, 1998,
180(3), 667-673); followed by conversion to glutaryl-CoA methyl
ester by a trans-2-enoyl-CoA reductase classified, for example, EC
1.3.1.- such as EC 1.3.1.38, EC 1.3.1.8, EC 1.3.1.10 or EC
1.3.1.44; followed by (i) conversion to glutarate methyl ester by a
thioesterase classified, for example, under EC 3.1.2.- such as the
tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a Bacteroides
thetaiotaomicron acyl-ACP thioesterase (GenBank Accession No.
AAO77182) or a Lactobacillus plantarum acyl-ACP thioesterase
(GenBank Accession No. CCC78182.1) or (ii) conversion to
glutaryl-CoA by a pimeloyl-[acp] methyl ester methylesterase
classified, for example, under EC 3.1.1.85 such as bioH (SEQ ID NO:
1).
[0136] As shown in FIG. 3, glutarate methyl ester can be
synthesized from malonyl-CoA by conversion of malonyl-CoA to
malonyl-CoA methyl ester by a malonyl-CoA O-methyltransferase
classified, for example, under EC 2.1.1.197 such as the gene
product of bioC; followed by conversion to 3-oxoglutaryl-CoA methyl
ester by condensation with acetyl-CoA by a .beta.-ketothiolase
classified, for example, under EC 2.3.1.16 such as the gene product
of bktB or by condensation with malonyl-CoA by a
.beta.-ketoacyl-[acp] synthase classified, for example, under EC
2.3.1.180 such as the gene product of fabH; followed by conversion
to 3-hydroxy-glutaryl-CoA methyl ester by a 3-hydroxyacyl-CoA
dehydrogenase classified, for example, under EC 1.1.1.- such as EC
1.1.1.35 or EC 1.1.1.157 (e.g., the gene product of fadB or hbd);
followed by conversion to 2,3-dehydroglutaryl-CoA methyl ester by
an enoyl-CoA hydratase classified, for example, under EC 4.2.1.17
such as the gene product of crt; followed by conversion to
glutaryl-CoA methyl ester by a trans-2-enoyl-CoA reductase
classified, for example, under EC 1.3.1.44 such as the gene product
of ter or tdter; followed by (i) conversion to glutarate methyl
ester by a thioesterase classified, for example, under EC 3.1.2.-
such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a
Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank
Accession No. AAO77182) or a Lactobacillus plantarum acyl-CoA
thioesterase (GenBank Accession No. CCC78182.1) or (ii) conversion
to glutaryl-CoA by a pimeloyl-[acp] methyl ester methylesterase
classified, for example, under EC 3.1.1.85 such as bioH (SEQ ID NO:
1).
Pathway to Glutarate or 5-Oxopentanoate Using Glutarate Methyl
Ester, Glutaryl-[acp] or Glutaryl-CoA as a Central Precursor
[0137] As depicted in FIG. 4, glutarate methyl ester can be
converted to glutarate by an esterase classified, for example, EC
3.1.1.-, such as EC 3.1.1.1 or EC 3.1.1.6 such as estC (SEQ ID NO:
8).
[0138] As depicted in FIG. 4, glutaryl-CoA can be converted to
glutarate by a (i) a thioesterase classified, for example, EC
3.1.2.-, such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or
Acot13, a Bacteroides thetaiotaomicron acyl ACP thioesterase
(GenBank Accession No. AAO77182) or a Lactobacillus plantarum
acyl-CoA thioesterase (GenBank Accession No. CCC78182.1) (ii) a
reversible CoA-ligase classified, for example, under EC 6.2.1.5,
(iii) a CoA-transferase classified, for example, under EC 2.8.3.-
such as EC 2.8.3.12, or (iv) an acylating dehydrogenase classified
under, for example, EC 1.2.1.10 or EC 1.2.1.76 such as encoded by
PduB or PduP and an aldehyde dehydrogenase classified under EC
1.2.1.- such as a glutarate semialdehyde dehydrogenase classified,
for example, under EC 1.2.1.20, a succinate-semialdehyde
dehydrogenase classified, for example, under EC 1.2.1.16 or EC
1.2.1.79, or an aldehyde dehydrogenase classified under EC 1.2.1.3.
For example, a 5-oxovalerate dehydrogenase such as the gene product
of CpnE, a 6-oxohexanoate dehydrogenase such as the gene product of
ChnE, or a 7-oxoheptanoate dehydrogenase (e.g., the gene product of
ThnG from Sphingomonas macrogolitabida) can be used to convert
5-oxopentanoic acid to glutarate.
[0139] As depicted in FIG. 4, glutaryl-{acp] can be converted to
glutarate by a thioesterase classified, for example, EC 3.1.2.-,
such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a
Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank
Accession No. AAO77182) or a Lactobacillus plantarum acyl-CoA
thioesterase (GenBank Accession No. CCC78182.1).
Pathway to 5-Hydroxypentanoate Using Glutarate Methyl Ester as a
Central Precursor
[0140] As depicted in FIG. 5, 5-hydroxypentanoate can be
synthesized from the central precursor glutarate methyl ester by
conversion of glutarate methyl ester to glutaric acid by an
esterase classified under EC 3.1.1.- (e.g., the gene product of
estC) such as a carboxyl esterase classified under EC 3.1.1.1 or an
acetylesterase classified under EC 3.1.1.6; followed by conversion
of glutaric acid to glutarate semialdehyde by a carboxylate
reductase classified, for example, under EC 1.2.99.6 such as the
gene product of car in combination with a phosphopantetheine
transferase enhancer (e.g., encoded by a sfp (Genbank Accession No.
CAA44858.1, SEQ ID NO:16) gene from Bacillus subtilis or npt
(Genbank Accession No. ABI83656.1, SEQ ID NO:17) gene from
Nocardia), or the gene product of GriC & GriD (Suzuki et al.,
J. Antibiot., 2007, 60(6), 380-387); followed by conversion to
5-hydroxypentanoate by a dehydrogenase classified, for example,
under EC 1.1.1.- such as a 6-hydroxyhexanoate dehydrogenase
classified, for example, under EC 1.1.1.258 (e.g., the gene from of
ChnD), a 5-hydroxypentanoate dehydrogenase classified, for example,
under EC 1.1.1.- such as the gene product of CpnD (see, for
example, Iwaki et al., 2002, Appl. Environ. Microbiol.,
68(11):5671-5684), or a 4-hydroxybutyrate dehydrogenase such as
gabD (see, for example, Lutke-Eversloh & Steinbuchel, 1999,
FEMS Microbiology Letters, 181(1):63-71). See, FIG. 5.
[0141] As depicted in FIG. 5, 5-hydroxypentanoate can be
synthesized from the central precursor glutarate methyl ester by
conversion of glutarate methyl ester to glutarate semialdehyde
methyl ester by a carboxylate reductase classified, for example,
under EC 1.2.99.6 such as from a Mycobacterium marinum (see Genbank
Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis
(see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a
Mycobacterium massiliense (see Genbank Accession No. EIV11143.1,
SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession
No. ADG98140.1, SEQ ID NO: 7), in combination with a
phosphopantetheine transferase enhancer (e.g., encoded by a sfp
(Genbank Accession No. CAA44858.1, SEQ ID NO:16) gene from Bacillus
subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:17)
gene from Nocardia), or the gene product of GriC & GriD (Suzuki
et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion
to glutarate semialdehyde by an esterase classified under EC
3.1.1.- (e.g., the gene product of estC) such as a carboxyl
esterase classified under EC 3.1.1.1 or an acetylesterase
classified under EC 3.1.1.6; followed by conversion to
5-hydroxypentanoate by a dehydrogenase classified, for example,
under EC 1.1.1.- such as a 6-hydroxyhexanoate dehydrogenase
classified, for example, under EC 1.1.1.258 (e.g., the gene from of
ChnD), a 5-hydroxypentanoate dehydrogenase classified, for example,
under EC 1.1.1.- such as the gene product of CpnD, or a
4-hydroxybutyrate dehydrogenase such as gabD.
[0142] As depicted in FIG. 5, 5-hydroxypentanoate can be
synthesized from the central precursor glutarate methyl ester by
conversion of glutarate methyl ester to glutarate semialdehyde
methyl ester by a carboxylate reductase classified, for example,
under EC 1.2.99.6 such as from a Mycobacterium marinum (see Genbank
Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis
(see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a
Mycobacterium massiliense (see Genbank Accession No. EIV11143.1,
SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession
No. ADG98140.1, SEQ ID NO: 7), in combination with
aphosphopantetheine transferase enhancer (e.g., encoded by a sfp
(Genbank Accession No. CAA44858.1, SEQ ID NO:16) gene from Bacillus
subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:17)
gene from Nocardia), or the gene product of GriC & GriD (Suzuki
et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion
to 5-hydroxypentanoate methyl ester by an alcohol dehydrogenase
classified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC
1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of
YMR318C, YqhD, or the protein having GenBank Accession No.
CAA81612.1; followed by conversion to 5-hydroxypentanoate by an
esterase classified under EC 3.1.1.- (e.g., the gene product of
estC) such as a carboxyl esterase classified under EC 3.1.1.1 or an
acetylesterase classified under EC 3.1.1.6.
Pathway to 2,4-Pentadienoyl-CoA Using 5-Hydroxypentanoate as a
Central Precursor
[0143] As depicted in FIG. 6, 2,4-pentadienoyl-CoA can be
synthesized from 5-hydroxypentanoate by conversion of
5-hydroxypentanoate to 5-hydroxypentanoyl-CoA by a
5-hydroxypentanoate CoA-transferase or 4-hydroxybutryrate
CoA-transferase classified, for example, under EC 2.8.3.- such as
EC 2.8.3.14 or EC 2.8.3.9 or by a synthase classified, for example,
under EC 6.2.1.- such as a 3-hydroxypropionyl-CoA synthase
classified under EC 6.2.1.36; followed by conversion to
2,4-pentadienoyl-CoA by a dehydratase such as 5-hydroxypentanoy-CoA
dehydratase classified, for example, under EC 4.2.1.- obtained from
Clostridium virile.
Cultivation Strategy
[0144] In some embodiments, the cultivation strategy entails
achieving an aerobic, anaerobic, micro-aerobic, or mixed
oxygen/denitrification cultivation condition. Enzymes characterized
in vitro as being oxygen sensitive require a micro-aerobic
cultivation strategy maintaining a very low dissolved oxygen
concentration (See, for example, Chayabatra & Lu-Kwang, Appl.
Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson and Bouwer,
1997, Journal of Industrial Microbiology and Biotechnology,
18(2-3), 116-130).
[0145] In some embodiments, a cyclical cultivation strategy entails
alternating between achieving an anaerobic cultivation condition
and achieving an aerobic cultivation condition.
[0146] In some embodiments, the cultivation strategy entails
nutrient limitation such as nitrogen, phosphate or oxygen
limitation.
[0147] In some embodiments, a final electron acceptor other than
oxygen such as nitrates can be utilized. In some embodiments, a
cell retention strategy using, for example, ceramic membranes can
be employed to achieve and maintain a high cell density during
either fed-batch or continuous fermentation.
[0148] In some embodiments, the principal carbon source fed to the
fermentation in the synthesis of one or more CS building blocks can
derive from biological or non-biological feedstocks.
[0149] In some embodiments, the biological feedstock can be or can
derive from, monosaccharides, disaccharides, lignocellulose,
hemicellulose, cellulose, lignin, levulinic acid and formic acid,
triglycerides, glycerol, fatty acids, agricultural waste, condensed
distillers' solubles, or municipal waste.
[0150] The efficient catabolism of crude glycerol stemming from the
production of biodiesel has been demonstrated in several
microorganisms such as Escherichia coli, Cupriavidus necator,
Pseudomonas oleavorans, Pseudomonas putida and Yarrowia lipolytica
(Lee et al., Appl. Biochem. Biotechnol., 2012, 166:1801-1813; Yang
et al., Biotechnology for Biofuels, 2012, 5:13; Meijnen et al.,
Appl. Microbiol. Biotechnol., 2011, 90:885-893).
[0151] The efficient catabolism of lignocellulosic-derived
levulinic acid has been demonstrated in several organisms such as
Cupriavidus necator and Pseudomonas putida in the synthesis of
3-hydroxyvalerate via the precursor propanoyl-CoA (Jaremko and Yu,
2011, supra; Martin and Prather, J. Biotechnol., 2009,
139:61-67).
[0152] The efficient catabolism of lignin-derived aromatic
compounds such as benzoate analogues has been demonstrated in
several microorganisms such as Pseudomonas putida, Cupriavidus
necator (Bugg et al., Current Opinion in Biotechnology, 2011, 22,
394-400; Perez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32,
736-794).
[0153] The efficient utilization of agricultural waste, such as
olive mill waste water has been demonstrated in several
microorganisms, including Yarrowia lipolytica (Papanikolaou et al.,
Bioresour. Technol, 2008, 99(7):2419-2428).
[0154] The efficient utilization of fermentable sugars such as
monosaccharides and disaccharides derived from cellulosic,
hemicellulosic, cane and beet molasses, cassava, corn and other
agricultural sources has been demonstrated for several
microorganism such as Escherichia coli, Corynebacterium glutamicum
and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g.,
Hermann et al, J. Biotechnol., 2003, 104:155-172; Wee et al., Food
Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al., J.
Bioscience and Bioengineering, 1999, 87(5):647-654).
[0155] The efficient utilization of furfural, derived from a
variety of agricultural lignocellulosic sources, has been
demonstrated for Cupriavidus necator (Li et al., Biodegradation,
2011, 22:1215-1225).
[0156] In some embodiments, the non-biological feedstock can be or
can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol,
ethanol, benzoate, non-volatile residue (NVR) or a caustic wash
waste stream from cyclohexane oxidation processes, or terephthalic
acid/isophthalic acid mixture waste streams.
[0157] The efficient catabolism of methanol has been demonstrated
for the methylotrophic yeast Pichia pastoris.
[0158] The efficient catabolism of ethanol has been demonstrated
for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci.
USA, 2008, 105(6) 2128-2133).
[0159] The efficient catabolism of CO.sub.2 and H.sub.2, which may
be derived from natural gas and other chemical and petrochemical
sources, has been demonstrated for Cupriavidus necator (Prybylski
et al., Energy, Sustainability and Society, 2012, 2:11).
[0160] The efficient catabolism of syngas has been demonstrated for
numerous microorganisms, such as Clostridium ljungdahlii and
Clostridium autoethanogenum (Kopke et al., Applied and
Environmental Microbiology, 2011, 77(15):5467-5475).
[0161] The efficient catabolism of the non-volatile residue waste
stream from cyclohexane processes has been demonstrated for
numerous microorganisms, such as Delftia acidovorans and
Cupriavidus necator (Ramsay et al., Applied and Environmental
Microbiology, 1986, 52(1):152-156).
Metabolic Engineering
[0162] The present document provides methods involving less than
all the steps described for all the above pathways. Such methods
can involve, for example, one, two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve or more of such steps. Where less
than all the steps are included in such a method, the first, and in
some embodiments the only, step can be any one of the steps
listed.
[0163] Furthermore, recombinant hosts described herein can include
any combination of the above enzymes such that one or more of the
steps, e.g., one, two, three, four, five, six, seven, eight, nine,
ten, or more of such steps, can be performed within a recombinant
host. This document provides host cells of any of the genera and
species listed and genetically engineered to express one or more
(e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12
or more) recombinant forms of any of the enzymes recited in the
document. Thus, for example, the host cells can contain exogenous
nucleic acids encoding enzymes catalyzing one or more of the steps
of any of the pathways described herein.
[0164] In addition, this document recognizes that where enzymes
have been described as accepting CoA-activated substrates,
analogous enzyme activities associated with [acp]-bound substrates
exist that are not necessarily in the same enzyme class.
[0165] Also, this document recognizes that where enzymes have been
described accepting (R)-enantiomers of substrate, analogous enzyme
activities associated with (S)-enantiomer substrates exist that are
not necessarily in the same enzyme class.
[0166] This document also recognizes that where an enzyme is shown
to accept a particular co-factor, such as NADPH, or co-substrate,
such as acetyl-CoA, many enzymes are promiscuous in terms of
accepting a number of different co-factors or co-substrates in
catalyzing a particular enzyme activity. Also, this document
recognizes that where enzymes have high specificity for e.g., a
particular co-factor such as NADH, an enzyme with similar or
identical activity that has high specificity for the co-factor
NADPH may be in a different enzyme class.
[0167] In some embodiments, the enzymes in the pathways outlined
herein are the result of enzyme engineering via non-direct or
rational enzyme design approaches with aims of improving activity,
improving specificity, reducing feedback inhibition, reducing
repression, improving enzyme solubility, changing
stereo-specificity, or changing co-factor specificity.
[0168] In some embodiments, the enzymes in the pathways outlined
here can be gene dosed, i.e., overexpressed, into the resulting
genetically modified organism via episomal or chromosomal
integration approaches.
[0169] In some embodiments, genome-scale system biology techniques
such as Flux Balance Analysis can be utilized to devise genome
scale attenuation or knockout strategies for directing carbon flux
to a 2,4-pentadienoyl-CoA.
[0170] Attenuation strategies include, but are not limited to; the
use of transposons, homologous recombination (double cross-over
approach), mutagenesis, enzyme inhibitors and RNAi
interference.
[0171] In some embodiments, fluxomic, metabolomic and
transcriptomal data can be utilized to inform or support
genome-scale system biology techniques, thereby devising genome
scale attenuation or knockout strategies in directing carbon flux
to 2,4-pentadienoyl-CoA.
[0172] In some embodiments, the host microorganism's tolerance to
high concentrations of 2,4-pentadienoyl-CoA can be improved through
continuous cultivation in a selective environment.
[0173] In some embodiments, the host microorganism's endogenous
biochemical network can be attenuated or augmented to (1) ensure
the intracellular availability of acetyl-CoA, (2) create a NADH or
NADPH imbalance that may be balanced via the formation of
2,4-pentadienoyl-CoA, and/or (3) prevent degradation of central
metabolites, central precursors leading to and including
2,4-pentadienoyl-CoA.
[0174] In some embodiments requiring intracellular availability of
acetyl-CoA-CoA for C5 building block synthesis, endogenous enzymes
catalyzing the hydrolysis of acetyl-CoA such as short-chain length
thioesterases can be attenuated in the host organism.
[0175] In some embodiments requiring condensation of acetyl-CoA and
malonyl-CoA for 2,4-pentadienoyl-CoA synthesis, one or more
endogenous .beta.-ketothiolases catalyzing the condensation of only
acetyl-CoA to acetoacetyl-CoA such as the endogenous gene products
of AtoB or phaA can be attenuated.
[0176] In some embodiments requiring the intracellular availability
of acetyl-CoA for 2,4-pentadienoyl-CoA synthesis, an endogenous
phosphotransacetylase generating acetate such as pta can be
attenuated (Shen et al., Appl. Environ. Microbiol., 2011,
77(9):2905-2915).
[0177] In some embodiments requiring the intracellular availability
of acetyl-CoA for 2,4-pentadienoyl-CoA synthesis, an endogenous
gene in an acetate synthesis pathway encoding an acetate kinase,
such as ack, can be attenuated.
[0178] In some embodiments requiring the intracellular availability
of acetyl-CoA and NADH for 2,4-pentadienoyl-CoA synthesis, an
endogenous gene encoding an enzyme that catalyzes the degradation
of pyruvate to lactate such as a lactate dehydrogenase encoded by
ldhA can be attenuated (Shen et al., 2011, supra).
[0179] In some embodiments requiring the intracellular availability
of acetyl-CoA and NADH for 2,4-pentadienoyl-CoA synthesis,
endogenous genes encoding enzymes, such as menaquinol-fumarate
oxidoreductase, that catalyze the degradation of phophoenolpyruvate
to succinate such as frdBC can be attenuated (see, e.g., Shen et
al., 2011, supra).
[0180] In some embodiments requiring the intracellular availability
of acetyl-CoA and NADH for 2,4-pentadienoyl-CoAsynthesis, an
endogenous gene encoding an enzyme that catalyzes the degradation
of acetyl-CoA to ethanol such as the alcohol dehydrogenase encoded
by adhE can be attenuated (Shen et al., 2011, supra).
[0181] In some embodiments, where pathways require excess NADH
co-factor for 2,4-pentadienoyl-CoA synthesis, a recombinant formate
dehydrogenase gene can be overexpressed in the host organism (Shen
et al., 2011, supra).
[0182] In some embodiments, acetyl-CoA carboxylase can be
overexpressed in the host organisms.
[0183] In some embodiments, one or more of 3-phosphoglycerate
dehydrogenase, 3-phosphoserine aminotransferase and phosphoserine
phosphatase can be overexpressed in the host to generate serine as
a methyl donor for the S-Adenosyl-L-methionine cycle.
[0184] In some embodiments, a methanol dehydrogenase or a
formaldehyde dehydrogenase can be overexpressed in the host to
allow methanol catabolism via formate.
[0185] In some embodiments, where pathways require excess NADH or
NADPH co-factor for 2,4-pentadienoyl-CoA synthesis, a
transhydrogenase dissipating the cofactor imbalance can be
attenuated.
[0186] In some embodiments, an endogenous gene encoding an enzyme
that catalyzes the degradation of pyruvate to ethanol such as
pyruvate decarboxylase can be attenuated.
[0187] In some embodiments, an endogenous gene encoding an enzyme
that catalyzes the generation of isobutanol such as a 2-oxoacid
decarboxylase can be attenuated.
[0188] In some embodiments requiring the intracellular availability
of acetyl-CoA for 2,4-pentadienoyl-CoA synthesis, a recombinant
acetyl-CoA synthetase such as the gene product of acs can be
overexpressed in the microorganism (Satoh et al., J. Bioscience and
Bioengineering, 2003, 95(4):335-341).
[0189] In some embodiments, carbon flux can be directed into the
pentose phosphate cycle to increase the supply of NADPH by
attenuating an endogenous glucose-6-phosphate isomerase (EC
5.3.1.9).
[0190] In some embodiments, carbon flux can be redirected into the
pentose phosphate cycle to increase the supply of NADPH by
overexpression a 6-phosphogluconate dehydrogenase and/or a
transketolase (Lee et al., 2003, Biotechnology Progress, 19(5),
1444-1449).
[0191] In some embodiments, where pathways require excess NADPH
co-factor in the synthesis of 2,4-pentadienoyl-CoA, a gene such as
UdhA encoding a puridine nucleotide transhydrogenase can be
overexpressed in the host organisms (Brigham et al., Advanced
Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).
[0192] In some embodiments, where pathways require excess NADPH
co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant
glyceraldehyde-3-phosphate-dehydrogenase gene such as GapN can be
overexpressed in the host organisms (Brigham et al., 2012,
supra).
[0193] In some embodiments, where pathways require excess NADPH
co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant
malic enzyme gene such as maeA or maeB can be overexpressed in the
host organisms (Brigham et al., 2012, supra).
[0194] In some embodiments, where pathways require excess NADPH
co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant
glucose-6-phosphate dehydrogenase gene such as zwf can be
overexpressed in the host organisms (Lim et al., J. Bioscience and
Bioengineering, 2002, 93(6), 543-549).
[0195] In some embodiments, where pathways require excess NADPH
co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant
fructose 1,6 diphosphatase gene such as fbp can be overexpressed in
the host organisms (Becker et al., J. Biotechnol., 2007,
132:99-109).
[0196] In some embodiments, where pathways require excess NADPH
co-factor in the synthesis of a C5 building block, endogenous
triose phosphate isomerase (EC 5.3.1.1) can be attenuated.
[0197] In some embodiments, where pathways require excess NADPH
co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant
glucose dehydrogenase such as the gene product of gdh can be
overexpressed in the host organism (Satoh et al., J. Bioscience and
Bioengineering, 2003, 95(4):335-341).
[0198] In some embodiments, endogenous enzymes facilitating the
conversion of NADPH to NADH can be attenuated, such as the NADH
generation cycle that may be generated via inter-conversion of
glutamate dehydrogenases classified under EC 1.4.1.2
(NADH-specific) and EC 1.4.1.4 (NADPH-specific).
[0199] In some embodiments, an endogenous glutamate dehydrogenase
(EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be
attenuated.
[0200] In some embodiments, a membrane-bound enoyl-CoA reductase
can be solubilized via expression as a fusion protein to a small
soluble protein such as a maltose binding protein (Gloerich et al.,
FEBS Letters, 2006, 580, 2092-2096).
[0201] In some embodiments using hosts that naturally accumulate
polyhydroxyalkanoates, the endogenous polyhydroxyalkanoate synthase
enzymes can be attenuated in the host strain.
[0202] In some embodiments using hosts that naturally accumulate
lipid bodies, the genes encoding enzymes involved with lipid body
synthesis are attenuated.
[0203] In some embodiments, a L-glutamate dehydrogenase, a
L-glutamine synthetase, or a glutamate synthase can be
overexpressed in the host to regenerate L-glutamate from
2-oxoglutarate as an amino donor for .omega.-transaminase
reactions.
[0204] In some embodiments, enzymes such as pimeloyl-CoA
dehydrogenase classified under, EC 1.3.1.62; an acyl-CoA
dehydrogenase classified, for example, under EC 1.3.8.7 or EC
1.3.8.1; and/or a glutaryl-CoA dehydrogenase classified, for
example, under EC 1.3.8.6 that degrade central metabolites and
central precursors leading to and including C5 building blocks can
be attenuated.
[0205] In some embodiments, endogenous enzymes activating C5
building blocks via Coenzyme A esterification such as CoA-ligases
(e.g., a glutaryl-CoA synthetase) classified under, for example, EC
6.2.1.6 can be attenuated.
Producing 2,4-Pentadienoyl-CoA Using a Recombinant Host
[0206] Typically, 2,4-pentadienoyl-CoA can be produced by providing
a host microorganism and culturing the provided microorganism with
a culture medium containing a suitable carbon source as described
above. In general, the culture media and/or culture conditions can
be such that the microorganisms grow to an adequate density and
produce 2,4-pentadienoyl-CoA efficiently. For large-scale
production processes, any method can be used such as those
described elsewhere (Manual of Industrial Microbiology and
Biotechnology, 2.sup.nd Edition, Editors: A. L. Demain and J. E.
Davies, ASM Press; and Principles of Fermentation Technology, P. F.
Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g., a
100 gallon, 200 gallon, 500 gallon, or more tank) containing an
appropriate culture medium is inoculated with a particular
microorganism. After inoculation, the microorganism is incubated to
allow biomass to be produced. Once a desired biomass is reached,
the broth containing the microorganisms can be transferred to a
second tank. This second tank can be any size. For example, the
second tank can be larger, smaller, or the same size as the first
tank. Typically, the second tank is larger than the first such that
additional culture medium can be added to the broth from the first
tank. In addition, the culture medium within this second tank can
be the same as, or different from, that used in the first tank.
[0207] Once transferred, the microorganisms can be incubated to
allow for the production of 2,4-pentadienoyl-CoA. Once produced,
any method can be used to produce 1,3-butadiene from
2,4-pentadienoyl-CoA such as depicted in FIG. 7. The invention is
further described in the following examples, which do not limit the
scope of the invention described in the claims.
EXAMPLES
Example 1
Enzyme Activity of Carboxylate Reductase Using Glutarate Methyl
Ester as Substrate and Forming Glutarate Semialdehyde Methyl
Ester
[0208] A nucleotide sequence encoding a His-tag was added to the
genes from Mycobacterium marinum, Mycobacterium smegmatis,
Segniliparus rugosus, Mycobacterium massiliense, and Segniliparus
rotundus that encode the carboxylate reductases of SEQ ID NOs: 2-4,
6 and 7, respectively (GenBank Accession Nos. ACC40567.1,
ABK71854.1, EFV11917.1, EIV11143.1, and ADG98140.1, respectively)
(see FIG. 8) such that N-terminal HIS tagged carboxylate reductases
could be produced. Each of the modified genes was cloned into a pET
Duet expression vector alongside a sfp gene encoding a His-tagged
phosphopantetheine transferase from Bacillus subtilis, both under
control of the T7 promoter. Each expression vector was transformed
into a BL21[DE3] E. coli host along with the expression vectors
from Example 2. Each resulting recombinant E. coli strain was
cultivated at 37.degree. C. in a 250 mL shake flask culture
containing 50 mL LB media and antibiotic selection pressure, with
shaking at 230 rpm. Each culture was induced overnight at
37.degree. C. using an auto-induction media.
[0209] The pellet from each induced shake flask culture was
harvested via centrifugation. Each pellet was resuspended and lysed
via sonication. The cell debris was separated from the supernatant
via centrifugation. The carboxylate reductases and
phosphopantetheine transferase were purified from the supernatant
using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES
buffer (pH=7.5) and concentrated via ultrafiltration.
[0210] The enzyme activity assay was performed in triplicate in a
buffer composed of a final concentration of 50 mM HEPES buffer
(pH=7.5), 2 mM glutarate methyl ester, 10 mM MgCl.sub.2, 1 mM ATP
and 1 mM NADPH. The enzyme activity assay reaction was initiated by
adding purified carboxylate reductase and phosphopantetheine
transferase or the empty vector control to the assay buffer
containing the glutarate methyl ester and then incubated at room
temperature for 20 min. The consumption of NADPH was monitored by
absorbance at 340 nm. The enzyme only control without glutarate
methyl ester demonstrated low base line consumption of NADPH. See
FIG. 9.
[0211] The gene product of SEQ ID NO 2, 3, 6 and 7, enhanced by the
gene product of sfp, accepted glutarate methyl ester as substrate
as confirmed against the empty vector control (see FIG. 10) and
synthesized glutarate semialdehyde methyl ester.
Example 2
Enzyme Activity of Pimeloyl-[acp] Methyl Ester Methylesterase Using
Glutaryl-CoA Methyl Ester as Substrate and Forming Glutaryl-CoA
[0212] A sequence encoding an C-terminal His-tag was added to the
gene from Escherichia coli encoding the pimeloyl-[acp] methyl ester
methylesterase of SEQ ID NO: 1 (see FIG. 8) such that C-terminal
HIS tagged pimeloyl-[acp] methyl ester methylesterase could be
produced. The resulting modified gene was cloned into a pET28b+
expression vector under control of the T7 promoter and the
expression vector was transformed into a BL21[DE3] E. coli host.
The resulting recombinant E. coli strain was cultivated at
37.degree. C. in a 500 mL shake flask culture containing 100 mL LB
media and antibiotic selection pressure, with shaking at 230 rpm.
Each culture was induced overnight at 18.degree. C. using 0.3 mM
IPTG.
[0213] The pellet from each induced shake flask culture was
harvested via centrifugation. Each pellet was resuspended and lysed
via sonication. The cell debris was separated from the supernatant
via centrifugation. The pimeloyl-[acp] methyl ester methylesterase
was purified from the supernatant using Ni-affinity chromatography,
buffer exchanged and concentrated into 20 mM HEPES buffer (pH=7.5)
via ultrafiltration and stored at 4.degree. C.
[0214] Enzyme activity assays converting glutaryl-CoA methyl ester
to glutaryl-CoA were performed in triplicate in a buffer composed
of a final concentration of 25 mM Tris.HCl buffer (pH=7.0) and 5
[mM] glutaryl-CoA methyl ester. The enzyme activity assay reaction
was initiated by adding pimeloyl-[acp] methyl ester methylesterase
to a final concentration of 10 [.mu.M] to the assay buffer
containing the glutaryl-CoA methyl ester and incubated at
30.degree. C. for 1 h, with shaking at 250 rpm. The formation of
glutaryl-CoA was quantified via LC-MS.
[0215] The substrate only control without enzyme showed no trace
quantities of the substrate glutaryl-CoA. See FIG. 11. The
pimeloyl-[acp] methyl ester methylesterase of SEQ ID NO. 1 accepted
glutaryl-CoA methyl ester as substrate and synthesized glutaryl-CoA
as reaction product as confirmed via LC-MS. See FIG. 11.
Other Embodiments
[0216] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
171256PRTEscherichia coli 1Met Asn Asn Ile Trp Trp Gln Thr Lys Gly
Gln Gly Asn Val His Leu1 5 10 15 Val Leu Leu His Gly Trp Gly Leu
Asn Ala Glu Val Trp Arg Cys Ile 20 25 30 Asp Glu Glu Leu Ser Ser
His Phe Thr Leu His Leu Val Asp Leu Pro 35 40 45 Gly Phe Gly Arg
Ser Arg Gly Phe Gly Ala Leu Ser Leu Ala Asp Met 50 55 60 Ala Glu
Ala Val Leu Gln Gln Ala Pro Asp Lys Ala Ile Trp Leu Gly65 70 75 80
Trp Ser Leu Gly Gly Leu Val Ala Ser Gln Ile Ala Leu Thr His Pro 85
90 95 Glu Arg Val Gln Ala Leu Val Thr Val Ala Ser Ser Pro Cys Phe
Ser 100 105 110 Ala Arg Asp Glu Trp Pro Gly Ile Lys Pro Asp Val Leu
Ala Gly Phe 115 120 125 Gln Gln Gln Leu Ser Asp Asp Phe Gln Arg Thr
Val Glu Arg Phe Leu 130 135 140 Ala Leu Gln Thr Met Gly Thr Glu Thr
Ala Arg Gln Asp Ala Arg Ala145 150 155 160 Leu Lys Lys Thr Val Leu
Ala Leu Pro Met Pro Glu Val Asp Val Leu 165 170 175 Asn Gly Gly Leu
Glu Ile Leu Lys Thr Val Asp Leu Arg Gln Pro Leu 180 185 190 Gln Asn
Val Ser Met Pro Phe Leu Arg Leu Tyr Gly Tyr Leu Asp Gly 195 200 205
Leu Val Pro Arg Lys Val Val Pro Met Leu Asp Lys Leu Trp Pro His 210
215 220 Ser Glu Ser Tyr Ile Phe Ala Lys Ala Ala His Ala Pro Phe Ile
Ser225 230 235 240 His Pro Ala Glu Phe Cys His Leu Leu Val Ala Leu
Lys Gln Arg Val 245 250 255 21174PRTMycobacterium marinum 2Met Ser
Pro Ile Thr Arg Glu Glu Arg Leu Glu Arg Arg Ile Gln Asp1 5 10 15
Leu Tyr Ala Asn Asp Pro Gln Phe Ala Ala Ala Lys Pro Ala Thr Ala 20
25 30 Ile Thr Ala Ala Ile Glu Arg Pro Gly Leu Pro Leu Pro Gln Ile
Ile 35 40 45 Glu Thr Val Met Thr Gly Tyr Ala Asp Arg Pro Ala Leu
Ala Gln Arg 50 55 60 Ser Val Glu Phe Val Thr Asp Ala Gly Thr Gly
His Thr Thr Leu Arg65 70 75 80 Leu Leu Pro His Phe Glu Thr Ile Ser
Tyr Gly Glu Leu Trp Asp Arg 85 90 95 Ile Ser Ala Leu Ala Asp Val
Leu Ser Thr Glu Gln Thr Val Lys Pro 100 105 110 Gly Asp Arg Val Cys
Leu Leu Gly Phe Asn Ser Val Asp Tyr Ala Thr 115 120 125 Ile Asp Met
Thr Leu Ala Arg Leu Gly Ala Val Ala Val Pro Leu Gln 130 135 140 Thr
Ser Ala Ala Ile Thr Gln Leu Gln Pro Ile Val Ala Glu Thr Gln145 150
155 160 Pro Thr Met Ile Ala Ala Ser Val Asp Ala Leu Ala Asp Ala Thr
Glu 165 170 175 Leu Ala Leu Ser Gly Gln Thr Ala Thr Arg Val Leu Val
Phe Asp His 180 185 190 His Arg Gln Val Asp Ala His Arg Ala Ala Val
Glu Ser Ala Arg Glu 195 200 205 Arg Leu Ala Gly Ser Ala Val Val Glu
Thr Leu Ala Glu Ala Ile Ala 210 215 220 Arg Gly Asp Val Pro Arg Gly
Ala Ser Ala Gly Ser Ala Pro Gly Thr225 230 235 240 Asp Val Ser Asp
Asp Ser Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser 245 250 255 Thr Gly
Ala Pro Lys Gly Ala Met Tyr Pro Arg Arg Asn Val Ala Thr 260 265 270
Phe Trp Arg Lys Arg Thr Trp Phe Glu Gly Gly Tyr Glu Pro Ser Ile 275
280 285 Thr Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gln Ile
Leu 290 295 300 Tyr Gly Thr Leu Cys Asn Gly Gly Thr Ala Tyr Phe Val
Ala Lys Ser305 310 315 320 Asp Leu Ser Thr Leu Phe Glu Asp Leu Ala
Leu Val Arg Pro Thr Glu 325 330 335 Leu Thr Phe Val Pro Arg Val Trp
Asp Met Val Phe Asp Glu Phe Gln 340 345 350 Ser Glu Val Asp Arg Arg
Leu Val Asp Gly Ala Asp Arg Val Ala Leu 355 360 365 Glu Ala Gln Val
Lys Ala Glu Ile Arg Asn Asp Val Leu Gly Gly Arg 370 375 380 Tyr Thr
Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Asp Glu Met Lys385 390 395
400 Ala Trp Val Glu Glu Leu Leu Asp Met His Leu Val Glu Gly Tyr Gly
405 410 415 Ser Thr Glu Ala Gly Met Ile Leu Ile Asp Gly Ala Ile Arg
Arg Pro 420 425 430 Ala Val Leu Asp Tyr Lys Leu Val Asp Val Pro Asp
Leu Gly Tyr Phe 435 440 445 Leu Thr Asp Arg Pro His Pro Arg Gly Glu
Leu Leu Val Lys Thr Asp 450 455 460 Ser Leu Phe Pro Gly Tyr Tyr Gln
Arg Ala Glu Val Thr Ala Asp Val465 470 475 480 Phe Asp Ala Asp Gly
Phe Tyr Arg Thr Gly Asp Ile Met Ala Glu Val 485 490 495 Gly Pro Glu
Gln Phe Val Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys 500 505 510 Leu
Ser Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe 515 520
525 Gly Asp Ser Pro Leu Val Arg Gln Ile Tyr Ile Tyr Gly Asn Ser Ala
530 535 540 Arg Ala Tyr Leu Leu Ala Val Ile Val Pro Thr Gln Glu Ala
Leu Asp545 550 555 560 Ala Val Pro Val Glu Glu Leu Lys Ala Arg Leu
Gly Asp Ser Leu Gln 565 570 575 Glu Val Ala Lys Ala Ala Gly Leu Gln
Ser Tyr Glu Ile Pro Arg Asp 580 585 590 Phe Ile Ile Glu Thr Thr Pro
Trp Thr Leu Glu Asn Gly Leu Leu Thr 595 600 605 Gly Ile Arg Lys Leu
Ala Arg Pro Gln Leu Lys Lys His Tyr Gly Glu 610 615 620 Leu Leu Glu
Gln Ile Tyr Thr Asp Leu Ala His Gly Gln Ala Asp Glu625 630 635 640
Leu Arg Ser Leu Arg Gln Ser Gly Ala Asp Ala Pro Val Leu Val Thr 645
650 655 Val Cys Arg Ala Ala Ala Ala Leu Leu Gly Gly Ser Ala Ser Asp
Val 660 665 670 Gln Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser
Leu Ser Ala 675 680 685 Leu Ser Phe Thr Asn Leu Leu His Glu Ile Phe
Asp Ile Glu Val Pro 690 695 700 Val Gly Val Ile Val Ser Pro Ala Asn
Asp Leu Gln Ala Leu Ala Asp705 710 715 720 Tyr Val Glu Ala Ala Arg
Lys Pro Gly Ser Ser Arg Pro Thr Phe Ala 725 730 735 Ser Val His Gly
Ala Ser Asn Gly Gln Val Thr Glu Val His Ala Gly 740 745 750 Asp Leu
Ser Leu Asp Lys Phe Ile Asp Ala Ala Thr Leu Ala Glu Ala 755 760 765
Pro Arg Leu Pro Ala Ala Asn Thr Gln Val Arg Thr Val Leu Leu Thr 770
775 780 Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu
Glu785 790 795 800 Arg Met Asp Leu Val Asp Gly Lys Leu Ile Cys Leu
Val Arg Ala Lys 805 810 815 Ser Asp Thr Glu Ala Arg Ala Arg Leu Asp
Lys Thr Phe Asp Ser Gly 820 825 830 Asp Pro Glu Leu Leu Ala His Tyr
Arg Ala Leu Ala Gly Asp His Leu 835 840 845 Glu Val Leu Ala Gly Asp
Lys Gly Glu Ala Asp Leu Gly Leu Asp Arg 850 855 860 Gln Thr Trp Gln
Arg Leu Ala Asp Thr Val Asp Leu Ile Val Asp Pro865 870 875 880 Ala
Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu Phe Gly Pro 885 890
895 Asn Ala Leu Gly Thr Ala Glu Leu Leu Arg Leu Ala Leu Thr Ser Lys
900 905 910 Ile Lys Pro Tyr Ser Tyr Thr Ser Thr Ile Gly Val Ala Asp
Gln Ile 915 920 925 Pro Pro Ser Ala Phe Thr Glu Asp Ala Asp Ile Arg
Val Ile Ser Ala 930 935 940 Thr Arg Ala Val Asp Asp Ser Tyr Ala Asn
Gly Tyr Ser Asn Ser Lys945 950 955 960 Trp Ala Gly Glu Val Leu Leu
Arg Glu Ala His Asp Leu Cys Gly Leu 965 970 975 Pro Val Ala Val Phe
Arg Cys Asp Met Ile Leu Ala Asp Thr Thr Trp 980 985 990 Ala Gly Gln
Leu Asn Val Pro Asp Met Phe Thr Arg Met Ile Leu Ser 995 1000 1005
Leu Ala Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glu Leu Ala Ala
1010 1015 1020 Asp Gly Ala Arg Gln Arg Ala His Tyr Asp Gly Leu Pro
Val Glu Phe1025 1030 1035 1040 Ile Ala Glu Ala Ile Ser Thr Leu Gly
Ala Gln Ser Gln Asp Gly Phe 1045 1050 1055 His Thr Tyr His Val Met
Asn Pro Tyr Asp Asp Gly Ile Gly Leu Asp 1060 1065 1070 Glu Phe Val
Asp Trp Leu Asn Glu Ser Gly Cys Pro Ile Gln Arg Ile 1075 1080 1085
Ala Asp Tyr Gly Asp Trp Leu Gln Arg Phe Glu Thr Ala Leu Arg Ala
1090 1095 1100 Leu Pro Asp Arg Gln Arg His Ser Ser Leu Leu Pro Leu
Leu His Asn1105 1110 1115 1120 Tyr Arg Gln Pro Glu Arg Pro Val Arg
Gly Ser Ile Ala Pro Thr Asp 1125 1130 1135 Arg Phe Arg Ala Ala Val
Gln Glu Ala Lys Ile Gly Pro Asp Lys Asp 1140 1145 1150 Ile Pro His
Val Gly Ala Pro Ile Ile Val Lys Tyr Val Ser Asp Leu 1155 1160 1165
Arg Leu Leu Gly Leu Leu 1170 31173PRTMycobacterium smegmatis 3Met
Thr Ser Asp Val His Asp Ala Thr Asp Gly Val Thr Glu Thr Ala1 5 10
15 Leu Asp Asp Glu Gln Ser Thr Arg Arg Ile Ala Glu Leu Tyr Ala Thr
20 25 30 Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val
Asp Ala 35 40 45 Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu
Gln Thr Leu Phe 50 55 60 Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly
Tyr Arg Ala Arg Glu Leu65 70 75 80 Ala Thr Asp Glu Gly Gly Arg Thr
Val Thr Arg Leu Leu Pro Arg Phe 85 90 95 Asp Thr Leu Thr Tyr Ala
Gln Val Trp Ser Arg Val Gln Ala Val Ala 100 105 110 Ala Ala Leu Arg
His Asn Phe Ala Gln Pro Ile Tyr Pro Gly Asp Ala 115 120 125 Val Ala
Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu Thr Leu Asp Leu 130 135 140
Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu Gln His Asn Ala145
150 155 160 Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val Glu Pro
Arg Ile 165 170 175 Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val
Glu Ser Val Arg 180 185 190 Asp Val Asn Ser Val Ser Gln Leu Val Val
Phe Asp His His Pro Glu 195 200 205 Val Asp Asp His Arg Asp Ala Leu
Ala Arg Ala Arg Glu Gln Leu Ala 210 215 220 Gly Lys Gly Ile Ala Val
Thr Thr Leu Asp Ala Ile Ala Asp Glu Gly225 230 235 240 Ala Gly Leu
Pro Ala Glu Pro Ile Tyr Thr Ala Asp His Asp Gln Arg 245 250 255 Leu
Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly 260 265
270 Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp Thr Met Ser Phe
275 280 285 Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met
Pro Leu 290 295 300 Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala
Val Gln Asn Gly305 310 315 320 Gly Thr Ser Tyr Phe Val Pro Glu Ser
Asp Met Ser Thr Leu Phe Glu 325 330 335 Asp Leu Ala Leu Val Arg Pro
Thr Glu Leu Gly Leu Val Pro Arg Val 340 345 350 Ala Asp Met Leu Tyr
Gln His His Leu Ala Thr Val Asp Arg Leu Val 355 360 365 Thr Gln Gly
Ala Asp Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala Glu 370 375 380 Leu
Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr Gly Phe Val Ser385 390
395 400 Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr
Leu 405 410 415 Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr
Gly Ala Val 420 425 430 Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val
Ile Asp Tyr Lys Leu 435 440 445 Ile Asp Val Pro Glu Leu Gly Tyr Phe
Ser Thr Asp Lys Pro Tyr Pro 450 455 460 Arg Gly Glu Leu Leu Val Arg
Ser Gln Thr Leu Thr Pro Gly Tyr Tyr465 470 475 480 Lys Arg Pro Glu
Val Thr Ala Ser Val Phe Asp Arg Asp Gly Tyr Tyr 485 490 495 His Thr
Gly Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val Tyr 500 505 510
Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val 515
520 525 Ala Val Ala Asn Leu Glu Ala Val Phe Ser Gly Ala Ala Leu Val
Arg 530 535 540 Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe Leu
Leu Ala Val545 550 555 560 Val Val Pro Thr Pro Glu Ala Leu Glu Gln
Tyr Asp Pro Ala Ala Leu 565 570 575 Lys Ala Ala Leu Ala Asp Ser Leu
Gln Arg Thr Ala Arg Asp Ala Glu 580 585 590 Leu Gln Ser Tyr Glu Val
Pro Ala Asp Phe Ile Val Glu Thr Glu Pro 595 600 605 Phe Ser Ala Ala
Asn Gly Leu Leu Ser Gly Val Gly Lys Leu Leu Arg 610 615 620 Pro Asn
Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr Ala625 630 635
640 Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu Leu Arg Arg Ala
645 650 655 Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln Ala Ala
Ala Thr 660 665 670 Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala
His Phe Thr Asp 675 680 685 Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr
Leu Ser Asn Leu Leu Ser 690 695 700 Asp Phe Phe Gly Phe Glu Val Pro
Val Gly Thr Ile Val Asn Pro Ala705 710 715 720 Thr Asn Leu Ala Gln
Leu Ala Gln His Ile Glu Ala Gln Arg Thr Ala 725 730 735 Gly Asp Arg
Arg Pro Ser Phe Thr Thr Val His Gly Ala Asp Ala Thr 740 745 750 Glu
Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala Glu 755 760
765 Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr Thr Glu Pro Arg
770 775 780 Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe
Leu Thr785 790 795 800 Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly
Gly Thr Leu Ile Thr 805 810 815 Ile Val Arg Gly Arg Asp Asp Ala Ala
Ala Arg Ala Arg Leu Thr Gln 820 825 830 Ala Tyr Asp Thr Asp Pro Glu
Leu Ser Arg Arg Phe Ala Glu Leu Ala 835 840 845 Asp Arg His Leu Arg
Val Val Ala Gly Asp Ile Gly Asp Pro Asn Leu 850 855 860 Gly Leu Thr
Pro Glu Ile Trp His Arg Leu Ala Ala Glu Val Asp Leu865
870 875 880 Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr
Arg Gln 885 890 895 Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val
Ile Lys Leu Ala 900 905 910 Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr
Leu Ser Thr Val Ser Val 915 920 925 Ala Met Gly Ile Pro Asp Phe Glu
Glu Asp Gly Asp Ile Arg Thr Val 930 935 940 Ser Pro Val Arg Pro Leu
Asp Gly Gly Tyr Ala Asn Gly Tyr Gly Asn945 950 955 960 Ser Lys Trp
Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys 965 970 975 Gly
Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His Pro 980 985
990 Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met Phe Thr Arg Leu Leu
995 1000 1005 Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe
Tyr Ile Gly 1010 1015 1020 Asp Gly Glu Arg Pro Arg Ala His Tyr Pro
Gly Leu Thr Val Asp Phe1025 1030 1035 1040 Val Ala Glu Ala Val Thr
Thr Leu Gly Ala Gln Gln Arg Glu Gly Tyr 1045 1050 1055 Val Ser Tyr
Asp Val Met Asn Pro His Asp Asp Gly Ile Ser Leu Asp 1060 1065 1070
Val Phe Val Asp Trp Leu Ile Arg Ala Gly His Pro Ile Asp Arg Val
1075 1080 1085 Asp Asp Tyr Asp Asp Trp Val Arg Arg Phe Glu Thr Ala
Leu Thr Ala 1090 1095 1100 Leu Pro Glu Lys Arg Arg Ala Gln Thr Val
Leu Pro Leu Leu His Ala1105 1110 1115 1120 Phe Arg Ala Pro Gln Ala
Pro Leu Arg Gly Ala Pro Glu Pro Thr Glu 1125 1130 1135 Val Phe His
Ala Ala Val Arg Thr Ala Lys Val Gly Pro Gly Asp Ile 1140 1145 1150
Pro His Leu Asp Glu Ala Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg
1155 1160 1165 Glu Phe Gly Leu Ile 1170 41148PRTSegniliparus
rugosus 4Met Gly Asp Gly Glu Glu Arg Ala Lys Arg Phe Phe Gln Arg
Ile Gly1 5 10 15 Glu Leu Ser Ala Thr Asp Pro Gln Phe Ala Ala Ala
Ala Pro Asp Pro 20 25 30 Ala Val Val Glu Ala Val Ser Asp Pro Ser
Leu Ser Phe Thr Arg Tyr 35 40 45 Leu Asp Thr Leu Met Arg Gly Tyr
Ala Glu Arg Pro Ala Leu Ala His 50 55 60 Arg Val Gly Ala Gly Tyr
Glu Thr Ile Ser Tyr Gly Glu Leu Trp Ala65 70 75 80 Arg Val Gly Ala
Ile Ala Ala Ala Trp Gln Ala Asp Gly Leu Ala Pro 85 90 95 Gly Asp
Phe Val Ala Thr Val Gly Phe Thr Ser Pro Asp Tyr Val Ala 100 105 110
Val Asp Leu Ala Ala Ala Arg Ser Gly Leu Val Ser Val Pro Leu Gln 115
120 125 Ala Gly Ala Ser Leu Ala Gln Leu Val Gly Ile Leu Glu Glu Thr
Glu 130 135 140 Pro Lys Val Leu Ala Ala Ser Ala Ser Ser Leu Glu Gly
Ala Val Ala145 150 155 160 Cys Ala Leu Ala Ala Pro Ser Val Gln Arg
Leu Val Val Phe Asp Leu 165 170 175 Arg Gly Pro Asp Ala Ser Glu Ser
Ala Ala Asp Glu Arg Arg Gly Ala 180 185 190 Leu Ala Asp Ala Glu Glu
Gln Leu Ala Arg Ala Gly Arg Ala Val Val 195 200 205 Val Glu Thr Leu
Ala Asp Leu Ala Ala Arg Gly Glu Ala Leu Pro Glu 210 215 220 Ala Pro
Leu Phe Glu Pro Ala Glu Gly Glu Asp Pro Leu Ala Leu Leu225 230 235
240 Ile Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Ala Met Tyr Ser
245 250 255 Gln Arg Leu Val Ser Gln Leu Trp Gly Arg Thr Pro Val Val
Pro Gly 260 265 270 Met Pro Asn Ile Ser Leu His Tyr Met Pro Leu Ser
His Ser Tyr Gly 275 280 285 Arg Ala Val Leu Ala Gly Ala Leu Ser Ala
Gly Gly Thr Ala His Phe 290 295 300 Thr Ala Asn Ser Asp Leu Ser Thr
Leu Phe Glu Asp Ile Ala Leu Ala305 310 315 320 Arg Pro Thr Phe Leu
Ala Leu Val Pro Arg Val Cys Glu Met Leu Phe 325 330 335 Gln Glu Ser
Gln Arg Gly Gln Asp Val Ala Glu Leu Arg Glu Arg Val 340 345 350 Leu
Gly Gly Arg Leu Leu Val Ala Val Cys Gly Ser Ala Pro Leu Ser 355 360
365 Pro Glu Met Arg Ala Phe Met Glu Glu Val Leu Gly Phe Pro Leu Leu
370 375 380 Asp Gly Tyr Gly Ser Thr Glu Ala Leu Gly Val Met Arg Asn
Gly Ile385 390 395 400 Ile Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu
Val Asp Val Pro Glu 405 410 415 Leu Gly Tyr Arg Thr Thr Asp Lys Pro
Tyr Pro Arg Gly Glu Leu Cys 420 425 430 Ile Arg Ser Thr Ser Leu Ile
Ser Gly Tyr Tyr Lys Arg Pro Glu Ile 435 440 445 Thr Ala Glu Val Phe
Asp Ala Gln Gly Tyr Tyr Lys Thr Gly Asp Val 450 455 460 Met Ala Glu
Ile Ala Pro Asp His Leu Val Tyr Val Asp Arg Ser Lys465 470 475 480
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Lys Leu 485
490 495 Glu Ala Ala Tyr Gly Thr Ser Pro Tyr Val Lys Gln Ile Phe Val
Tyr 500 505 510 Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val
Pro Asn Ala 515 520 525 Glu Val Leu Gly Ala Arg Asp Gln Glu Glu Ala
Lys Pro Leu Ile Ala 530 535 540 Ala Ser Leu Gln Lys Ile Ala Lys Glu
Ala Gly Leu Gln Ser Tyr Glu545 550 555 560 Val Pro Arg Asp Phe Leu
Ile Glu Thr Glu Pro Phe Thr Thr Gln Asn 565 570 575 Gly Leu Leu Ser
Glu Val Gly Lys Leu Leu Arg Pro Lys Leu Lys Ala 580 585 590 Arg Tyr
Gly Glu Ala Leu Glu Ala Arg Tyr Asp Glu Ile Ala His Gly 595 600 605
Gln Ala Asp Glu Leu Arg Ala Leu Arg Asp Gly Ala Gly Gln Arg Pro 610
615 620 Val Val Glu Thr Val Val Arg Ala Ala Val Ala Ile Ser Gly Ser
Glu625 630 635 640 Gly Ala Glu Val Gly Pro Glu Ala Asn Phe Ala Asp
Leu Gly Gly Asp 645 650 655 Ser Leu Ser Ala Leu Ser Leu Ala Asn Leu
Leu His Asp Val Phe Glu 660 665 670 Val Glu Val Pro Val Arg Ile Ile
Ile Gly Pro Thr Ala Ser Leu Ala 675 680 685 Gly Ile Ala Lys His Ile
Glu Ala Glu Arg Ala Gly Ala Ser Ala Pro 690 695 700 Thr Ala Ala Ser
Val His Gly Ala Gly Ala Thr Arg Ile Arg Ala Ser705 710 715 720 Glu
Leu Thr Leu Glu Lys Phe Leu Pro Glu Asp Leu Leu Ala Ala Ala 725 730
735 Lys Gly Leu Pro Ala Ala Asp Gln Val Arg Thr Val Leu Leu Thr Gly
740 745 750 Ala Asn Gly Trp Leu Gly Arg Phe Leu Ala Leu Glu Gln Leu
Glu Arg 755 760 765 Leu Ala Arg Ser Gly Gln Asp Gly Gly Lys Leu Ile
Cys Leu Val Arg 770 775 780 Gly Lys Asp Ala Ala Ala Ala Arg Arg Arg
Ile Glu Glu Thr Leu Gly785 790 795 800 Thr Asp Pro Ala Leu Ala Ala
Arg Phe Ala Glu Leu Ala Glu Gly Arg 805 810 815 Leu Glu Val Val Pro
Gly Asp Val Gly Glu Pro Lys Phe Gly Leu Asp 820 825 830 Asp Ala Ala
Trp Asp Arg Leu Ala Glu Glu Val Asp Val Ile Val His 835 840 845 Pro
Ala Ala Leu Val Asn His Val Leu Pro Tyr His Gln Leu Phe Gly 850 855
860 Pro Asn Val Val Gly Thr Ala Glu Ile Ile Arg Leu Ala Ile Thr
Ala865 870 875 880 Lys Arg Lys Pro Val Thr Tyr Leu Ser Thr Val Ala
Val Ala Ala Gly 885 890 895 Val Glu Pro Ser Ser Phe Glu Glu Asp Gly
Asp Ile Arg Ala Val Val 900 905 910 Pro Glu Arg Pro Leu Gly Asp Gly
Tyr Ala Asn Gly Tyr Gly Asn Ser 915 920 925 Lys Trp Ala Gly Glu Val
Leu Leu Arg Glu Ala His Glu Leu Val Gly 930 935 940 Leu Pro Val Ala
Val Phe Arg Ser Asp Met Ile Leu Ala His Thr Arg945 950 955 960 Tyr
Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu Val Leu 965 970
975 Ser Leu Leu Ala Thr Gly Ile Ala Pro Lys Ser Phe Tyr Gln Gln Gly
980 985 990 Ala Ala Gly Glu Arg Gln Arg Ala His Tyr Asp Gly Ile Pro
Val Asp 995 1000 1005 Phe Thr Ala Glu Ala Ile Thr Thr Leu Gly Ala
Glu Pro Ser Trp Phe 1010 1015 1020 Asp Gly Gly Ala Gly Phe Arg Ser
Phe Asp Val Phe Asn Pro His His1025 1030 1035 1040 Asp Gly Val Gly
Leu Asp Glu Phe Val Asp Trp Leu Ile Glu Ala Gly 1045 1050 1055 His
Pro Ile Ser Arg Ile Asp Asp His Lys Glu Trp Phe Ala Arg Phe 1060
1065 1070 Glu Thr Ala Val Arg Gly Leu Pro Glu Ala Gln Arg Gln His
Ser Leu 1075 1080 1085 Leu Pro Leu Leu Arg Ala Tyr Ser Phe Pro His
Pro Pro Val Asp Gly 1090 1095 1100 Ser Val Tyr Pro Thr Gly Lys Phe
Gln Gly Ala Val Lys Ala Ala Gln1105 1110 1115 1120 Val Gly Ser Asp
His Asp Val Pro His Leu Gly Lys Ala Leu Ile Val 1125 1130 1135 Lys
Tyr Ala Asp Asp Leu Lys Ala Leu Gly Leu Leu 1140 1145
51168PRTMycobacterium smegmatis 5Met Thr Ile Glu Thr Arg Glu Asp
Arg Phe Asn Arg Arg Ile Asp His1 5 10 15 Leu Phe Glu Thr Asp Pro
Gln Phe Ala Ala Ala Arg Pro Asp Glu Ala 20 25 30 Ile Ser Ala Ala
Ala Ala Asp Pro Glu Leu Arg Leu Pro Ala Ala Val 35 40 45 Lys Gln
Ile Leu Ala Gly Tyr Ala Asp Arg Pro Ala Leu Gly Lys Arg 50 55 60
Ala Val Glu Phe Val Thr Asp Glu Glu Gly Arg Thr Thr Ala Lys Leu65
70 75 80 Leu Pro Arg Phe Asp Thr Ile Thr Tyr Arg Gln Leu Ala Gly
Arg Ile 85 90 95 Gln Ala Val Thr Asn Ala Trp His Asn His Pro Val
Asn Ala Gly Asp 100 105 110 Arg Val Ala Ile Leu Gly Phe Thr Ser Val
Asp Tyr Thr Thr Ile Asp 115 120 125 Ile Ala Leu Leu Glu Leu Gly Ala
Val Ser Val Pro Leu Gln Thr Ser 130 135 140 Ala Pro Val Ala Gln Leu
Gln Pro Ile Val Ala Glu Thr Glu Pro Lys145 150 155 160 Val Ile Ala
Ser Ser Val Asp Phe Leu Ala Asp Ala Val Ala Leu Val 165 170 175 Glu
Ser Gly Pro Ala Pro Ser Arg Leu Val Val Phe Asp Tyr Ser His 180 185
190 Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala Ala Lys Gly Lys Leu
195 200 205 Ala Gly Thr Gly Val Val Val Glu Thr Ile Thr Asp Ala Leu
Asp Arg 210 215 220 Gly Arg Ser Leu Ala Asp Ala Pro Leu Tyr Val Pro
Asp Glu Ala Asp225 230 235 240 Pro Leu Thr Leu Leu Ile Tyr Thr Ser
Gly Ser Thr Gly Thr Pro Lys 245 250 255 Gly Ala Met Tyr Pro Glu Ser
Lys Thr Ala Thr Met Trp Gln Ala Gly 260 265 270 Ser Lys Ala Arg Trp
Asp Glu Thr Leu Gly Val Met Pro Ser Ile Thr 275 280 285 Leu Asn Phe
Met Pro Met Ser His Val Met Gly Arg Gly Ile Leu Cys 290 295 300 Ser
Thr Leu Ala Ser Gly Gly Thr Ala Tyr Phe Ala Ala Arg Ser Asp305 310
315 320 Leu Ser Thr Phe Leu Glu Asp Leu Ala Leu Val Arg Pro Thr Gln
Leu 325 330 335 Asn Phe Val Pro Arg Ile Trp Asp Met Leu Phe Gln Glu
Tyr Gln Ser 340 345 350 Arg Leu Asp Asn Arg Arg Ala Glu Gly Ser Glu
Asp Arg Ala Glu Ala 355 360 365 Ala Val Leu Glu Glu Val Arg Thr Gln
Leu Leu Gly Gly Arg Phe Val 370 375 380 Ser Ala Leu Thr Gly Ser Ala
Pro Ile Ser Ala Glu Met Lys Ser Trp385 390 395 400 Val Glu Asp Leu
Leu Asp Met His Leu Leu Glu Gly Tyr Gly Ser Thr 405 410 415 Glu Ala
Gly Ala Val Phe Ile Asp Gly Gln Ile Gln Arg Pro Pro Val 420 425 430
Ile Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe Ala Thr 435
440 445 Asp Arg Pro Tyr Pro Arg Gly Glu Leu Leu Val Lys Ser Glu Gln
Met 450 455 460 Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Ile Thr Ala Glu
Met Phe Asp465 470 475 480 Glu Asp Gly Tyr Tyr Arg Thr Gly Asp Ile
Val Ala Glu Leu Gly Pro 485 490 495 Asp His Leu Glu Tyr Leu Asp Arg
Arg Asn Asn Val Leu Lys Leu Ser 500 505 510 Gln Gly Glu Phe Val Thr
Val Ser Lys Leu Glu Ala Val Phe Gly Asp 515 520 525 Ser Pro Leu Val
Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala Arg Ser 530 535 540 Tyr Leu
Leu Ala Val Val Val Pro Thr Glu Glu Ala Leu Ser Arg Trp545 550 555
560 Asp Gly Asp Glu Leu Lys Ser Arg Ile Ser Asp Ser Leu Gln Asp Ala
565 570 575 Ala Arg Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp
Phe Leu 580 585 590 Val Glu Thr Thr Pro Phe Thr Leu Glu Asn Gly Leu
Leu Thr Gly Ile 595 600 605 Arg Lys Leu Ala Arg Pro Lys Leu Lys Ala
His Tyr Gly Glu Arg Leu 610 615 620 Glu Gln Leu Tyr Thr Asp Leu Ala
Glu Gly Gln Ala Asn Glu Leu Arg625 630 635 640 Glu Leu Arg Arg Asn
Gly Ala Asp Arg Pro Val Val Glu Thr Val Ser 645 650 655 Arg Ala Ala
Val Ala Leu Leu Gly Ala Ser Val Thr Asp Leu Arg Ser 660 665 670 Asp
Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser 675 680
685 Phe Ser Asn Leu Leu His Glu Ile Phe Asp Val Asp Val Pro Val Gly
690 695 700 Val Ile Val Ser Pro Ala Thr Asp Leu Ala Gly Val Ala Ala
Tyr Ile705 710 715 720 Glu Gly Glu Leu Arg Gly Ser Lys Arg Pro Thr
Tyr Ala Ser Val His 725 730 735 Gly Arg Asp Ala Thr Glu Val Arg Ala
Arg Asp Leu Ala Leu Gly Lys 740 745 750 Phe Ile Asp Ala Lys Thr Leu
Ser Ala Ala Pro Gly Leu Pro Arg Ser 755 760 765 Gly Thr Glu Ile Arg
Thr Val Leu Leu Thr Gly Ala Thr Gly Phe Leu 770 775 780 Gly Arg Tyr
Leu Ala Leu Glu Trp Leu Glu Arg Met Asp Leu Val Asp785 790 795 800
Gly Lys Val Ile Cys Leu Val Arg Ala Arg Ser Asp Asp Glu Ala Arg 805
810 815 Ala Arg Leu Asp Ala Thr Phe Asp Thr Gly Asp Ala Thr Leu Leu
Glu 820 825 830 His Tyr Arg Ala Leu Ala Ala Asp His Leu Glu Val Ile
Ala Gly Asp 835 840 845 Lys Gly Glu Ala Asp Leu Gly Leu Asp His Asp
Thr Trp Gln Arg Leu 850
855 860 Ala Asp Thr Val Asp Leu Ile Val Asp Pro Ala Ala Leu Val Asn
His865 870 875 880 Val Leu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala
Leu Gly Thr Ala 885 890 895 Glu Leu Ile Arg Ile Ala Leu Thr Thr Thr
Ile Lys Pro Tyr Val Tyr 900 905 910 Val Ser Thr Ile Gly Val Gly Gln
Gly Ile Ser Pro Glu Ala Phe Val 915 920 925 Glu Asp Ala Asp Ile Arg
Glu Ile Ser Ala Thr Arg Arg Val Asp Asp 930 935 940 Ser Tyr Ala Asn
Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu945 950 955 960 Leu
Arg Glu Ala His Asp Trp Cys Gly Leu Pro Val Ser Val Phe Arg 965 970
975 Cys Asp Met Ile Leu Ala Asp Thr Thr Tyr Ser Gly Gln Leu Asn Leu
980 985 990 Pro Asp Met Phe Thr Arg Leu Met Leu Ser Leu Val Ala Thr
Gly Ile 995 1000 1005 Ala Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp
Gly Asn Arg Gln Arg 1010 1015 1020 Ala His Tyr Asp Gly Leu Pro Val
Glu Phe Ile Ala Glu Ala Ile Ser1025 1030 1035 1040 Thr Ile Gly Ser
Gln Val Thr Asp Gly Phe Glu Thr Phe His Val Met 1045 1050 1055 Asn
Pro Tyr Asp Asp Gly Ile Gly Leu Asp Glu Tyr Val Asp Trp Leu 1060
1065 1070 Ile Glu Ala Gly Tyr Pro Val His Arg Val Asp Asp Tyr Ala
Thr Trp 1075 1080 1085 Leu Ser Arg Phe Glu Thr Ala Leu Arg Ala Leu
Pro Glu Arg Gln Arg 1090 1095 1100 Gln Ala Ser Leu Leu Pro Leu Leu
His Asn Tyr Gln Gln Pro Ser Pro1105 1110 1115 1120 Pro Val Cys Gly
Ala Met Ala Pro Thr Asp Arg Phe Arg Ala Ala Val 1125 1130 1135 Gln
Asp Ala Lys Ile Gly Pro Asp Lys Asp Ile Pro His Val Thr Ala 1140
1145 1150 Asp Val Ile Val Lys Tyr Ile Ser Asn Leu Gln Met Leu Gly
Leu Leu 1155 1160 1165 61185PRTMycobacterium massiliense 6Met Thr
Asn Glu Thr Asn Pro Gln Gln Glu Gln Leu Ser Arg Arg Ile1 5 10 15
Glu Ser Leu Arg Glu Ser Asp Pro Gln Phe Arg Ala Ala Gln Pro Asp 20
25 30 Pro Ala Val Ala Glu Gln Val Leu Arg Pro Gly Leu His Leu Ser
Glu 35 40 45 Ala Ile Ala Ala Leu Met Thr Gly Tyr Ala Glu Arg Pro
Ala Leu Gly 50 55 60 Glu Arg Ala Arg Glu Leu Val Ile Asp Gln Asp
Gly Arg Thr Thr Leu65 70 75 80 Arg Leu Leu Pro Arg Phe Asp Thr Thr
Thr Tyr Gly Glu Leu Trp Ser 85 90 95 Arg Thr Thr Ser Val Ala Ala
Ala Trp His His Asp Ala Thr His Pro 100 105 110 Val Lys Ala Gly Asp
Leu Val Ala Thr Leu Gly Phe Thr Ser Ile Asp 115 120 125 Tyr Thr Val
Leu Asp Leu Ala Ile Met Ile Leu Gly Gly Val Ala Val 130 135 140 Pro
Leu Gln Thr Ser Ala Pro Ala Ser Gln Trp Thr Thr Ile Leu Ala145 150
155 160 Glu Ala Glu Pro Asn Thr Leu Ala Val Ser Ile Glu Leu Ile Gly
Ala 165 170 175 Ala Met Glu Ser Val Arg Ala Thr Pro Ser Ile Lys Gln
Val Val Val 180 185 190 Phe Asp Tyr Thr Pro Glu Val Asp Asp Gln Arg
Glu Ala Phe Glu Ala 195 200 205 Ala Ser Thr Gln Leu Ala Gly Thr Gly
Ile Ala Leu Glu Thr Leu Asp 210 215 220 Ala Val Ile Ala Arg Gly Ala
Ala Leu Pro Ala Ala Pro Leu Tyr Ala225 230 235 240 Pro Ser Ala Gly
Asp Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly 245 250 255 Ser Thr
Gly Ala Pro Lys Gly Ala Met His Ser Glu Asn Ile Val Arg 260 265 270
Arg Trp Trp Ile Arg Glu Asp Val Met Ala Gly Thr Glu Asn Leu Pro 275
280 285 Met Ile Gly Leu Asn Phe Met Pro Met Ser His Ile Met Gly Arg
Gly 290 295 300 Thr Leu Thr Ser Thr Leu Ser Thr Gly Gly Thr Gly Tyr
Phe Ala Ala305 310 315 320 Ser Ser Asp Met Ser Thr Leu Phe Glu Asp
Met Glu Leu Ile Arg Pro 325 330 335 Thr Ala Leu Ala Leu Val Pro Arg
Val Cys Asp Met Val Phe Gln Arg 340 345 350 Phe Gln Thr Glu Val Asp
Arg Arg Leu Ala Ser Gly Asp Thr Ala Ser 355 360 365 Ala Glu Ala Val
Ala Ala Glu Val Lys Ala Asp Ile Arg Asp Asn Leu 370 375 380 Phe Gly
Gly Arg Val Ser Ala Val Met Val Gly Ser Ala Pro Leu Ser385 390 395
400 Glu Glu Leu Gly Glu Phe Ile Glu Ser Cys Phe Glu Leu Asn Leu Thr
405 410 415 Asp Gly Tyr Gly Ser Thr Glu Ala Gly Met Val Phe Arg Asp
Gly Ile 420 425 430 Val Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu Val
Asp Val Pro Glu 435 440 445 Leu Gly Tyr Phe Ser Thr Asp Lys Pro His
Pro Arg Gly Glu Leu Leu 450 455 460 Leu Lys Thr Asp Gly Met Phe Leu
Gly Tyr Tyr Lys Arg Pro Glu Val465 470 475 480 Thr Ala Ser Val Phe
Asp Ala Asp Gly Phe Tyr Met Thr Gly Asp Ile 485 490 495 Val Ala Glu
Leu Ala His Asp Asn Ile Glu Ile Ile Asp Arg Arg Asn 500 505 510 Asn
Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Thr Leu 515 520
525 Glu Ala Glu Tyr Ala Asn Ser Pro Val Val His Gln Ile Tyr Val Tyr
530 535 540 Gly Ser Ser Glu Arg Ser Tyr Leu Leu Ala Val Val Val Pro
Thr Pro545 550 555 560 Glu Ala Val Ala Ala Ala Lys Gly Asp Ala Ala
Ala Leu Lys Thr Thr 565 570 575 Ile Ala Asp Ser Leu Gln Asp Ile Ala
Lys Glu Ile Gln Leu Gln Ser 580 585 590 Tyr Glu Val Pro Arg Asp Phe
Ile Ile Glu Pro Gln Pro Phe Thr Gln 595 600 605 Gly Asn Gly Leu Leu
Thr Gly Ile Ala Lys Leu Ala Arg Pro Asn Leu 610 615 620 Lys Ala His
Tyr Gly Pro Arg Leu Glu Gln Met Tyr Ala Glu Ile Ala625 630 635 640
Glu Gln Gln Ala Ala Glu Leu Arg Ala Leu His Gly Val Asp Pro Asp 645
650 655 Lys Pro Ala Leu Glu Thr Val Leu Lys Ala Ala Gln Ala Leu Leu
Gly 660 665 670 Val Ser Ser Ala Glu Leu Ala Ala Asp Ala His Phe Thr
Asp Leu Gly 675 680 685 Gly Asp Ser Leu Ser Ala Leu Ser Phe Ser Asp
Leu Leu Arg Asp Ile 690 695 700 Phe Ala Val Glu Val Pro Val Gly Val
Ile Val Ser Ala Ala Asn Asp705 710 715 720 Leu Gly Gly Val Ala Lys
Phe Val Asp Glu Gln Arg His Ser Gly Gly 725 730 735 Thr Arg Pro Thr
Ala Glu Thr Val His Gly Ala Gly His Thr Glu Ile 740 745 750 Arg Ala
Ala Asp Leu Thr Leu Asp Lys Phe Ile Asp Glu Ala Thr Leu 755 760 765
His Ala Ala Pro Ser Leu Pro Lys Ala Ala Gly Ile Pro His Thr Val 770
775 780 Leu Leu Thr Gly Ser Asn Gly Tyr Leu Gly His Tyr Leu Ala Leu
Glu785 790 795 800 Trp Leu Glu Arg Leu Asp Lys Thr Asp Gly Lys Leu
Ile Val Ile Val 805 810 815 Arg Gly Lys Asn Ala Glu Ala Ala Tyr Gly
Arg Leu Glu Glu Ala Phe 820 825 830 Asp Thr Gly Asp Thr Glu Leu Leu
Ala His Phe Arg Ser Leu Ala Asp 835 840 845 Lys His Leu Glu Val Leu
Ala Gly Asp Ile Gly Asp Pro Asn Leu Gly 850 855 860 Leu Asp Ala Asp
Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Val Ile865 870 875 880 Val
His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Asn Gln Leu 885 890
895 Phe Gly Pro Asn Val Val Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910 Thr Thr Lys Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ala
Val Ala 915 920 925 Ala Tyr Val Asp Pro Thr Thr Phe Asp Glu Glu Ser
Asp Ile Arg Leu 930 935 940 Ile Ser Ala Val Arg Pro Ile Asp Asp Gly
Tyr Ala Asn Gly Tyr Gly945 950 955 960 Asn Ala Lys Trp Ala Gly Glu
Val Leu Leu Arg Glu Ala His Asp Leu 965 970 975 Cys Gly Leu Pro Val
Ala Val Phe Arg Ser Asp Met Ile Leu Ala His 980 985 990 Ser Arg Tyr
Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu 995 1000 1005
Ile Leu Ser Leu Ile Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Gln
1010 1015 1020 Ala Gln Thr Thr Gly Glu Arg Pro Leu Ala His Tyr Asp
Gly Leu Pro1025 1030 1035 1040 Gly Asp Phe Thr Ala Glu Ala Ile Thr
Thr Leu Gly Thr Gln Val Pro 1045 1050 1055 Glu Gly Ser Glu Gly Phe
Val Thr Tyr Asp Cys Val Asn Pro His Ala 1060 1065 1070 Asp Gly Ile
Ser Leu Asp Asn Phe Val Asp Trp Leu Ile Glu Ala Gly 1075 1080 1085
Tyr Pro Ile Ala Arg Ile Asp Asn Tyr Thr Glu Trp Phe Thr Arg Phe
1090 1095 1100 Asp Thr Ala Ile Arg Gly Leu Ser Glu Lys Gln Lys Gln
His Ser Leu1105 1110 1115 1120 Leu Pro Leu Leu His Ala Phe Glu Gln
Pro Ser Ala Ala Glu Asn His 1125 1130 1135 Gly Val Val Pro Ala Lys
Arg Phe Gln His Ala Val Gln Ala Ala Gly 1140 1145 1150 Ile Gly Pro
Val Gly Gln Asp Gly Thr Thr Asp Ile Pro His Leu Ser 1155 1160 1165
Arg Arg Leu Ile Val Lys Tyr Ala Lys Asp Leu Glu Gln Leu Gly Leu
1170 1175 1180 Leu118571186PRTSegniliparus rotundus 7Met Thr Gln
Ser His Thr Gln Gly Pro Gln Ala Ser Ala Ala His Ser1 5 10 15 Arg
Leu Ala Arg Arg Ala Ala Glu Leu Leu Ala Thr Asp Pro Gln Ala 20 25
30 Ala Ala Thr Leu Pro Asp Pro Glu Val Val Arg Gln Ala Thr Arg Pro
35 40 45 Gly Leu Arg Leu Ala Glu Arg Val Asp Ala Ile Leu Ser Gly
Tyr Ala 50 55 60 Asp Arg Pro Ala Leu Gly Gln Arg Ser Phe Gln Thr
Val Lys Asp Pro65 70 75 80 Ile Thr Gly Arg Ser Ser Val Glu Leu Leu
Pro Thr Phe Asp Thr Ile 85 90 95 Thr Tyr Arg Glu Leu Arg Glu Arg
Ala Thr Ala Ile Ala Ser Asp Leu 100 105 110 Ala His His Pro Gln Ala
Pro Ala Lys Pro Gly Asp Phe Leu Ala Ser 115 120 125 Ile Gly Phe Ile
Ser Val Asp Tyr Val Ala Ile Asp Ile Ala Gly Val 130 135 140 Phe Ala
Gly Leu Thr Ala Val Pro Leu Gln Thr Gly Ala Thr Leu Ala145 150 155
160 Thr Leu Thr Ala Ile Thr Ala Glu Thr Ala Pro Thr Leu Phe Ala Ala
165 170 175 Ser Ile Glu His Leu Pro Thr Ala Val Asp Ala Val Leu Ala
Thr Pro 180 185 190 Ser Val Arg Arg Leu Leu Val Phe Asp Tyr Arg Ala
Gly Ser Asp Glu 195 200 205 Asp Arg Glu Ala Val Glu Ala Ala Lys Arg
Lys Ile Ala Asp Ala Gly 210 215 220 Ser Ser Val Leu Val Asp Val Leu
Asp Glu Val Ile Ala Arg Gly Lys225 230 235 240 Ser Ala Pro Lys Ala
Pro Leu Pro Pro Ala Thr Asp Ala Gly Asp Asp 245 250 255 Ser Leu Ser
Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys 260 265 270 Gly
Ala Met Tyr Pro Glu Arg Asn Val Ala His Phe Trp Gly Gly Val 275 280
285 Trp Ala Ala Ala Phe Asp Glu Asp Ala Ala Pro Pro Val Pro Ala Ile
290 295 300 Asn Ile Thr Phe Leu Pro Leu Ser His Val Ala Ser Arg Leu
Ser Leu305 310 315 320 Met Pro Thr Leu Ala Arg Gly Gly Leu Met His
Phe Val Ala Lys Ser 325 330 335 Asp Leu Ser Thr Leu Phe Glu Asp Leu
Lys Leu Ala Arg Pro Thr Asn 340 345 350 Leu Phe Leu Val Pro Arg Val
Val Glu Met Leu Tyr Gln His Tyr Gln 355 360 365 Ser Glu Leu Asp Arg
Arg Gly Val Gln Asp Gly Thr Arg Glu Ala Glu 370 375 380 Ala Val Lys
Asp Asp Leu Arg Thr Gly Leu Leu Gly Gly Arg Ile Leu385 390 395 400
Thr Ala Gly Phe Gly Ser Ala Pro Leu Ser Ala Glu Leu Ala Gly Phe 405
410 415 Ile Glu Ser Leu Leu Gln Ile His Leu Val Asp Gly Tyr Gly Ser
Thr 420 425 430 Glu Ala Gly Pro Val Trp Arg Asp Gly Tyr Leu Val Lys
Pro Pro Val 435 440 445 Thr Asp Tyr Lys Leu Ile Asp Val Pro Glu Leu
Gly Tyr Phe Ser Thr 450 455 460 Asp Ser Pro His Pro Arg Gly Glu Leu
Ala Ile Lys Thr Gln Thr Ile465 470 475 480 Leu Pro Gly Tyr Tyr Lys
Arg Pro Glu Thr Thr Ala Glu Val Phe Asp 485 490 495 Glu Asp Gly Phe
Tyr Leu Thr Gly Asp Val Val Ala Gln Ile Gly Pro 500 505 510 Glu Gln
Phe Ala Tyr Val Asp Arg Arg Lys Asn Val Leu Lys Leu Ser 515 520 525
Gln Gly Glu Phe Val Thr Leu Ala Lys Leu Glu Ala Ala Tyr Ser Ser 530
535 540 Ser Pro Leu Val Arg Gln Leu Phe Val Tyr Gly Ser Ser Glu Arg
Ser545 550 555 560 Tyr Leu Leu Ala Val Ile Val Pro Thr Pro Asp Ala
Leu Lys Lys Phe 565 570 575 Gly Val Gly Glu Ala Ala Lys Ala Ala Leu
Gly Glu Ser Leu Gln Lys 580 585 590 Ile Ala Arg Asp Glu Gly Leu Gln
Ser Tyr Glu Val Pro Arg Asp Phe 595 600 605 Ile Ile Glu Thr Asp Pro
Phe Thr Val Glu Asn Gly Leu Leu Ser Asp 610 615 620 Ala Arg Lys Ser
Leu Arg Pro Lys Leu Lys Glu His Tyr Gly Glu Arg625 630 635 640 Leu
Glu Ala Met Tyr Lys Glu Leu Ala Asp Gly Gln Ala Asn Glu Leu 645 650
655 Arg Asp Ile Arg Arg Gly Val Gln Gln Arg Pro Thr Leu Glu Thr Val
660 665 670 Arg Arg Ala Ala Ala Ala Met Leu Gly Ala Ser Ala Ala Glu
Ile Lys 675 680 685 Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser
Leu Ser Ala Leu 690 695 700 Thr Phe Ser Asn Phe Leu His Asp Leu Phe
Glu Val Asp Val Pro Val705 710 715 720 Gly Val Ile Val Ser Ala Ala
Asn Thr Leu Gly Ser Val Ala Glu His 725 730 735 Ile Asp Ala Gln Leu
Ala Gly Gly Arg Ala Arg Pro Thr Phe Ala Thr 740 745 750 Val His Gly
Lys Gly Ser Thr Thr Ile Lys Ala Ser Asp Leu Thr Leu 755 760 765 Asp
Lys Phe Ile Asp Glu Gln Thr Leu Glu Ala Ala Lys His Leu Pro 770 775
780 Lys Pro Ala Asp Pro Pro Arg Thr Val Leu Leu Thr Gly Ala Asn
Gly785 790 795 800 Trp Leu Gly Arg Phe Leu Ala Leu Glu Trp Leu
Glu Arg Leu Ala Pro 805 810 815 Ala Gly Gly Lys Leu Ile Thr Ile Val
Arg Gly Lys Asp Ala Ala Gln 820 825 830 Ala Lys Ala Arg Leu Asp Ala
Ala Tyr Glu Ser Gly Asp Pro Lys Leu 835 840 845 Ala Gly His Tyr Gln
Asp Leu Ala Ala Thr Thr Leu Glu Val Leu Ala 850 855 860 Gly Asp Phe
Ser Glu Pro Arg Leu Gly Leu Asp Glu Ala Thr Trp Asn865 870 875 880
Arg Leu Ala Asp Glu Val Asp Phe Ile Ser His Pro Gly Ala Leu Val 885
890 895 Asn His Val Leu Pro Tyr Asn Gln Leu Phe Gly Pro Asn Val Ala
Gly 900 905 910 Val Ala Glu Ile Ile Lys Leu Ala Ile Thr Thr Arg Ile
Lys Pro Val 915 920 925 Thr Tyr Leu Ser Thr Val Ala Val Ala Ala Gly
Val Glu Pro Ser Ala 930 935 940 Leu Asp Glu Asp Gly Asp Ile Arg Thr
Val Ser Ala Glu Arg Ser Val945 950 955 960 Asp Glu Gly Tyr Ala Asn
Gly Tyr Gly Asn Ser Lys Trp Gly Gly Glu 965 970 975 Val Leu Leu Arg
Glu Ala His Asp Arg Thr Gly Leu Pro Val Arg Val 980 985 990 Phe Arg
Ser Asp Met Ile Leu Ala His Gln Lys Tyr Thr Gly Gln Val 995 1000
1005 Asn Ala Thr Asp Gln Phe Thr Arg Leu Val Gln Ser Leu Leu Ala
Thr 1010 1015 1020 Gly Leu Ala Pro Lys Ser Phe Tyr Glu Leu Asp Ala
Gln Gly Asn Arg1025 1030 1035 1040 Gln Arg Ala His Tyr Asp Gly Ile
Pro Val Asp Phe Thr Ala Glu Ser 1045 1050 1055 Ile Thr Thr Leu Gly
Gly Asp Gly Leu Glu Gly Tyr Arg Ser Tyr Asn 1060 1065 1070 Val Phe
Asn Pro His Arg Asp Gly Val Gly Leu Asp Glu Phe Val Asp 1075 1080
1085 Trp Leu Ile Glu Ala Gly His Pro Ile Thr Arg Ile Asp Asp Tyr
Asp 1090 1095 1100 Gln Trp Leu Ser Arg Phe Glu Thr Ser Leu Arg Gly
Leu Pro Glu Ser1105 1110 1115 1120 Lys Arg Gln Ala Ser Val Leu Pro
Leu Leu His Ala Phe Ala Arg Pro 1125 1130 1135 Gly Pro Ala Val Asp
Gly Ser Pro Phe Arg Asn Thr Val Phe Arg Thr 1140 1145 1150 Asp Val
Gln Lys Ala Lys Ile Gly Ala Glu His Asp Ile Pro His Leu 1155 1160
1165 Gly Lys Ala Leu Val Leu Lys Tyr Ala Asp Asp Ile Lys Gln Leu
Gly 1170 1175 1180 Leu Leu1185 8382PRTPseudomonas fluorescens 8Met
Gln Ile Gln Gly His Tyr Glu Leu Gln Phe Glu Ala Val Arg Glu1 5 10
15 Ala Phe Ala Ala Leu Phe Asp Asp Pro Gln Glu Arg Gly Ala Gly Leu
20 25 30 Cys Ile Gln Ile Gly Gly Glu Thr Val Val Asp Leu Trp Ala
Gly Thr 35 40 45 Ala Asp Lys Asp Gly Thr Glu Ala Trp His Ser Asp
Thr Ile Val Asn 50 55 60 Leu Phe Ser Cys Thr Lys Thr Phe Thr Ala
Val Thr Ala Leu Gln Leu65 70 75 80 Val Ala Glu Gly Lys Leu Gln Leu
Asp Ala Pro Val Ala Asn Tyr Trp 85 90 95 Pro Glu Phe Ala Ala Ala
Gly Lys Glu Ala Ile Thr Leu Arg Gln Leu 100 105 110 Leu Cys His Gln
Ala Gly Leu Pro Ala Ile Arg Glu Met Leu Pro Thr 115 120 125 Glu Ala
Leu Tyr Asp Trp Arg Leu Met Val Asp Thr Leu Ala Ala Glu 130 135 140
Ala Pro Trp Trp Thr Pro Gly Gln Gly His Gly Tyr Glu Ala Ile Thr145
150 155 160 Tyr Gly Trp Leu Val Gly Glu Leu Leu Arg Arg Ala Asp Gly
Arg Gly 165 170 175 Pro Gly Glu Ser Ile Val Ala Arg Val Ala Arg Pro
Leu Gly Leu Asp 180 185 190 Phe His Val Gly Leu Ala Asp Glu Glu Phe
Tyr Arg Val Ala His Ile 195 200 205 Ala Arg Ser Lys Gly Asn Met Gly
Asp Glu Ala Ala Gln Arg Leu Leu 210 215 220 Gln Val Met Met Arg Glu
Pro Thr Ala Met Thr Thr Arg Ala Phe Ala225 230 235 240 Asn Pro Pro
Ser Ile Leu Thr Ser Thr Asn Lys Pro Glu Trp Arg Arg 245 250 255 Met
Gln Gln Pro Ala Ala Asn Gly His Gly Asn Ala Arg Ser Leu Ala 260 265
270 Gly Phe Tyr Ser Gly Leu Leu Asp Gly Ser Leu Leu Glu Ala Asp Met
275 280 285 Leu Glu Gln Leu Thr Arg Glu His Ser Ile Gly Pro Asp Lys
Thr Leu 290 295 300 Leu Thr Gln Thr Arg Phe Gly Leu Gly Cys Met Leu
Asp Gln Gln Pro305 310 315 320 Gln Leu Pro Asn Ala Thr Phe Gly Leu
Gly Pro Arg Ala Phe Gly His 325 330 335 Pro Arg Ser Ala Pro Val Val
Arg Trp Val Leu Pro Glu His Asp Val 340 345 350 Ala Phe Gly Phe Val
Thr Asn Thr Leu Gly Pro Tyr Val Leu Met Asp 355 360 365 Pro Arg Ala
Gln Lys Leu Val Gly Ile Leu Ala Gly Cys Leu 370 375 380
9246PRTLactobacillus brevis 9Met Ala Ala Asn Glu Phe Ser Glu Thr
His Arg Val Val Tyr Tyr Glu1 5 10 15 Ala Asp Asp Thr Gly Gln Leu
Thr Leu Ala Met Leu Ile Asn Leu Phe 20 25 30 Val Leu Val Ser Glu
Asp Gln Asn Asp Ala Leu Gly Leu Ser Thr Ala 35 40 45 Phe Val Gln
Ser His Gly Val Gly Trp Val Val Thr Gln Tyr His Leu 50 55 60 His
Ile Asp Glu Leu Pro Arg Thr Gly Ala Gln Val Thr Ile Lys Thr65 70 75
80 Arg Ala Thr Ala Tyr Asn Arg Tyr Phe Ala Tyr Arg Glu Tyr Trp Leu
85 90 95 Leu Asp Asp Ala Gly Gln Val Leu Ala Tyr Gly Glu Gly Ile
Trp Val 100 105 110 Thr Met Ser Tyr Ala Thr Arg Lys Ile Thr Thr Ile
Pro Ala Glu Val 115 120 125 Met Ala Pro Tyr His Ser Glu Glu Gln Thr
Arg Leu Pro Arg Leu Pro 130 135 140 Arg Pro Asp His Phe Asp Glu Ala
Val Asn Gln Thr Leu Lys Pro Tyr145 150 155 160 Thr Val Arg Tyr Phe
Asp Ile Asp Gly Asn Gly His Val Asn Asn Ala 165 170 175 His Tyr Phe
Asp Trp Met Leu Asp Val Leu Pro Ala Thr Phe Leu Arg 180 185 190 Ala
His His Pro Thr Asp Val Lys Ile Arg Phe Glu Asn Glu Val Gln 195 200
205 Tyr Gly His Gln Val Thr Ser Glu Leu Ser Gln Ala Ala Ala Leu Thr
210 215 220 Thr Gln His Met Ile Lys Val Gly Asp Leu Thr Ala Val Lys
Ala Thr225 230 235 240 Ile Gln Trp Asp Asn Arg 245
10261PRTLactobacillus plantarum 10Met Ala Thr Leu Gly Ala Asn Ala
Ser Leu Tyr Ser Glu Gln His Arg1 5 10 15 Ile Thr Tyr Tyr Glu Cys
Asp Arg Thr Gly Arg Ala Thr Leu Thr Thr 20 25 30 Leu Ile Asp Ile
Ala Val Leu Ala Ser Glu Asp Gln Ser Asp Ala Leu 35 40 45 Gly Leu
Thr Thr Glu Met Val Gln Ser His Gly Val Gly Trp Val Val 50 55 60
Thr Gln Tyr Ala Ile Asp Ile Thr Arg Met Pro Arg Gln Asp Glu Val65
70 75 80 Val Thr Ile Ala Val Arg Gly Ser Ala Tyr Asn Pro Tyr Phe
Ala Tyr 85 90 95 Arg Glu Phe Trp Ile Arg Asp Ala Asp Gly Gln Gln
Leu Ala Tyr Ile 100 105 110 Thr Ser Ile Trp Val Met Met Ser Gln Thr
Thr Arg Arg Ile Val Lys 115 120 125 Ile Leu Pro Glu Leu Val Ala Pro
Tyr Gln Ser Glu Val Val Lys Arg 130 135 140 Ile Pro Arg Leu Pro Arg
Pro Ile Ser Phe Glu Ala Thr Asp Thr Thr145 150 155 160 Ile Thr Lys
Pro Tyr His Val Arg Phe Phe Asp Ile Asp Pro Asn Arg 165 170 175 His
Val Asn Asn Ala His Tyr Phe Asp Trp Leu Val Asp Thr Leu Pro 180 185
190 Ala Thr Phe Leu Leu Gln His Asp Leu Val His Val Asp Val Arg Tyr
195 200 205 Glu Asn Glu Val Lys Tyr Gly Gln Thr Val Thr Ala His Ala
Asn Ile 210 215 220 Leu Pro Ser Glu Val Ala Asp Gln Val Thr Thr Ser
His Leu Ile Glu225 230 235 240 Val Asp Asp Glu Lys Cys Cys Glu Val
Thr Ile Gln Trp Arg Thr Leu 245 250 255 Pro Glu Pro Ile Gln 260
11397PRTTreponema denticola 11Met Ile Val Lys Pro Met Val Arg Asn
Asn Ile Cys Leu Asn Ala His1 5 10 15 Pro Gln Gly Cys Lys Lys Gly
Val Glu Asp Gln Ile Glu Tyr Thr Lys 20 25 30 Lys Arg Ile Thr Ala
Glu Val Lys Ala Gly Ala Lys Ala Pro Lys Asn 35 40 45 Val Leu Val
Leu Gly Cys Ser Asn Gly Tyr Gly Leu Ala Ser Arg Ile 50 55 60 Thr
Ala Ala Phe Gly Tyr Gly Ala Ala Thr Ile Gly Val Ser Phe Glu65 70 75
80 Lys Ala Gly Ser Glu Thr Lys Tyr Gly Thr Pro Gly Trp Tyr Asn Asn
85 90 95 Leu Ala Phe Asp Glu Ala Ala Lys Arg Glu Gly Leu Tyr Ser
Val Thr 100 105 110 Ile Asp Gly Asp Ala Phe Ser Asp Glu Ile Lys Ala
Gln Val Ile Glu 115 120 125 Glu Ala Lys Lys Lys Gly Ile Lys Phe Asp
Leu Ile Val Tyr Ser Leu 130 135 140 Ala Ser Pro Val Arg Thr Asp Pro
Asp Thr Gly Ile Met His Lys Ser145 150 155 160 Val Leu Lys Pro Phe
Gly Lys Thr Phe Thr Gly Lys Thr Val Asp Pro 165 170 175 Phe Thr Gly
Glu Leu Lys Glu Ile Ser Ala Glu Pro Ala Asn Asp Glu 180 185 190 Glu
Ala Ala Ala Thr Val Lys Val Met Gly Gly Glu Asp Trp Glu Arg 195 200
205 Trp Ile Lys Gln Leu Ser Lys Glu Gly Leu Leu Glu Glu Gly Cys Ile
210 215 220 Thr Leu Ala Tyr Ser Tyr Ile Gly Pro Glu Ala Thr Gln Ala
Leu Tyr225 230 235 240 Arg Lys Gly Thr Ile Gly Lys Ala Lys Glu His
Leu Glu Ala Thr Ala 245 250 255 His Arg Leu Asn Lys Glu Asn Pro Ser
Ile Arg Ala Phe Val Ser Val 260 265 270 Asn Lys Gly Leu Val Thr Arg
Ala Ser Ala Val Ile Pro Val Ile Pro 275 280 285 Leu Tyr Leu Ala Ser
Leu Phe Lys Val Met Lys Glu Lys Gly Asn His 290 295 300 Glu Gly Cys
Ile Glu Gln Ile Thr Arg Leu Tyr Ala Glu Arg Leu Tyr305 310 315 320
Arg Lys Asp Gly Thr Ile Pro Val Asp Glu Glu Asn Arg Ile Arg Ile 325
330 335 Asp Asp Trp Glu Leu Glu Glu Asp Val Gln Lys Ala Val Ser Ala
Leu 340 345 350 Met Glu Lys Val Thr Gly Glu Asn Ala Glu Ser Leu Thr
Asp Leu Ala 355 360 365 Gly Tyr Arg His Asp Phe Leu Ala Ser Asn Gly
Phe Asp Val Glu Gly 370 375 380 Ile Asn Tyr Glu Ala Glu Val Glu Arg
Phe Asp Arg Ile385 390 395 12539PRTEuglena gracilis 12Met Ser Cys
Pro Ala Ser Pro Ser Ala Ala Val Val Ser Ala Gly Ala1 5 10 15 Leu
Cys Leu Cys Val Ala Thr Val Leu Leu Ala Thr Gly Ser Asn Pro 20 25
30 Thr Ala Leu Ser Thr Ala Ser Thr Arg Ser Pro Thr Ser Leu Val Arg
35 40 45 Gly Val Asp Arg Gly Leu Met Arg Pro Thr Thr Ala Ala Ala
Leu Thr 50 55 60 Thr Met Arg Glu Val Pro Gln Met Ala Glu Gly Phe
Ser Gly Glu Ala65 70 75 80 Thr Ser Ala Trp Ala Ala Ala Gly Pro Gln
Trp Ala Ala Pro Leu Val 85 90 95 Ala Ala Ala Ser Ser Ala Leu Ala
Leu Trp Trp Trp Ala Ala Arg Arg 100 105 110 Ser Val Arg Arg Pro Leu
Ala Ala Leu Ala Glu Leu Pro Thr Ala Val 115 120 125 Thr His Leu Ala
Pro Pro Met Ala Met Phe Thr Thr Thr Ala Lys Val 130 135 140 Ile Gln
Pro Lys Ile Arg Gly Phe Ile Cys Thr Thr Thr His Pro Ile145 150 155
160 Gly Cys Glu Lys Arg Val Gln Glu Glu Ile Ala Tyr Ala Arg Ala His
165 170 175 Pro Pro Thr Ser Pro Gly Pro Lys Arg Val Leu Val Ile Gly
Cys Ser 180 185 190 Thr Gly Tyr Gly Leu Ser Thr Arg Ile Thr Ala Ala
Phe Gly Tyr Gln 195 200 205 Ala Ala Thr Leu Gly Val Phe Leu Ala Gly
Pro Pro Thr Lys Gly Arg 210 215 220 Pro Ala Ala Ala Gly Trp Tyr Asn
Thr Val Ala Phe Glu Lys Ala Ala225 230 235 240 Leu Glu Ala Gly Leu
Tyr Ala Arg Ser Leu Asn Gly Asp Ala Phe Asp 245 250 255 Ser Thr Thr
Lys Ala Arg Thr Val Glu Ala Ile Lys Arg Asp Leu Gly 260 265 270 Thr
Val Asp Leu Val Val Tyr Ser Ile Ala Ala Pro Lys Arg Thr Asp 275 280
285 Pro Ala Thr Gly Val Leu His Lys Ala Cys Leu Lys Pro Ile Gly Ala
290 295 300 Thr Tyr Thr Asn Arg Thr Val Asn Thr Asp Lys Ala Glu Val
Thr Asp305 310 315 320 Val Ser Ile Glu Pro Ala Ser Pro Glu Glu Ile
Ala Asp Thr Val Lys 325 330 335 Val Met Gly Gly Glu Asp Trp Glu Leu
Trp Ile Gln Ala Leu Ser Glu 340 345 350 Ala Gly Val Leu Ala Glu Gly
Ala Lys Thr Val Ala Tyr Ser Tyr Ile 355 360 365 Gly Pro Glu Met Thr
Trp Pro Val Tyr Trp Ser Gly Thr Ile Gly Glu 370 375 380 Ala Lys Lys
Asp Val Glu Lys Ala Ala Lys Arg Ile Thr Gln Gln Tyr385 390 395 400
Gly Cys Pro Ala Tyr Pro Val Val Ala Lys Ala Leu Val Thr Gln Ala 405
410 415 Ser Ser Ala Ile Pro Val Val Pro Leu Tyr Ile Cys Leu Leu Tyr
Arg 420 425 430 Val Met Lys Glu Lys Gly Thr His Glu Gly Cys Ile Glu
Gln Met Val 435 440 445 Arg Leu Leu Thr Thr Lys Leu Tyr Pro Glu Asn
Gly Ala Pro Ile Val 450 455 460 Asp Glu Ala Gly Arg Val Arg Val Asp
Asp Trp Glu Met Ala Glu Asp465 470 475 480 Val Gln Gln Ala Val Lys
Asp Leu Trp Ser Gln Val Ser Thr Ala Asn 485 490 495 Leu Lys Asp Ile
Ser Asp Phe Ala Gly Tyr Gln Thr Glu Phe Leu Arg 500 505 510 Leu Phe
Gly Phe Gly Ile Asp Gly Val Asp Tyr Asp Gln Pro Val Asp 515 520 525
Val Glu Ala Asp Leu Pro Ser Ala Ala Gln Gln 530 535
13269PRTBacillus cereus 13Met Ile Asn Lys Thr Leu Leu Gln Lys Arg
Phe Asn Gly Ala Ala Val1 5 10 15 Ser Tyr Asp Arg Tyr Ala Asn Val
Gln Lys Lys Met Ala His Ser Leu 20 25 30 Leu Ser Ile Leu Lys Glu
Arg Tyr Ser Glu Thr Ala Ser Ile Arg Ile 35 40 45 Leu Glu Leu Gly
Cys Gly Thr Gly Tyr Val Thr Glu Gln Leu Ser Lys 50 55 60 Leu Phe
Pro Lys Ser His Ile Thr Ala Val Asp Phe Ala Glu Ser Met65 70 75 80
Ile Ala Ile Ala Gln Thr Arg Gln Asn Val Lys Asn Val Thr Phe
His 85 90 95 Cys Glu Asp Ile Glu Arg Leu Arg Leu Glu Glu Ser Tyr
Asp Val Ile 100 105 110 Ile Ser Asn Ala Thr Phe Gln Trp Leu Asn Asn
Leu Gln Gln Val Leu 115 120 125 Arg Asn Leu Phe Gln His Leu Ser Ile
Asp Gly Ile Leu Leu Phe Ser 130 135 140 Thr Phe Gly His Glu Thr Phe
Gln Glu Leu His Ala Ser Phe Gln Arg145 150 155 160 Ala Lys Glu Glu
Arg Asn Ile Lys Asn Glu Thr Ser Ile Gly Gln Arg 165 170 175 Phe Tyr
Ser Lys Asp Gln Leu Leu His Ile Cys Lys Ile Glu Thr Gly 180 185 190
Asp Val His Val Ser Glu Thr Cys Tyr Ile Glu Ser Phe Thr Glu Val 195
200 205 Lys Glu Phe Leu His Ser Ile Arg Lys Val Gly Ala Thr Asn Ser
Asn 210 215 220 Glu Gly Ser Tyr Cys Gln Ser Pro Ser Leu Phe Arg Ala
Met Leu Arg225 230 235 240 Ile Tyr Glu Arg Asp Phe Thr Gly Asn Glu
Gly Ile Met Ala Thr Tyr 245 250 255 His Ala Leu Phe Ile His Ile Thr
Lys Glu Gly Lys Arg 260 265 14132PRTEscherichia coli 14Met Ser Thr
Thr His Asn Val Pro Gln Gly Asp Leu Val Leu Arg Thr1 5 10 15 Leu
Ala Met Pro Ala Asp Thr Asn Ala Asn Gly Asp Ile Phe Gly Gly 20 25
30 Trp Leu Met Ser Gln Met Asp Ile Gly Gly Ala Ile Leu Ala Lys Glu
35 40 45 Ile Ala His Gly Arg Val Val Thr Val Arg Val Glu Gly Met
Thr Phe 50 55 60 Leu Arg Pro Val Ala Val Gly Asp Val Val Cys Cys
Tyr Ala Arg Cys65 70 75 80 Val Gln Lys Gly Thr Thr Ser Val Ser Ile
Asn Ile Glu Val Trp Val 85 90 95 Lys Lys Val Ala Ser Glu Pro Ile
Gly Gln Arg Tyr Lys Ala Thr Glu 100 105 110 Ala Leu Phe Lys Tyr Val
Ala Val Asp Pro Glu Gly Lys Pro Arg Ala 115 120 125 Leu Pro Val Glu
130 15286PRTEscherichia coli 15Met Ser Gln Ala Leu Lys Asn Leu Leu
Thr Leu Leu Asn Leu Glu Lys1 5 10 15 Ile Glu Glu Gly Leu Phe Arg
Gly Gln Ser Glu Asp Leu Gly Leu Arg 20 25 30 Gln Val Phe Gly Gly
Gln Val Val Gly Gln Ala Leu Tyr Ala Ala Lys 35 40 45 Glu Thr Val
Pro Glu Glu Arg Leu Val His Ser Phe His Ser Tyr Phe 50 55 60 Leu
Arg Pro Gly Asp Ser Lys Lys Pro Ile Ile Tyr Asp Val Glu Thr65 70 75
80 Leu Arg Asp Gly Asn Ser Phe Ser Ala Arg Arg Val Ala Ala Ile Gln
85 90 95 Asn Gly Lys Pro Ile Phe Tyr Met Thr Ala Ser Phe Gln Ala
Pro Glu 100 105 110 Ala Gly Phe Glu His Gln Lys Thr Met Pro Ser Ala
Pro Ala Pro Asp 115 120 125 Gly Leu Pro Ser Glu Thr Gln Ile Ala Gln
Ser Leu Ala His Leu Leu 130 135 140 Pro Pro Val Leu Lys Asp Lys Phe
Ile Cys Asp Arg Pro Leu Glu Val145 150 155 160 Arg Pro Val Glu Phe
His Asn Pro Leu Lys Gly His Val Ala Glu Pro 165 170 175 His Arg Gln
Val Trp Ile Arg Ala Asn Gly Ser Val Pro Asp Asp Leu 180 185 190 Arg
Val His Gln Tyr Leu Leu Gly Tyr Ala Ser Asp Leu Asn Phe Leu 195 200
205 Pro Val Ala Leu Gln Pro His Gly Ile Gly Phe Leu Glu Pro Gly Ile
210 215 220 Gln Ile Ala Thr Ile Asp His Ser Met Trp Phe His Arg Pro
Phe Asn225 230 235 240 Leu Asn Glu Trp Leu Leu Tyr Ser Val Glu Ser
Thr Ser Ala Ser Ser 245 250 255 Ala Arg Gly Phe Val Arg Gly Glu Phe
Tyr Thr Gln Asp Gly Val Leu 260 265 270 Val Ala Ser Thr Val Gln Glu
Gly Val Met Arg Asn His Asn 275 280 285 16224PRTBacillus subtilis
16Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu1
5 10 15 Asn Glu Arg Phe Met Ser Phe Ile Ser Pro Glu Lys Arg Glu Lys
Cys 20 25 30 Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu
Leu Gly Asp 35 40 45 Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr
Gln Leu Asp Lys Ser 50 55 60 Asp Ile Arg Phe Ser Thr Gln Glu Tyr
Gly Lys Pro Cys Ile Pro Asp65 70 75 80 Leu Pro Asp Ala His Phe Asn
Ile Ser His Ser Gly Arg Trp Val Ile 85 90 95 Cys Ala Phe Asp Ser
Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys 100 105 110 Pro Ile Ser
Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr 115 120 125 Ser
Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr 130 135
140 His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys
Gly145 150 155 160 Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu
His Gln Asp Gly 165 170 175 Gln Val Ser Ile Glu Leu Pro Asp Ser His
Ser Pro Cys Tyr Ile Lys 180 185 190 Thr Tyr Glu Val Asp Pro Gly Tyr
Lys Met Ala Val Cys Ala Ala His 195 200 205 Pro Asp Phe Pro Glu Asp
Ile Thr Met Val Ser Tyr Glu Glu Leu Leu 210 215 220
17222PRTNocardia sp. NRRL 5646 17Met Ile Glu Thr Ile Leu Pro Ala
Gly Val Glu Ser Ala Glu Leu Leu1 5 10 15 Glu Tyr Pro Glu Asp Leu
Lys Ala His Pro Ala Glu Glu His Leu Ile 20 25 30 Ala Lys Ser Val
Glu Lys Arg Arg Arg Asp Phe Ile Gly Ala Arg His 35 40 45 Cys Ala
Arg Leu Ala Leu Ala Glu Leu Gly Glu Pro Pro Val Ala Ile 50 55 60
Gly Lys Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg Gly Val Val Gly65
70 75 80 Ser Leu Thr His Cys Asp Gly Tyr Arg Ala Ala Ala Val Ala
His Lys 85 90 95 Met Arg Phe Arg Ser Ile Gly Ile Asp Ala Glu Pro
His Ala Thr Leu 100 105 110 Pro Glu Gly Val Leu Asp Ser Val Ser Leu
Pro Pro Glu Arg Glu Trp 115 120 125 Leu Lys Thr Thr Asp Ser Ala Leu
His Leu Asp Arg Leu Leu Phe Cys 130 135 140 Ala Lys Glu Ala Thr Tyr
Lys Ala Trp Trp Pro Leu Thr Ala Arg Trp145 150 155 160 Leu Gly Phe
Glu Glu Ala His Ile Thr Phe Glu Ile Glu Asp Gly Ser 165 170 175 Ala
Asp Ser Gly Asn Gly Thr Phe His Ser Glu Leu Leu Val Pro Gly 180 185
190 Gln Thr Asn Asp Gly Gly Thr Pro Leu Leu Ser Phe Asp Gly Arg Trp
195 200 205 Leu Ile Ala Asp Gly Phe Ile Leu Thr Ala Ile Ala Tyr Ala
210 215 220
* * * * *
References