U.S. patent application number 15/459116 was filed with the patent office on 2017-07-06 for methods of producing 7-carbon chemicals via carbon chain elongation associated with cyclohexane carboxylate synthesis.
The applicant listed for this patent is INVISTA North America S.a.r.l.. Invention is credited to Adriana Leonora Botes, Changlin Chen, Alex Van Eck Conradie, Paul S. Pearlman.
Application Number | 20170191096 15/459116 |
Document ID | / |
Family ID | 50001269 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170191096 |
Kind Code |
A1 |
Botes; Adriana Leonora ; et
al. |
July 6, 2017 |
METHODS OF PRODUCING 7-CARBON CHEMICALS VIA CARBON CHAIN ELONGATION
ASSOCIATED WITH CYCLOHEXANE CARBOXYLATE SYNTHESIS
Abstract
This document describes biochemical pathways for producing
pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid,
heptamethylenediamine or 1,7-heptanediol by forming two terminal
functional groups, comprised of carboxyl, amine or hydroxyl group,
in a C7 aliphatic backbone substrate. These pathways, metabolic
engineering and cultivation strategies described herein rely on the
carbon chain elongation enzymes or homologs thereof associated with
the cyclohexane carboxylate biosynthesis from Syntrophus
aciditrophicus or 2-aminoadipate lysine biosynthesis.
Inventors: |
Botes; Adriana Leonora;
(Rosedale East, GB) ; Conradie; Alex Van Eck;
(Eaglescliffe, GB) ; Chen; Changlin; (Ingleby
Barwick, GB) ; Pearlman; Paul S.; (Thornton,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INVISTA North America S.a.r.l. |
Wilmington |
DE |
US |
|
|
Family ID: |
50001269 |
Appl. No.: |
15/459116 |
Filed: |
March 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14138904 |
Dec 23, 2013 |
9637764 |
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15459116 |
|
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61747409 |
Dec 31, 2012 |
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61829092 |
May 30, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 7/18 20130101; C12P
7/42 20130101; C12N 9/16 20130101; C12Y 102/99006 20130101; C12N
9/1096 20130101; C12Y 301/02002 20130101; C12N 9/0008 20130101;
C12P 13/002 20130101; C12P 7/44 20130101; C12P 13/001 20130101 |
International
Class: |
C12P 13/00 20060101
C12P013/00; C12N 9/02 20060101 C12N009/02; C12N 9/16 20060101
C12N009/16; C12N 9/10 20060101 C12N009/10; C12P 7/44 20060101
C12P007/44; C12P 7/42 20060101 C12P007/42 |
Claims
1. A method for biosynthesizing a product selected from the group
consisting of pimelic acid, 7-aminoheptanoic acid,
7-hydroxyheptanoic acid, heptamethylenediamine, and
1,7-heptanediol, said method comprising enzymatically synthesizing
a seven carbon chain aliphatic backbone using enzymes associated
with cyclohexane carboxylate biosynthesis or the 2-aminoadipate
lysine biosynthesis pathway, and enzymatically forming one or two
terminal functional groups selected from the group consisting of
carboxyl, amine, and hydroxyl groups in said backbone, thereby
forming the product.
2. The method of claim 1, wherein the seven carbon chain aliphatic
backbone is pimeloyl-CoA.
3. The method of claim 1 or 2, wherein pimeloyl-CoA is
enzymatically synthesized from acetyl-CoA or 2-oxoglutarate via
3-ketopimeloyl-CoA.
4. The method of claim 3, wherein a .beta.-ketothiolase or a
.beta.-ketoacyl [acp] synthase converts glutaryl-CoA to
3-ketopimeloyl-CoA.
5. The method of claim 4, wherein glutaryl-CoA is produced from
crotonyl-CoA using a glutaconyl-CoA decarboxylase or a glutaryl-CoA
dehydrogenase.
6. The method of any one of claims 3-5, wherein 3-ketopimeloyl-CoA
is converted to 3-hydroxypimeloyl-CoA using a 3-hydroxyadipyl-CoA
dehydrogenase.
7. The method of claim 6, wherein 3-hydroxypimeloyl-CoA is
converted to 6-ketocyclohex-1-ene-1-carboxyl-CoA by a
6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase.
8. The method of claim 7, wherein 3-hydroxypimeloyl-CoA is
converted to 2,3-dehydropimeloyl-CoA by an enoyl-CoA hydratase.
9. The method of any one of claims 1-8, wherein said two terminal
functional groups are the same.
10. The method of any one of claims 1-8, wherein said two terminal
functional groups are different.
11. The method of claim 10, wherein said product comprises a
terminal amine and a terminal carboxyl group.
12. The method of claim 10, wherein said product comprises a
terminal hydroxyl group and a terminal carboxyl group.
13. The method of any one of claims 1-9, wherein said two terminal
functional groups are amine.
14. The method of any one of claims 1-9, wherein said two terminal
functional groups are hydroxyl groups.
15. The method of claim 12 or claim 14, wherein a
6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate
dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol
dehydrogenase enzymatically forms the two hydroxyl groups.
16. The method of any one of claims 1-14, wherein a thioesterase,
an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a
6-oxohexanoate dehydrogenase, a CoA-transferase, or a reversible
CoA-ligase enzymatically forms a terminal carboxyl group.
17. The method of claim 16, wherein said thioesterase has at least
70% sequence identity to the amino acid sequence set forth in SEQ
ID NO: 1.
18. The method of claim 11 or claim 13, wherein a
.omega.-transaminase or a deacetylase enzymatically forms the amine
group.
19. The method of claim 18, wherein said .omega.-transaminase has
at least 70% sequence identity to any one of the amino acid
sequences set forth in SEQ ID NO. 8-13.
20. The method of any one of claims 1-14, wherein a carboxylate
reductase in combination with a phosphopantetheine transferase
enhancer form a terminal aldehyde group as an intermediate in
forming the product.
21. The method of claim 20, wherein said carboxylate reductase has
at least 70% sequence identity to any one of the amino acid
sequences set forth in SEQ ID NO. 2-7.
22. The method of any of the preceding claims, wherein said method
is performed in a recombinant host by fermentation.
23. The method of claim 22, wherein said host is subjected to a
cultivation strategy under anaerobic, micro-aerobic or mixed
oxygen/denitrification cultivation conditions.
24. The method of claim 22 or claim 23, wherein said host is
cultured under conditions of nutrient limitation.
25. The method according to any one of claims 22-24, wherein said
host is retained using a ceramic hollow fiber membrane to maintain
a high cell density during fermentation.
26. The method of any one of claims 22-25, wherein the final
electron acceptor is nitrate.
27. The method of any one of claims 22-26, wherein the principal
carbon source fed to the fermentation derives from biological or
non-biological feedstocks.
28. The method of claim 27, wherein the biological feedstock is, or
derives from, monosaccharides, disaccharides, lignocellulose,
hemicellulose, cellulose, lignin, levulinic acid, formic acid,
triglycerides, glycerol, fatty acids, agricultural waste, condensed
distillers'solubles, or municipal waste.
29. The method of claim 27, wherein the non-biological feedstock
is, or derives from, natural gas, syngas, CO.sub.2/H.sub.2,
methanol, ethanol, benzoate, non-volatile residue (NVR) caustic
wash waste stream from cyclohexane oxidation processes, or
terephthalic acid/isophthalic acid mixture waste streams.
30. The method of any one of claims 22-29, wherein the host is a
prokaryote.
31. The method of claim 30, wherein said prokaryote is from the
genus Escherichia such as Escherichia coli; from the genus
Clostridia such as Clostridium ljungdahlii, Clostridium
autoethanogenum or Clostridium kluyveri; from the genus
Corynebacteria such as Corynebacterium glutamicum; from the genus
Cupriavidus such as Cupriavidus necator or Cupriavidus
metallidurans; from the genus Pseudomonas such as Pseudomonas
fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the
genus Delftia acidovorans, from the genus Bacillus such as Bacillus
subtillis; from the genes Lactobacillus such as Lactobacillus
delbrueckii; from the genus Lactococcus such as Lactococcus lactis
or from the genus Rhodococcus such as Rhodococcus equi.
32. The method of any one of claims 22-29, wherein the host is a
eukaryote.
33. The method of claim 32, wherein said eukaryote is from the
genus Aspergillus such as Aspergillus niger; from the genus
Saccharomyces such as Saccharomyces cerevisiae; from the genus
Pichia such as Pichia pastoris; from the genus Yarrowia such as
Yarrowia lipolytica, from the genus Issatchenkia such as
Issathenkia orientalis, from the genus Debaryomyces such as
Debaryomyces hansenii, from the genus Arxula such as Arxula
adenoinivorans, or from the genus Kluyveromyces such as
Kluyveromyces lactis .
34. The method of any one of claims 22-33, wherein the host's
tolerance to high concentrations of a C7 building block is improved
through continuous cultivation in a selective environment.
35. The method of any one of claims 22-34, wherein said host
comprises one or more of the following attenuated enzymes: a
phosphotransacetylase generating acetate, an acetate kinase; a
lactate dehydrogenase; a menaquinol-fumarate oxidoreductase; an
alcohol dehydrogenase producing ethanol; a pyruvate decarboxylase;
a 2-oxoacid decarboxylase generating isobutanol; a polymer
synthase; a NADPH-specific L-glutamate dehydrogenase; a NADPH/NADH
L-glutamate dehydrogenase; a NADH-consuming transhydrogenase, a
pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase that degrades
C7 building blocks and their precursors; a glutaryl-CoA
dehydrogenase; or a pimeloyl-CoA synthetase.
36. The method of any one of claims 22-35, wherein said host
overexpresses one or more genes encoding: an acetyl-CoA synthetase,
a formate dehydrogenase, a PEP carboxykinase, a PEP carboxylase, a
pyruvate carboxylase, a PEP synthase, a L-alanine dehydrogenase; a
NADH-specific L-glutamate dehydrogenase; a diamine transporter; a
dicarboxylate transporter; and/or a multidrug transporter.
37. A recombinant host comprising at least one exogenous nucleic
acid encoding i) a .beta.-ketothiolase or a) .beta.-ketoacyl [acp]
synthase, (ii) a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or an
enoyl-CoA hydratase, and (iii) a trans-2-enoyl-CoA reductase, an
enoyl-[acp] reductase, or a 2-ketocyclohexanecarboxyl-CoA
hydrolase, said host producing pimeloyl-CoA.
38. The recombinant host of claim 37, further comprising a
glutaconyl-CoA decarboxylase or a glutaryl-CoA dehydrogenase.
39. The recombinant host of claim 37 or claim 38, said host further
comprising one or more of an aldehyde dehydrogenase, a
6-oxohexanoate dehydrogenase, a thioesterase, a 7-oxoheptanoate
dehydrogenase, a reversible succinyl-CoA-ligase, a glutaconate CoA
transferase, a carboxylate reductase, or an acetylating aldehyde
dehydrogenase said host producing pimelate or pimelate
semialdehyde.
40. The recombinant host of claim 39, said host further comprising
a .omega.-transaminase, said host producing 7-aminoheptanoate.
41. The recombinant host of claim 39, said host further comprising
one or more of a 6-hydroxyhexanoate dehydrogenase, a
5-hydroxypentanoate dehydrogenase, or a 4-hydroxybutyrate
dehydrogenase, said host producing 7-hydroxyheptanoic acid.
42. The recombinant host of any one of claims 39-41, said host
further comprising one or more of a deacetylase, an N-acetyl
transferase, or an alcohol dehydrogenase, said host producing
heptamethylenediamine.
43. The recombinant host of claim 41, said host further comprising
a carboxylate reductase or an alcohol dehydrogenase, said host
producing 1,7-heptanediol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. Provisional
Application Ser. No. 61/747,409, filed Dec. 31, 2012, and U.S.
Provisional Application Ser. No. 61/829,092, filed May 30, 2013.
The contents of the prior applications are incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0002] This invention relates to methods for biosynthesizing one or
more of pimelic acid, 7-hydroxyheptanoate, 7-aminoheptanoate,
heptamethylenediamine and 1,7-heptanediol (hereafter "C7 building
blocks") using one or more isolated enzymes such as
.beta.-ketothiolases, dehydrogenases, reductases, thioesterases,
decarboxylases, hydratases, synthases, thioesterases, CoA-ligases,
CoA-transferases, deacetylases, or transaminases or using
recombinant host cells expressing one or more such enzymes or using
recombinant host cells expressing one or more such enzymes.
BACKGROUND
[0003] Nylons are polyamides which are generally synthesized by the
condensation polymerisation of a diamine with a dicarboxylic acid.
Similarly, Nylons may be produced by the condensation
polymerisation of lactams. A ubiquitous Nylon is Nylon 6,6, which
is produced by reaction of hexamethylenediamine (HMD) and adipic
acid. Nylon 6 can be produced by a ring opening polymerisation of
caprolactam (Anton & Baird, Polyamides Fibers, Encyclopedia of
Polymer Science and Technology, 2001).
[0004] Nylon 7 and Nylon 7,7 represent novel polyamides with
value-added characteristics compared to Nylon 6 and Nylon 6,6.
Nylon 7 is produced by polymerisation of 7-aminoheptanoic acid,
whereas Nylon 7,7 is produced by condensation polymerisation of
pimelic acid and heptamethylenediamine. No economically viable
petrochemical routes exist to producing the monomers for Nylon 7
and Nylon 7,7.
[0005] Given no economically viable petrochemical monomer
feedstocks; biotechnology offers an alternative approach via
biocatalysis. Biocatalysis is the use of biological catalysts, such
as enzymes, to perform biochemical transformations of organic
compounds.
[0006] Both bioderived feedstocks and petrochemical feedstocks are
viable starting materials for the biocatalysis processes.
[0007] Accordingly, against this background, it is clear that there
is a need for sustainable methods for producing one or more of
pimelic acid, 7-hydroxyheptanoate, 7-aminoheptanoate,
heptamethylenediamine and 1,7-heptanediol (hereafter "C7 building
blocks") wherein the methods are biocatalyst based.
[0008] However, no wild-type prokaryote or eukaryote naturally
overproduces or excretes such C7 building blocks to the
extracellular environment. Nevertheless, the metabolism of pimelic
acid has been reported.
[0009] The dicarboxylic acid, pimelic acid, is converted
efficiently as a carbon source by a number of bacteria and yeasts
via .beta.-oxidation into central metabolites. .beta.-oxidation of
Coenzyme A (CoA) activated pimelate to CoA activated 3-oxopimelate
facilitates further catabolism via, for example, pathways
associated with aromatic substrate degradation. The catabolism of
3-oxopimeloyl-CoA to acetyl-CoA and glutaryl-CoA by several
bacteria has been characterized comprehensively (Harwood and
Parales, Annual Review of Microbiology, 1996, 50:553-590).
[0010] The optimality principle states that microorganisms regulate
their biochemical networks to support maximum biomass growth.
Beyond the need for expressing heterologous pathways in a host
organism, directing carbon flux towards C7 building blocks that
serve as carbon sources rather than as biomass growth constituents,
contradicts the optimality principle. For example, transferring the
1-butanol pathway from Clostridium species into other production
strains has often fallen short by an order of magnitude compared to
the production performance of native producers (Shen et al., Appl.
Environ. Microbiol., 2011, 77(9):2905-2915).
[0011] The efficient synthesis of the seven carbon aliphatic
backbone precursor is a key consideration in synthesizing one or
more C7 building blocks prior to forming terminal functional
groups, such as carboxyl, amine or hydroxyl groups, on the C7
aliphatic backbone.
SUMMARY
[0012] This document is based at least in part on the discovery
that it is possible to construct biochemical pathways for producing
a seven carbon chain aliphatic backbone precursor in which one or
two functional groups, i.e., carboxyl, amine or hydroxyl, can be
formed, leading to the synthesis of one or more of pimelic acid,
7-aminoheptanoic acid, 7-hydroxyheptanoic acid,
heptamethylenediamine, and 1,7-heptanediol (hereafter "C7 building
blocks). Pimelic acid and pimilate, 7-hydroxyheptanoic acid and
7-hydroxyheptanoate, and 7-aminoheptanoic and 7-aminoheptanoate are
used interchangeably herein to refer to the compound in any of its
neutral or ionized forms, including any salt forms thereof. It is
understood by those skilled in the art that the specific form will
depend on pH. These pathways, metabolic engineering and cultivation
strategies described herein rely on carbon chain elongation enzymes
or homologs associated with cyclohexane carboxylate biosynthesis in
Synthrophus aciditrophicus or 2-aminoadipate lysine
biosynthesis.
[0013] In the face of the optimality principle, it surprisingly has
been discovered that appropriate non-natural pathways, feedstocks,
host microorganisms, attenuation strategies to the host's
biochemical network, and cultivation strategies may be combined to
efficiently produce one or more C7 building blocks.
[0014] In some embodiments, the C7 aliphatic backbone for
conversion to a C7 building block is 3-ketopimeloyl-CoA (also can
be referred to as 3-oxopimeloyl-CoA) or pimeloyl-CoA, which can be
formed from acetyl-CoA using enzymes associated with cyclohexane
carboxylate biosynthesis in in Synthrophus aciditrophicus or
2-aminoadipate lysine biosynthesis. See FIG. 1 and FIG. 2.
[0015] In some embodiments, a terminal carboxyl group can be
enzymatically formed using a thioesterase, an aldehyde
dehydrogenase, a 7-oxoheptanoate dehydrogenase, a CoA-transferase,
or a reversible CoA-ligase. See FIG. 1, FIG.2 and FIG. 3.
[0016] In some embodiments, a terminal amine group can be
enzymatically formed using a .omega.-transaminase or a deacetylase.
See FIG. 4 and FIG. 5.
[0017] In some embodiments, a terminal hydroxyl group can be
enzymatically formed using an alcohol dehydrogenase. See FIG. 6 and
FIG. 7.
[0018] In one aspect, this document features a method for
biosynthesizing a product selected from the group consisting of
pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid,
heptamethylenediamine, and 1,7-heptanediol. The method includes
enzymatically synthesizing a seven carbon chain aliphatic backbone
using enzymes associated with cyclohexane carboxylate biosynthesis
or the 2-aminoadipate lysine biosynthesis pathway, and
enzymatically forming one or two terminal functional groups
selected from the group consisting of carboxyl, amine, and hydroxyl
groups in the backbone, thereby forming the product. The seven
carbon chain aliphatic backbone can be pimeloyl-CoA. Pimeloyl-CoA
can be enzymatically synthesized from acetyl-CoA or 2-oxoglutarate
via 3-ketopimeloyl-CoA. A .beta.-ketothiolase or a .beta.-ketoacyl
[acp] synthase can convert glutaryl-CoA to 3-ketopimeloyl-CoA.
Glutaryl-CoA can be produced from crotonyl-CoA using a
glutaconyl-CoA decarboxylase or a glutaryl-CoA dehydrogenase.
3-ketopimeloyl-CoA can be converted to 3-hydroxypimeloyl-CoA using
a 3-hydroxyadipyl-CoA dehydrogenase; 3-hydroxypimeloyl-CoA can be
converted to 6-ketocyclohex-1-ene-1-carboxyl-CoA by a
6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or
3-hydroxypimeloyl-CoA can be converted to 2,3-dehydropimeloyl-CoA
by an enoyl-CoA hydratase.
[0019] The two terminal functional groups can be the same (e.g.,
amine or hydroxyl) or can be different (e.g., a terminal amine and
a terminal carboxyl group; or a terminal hydroxyl group and a
terminal carboxyl group).
[0020] A .omega.-transaminase or a deacetylase can enzymatically
form an amine group. The .omega.-transaminase can have at least 70%
sequence identity to any one of the amino acid sequences set forth
in SEQ ID NO. 8-13.
[0021] A 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate
dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol
dehydrogenase can enzymatically form a hydroxyl group.
[0022] A thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate
dehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA-transferase
(e.g. a glutaconate CoA transferase), or a reversible CoA-ligase
(e.g., a reversible succinate-CoA ligase) can enzymatically forms a
terminal carboxyl group. The thioesterase can have at least 70%
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 1.
[0023] A carboxylate reductase and a phosphopantetheinyl
transferase can form a terminal aldehyde group as an intermediate
in forming the product. The carboxylate reductase can have at least
70% sequence identity to any one of the amino acid sequences set
forth in SEQ ID NO. 2-7.
[0024] 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, micro-aerobic or mixed
oxygen/denitrification cultivation conditions. The host can be
cultured under conditions of nutrient limitation. The host can be
retained using a ceramic hollow fiber membrane to maintain a high
cell density during fermentation.
[0025] In any of the methods, the host's tolerance to high
concentrations of a C7 building block can be improved through
continuous cultivation in a selective environment.
[0026] The principal carbon source fed to the fermentation can
derive from biological or non-biological feedstocks. In some
embodiments, the biological feedstock is, includes, 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.
[0027] In some embodiments, the non-biological feedstock 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 a
terephthalic acid/isophthalic acid mixture waste stream.
[0028] In another aspect, this document features a recombinant host
that includes at least one exogenous nucleic acid encoding i) a
.beta.-ketothiolase or a .beta.-ketoacyl [acp] synthase, (ii) a
6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or an enoyl-CoA
hydratase, and (iii) a trans-2-enoyl-CoA reductase, an enoyl-[acp]
reductase, or a 2-ketocyclohexanecarboxyl-CoA hydrolase, the host
producing pimeloyl-CoA. The host further can include a
glutaconyl-CoA decarboxylase or a glutaryl-CoA dehydrogenase.
[0029] A recombinant host producing pimeloyl-CoA further can
include at least one exogenous nucleic acid encoding one or more of
a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate
dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate
CoA-transferase, a reversible succinyl-CoA ligase, an acetylating
aldehyde dehydrogenase, or a carboxylate reductase, the host
producing pimelic acid or pimelate semialdehyde.
[0030] A recombinant host producing pimelate semialdehyde further
can include at least one exogenous nucleic acid encoding a
.omega.-transaminase, and producing 7-aminoheptanoate.
[0031] A recombinant host producing pimelate semialdehyde further
can include at least one exogenous nucleic acid encoding a
4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate
dehydrogenase or a 6-hydroxyhexanoate dehydrogenase, the host
producing 7-hydroxyheptanoic acid.
[0032] A recombinant host producing pimelate semialdehyde,
7-aminoheptanoate, or 7-hydroxyheptanoic acid further can include a
carboxylate reductase, a .omega.-transaminase, a deacetylase, an
N-acetyl transferase, or an alcohol dehydrogenase, the host
producing heptamethylenediamine.
[0033] A recombinant host producing 7-hydroxyheptanoic acid further
can include at least one exogenous nucleic acid encoding a
carboxylate reductase or an alcohol dehydrogenase, the host
producing 1,7-heptanediol.
[0034] The recombinant host can be a prokaryote, e.g., from the
genus Escherichia such as Escherichia coli; from the genus
Clostridia such as Clostridium ljungdahlii, Clostridium
autoethanogenum or Clostridium kluyveri; from the genus
Corynebacteria such as Corynebacterium glutamicum; from the genus
Cupriavidus such as Cupriavidus necator or Cupriavidus
metallidurans; from the genus Pseudomonas such as Pseudomonas
fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the
genus Delftia acidovorans, from the genus Bacillus such as Bacillus
subtillis; from the genes Lactobacillus such as Lactobacillus
delbrueckii; from the genus Lactococcus such as Lactococcus lactis
or from the genus Rhodococcus such as Rhodococcus equi.
[0035] The recombinant host can be a eukaryote, e.g., a eukaryote
from the genus Aspergillus such as Aspergillus niger; from the
genus Saccharomyces such as Saccharomyces cerevisiae; from the
genus Pichia such as Pichia pastoris; from the genus Yarrowia such
as Yarrowia lipolytica, from the genus Issatchenkia such as
Issathenkia orientalis, from the genus Debaryomyces such as
Debaryomyces hansenii, from the genus Arxula such as Arxula
adenoinivorans, or from the genus Kluyveromyces such as
Kluyveromyces lactis.
[0036] Any of the recombinant hosts described herein further can
include one or more of the following attenuated enzymes: a
phosphotransacetylase generating acetate, an acetate kinase; a
lactate dehydrogenase; a menaquinol-fumarate oxidoreductase; an
alcohol dehydrogenase producing ethanol; a pyruvate decarboxylase;
a 2-oxoacid decarboxylase generating isobutanol; a polymer
synthase; a NADPH-specific L-glutamate dehydrogenase; a NADPH/NADH
L-glutamate dehydrogenase; a NADH-consuming transhydrogenase, a
pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase that degrades
C7 building blocks and their precursors; a glutaryl-CoA
dehydrogenase; or a pimeloyl-CoA synthetase.
[0037] Any of the recombinant hosts described herein further can
overexpress one or more genes encoding: an acetyl-CoA synthetase, a
formate dehydrogenase, a PEP carboxykinase, a PEP carboxylase, a
pyruvate carboxylase, a PEP synthase, a L-alanine dehydrogenase; a
NADH-specific L-glutamate dehydrogenase; a diamine transporter; a
dicarboxylate transporter; and/or a multidrug transporter.
[0038] 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 any of the above types of
host cells and used in a purified or semi-purified form. Extracted
enzymes can optionally be immobilized on a solid substrate such as
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.
[0039] Many of the enzymes described herein catalyze reversible
reactions, and the reaction of interest may be the reverse of the
described reaction. The schematic pathways shown in FIGS. 1-6
illustrate the reaction of interest for each of the
intermediates.
[0040] In some embodiments, the host microorganism's endogenous
biochemical network is attenuated or augmented to (1) ensure the
intracellular availability of acetyl-CoA (2) create an NADH
imbalance that may only be balanced via the formation of a C7
building block, (3) prevent degradation of central metabolites or
central precursors leading to and including C7 building blocks and
(4) ensure efficient efflux from the cell.
[0041] 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 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.
[0042] 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
[0043] FIG. 1 is a schematic of an exemplary biochemical pathway
leading to pimeloyl-CoA from acetyl-CoA or 2-oxo-glutarate as a
central metabolite.
[0044] FIG. 2 is a schematic of exemplary biochemical pathways
leading to pimelate using pimeloyl-CoA as a central precursor.
[0045] FIG. 3 is a schematic of exemplary biochemical pathways
leading to 7-aminoheptanoate using pimeloyl-CoA or pimelate as a
central precursor.
[0046] FIG. 4 is a schematic of an exemplary biochemical pathway
leading to heptamethylenediamine using 7-aminheptanoate,
7-hydroxyheptanoate, or pimelate semialdehyde as a central
precursor.
[0047] FIG. 5 is a schematic of an exemplary biochemical pathway
leading to 7-hydroxyheptanoate using pimeloyl-CoA or pimelic acid
as a central precursor.
[0048] FIG. 6 is a schematic of an exemplary biochemical pathway
leading to 1,7-heptanediol using 7-hydroxyheptanoate as a central
precursor.
[0049] FIG. 7 contains the amino acid sequences of an Escherichia
coli thioesterase encoded by tesB (see GenBank Accession No.
AAA24665.1, 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), a
Chromobacterium violaceum .omega.-transaminase (see Genbank
Accession No. AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa
.omega.-transaminase (see Genbank Accession No. AAG08191.1, SEQ ID
NO: 9), a Pseudomonas syringae .omega.-transaminase (see Genbank
Accession No. AAY39893.1, SEQ ID NO: 10), a Rhodobacter sphaeroides
.omega.-transaminase (see Genbank Accession No. ABA81135.1, SEQ ID
NO: 11), an Escherichia coli .omega.-transaminase (see Genbank
Accession No. AAA57874.1, SEQ ID NO: 12), a Vibrio fluvialis
.omega.-transaminase (see Genbank Accession No. AEA39183.1, SEQ ID
NO: 13), a Bacillus subtilis phosphopantetheinyl transferase (see
Genbank Accession No. CAA44858.1, SEQ ID NO:14), or a Nocardia sp.
NRRL 5646 phosphopantetheinyl transferase (see Genbank Accession
No. ABI83656.1, SEQ ID NO:15).
[0050] FIG. 8 is a bar graph of the relative absorbance at 412 nm
of released CoA as a measure of the activity of a thioesterase for
converting pimeloyl-CoA to pimelate relative to the empty vector
control.
[0051] 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 carboxylate reductases relative to the
enzyme only controls (no substrate).
[0052] 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 the activity of carboxylate reductases for converting pimelate
to pimelate semialdehyde relative to the empty vector control.
[0053] FIG. 11 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 the activity of carboxylate reductases for converting
7-hydroxyheptanoate to 7-hydroxyheptanal relative to the empty
vector control.
[0054] FIG. 12 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 the activity of carboxylate reductases for converting
N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal relative
to the empty vector control.
[0055] FIG. 13 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 carboxylate reductases for converting pimelate
semialdehyde to heptanedial relative to the empty vector
control.
[0056] FIG. 14 is a bar graph summarizing the percent conversion
after 4 hours of pyruvate to L-alanine (mol/mol) as a measure of
the .omega.-transaminase activity of the enzyme only controls (no
substrate) or the empty vector.
[0057] FIG. 15 is a bar graph of the percent conversion after 4
hours of pyruvate to L-alanine (mol/mol) as a measure of the
.omega.-transaminase activity for converting 7-aminoheptanoate to
pimelate semialdehyde relative to the empty vector control.
[0058] FIG. 16 is a bar graph of the percent conversion after 4
hours of L-alanine to pyruvate (mol/mol) as a measure of the
.omega.-transaminase activity for converting pimelate semialdehyde
to 7-aminoheptanoate relative to the empty vector control.
[0059] FIG. 17 is a bar graph of the percent conversion after 4
hours of pyruvate to L-alanine (mol/mol) as a measure of the
.omega.-transaminase activity for converting heptamethylene diamine
to 7-aminoheptanal relative to the empty vector control.
[0060] FIG. 18 is a bar graph of the percent conversion after 4
hours of pyruvate to L-alanine (mol/mol) as a measure of the
.omega.-transaminase activity for converting
N7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal relative
to the empty vector control.
[0061] FIG. 19 is a bar graph of the percent conversion after 4
hours of pyruvate to L-alanine (mol/mol) as a measure of the
.omega.-transaminase activity for converting 7-aminoheptanol to
7-hydroxyheptanal relative to the empty vector control.
DETAILED DESCRIPTION
[0062] This document provides enzymes, non-natural pathways,
cultivation strategies, feedstocks, host microorganisms and
attenuations to the host's biochemical network, which generates a
seven carbon chain aliphatic backbone from central metabolites in
which one or two terminal functional groups may be formed leading
to the synthesis of one or more of pimelic acid, 7-hydroxyheptanoic
acid, 7-aminoheptanoic acid, heptamethylenediamine, and
1,7-heptanediol (referred to as "C7 building blocks" herein). As
used herein, the term "central precursor" is used to denote any
metabolite in any metabolic pathway shown herein leading to the
synthesis of a C7 building block. The term "central metabolite" is
used herein to denote a metabolite that is produced in all
microorganisms to support growth.
[0063] Host microorganisms described herein can include endogenous
pathways that can be manipulated such that one or more C7 building
blocks 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.
[0064] 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.
[0065] 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.
[0066] 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 in addition to (i) a .beta.-ketothiolase or a
.beta.-ketoacyl [acp] synthase; (ii) a
6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or an enoyl-CoA
hydratase accepting 3-hydroxypimeloyl-CoA as substrate, and (iii) a
trans-2-enoyl-CoA reductase, an enoyl-[acp] reductase, or a
2-ketocyclohexanecarboxyl-CoA hydrolase: a glutaconyl-CoA
decarboxylase, a glutaryl-CoA dehydrogenase, a 3-hydroxyacyl-CoA
dehydrogenase, a 6-hydroxycyclohex-1-ene-1-carboxyl-CoA
dehydrogenase, a cyclohexa-1,5-dienecarbonyl-CoA hydratase, a
cyclohex-1-ene-1-carboxyl-CoA dehydrogenase, a
cyclohex-1-ene-1-carboxyl-CoA hydratase, a
2-hydroxycyclohexanecarboxyl-CoA dehydrogenase, a thioesterase, an
aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a
6-oxohexanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a
5-hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate
dehydrogenase, a CoA-transferase, a reversible CoA-ligase, an
acetylating aldehyde dehydrogenase, a .omega.-transaminase, a
carboxylate reductase, a deacetylase, a lysine N-acetyl
transferase, or an alcohol dehydrogenase. In recombinant hosts
expressing a carboxylate reductase, a phosphopantetheinyl
transferase also can be expressed as it enhances activity of the
carboxylate reductase.
[0067] For example, a recombinant host can include a
.beta.-ketothiolase or a .beta.-ketoacyl [acp] synthase; a
6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or an enoyl-CoA
hydratase accepting 3-hydroxypimeloyl-CoA as substrate; and a
trans-2-enoyl-CoA reductase, an enoyl-[acp] reductase, or a
2-ketocyclohexanecarboxyl-CoA hydrolase, and produce pimeloyl-CoA.
The host further can include a glutaconyl-CoA decarboxylase or a
glutaryl-CoA dehydrogenase.
[0068] A recombinant host producing pimeloyl-CoA further can
include one or more of an aldehyde dehydrogenase, a 6-oxohexanoate
dehydrogenase, a thioesterase, a 7-oxoheptanoate dehydrogenase, a
reversible succinyl-CoA-ligase, a glutaconate CoA transferase, a
carboxylate reductase, or an acetylating aldehyde dehydrogenase,
and produce pimelate or pimelate semialdehyde. For example, a
recombinant host producing pimeloyl-CoA can include a thioesterase,
a CoA-transferase, or a reversible CoA-ligase and produce pimelic
acid. For example, a recombinant host producing pimeloyl-CoA can
include an acetylating aldehyde dehydrogenase and either an
aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a
6-oxohexanoate dehydrogenase and produce pimelic acid. For example,
a recombinant host producing pimeloyl-CoA can include an
acetylating aldehyde dehydrogenase or a carboxylate reductase and
produce pimelate semialdehyde.
[0069] A recombinant host producing pimelic acid or pimelate
semialdehyde further can include at least one exogenous nucleic
acid encoding a .omega.-transaminase and produce
7-aminoheptanoate.
[0070] A recombinant host producing pimelate semialdehyde further
can include a 6-hydroxyhexanoate dehydrogenase, a
5-hydroxypentanoate dehydrogenase or a 4-hydroxybutyrate
dehydrogenase, and produce 7-hydroxyheptanoic acid.
[0071] A recombinant host producing pimelate semialdehyde,
7-aminoheptanoate, or 7-hydroxyheptanoate further can include at
least one exogenous nucleic acid encoding a carboxylate reductase,
a .omega.-transaminase, a deacetylase, a lysine N-acetyl
transferase, or an alcohol dehydrogenase, and produce
heptamethylenediamine. For example, a recombinant host producing
7-hydroxyheptanoate can include a carboxylate reductase, a
.omega.-transaminase and an alcohol dehydrogenase.
[0072] A recombinant host producing 7-hydroxyheptanoic acid further
can include a carboxylate reductase or an alcohol dehydrogenase,
and produce 1,7-heptanediol.
[0073] 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.
[0074] Any of the enzymes described herein that can be used for
production of one or more C7 building blocks can have at least 70%
sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,
95%, 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).
[0075] For example, a thioesterase described herein can have at
least 70% sequence identity (homology) (e.g., at least 75%, 80%,
85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence
of an Escherichia coli thioesterase encoded by tesB (see GenBank
Accession No. AAA24665.1, SEQ ID NO: 1). See FIG. 7.
[0076] For example, a carboxylate reductase described herein can
have at least 70% sequence identity (homology) (e.g., at least 75%,
80%, 85%, 90%, 95%, 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. 7.
[0077] For example, a .omega.-transaminase described herein can
have at least 70% sequence identity (homology) (e.g., at least 75%,
80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid
sequence of a Chromobacterium violaceum (see Genbank Accession No.
AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa (see Genbank
Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae
(see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), a
Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQ
ID NO: 11), an Escherichia coli (see Genbank Accession No.
AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank
Accession No. AEA39183.1, SEQ ID NO: 13) .omega.-transaminase. Some
of these .omega.-transaminases are diamine .omega.-transaminases.
See, FIG. 7.
[0078] For example, a phosphopantetheinyl transferase described
herein can have at least 70% sequence identity (homology) (e.g., at
least 75%, 80%, 85%, 90%, 95%, 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:14) or
a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see
Genbank Accession No. ABI83656.1, SEQ ID NO:15). See FIG. 7.
[0079] 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 (B12seq) 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
B12seq program can be found in the readme file accompanying BLASTZ.
B12seq performs a comparison between two amino acid sequences using
the BLASTP algorithm. To compare two amino acid sequences, the
options of B12seq 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:\B12seq -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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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 of a dehydrogenase, a .beta.-ketothiolase, a
.beta.-ketoacyl-[acp] synthase, a reductase, a hydratase, a
thioesterase, a CoA-ligase, a reversible CoA-ligase, a
CoA-transferase, an N-acetyl transferase, a deacetylase, or a
.omega.-transaminase as described in detail herein.
[0086] In addition, the production of one or more C7 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 C7 Aliphatic Backbone, pimeloyl-CoA, for
Conversion to C7 Building Blocks
[0087] As depicted in FIG. 1, pimeloyl-CoA, a C7 aliphatic backbone
for conversion to a C7 building block, can be formed from the
central metabolites acetyl-CoA or 2-oxoglutarate via carbon chain
elongation (i) associated with cyclohexane carboxylate biosynthesis
in Synthrophus aciditrophicus or (ii) associated with lysine
biosynthesis via .alpha.-aminoadipate.
[0088] In some embodiments, glutaryl-CoA is formed via
CoA-dependent carbon chain elongation associated with cyclohexane
carboxylate biosynthesis in Synthrophus aciditrophicus which
comprises using (i) a .beta.-ketothiolase or an acetyl-carboxylase
in combination with an acetoacetyl-CoA synthase, (ii) a
3-hydroxybutyryl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase,
and either (iv) a glutaryl-CoA dehydrogenase in combination with an
enoyl-CoA reductase or a trans-2-enoyl-CoA reductase or (v) a
glutaconyl-CoA decarboxylase.
[0089] In some embodiments, glutaryl-CoA is formed via C1 carbon
chain elongation associated with lysine biosynthesis via
.alpha.-aminoadipate, which comprises using (i) a homocitrate
synthase, (ii) a homocitrate dehydratase and a homoaconitate
hydratase, (iii) an isohomocitrate dehydrogenase, (iv) an
indolepyruvate decarboxylase, (vi) a glutarate-semialdehyde
dehydrogenase and (v) a glutarate: CoA ligase.
[0090] In some embodiments, pimeloyl-CoA is formed from
glutaryl-CoA via CoA dependent chain elongation using (i) a
.beta.-ketothiolase or .beta.-ketoacyl-[acp] synthase, (ii) a
3-hydroxyacyl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase, and
(iv) an enoyl-CoA reductase.
[0091] In some embodiments, pimeloyl-CoA is formed from
3-hydroxypimeloyl-CoA via aromatic intermediates associated with
cyclohexane carboxylate biosynthesis in Synthrophus aciditrophicus
using (i) a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase, (ii) a
6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase, (iii) a
cyclohexa-1,5-dienecarbonyl-CoA hydratase, (iv) a
cyclohex-1-ene-1-carboxyl-CoA dehydrogenase, (v) a
cyclohex-1-ene-1-carboxyl-CoA hydratase, (vi) a
2-hydroxycyclohexanecarboxyl-CoA dehydrogenase and (vi) a
2-ketocyclohexanecarboxyl-CoA hydrolase.
[0092] In some embodiments, a .beta.-ketothiolase may be classified
under EC 2.3.1.- (e.g., EC 2.3.1.9, EC 2.3.1.16, or EC 2.3.174).
For example, a .beta.-ketothiolase may be classified under EC
2.3.1.9, such as the gene product of atoB or phaA. The
.beta.-ketothiolase encoded by atoB or phaA accepts acetyl-CoA as
substrates, forming butanoyl-CoA (see, Haywood et al., 1988, supra;
Slater et al., 1998, supra). The .beta.-ketothiolase encoded by
paaJ can be classified under, for example, EC 2.3.1.174. The
.beta.-ketothiolase encoded by paaJ condenses acetyl-CoA and
succinyl-CoA to 3-oxoadipyl-CoA (see, for example, Fuchs et al.,
2011, Nature Reviews Microbiology, 9, 803-816). A homologue of paaJ
in Synthrophus aciditrophicus catalyses the condensation of
acetyl-CoA and glutaryl-CoA to 3-oxopimeloyl-CoA such as Genbank
Accession No. ABC78517.1 or Genbank Accession No. ABC78881.1.
Alternately, a .beta.-ketoacyl-[acp] homologue of paaJ in S.
aciditrophicus catalyses the condensation of acetyl-CoA and
glutaryl-CoA to 3-oxopimeloyl-CoA.
[0093] A .beta.-ketoacyl-[acp] synthase can be classified, for
example, under, EC 2.3.1.41, EC 2.3.1.179, or EC 2.3.1.180. The
.beta.-ketothiolases and .beta.-ketoacyl-[acp] synthases involved
in fatty acid synthesis in S. aciditrophicus likely accept CoA
activated dicarboxylic acids (Mouttaki et al., Appl. Environ.
Microbiol., 2007, 73(3), 930-938).
[0094] An acetyl-CoA carboxylase can be classified under EC 6.4.1.2
and an acetoacetyl-CoA synthase can be classified under EC
2.3.1.194. Conversion of acetyl-CoA to malonyl-CoA by an acetyl-CoA
carboxylase has been shown to increase the rate of fatty acid
synthesis (Davis et al., J. Biol. Chem., 2000, 275(37),
28593-28598). It has been demonstrated that acetoacetyl-CoA
synthase may be used as an irreversible substitute for the gene
product of phaA in the carbon chain elongation associated with
polyhydroxybutyrate synthesis (Matsumoto et al., Biosci.
Biotechnol. Biochem., 2011, 75(2), 364-366).
[0095] In some embodiments, a 3-hydroxybutyryl-CoA dehydrogenase
(also can be referred to as a 3-hydroxyacyl-CoA dehydrogenase) may
be classified under EC 1.1.1.157 such as the gene product hbd (see,
for example, Shen et al., 2011, supra; Budde et al., J. Bacteriol.,
2010, 192(20), 5319-5328).
[0096] In some embodiments, an enoyl-CoA hydratase can be
classified under EC 4.2.1.17, such as the gene product of crt (see,
for example, Shen et al., 2011, supra; Fukui et al., J. Bacteriol.,
1998, 180(3), 667-673) or the gene product of paaF (see, for
example, Fuchs et al., 2011, supra). Homologs of paaF in S.
aciditrophicus include the enoyl-CoA hydratase of Genbank Accession
No. ABC77794.1 or the enoyl-CoA dehydratase of Genbank Accession
No. ABC78950.1.
[0097] In some embodiments, an enoyl-[acp] reductase can be
classified under EC 1.3.1.- (e.g., EC 1.3.1.9) such as the
enoyl-[acp] reductase obtained from S. aciditrophicus or the gene
product of Fabl (Genbank Accession No: CAB13029.2) from Bacillus
subtillis (see, for example, Heath et al., 2000, J. Biol. Chem.,
275(51), 40128-33). The enoyl-[acp] reductase involved in fatty
acid synthesis in S. aciditrophicus likely accepts CoA activated
dicarboxylic acids (Mouttaki et al., 2007, supra).
[0098] In some embodiments, a trans-2-enoyl-CoA reductase can be
classified, for example, under EC 1.3.1.44, such as the gene
product of ter (Genbank Accession No. AAW66853.1) (Hoffmeister et
al., 2005, J. Biol. Chem., 280(6), 4329-4338; Shen et al., 2011,
supra) or tdter (Genbank Accession No. AAS11092.1) (Bond-Watts et
al., Biochemistry, 2012, 51, 6827-6837).
[0099] In some embodiments, a reversible glutaconyl-CoA
decarboxylase that relies on a Na.sup.+ membrane pump can be
classified, for example, under EC 4.1.1.70 (see Mouttaki et al.,
2007, supra). The EC 4.1.1.70 enzyme activity is associated with
the following subunits in S. aciditrophicus, viz. Genbank Accession
Nos. (1) ABC77900.1, (2) ABC76114.1 and (3) ABC77898.1.
[0100] In some embodiments, a glutaryl-CoA dehydrogenase from S.
aciditrophicus subject to electron bifurcation via NADH can be
classified under EC 1.3.8.6 such as the dehydrogenases in Genbank
Accession Nos. (1) ABC77899.1, (2) ABC76101.1, (3) ABC76260.1, (4)
ABC76949.1 or (5) ABC78863.1.
[0101] In some embodiments, a homocitrate synthase can be
classified, for example, under EC 2.3.3.14 or EC 2.3.3.13, such as
the gene product of LYS20 and LYS21 from Saccharomyces cerevisiae
or hcs from Thermus thermophiles.
[0102] In some embodiments, the combination of homocitrate
dehydratase and homoaconitate hydratase may be classified, for
example, under EC 4.2.1.- (e.g., EC 4.2.1.114, EC 4.2.1.36 or EC
4.2.1.33), such as the gene product of LYS4 from Saccharomyces
cerevisiae or lysT and LysU from Thermus thermophiles.
[0103] In some embodiments, an isohomocitrate dehydrogenase may be
classified, for example, under EC 1.1.1.- such as EC 1.1.1.85, EC
1.1.1.87 or EC 1.1.1.286, such as the gene product of LYS12 from
Saccharomyces cerevisiae or hicdh from Thermus thermophiles.
[0104] In some embodiments, a 5-oxopentanoate dehydrogenase may be
classified, for example under EC 1.2.1.- such as the gene product
of CpnE (see, for example, Iwaki et al., 2002, Appl. Environ.
Microbiol., 68(11):5671-5684).
[0105] In some embodiments, an indolepyruvate decarboxylase can be
classified, for example, under EC 4.1.1.43 or EC 4.1.1.74 such as
the indole-3-pyruvate decarboxylase from Salmonella typhimurium
(see, for example, Genbank Accession No. CAC48239.1 in which
residue 544 can be a leucine or an alanine). A mutant variant of
the indolepyruvate decarboxylase from Salmonella typhimurium has
been engineered successfully to selectively accept longer chain
length substrates. The L544A mutation of Genbank Accession No.
CAC48239.1 allowed for 567 times higher selectivity towards the C7
2-oxoacid than towards the C5 2-oxoacid (see, Xiong et al., 2012,
Scientific Reports, 2: 311).
[0106] In some embodiments, a 3-hydroxyadipyl-CoA dehydrogenase
(also can be referred to as 3-hydroxyacyl-CoA:NAD.sup.+
oxidoreducatase) may be classified under EC 1.1.1.157 such as the
gene product of paaH (see, for example, Fuchs et al., 2011, Nature
Reviews Microbiology, 9, 803-816). Homologues of paaH in S.
aciditrophicus include the 3-hydroxyacyl-CoA dehydrogenase of
Genbank Accession No. ABC77793.1.
[0107] In some embodiments, a 6-oxo-cyclohex-1-ene-carbonyl-CoA
hydrolase can be classified under EC 3.7.1.- such as the gene
product of Oah or BamA (see, for example, Fuchs et al., 2011,
supra).
[0108] In some embodiments, a
6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase can be
classified under EC 1.1.1.- such as the gene product of Had or BamA
(see, for example, Fuchs et al., 2011, supra).
[0109] In some embodiments, a cyclohexa-1,5-dienecarbonyl-CoA
hydratase can be classified under EC 4.2.1.100 such as the gene
product of Dch or BamR (see, for example, Fuchs et al., 2011,
supra).
[0110] In some embodiments, a cyclohex-1-ene-1-carboxyl-CoA
dehydrogenase can be classified under EC 1.3.8.- such as the
acyl-CoA dehydrogenase of Genbank Accession No. ABC76101.1 from S.
aciditrophicus (see, for example, Kung et al., 2013, J. Bacteriol.,
195(14), 3193-3200). This enzyme was reassigned to class EC 1.3.8.-
from EC 1.3.99.-.
[0111] In some embodiments, a cyclohex-1-ene-1-carboxyl-CoA
hydratase can be classified under EC 4.2.1.- such as the gene
product of badK (see, for example, Harwood et al., 1999, FEMS
Microbiol. Reviews, 22, 439-458). Homologues of badK in S.
aciditrophicus include the enoyl-CoA hydratase of Genbank Accession
No. ABC77794.1.
[0112] In some embodiments, a 2-hydroxycyclohexanecarboxyl-CoA
dehydrogenase can be classified under EC 1.1.1.- such as the gene
product of badH (see, for example, Harwood et al., 1999, FEMS
Microbiol. Reviews, 22, 439-458). Homologs of badH in S.
aciditrophicus include the 3-oxoacyl-ACP reductase of Genbank
Accession No. ABC76948.1.
[0113] In some embodiments, a 2-ketocyclohexanecarboxyl-CoA
hydrolase can be classified under EC 4.1.3.36 such as the gene
product of badI (see, for example, Harwood et al., 1999, supra).
Homologs of badI in S. aciditrophicus such as encoded by Genbank
Accession No. ABC78756.1.
Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis
of C7 Building Blocks
[0114] As depicted in FIG. 1 and FIG. 2, a terminal carboxyl group
can be enzymatically formed using a thioesterase, an aldehyde
dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate
dehydrogenase, a CoA-transferase, or a reversible CoA-ligase. In
some embodiments, only one terminal carboxyl group is added as the
central metabolite C7 building block (e.g., 3-ketopimeloyl-CoA)
already has a carboxyl group at one terminus.
[0115] In some embodiments, the second terminal carboxyl group
leading to the synthesis of a C7 building block (e.g., pimelic
acid) is enzymatically formed by a thioesterase classified under EC
3.1.2.-, such as the gene product of YciA, tesB (Genbank Accession
No. AAA24665.1, SEQ ID NO: 1) 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).
[0116] In some embodiments, the second terminal carboxyl group
leading to the synthesis of pimelic acid is enzymatically formed by
an aldehyde dehydrogenase classified, for example, under EC 1.2.1.3
(see, for example, Guerrillot & Vandecasteele, Eur. J.
Biochem., 1977, 81, 185-192).
[0117] In some embodiments, the second terminal carboxyl group
leading to the synthesis of pimelic acid is enzymatically formed by
a 6-oxohexanoate dehydrogenase or a 7-oxoheptanoate dehydrogenase
classified under EC 1.2.1.-, such as the gene product of ChnE from
Acinetobacter sp. or ThnG from Sphingomonas macrogolitabida (see,
for example, Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11),
5158-5162; or 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. For example, a 7-oxoheptanoate
dehydrogenase can be classified under EC 1.2.1.-.
[0118] In some embodiments, the second terminal carboxyl group
leading to the synthesis of pimelic 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.
[0119] In some embodiments, a terminal carboxyl group leading to
the synthesis of pimelic acid is enzymatically formed by a
reversible CoA-ligase classified under EC 6.2.1.5.
[0120] In some embodiments, the second terminal carboxyl group
leading to the synthesis of pimelic 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.
Enzymes Generating the Terminal Amine Groups in the Biosynthesis of
C7 Building Blocks
[0121] As depicted in FIG. 3 and FIG. 4, terminal amine groups can
be enzymatically formed using a .omega.-transaminase or a
deacetylase.
[0122] In some embodiments, the first terminal amine group leading
to the synthesis of 7-aminoheptanoic acid is enzymatically formed
by a .omega.-transaminase classified, for example, under EC
2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82
such as that obtained from Chromobacterium violaceum (Genbank
Accession No. AAQ59697.1, SEQ ID NO: 8), Pseudomonas aeruginosa
(Genbank Accession No. AAG08191.1, SEQ ID NO: 9), Pseudomonas
syringae (Genbank Accession No. AAY39893.1, SEQ ID NO: 10),
Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQ ID
NO: 11), Vibrio Fluvialis (Genbank Accession No. AAA57874.1, SEQ ID
NO: 13), Streptomyces griseus, or Clostridium viride. An additional
.omega.-transaminase that can be used in the methods and hosts
described herein is from Escherichia coli (Genbank Accession No.
AAA57874.1, SEQ ID NO: 12). Some of the .omega.-transaminases
classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are
diamine .omega.-transaminases (e.g., SEQ ID NO:12).
[0123] The reversible .omega.-transaminase from Chromobacterium
violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 8) has
demonstrated analogous activity accepting 6-aminohexanoic acid as
amino donor, thus forming the first terminal amine group in adipate
semialdehyde (Kaulmann et al., Enzyme and Microbial Technology,
2007, 41, 628-637).
[0124] The reversible 4-aminobubyrate:2-oxoglutarate transaminase
from Streptomyces griseus has demonstrated analogous activity for
the conversion of 6-aminohexanoate to adipate semialdehyde (Yonaha
et al., Eur. J. Biochem., 1985, 146, 101-106).
[0125] The reversible 5-aminovalerate transaminase from Clostridium
viride has demonstrated analogous activity for the conversion of
6-aminohexanoate to adipate semialdehyde (Barker et al., J. Biol.
Chem., 1987, 262(19), 8994-9003).
[0126] In some embodiments, the second terminal amine group leading
to the synthesis of heptamethylenediamine is enzymatically formed
by a diamine transaminase. For example, the second terminal amino
group can be enzymatically formed by a diamine transaminase
classified, for example, under EC 2.6.1.29 or classified, for
example, under EC 2.6.1.82, such as the gene product of YgjG from
E. coli (Genbank Accession No. AAA57874.1, SEQ ID NO: 12).
[0127] The gene product of ygiG accepts a broad range of diamine
carbon chain length substrates, such as putrescine, cadaverine and
spermidine (Samsonova et al., BMC Microbiology, 2003, 3:2).
[0128] The diamine transaminase from E.coli strain B has
demonstrated activity for 1,7 diaminoheptane (Kim, The Journal of
Chemistry, 1964, 239(3), 783-786).
[0129] In some embodiments, the second terminal amine group leading
to the synthesis of heptamethylenediamine is enzymatically formed
by a deacetylase classified, for example, under EC 3.5.1.62 such as
an acetylputrescine deacetylase. The acetylputrescine deacetylase
from Micrococcus luteus K-11 accepts a broad range of carbon chain
length substrates, such as acetylputrescine, acetylcadaverine and
N.sup.8. acetylspermidine (see, for example, Suzuki et al., 1986,
BBA--General Subjects, 882(1):140-142).
Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis
of C7 Building Blocks
[0130] As depicted in FIG. 5 and FIG. 6, the terminal hydroxyl
group can be enzymatically formed using an alcohol
dehydrogenase.
[0131] In some embodiments, the terminal hydroxyl group leading to
the synthesis of 1,7 heptanediol is enzymatically formed by an
alcohol dehydrogenase classified, for example, under EC 1.1.1.-
(e.g., EC 1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184).
Biochemical Pathways
[0132] Pathway Using Acetyl-CoA or 2-oxo-glutarate as Central
Metabolite In The Biosynthesis of C7 Backbone
[0133] In some embodiments, glutaryl-CoA is synthesized from the
central metabolite, acetyl-CoA, by conversion of acetyl-CoA to
acetoacetyl-CoA by a .beta.-ketothiolase classified, for example,
under EC 2.3.1.9 such as the gene product of atoB or phaA or by an
acetyl-CoA carboxylase classified under, for example, EC 6.4.1.2
and an acetoacetyl-CoA synthase classified, for example, under EC
2.3.1.194; followed by conversion to 3-hydroxybutanoyl-CoA by a
3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC
1.1.1.- such as EC 1.1.1.157 such as the gene product of hbd;
followed by conversion to crotonyl-CoA by an enoyl-CoA reductase
classified, for example, under EC 4.2.1.- (e.g., EC 4.2.1.17) such
as the gene product of crt, followed by conversion to either a)
glutaconyl-CoA by a glutaconyl-CoA decarboxylase classified, for
example, under EC 4.1.1.70; followed by conversion to glutaryl-CoA
by either (i) an enoyl-[acp] reductase classified, for example,
under EC 1.3.1.9 or (ii) a trans-2-enoyl-CoA reductase classified,
for example, under EC 1.3.1.44 such as the gene product of ter or
tdter or (b) glutaryl-CoA by a glutaryl-CoA dehydrogenase subject
to electron bifurcation from Synthrophus aciditrophicus such as the
dehydrogenases of Genbank Accession Nos. (1) ABC77899.1, (2)
ABC76101.1, (3) ABC76260.1, (4) ABC76949.1 or (5) ABC78863.1. See,
FIG. 1.
[0134] In some embodiments, glutaryl-CoA can be synthesized from
the central metabolite, 2-oxoglutarate, by conversion of
2-oxoglutrate to (Homo).sub.icitrate by a homocitrate synthase
classified, for example, under EC 2.3.3.14 or EC 2.3.3.13 such as
the gene product of LYS20 and LYS21 from Saccharomyces cerevisiae
or hcs from Thermus thermophiles; followed by conversion to
iso(homo).sub.1citrate by a homocitrate dehydratase and a
homoaconitate hydratase classified, for example, under EC
4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33 such as the gene product of
LYS4 from Saccharomyces cerevisiae or lysT and LysU from Thermus
thermophiles; followed by conversion to 2-oxoadipate by an
iso(homo).sub.ncitrate dehydrogenase classified, for example, under
EC 1.1.1.85, EC 1.1.1.87 or EC 1.1.1.286 such as the gene product
of LYS/2 from Saccharomyces cerevisiae or hicdh from Thermus
thermophiles; followed by conversion to glutarate semialdehyde by
an indolepyruvate decarboxylase classified under EC 4.1.1.43 or EC
4.1.1.74 (e.g., GenBank Accession No. CAC48239.1); followed by
conversion to glutarate by a glutarate semialdehyde dehydrogenase
classified, for example, under EC 1.2.1.20, EC 1.2.1.16 or EC
1.2.1.79 such as the gene product of CpnE; followed by conversion
to glutaryl-CoA by a glutarate:CoA ligase classified, for example,
under EC 6.2.1.6. See, e.g., FIG. 1.
[0135] In some embodiments, pimeloyl-CoA can be synthesized from
glutaryl-CoA produced as described above by conversion of
glutaryl-CoA to 3-ketopimeloyl-CoA by a .beta.-ketothiolase
classified under EC 2.3.1.-, e.g., EC 2.3.1.174 or EC 2.3.1.16 such
as the gene product of paaJ or homologs of paaJ (e.g., Genbank
Accession No. ABC78517.1 or Genbank Accession No. ABC78881.1) or by
a .beta.-ketoacyl-[acp] synthase classified, for example, under EC
2.3.1.41, EC 2.3.1.179, EC 2.3.1.180; followed by conversion to
3-hydroxypimeloyl-CoA by a 3-hydroxyadipyl-CoA dehydrogenase
classified, for example, under EC 1.1.1.157 such as the gene
product ofpaaH or homologs ofpaaH (e.g., Genbank Accession No.
ABC77793.1); followed by conversion to 2,3-dehydropimeloyl-CoA by
an enoyl-CoA hydratase such as the gene product of paaF or homologs
of paaF (e.g., Genbank Accession No. ABC77794.1 or Genbank
Accession No. ABC78950.1); followed by conversion to pimeloyl-CoA
by an enoyl-[acp] reductase from S. aciditrophicus classified, for
example, under EC 1.3.1.9 or a trans-2-enoyl-CoA reductase
classified, for example, under EC 1.3.1.44 such as the gene product
of ter or tdter. See, e.g., FIG. 1.
[0136] In some embodiments, pimeloyl-CoA can be synthesized from
glutaryl-CoA produced as described above by conversion of
glutaryl-CoA to 3-ketopimeloyl-CoA by a .beta.-ketothiolase
classified under EC 2.3.1.-, e.g., EC 2.3.1.174 or EC 2.3.1.16 such
as the gene product ofpaca or homologs ofpaca (e.g., Genbank
Accession No. ABC78517.1 or Genbank Accession No. ABC78881.1) or by
a .beta.-ketoacyl-[acp] synthase classified, for example, under EC
2.3.1.41, EC 2.3.1.179, EC 2.3.1.180; followed by conversion to
6-ketocyclohex-1-ene-1-carboxyl-CoA by a
6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase classified, for
example, under EC 3.7.1.- such as the gene product of Oah or BamA;
followed by conversion to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA by
a 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase classified,
for example, under EC 1.1.1.- such as the gene product of Had or
BamQ; followed by conversion to cyclohexa-1,5-diene-1-carbonyl-CoA
by a cyclohexa-1,5-dienecarbonyl-CoA hydratase classified under,
for example, EC 4.2.1.100 such as the gene product of Dch or BamR;
followed by conversion to cyclohex-1-ene-1-carboxyl-CoA by a
cyclohex-1-ene-1-Carboxyl-CoA dehydrogenase classified under EC
1.3.8.- such as Genbank Accession No. ABC76100.1; followed by
conversion to 2-hydroxycyclohexane-1-carboxyl-CoA by a
cyclohex-1-ene-1-carboxyl-CoA hydratase classified under, for
example, EC 4.2.1.- such as the gene product of badK or homologs of
badK (e.g., Genbank Accession No. ABC77794.1); followed by
conversion to 2-ketocyclohex-1-ene-1-carboxyl-CoA by a
2-hydroxycyclohexanecarboxyl-CoA dehydrogenase classified, for
example, under EC 1.1.1.- such as the gene product of badH or
homologs of badH (e.g., Genbank Accession No. ABC76948.1); followed
by conversion to pimeloyl-CoA by a 2-ketocyclohexanecarboxyl-CoA
hydrolase classified, for example, under, EC 4.1.3.36 such as the
gene product of badI or homologs of badI (e.g., Genbank Accession
No. ABC78756.1).
Pathways Using Pimeloyl-CoA as Central Precursor to Pimelic
Acid
[0137] In some embodiments, pimelic acid is synthesized from the
central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to
pimelate by a thioesterase classified under EC 3.1.2.- such as the
gene products of YciA, tesB (Genbank Accession No. AAA24665.1, SEQ
ID NO: 1) or Acot13. See FIG. 2.
[0138] In some embodiments, pimelic acid is synthesized from the
central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to
pimelate by a reversible CoA ligase such as a succinate CoA-ligase
classified, for example, under EC 6.2.1.5. See FIG. 2.
[0139] In some embodiments, pimelic acid is synthesized from the
central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to
pimelate by a CoA-transferase classified, for example, under EC
2.8.3.12 such as a glutaconate CoA-transferase. See FIG. 2.
[0140] In some embodiments, pimeloyl-CoA can be converted to
pimelate semialdehyde by an acetylating aldehyde dehydrogenase
classified, for example, under EC 1.2.1.10 such as the gene product
of PduB or PduP; followed by conversion to pimelic acid by a
dehydrogenase classified, for example, under EC 1.2.1.- such as a
7-oxoheptanoate dehydrogenase (e.g., the gene product of ThnG), a
6-oxohexanoate dehydrogenase (e.g., the gene product of ChnE), or
an aldehyde dehydrogenase classified under EC 1.2.1.3. See FIG.
2.
Pathways Using Pimeloyl-CoA as Central Precursor to
7-aminoheptanoate
[0141] In some embodiments, 7-aminoheptanoate is synthesized from
the central precursor, pimelate or pimeloyl-CoA. For example,
pimelate, formed as described above, can be converted to pimelate
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 gene from Bacillus subtilis or npt gene from Nocardia) or the
gene products of GriC and GriD from Streptomyces griseus (see
Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by
conversion of pimelate semialdehyde to 7-aminoheptanoate by a
.omega.-transaminase (classified, for example, under EC 2.6.1.- EC
2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.82, see SEQ ID
NOs:8-13). See FIG. 3. The carboxylate reductase can be obtained,
for example, from Mycobacterium marinum (Genbank Accession No.
ACC40567.1, SEQ ID NO: 2), Mycobacterium smegmatis (Genbank
Accession No. ABK71854.1, SEQ ID NO: 3), Segniliparus rugosus
(Genbank Accession No. EFV11917.1, SEQ ID NO: 4), Mycobacterium
smegmatis (Genbank Accession No. ABK75684.1, SEQ ID NO: 5),
Mycobacterium massiliense (Genbank Accession No. EIV11143.1, SEQ ID
NO: 6), or Segniliparus rotundus (Genbank Accession No. ADG98140.1,
SEQ ID NO: 7). See FIG. 7.
[0142] The carboxylate reductase encoded by the gene product of car
and enhancer npt or sfp has broad substrate specificity, including
terminal difunctional C4 and C5 carboxylic acids
(Venkitasubramanian et al., Enzyme and Microbial Technology, 2008,
42, 130-137).
[0143] In some embodiments, pimeloyl-CoA formed as described above
is converted to pimelate semialdehyde by an acetylating aldehyde
dehydrogenase classified, for example, under EC 1.2.1.10 such as
the gene product of PduB or PduP; followed by conversion of
pimelate semialdehyde to 7-aminoheptanoate by a
.omega.-transaminase classified, for example, under EC 2.6.1.- such
as EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48, see). See FIG. 3.
Pathway Using 7-aminoheptanoate as Central Precursor to
Heptamethylenediamine
[0144] In some embodiments, heptamethylenediamine is synthesized
from the central precursor, 7-aminoheptanoate, by conversion of
7-aminoheptanoate to 7-aminoheptanal by a carboxylate reductase
classified, for example, under EC 1.2.99.6 such as the gene product
of car (see above) in combination with a phosphopantetheine
transferase enhancer (e.g., encoded by a sfp gene from Bacillus
subtilis or npt gene from Nocardia) or the gene products of GriC
and GriD from Streptomyces griseus (Suzuki et al., 2007, supra);
followed by conversion of 7-aminoheptanal to heptamethylenediamine
by a .omega.-transaminase (e.g., EC 2.6.1.18, EC 2.6.1.19, EC
2.6.1.48, EC 2.6.1.82 such as SEQ ID NOs:8-13). . See FIG. 4.
[0145] The carboxylate reductase encoded by the gene product of car
and enhancer npt has broad substrate specificity, including
terminal difunctional C4 and C5 carboxylic acids
(Venkitasubramanian et al., Enzyme and Microbial Technology, 2008,
42, 130-137).
[0146] In some embodiments, heptamethylenediamine is synthesized
from the central precursor, 7-hydroxyheptanoate (which can be
produced as described in FIG. 5), by conversion of
7-hydroxyheptanoate to 7-hydroxyheptanal by a carboxylate reductase
classified, for example, under EC 1.2.99.6 such as the gene product
of car (see above) in combination with a phosphopantetheine
transferase enhancer (e.g., encoded by a sfp gene from Bacillus
subtilis or npt gene from Nocardia) or the gene product of GriC
& GriD (Suzuki et al., 2007, supra); followed by conversion of
7-aminoheptanal to 7-aminoheptanol by a .omega.-transaminase
classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC
2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see
above; followed by conversion to 7-aminoheptanal 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 or YqhD (Liu et al., Microbiology, 2009, 155,
2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172;
Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the
protein having GenBank Accession No. CAA81612.1; followed by
conversion to heptamethylenediamine by a .omega.-transaminase
classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC
2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see
above. See FIG. 4.
[0147] In some embodiments, heptamethylenediamine is synthesized
from the central precursor, 7-aminoheptanoate, by conversion of
7-aminoheptanoate to N7-acetyl-7-aminoheptanoate by an
N-acetyltransferase such as a lysine N-acetyltransferase
classified, for example, under EC 2.3.1.32; followed by conversion
to N7-acetyl-7-aminoheptanal by a carboxylate reductase classified,
for example, under EC 1.2.99.6 such as the gene product of car (see
above) in combination with a phosphopantetheine transferase
enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt
gene from Nocardia) or the gene product of GriC & GriD;
followed by conversion to N7-acetyl-1,7-diaminoheptane by a
.omega.-transaminase classified, for example, under EC 2.6.1.18, EC
2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID
NOs:8-13, see above; followed by conversion to
heptamethylenediamine by an acetylputrescine deacetylase
classified, for example, under EC 3.5.1.62. See, FIG. 4.
[0148] In some embodiments, heptamethylenediamine is synthesized
from the central precursor, pimelate semialdehyde, by conversion of
pimelate semialdehyde to heptanedial by a carboxylate reductase
classified, for example, under EC 1.2.99.6 such as the gene product
of car (see above) in combination with a phosphopantetheine
transferase enhancer (e.g., encoded by a sfp gene from Bacillus
subtilis or npt gene from Nocardia) or the gene product of GriC
& GriD; followed by conversion to 7-aminoheptanal by a
.omega.-transaminase classified, for example, under EC 2.6.1.18, EC
2.6.1.19, or EC 2.6.1.48; followed by conversion to
heptamethylenediamine by a .omega.-transaminase classified, for
example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48,
or EC 2.6.1.82 such as SEQ ID NOs:8-13. See FIG. 4.
Pathways using Pimelate or Pimelate Semialdehyde as Central
Precursors to 7-hydroxyheptanoate or 1,7-heptanediol
[0149] In some embodiments, 7-hydroxyheptanoate is synthesized from
the central precursor, pimelate, by conversion of pimelate to
pimelate semialdehyde by a carboxylate reductase classified, for
example, under EC 1.2.99.6 such as the gene product of car (see
above) in combination with a phosphopantetheine transferase
enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt
gene from Nocardia) or the gene product of GriC & GriD;
followed by conversion to 7-hydroxyheptanoate by a
6-hydroxyhexanoate dehydrogenase classified, for example, under EC
1.1.1.258 such as the gene product 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, Liitke-Eversloh &
Steinbuchel, 1999, FEMS Microbiology Letters, 181(1):63-71). See
FIG. 5.
[0150] In some embodiments, 7-hydroxyheptanoate is synthesized from
the central precursor, pimeloyl-CoA, by conversion of pimeloyl-CoA
to pimelate semialdehyde by an acetylating aldehyde dehydrogenase
classified, for example, under EC 1.2.1.10 such as the gene product
of PduB or PduP; followed by conversion to 7-hydroxyheptanoate by a
6-hydroxyhexanoate dehydrogenase classified, for example, under EC
1.1.1.258 such as the gene product 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, Liitke-Eversloh &
Steinbuchel, 1999, FEMS Microbiology Letters, 181(1):63-71). See
FIG. 5.
[0151] In some embodiments, pimeloyl-CoA formed as described above
is converted to pimelate semialdehyde by an acetylating aldehyde
dehydrogenase (classified, for example, under EC 1.2.1.10) such as
the gene product of PduB; followed by conversion of pimelate
semialdehyde to 7-hydroxyheptanoate 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). See FIG. 5.
[0152] In some embodiments, 1,7 heptanediol is synthesized from the
central precursor, 7-hydroxyheptanoate, by conversion of
7-hydroxyheptanoate to 7-hydroxyheptanal by a carboxylate reductase
classified, for example, under EC 1.2.99.6 such as the gene product
of car (see above) in combination with a phosphopantetheine
transferase enhancer (e.g., encoded by a sfp gene from Bacillus
subtilis or npt gene from Nocardia) or the gene products of GriC
and GriD from Streptomyces griseus (Suzuki et al., 2007, supra);
followed by conversion of 7-hydroxyheptanal to 1,7 heptanediol by
an alcohol dehydrogenase classified, for example, under EC 1.1.1.-
such as 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 or YqhD (from E. coli, GenBank
Accession No. AAA69178.1) (see, e.g., Liu et al., Microbiology,
2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1),
163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2),
249-257) or the protein having GenBank Accession No. CAA81612.1
(from Geobacillus stearothermophilus). See FIG. 6.
Cultivation Strategy
[0153] In some embodiments, the cultivation strategy entails
achieving an anaerobic cultivation, micro-aerobic, or mixed
oxygen/denitrification 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).
[0154] In some embodiments, the cultivation strategy entails
nutrient limitation such as nitrogen, phosphate or oxygen
limitation.
[0155] In some embodiments, a final electron acceptor other than
oxygen such as nitrates can be utilized.
[0156] In some embodiments, a cell retention strategy using, for
example, ceramic hollow fiber membranes can be employed to achieve
and maintain a high cell density during either fed-batch or
continuous fermentation.
[0157] In some embodiments, the principal carbon source fed to the
fermentation in the synthesis of one or more C7 building blocks can
derive from biological or non-biological feedstocks.
[0158] In some embodiments, the biological feedstock can be, can
include, 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.
[0159] 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).
[0160] 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,
Journal of Biotechnology, 2011, 155, 2011, 293-298; Martin and
Prather, Journal of Biotechnology, 2009, 139, 61-67).
[0161] 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).
[0162] 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).
[0163] 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, Journal of Biotechnology, 2003, 104, 155-172; Wee et
al., Food Technol. Biotechnol., 2006, 44(2), 163-172; Ohashi et
al., Journal of Bioscience and Bioengineering, 1999, 87(5),
647-654).
[0164] 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).
[0165] In some embodiments, the non-biological feedstock can be or
can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol,
ethanol, benzoic acid, non-volatile residue (NVR), a caustic wash
waste stream from cyclohexane oxidation processes, or terephthalic
acid/isophthalic acid mixture waste streams.
[0166] The efficient catabolism of methanol has been demonstrated
for the methylotrophic yeast Pichia pastoris.
[0167] The efficient catabolism of ethanol has been demonstrated
for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci.
USA, 2008, 105(6) 2128-2133).
[0168] 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).
[0169] 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).
[0170] 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).
[0171] In some embodiments, the host microorganism is a prokaryote.
For example, the prokaryote can be a bacterium from the genus
Escherichia such as Escherichia coli; from the genus Clostridia
such as Clostridium ljungdahlii, Clostridium autoethanogenum or
Clostridium kluyveri; from the genus Corynebacteria such as
Corynebacterium glutamicum; from the genus Cupriavidus such as
Cupriavidus necator or Cupriavidus metallidurans; from the genus
Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or
Pseudomonas oleavorans; from the genus Delftia such as Delftia
acidovorans; from the genus Bacillus such as Bacillus subtillis;
from the genus Lactobacillus such as Lactobacillus delbrueckii; or
from the genus Lactococcus such as Lactococcus lactis. Such
prokaryotes also can be a source of genes to construct recombinant
host cells described herein that are capable of producing one or
more C7 building blocks.
[0172] In some embodiments, the host microorganism is a eukaryote.
For example, the eukaryote can be a filamentous fungus, e.g., one
from the genus Aspergillus such as Aspergillus niger.
Alternatively, the eukaryote can be a yeast, e.g., one from the
genus Saccharomyces such as Saccharomyces cerevisiae; from the
genus Pichia such as Pichia pastoris; or from the genus Yarrowia
such as Yarrowia lipolytica; from the genus Issatchenkia such as
Issathenkia orientalis; from the genus Debaryomyces such as
Debaryomyces hansenii; from the genus Arxula such as Arxula
adenoinivorans; or from the genus Kluyveromyces such as
Kluyveromyces lactis. Such eukaryotes also can be a source of genes
to construct recombinant host cells described herein that are
capable of producing one or more C7 building blocks.
Metabolic Engineering
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] This document also recognizes that where an enzyme is shown
to accept a particular .omega.-factor, such as NADPH, or a
.omega.-substrate, such as acetyl-CoA, many enzymes are promiscuous
in terms of accepting a number of different .omega.-factors or
.omega.-substrates in catalyzing a particular enzyme activity.
Also, this document recognizes that where enzymes have high
specificity for e.g., a particular .omega.-factor such as NADH, an
enzyme with similar or identical activity that has high specificity
for the .omega.-factor NADPH may be in a different enzyme
class.
[0178] 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 .omega.-factor specificity.
[0179] In some embodiments, the enzymes in the pathways outlined
herein can be gene dosed (i.e., overexpressed by having a plurality
of copies of the gene in the host organism), into the resulting
genetically modified organism via episomal or chromosomal
integration approaches.
[0180] 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 C7 building block.
[0181] Attenuation strategies include, but are not limited to; the
use of transposons, homologous recombination (double cross-over
approach), mutagenesis, enzyme inhibitors and RNA interference
(RNAi).
[0182] 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 a C7 building block.
[0183] In some embodiments, the host microorganism's tolerance to
high concentrations of a C7 building block can be improved through
continuous cultivation in a selective environment.
[0184] In some embodiments, the host microorganism's endogenous
biochemical network can be attenuated or augmented to ensure the
intracellular availability of acetyl-CoA (2) create an NADH
imbalance that may only be balanced via the formation of a C7
building block, (3) prevent degradation of central metabolites or
central precursors leading to and including C7 building blocks and
(4) ensure efficient efflux from the cell.
[0185] In some embodiments requiring the intracellular availability
of acetyl-CoA for C7 building block synthesis, an endogenous gene
encoding a phosphotransacetylase, which generates acetate, such as
pta is attenuated (Shen et al., Appl. Environ. Microbiol., 2011,
77(9), 2905-2915).
[0186] In some embodiments requiring the intracellular availability
of acetyl-CoA for C7 building block synthesis, an endogenous gene
encoding an acetate kinase in an acetate synthesis pathway, such as
ack, is attenuated.
[0187] In some embodiments requiring the intracellular availability
of acetyl-CoA and NADH for C7 building block synthesis, an
endogenous gene encoding an enzyme that catalyzes the degradation
of pyruvate to lactate such as ldhA is attenuated (Shen et al.,
Appl. Environ. Microbiol., 2011, 77(9), 2905-2915).
[0188] In some embodiments requiring the intracellular availability
of acetyl-CoA and NADH for C7 building block synthesis, an
endogenous gene encoding an enzyme that catalyzes the degradation
of phophoenolpyruvate to succinate such as frdBC is attenuated
(see, e.g., Shen et al., 2011, supra).
[0189] In some embodiments requiring the intracellular availability
of acetyl-CoA and NADH for C7 building block synthesis, an
endogenous gene encoding an enzyme that catalyzes the degradation
of acetyl-CoA to ethanol such as the alcohol dehydrogenase encoded
by adhE is attenuated (Shen et al., 2011, supra).
[0190] In some embodiments, an endogenous gene encoding an enzyme
that catalyzes the degradation of pyruvate to ethanol such as
pyruvate decarboxylase is attenuated.
[0191] In some embodiments requiring the intracellular availability
of acetyl-CoA for C7 building block synthesis, a gene encoding an
acetyl-CoA synthetase such as the gene acs is overexpressed in the
microorganism (Satoh et al., Journal of Bioscience and
Bioengineering, 2003, 95(4), 335-341).
[0192] In some embodiments, where a pathway requires excess NADH
for C7 building block synthesis, carbon flux is directed into the
pentose phosphate cycle by attenuating an endogenous gene encoding
a glucose-6-phosphate isomerase (EC 5.3.1.9).
[0193] In some embodiments, where a pathway requires excess NADH
.omega.-factor for C7 building block synthesis, a gene encoding
aformate dehydrogenase is overexpressed in the host organism (Shen
et al., 2011, supra).
[0194] In some embodiments, endogenous enzymes facilitating the
conversion of NADH to NADPH are attenuated, such as the NADPH
generation cycle that may be generated via inter-conversion of
glutamate dehydrogenases in EC 1.4.1.2 (NADH-specific) and EC
1.4.1.4 (NADPH-specific). Similarly, transhydrogenases such as
classified, for example, under EC 1.6.1.1, EC 1.6.1.2 or EC
1.6.1.3, may be attenuated.
[0195] In some embodiments, an endogenous gene encoding a glutamate
dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as
.omega.-factors is attenuated.
[0196] In some embodiments, membrane-bound enoyl-CoA reductases are
solubilized via truncation of the amino acid residues that anchor
the protein in the membrane (Hoffmeister et al., J. Biol. Chem.,
2005, 280(6) 4329-4338).
[0197] In some embodiments, membrane-bound enoyl-CoA reductases are
solubilized via expression as a fusion protein (Gloerich et al.,
FEBS Letters, 2006, 580, 2092-2096).
[0198] In some embodiments, an enoyl-[acp] reductase from fatty
acid synthesis or a trans-2-enoyl-CoA reductase is subjected to
enzyme engineering with the aim of modifying the tunnel-like
hydrophobic cavity to accept CoA activated dicarboxylic acids as
substrate (Roujeinikova et al., J. Mol. Biol., 2007, 365,
135-145).
[0199] In some embodiments using hosts that naturally accumulate
polyhydroxyalkanoates, an endogenous gene encoding a polymer
synthase enzyme can be attenuated in the host strain.
[0200] In some embodiments, a L-alanine dehydrogenase can be
overexpressed in the host to regenerate L-alanine from pyruvate as
amino donor for .omega.-transaminase reactions.
[0201] In some embodiments, a NADH-specific L-glutamate
dehydrogenase can be overexpressed in the host to regenerate
L-glutamate from 2-oxoglutarate as amino donor for
.omega.-transaminase reactions.
[0202] In some embodiments, enzymes such as pimeloyl-CoA
dehydrogenase classified under, for example, EC 1.3.1.62 and/or an
acyl-CoA dehydrogenase classified under, for example, EC 1.3.8.7 or
EC 1.3.8.1 that degrade central metabolites and central precursors
leading to and including C7 building blocks can be attenuated.
[0203] In some embodiments, endogenous enzymes activating C7
building blocks via Coenzyme A esterification such as CoA-ligases
such as pimeloyl-CoA synthetase classified under, for example, EC
6.2.1.14 can be attenuated.
[0204] In some embodiments, the efflux of a C7 building block
across the cell membrane to the extracellular media can be enhanced
or amplified by genetically engineering structural modifications to
the cell membrane or increasing any associated transporter activity
for a C7 building block.
[0205] The efflux of heptamethylenediamine can be enhanced or
amplified by overexpressing broad substrate range multidrug
transporters such as Blt from Bacillus subtilis (Woolridge et al.,
1997, J. Biol. Chem., 272(14):8864-8866); AcrB and AcrD from
Escherichia coli (Elkins & Nikaido, 2002, J. Bacteriol.,
184(23), 6490-6499) or NorA from Staphylococcus aereus (Ng et al.,
1994, Antimicrob Agents Chemother, 38(6), 1345-1355) or Bmr from
Bacillus subtilis (Neyfakh, 1992, Antimicrob Agents Chemother,
36(2), 484-485).
[0206] The efflux of 7-aminoheptanoate and heptamethylenediamine
can be enhanced or amplified by overexpressing the solute
transporters such as the lysE transporter from Corynebacterium
glutamicum (Bellmann et al., 2001, Microbiology, 147,
1765-1774).
[0207] The efflux of pimelic acid can be enhanced or amplified by
overexpressing a dicarboxylate transporter such as the SucE
transporter from Corynebacterium glutamicum (Huhn et al., Appl.
Microbiol. & Biotech., 89(2), 327-335).
Producing C7 Building Blocks Using a Recombinant Host
[0208] Typically, one or more C7 building blocks 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 a C7 building block 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.
[0209] Once transferred, the microorganisms can be incubated to
allow for the production of a C7 building block. Once produced, any
method can be used to isolate C7 building blocks. For example, C7
building blocks can be recovered selectively from the fermentation
broth via adsorption processes. In the case of pimelic acid and
7-aminoheptanoic acid, the resulting eluate can be further
concentrated via evaporation, crystallized via evaporative and/or
cooling crystallization, and the crystals recovered via
centrifugation. In the case of heptamethylenediamine and
1,7-heptanediol, distillation may be employed to achieve the
desired product purity.
[0210] The invention is further described in the following example,
which does not limit the scope of the invention described in the
claims.
EXAMPLES
Example 1
Enzyme Activity of Thioesterases Using Pimeloyl-CoA as a Substrate
and Forming Pimelic Acid
[0211] A sequence encoding an N-terminal His tag was added to the
tesB gene from Escherichia coli that encodes a thioesterase (SEQ ID
NO 1, see FIG. 7), such that an N-terminal HIS tagged thioesterase
could be produced. The modified tesB gene was cloned into a pET15b
expression vector under control of the T7 promoter. 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 50 mL Luria Broth (LB) media and
antibiotic selection pressure, with shaking at 230 rpm. The culture
was induced overnight at 17.degree. C. using 0.5 mM IPTG.
[0212] The pellet from the induced shake flask culture was
harvested via centrifugation. The pellet was resuspended and lysed
in Y-perTM solution (ThermoScientific, Rockford, Ill.). The cell
debris was separated from the supernatant via centrifugation. The
thioesterase was purified from the supernatant using Ni-affinity
chromatography and the eluate was buffer exchanged and concentrated
via ultrafiltration.
[0213] The enzyme activity assay was performed in triplicate in a
buffer composed of 50 mM phosphate buffer (pH=7.4), 0.1 mM Ellman's
reagent, and 667 .mu.M of pimeloyl-CoA (as substrate). The enzyme
activity assay reaction was initiated by adding 0.8 .mu.M of the
tesB gene product to the assay buffer containing the pimeloyl-CoA
and incubating at 37.degree. C. for 20 min. The release of Coenzyme
A was monitored by absorbance at 412 nm. The absorbance associated
with the substrate only control, which contained boiled enzyme, was
subtracted from the active enzyme assay absorbance and compared to
the empty vector control. The gene product of tesB accepted
pimeloyl-CoA as substrate as confirmed via relative
spectrophotometry (see FIG. 8) and synthesized pimelate as a
reaction product.
Example 2
[0214] Enzyme Activity of .omega.-transaminase Using Pimelate
Semialdehyde as Substrate and Forming 7-aminoheptanoate
[0215] A sequence encoding an N-terminal His-tag was added to the
genes from Chromobacterium violaceum, Pseudomonas syringae,
Rhodobacter sphaeroides, and Vibrio Fluvialis encoding the
.omega.-transaminases of SEQ ID NOs: 8, 10, 11 and 13, respectively
(see FIG. 7) such that N-terminal HIS tagged .omega.-transaminases
could be produced. Each of the resulting modified genes was cloned
into a pET21a expression vector under control of the T7 promoter
and each expression vector was transformed into a BL21[DE3] E. coli
host. The resulting recombinant E. coli strains were 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 16.degree. C. using 1 mM
IPTG.
[0216] 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 and the cell free extract was used immediately
in enzyme activity assays.
[0217] Enzyme activity assays in the reverse direction (i.e.,
7-aminoheptanoate to pimelate semialdehyde) were performed in a
buffer composed of a final concentration of 50 mM HEPES buffer
(pH=7.5), 10 mM 7-aminoheptanoate, 10 mM pyruvate and 100 .mu.M
pyridoxyl 5' phosphate. Each enzyme activity assay reaction was
initiated by adding cell free extract of the .omega.-transaminase
gene product or the empty vector control to the assay buffer
containing the 7-aminoheptanoate and incubated at 25.degree. C. for
4 h, with shaking at 250 rpm. The formation of L-alanine from
pyruvate was quantified via RP-HPLC.
[0218] Each enzyme only control without 7-aminoheptanoate
demonstrated low base line conversion of pyruvate to L-alanine. See
FIG. 14. The gene product of SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO
11 and SEQ ID NO 13 accepted 7-aminoheptanote as substrate as
confirmed against the empty vector control. See FIG. 15.
[0219] Enzyme activity in the forward direction (i.e., pimelate
semialdehyde to 7-aminoheptanoate) was confirmed for the
transaminases of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13.
Enzyme activity assays were performed in a buffer composed of a
final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM pimelate
semialdehyde, 10 mM L-alanine and 100 .sub.ILIM pyridoxyl 5'
phosphate. Each enzyme activity assay reaction was initiated by
adding a cell free extract of the .omega.-transaminase gene product
or the empty vector control to the assay buffer containing the
pimelate semialdehyde and incubated at 25.degree. C. for 4 h, with
shaking at 250 rpm. The formation of pyruvate was quantified via
RP-HPLC.
[0220] The gene product of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO
13 accepted pimelate semialdehyde as substrate as confirmed against
the empty vector control. See FIG. 16. The reversibility of the
.omega.-transaminase activity was confirmed, demonstrating that the
.omega.-transaminases of SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11,
and SEQ ID NO 13 accepted pimelate semialdehyde as substrate and
synthesized 7-aminoheptanoate as a reaction product.
Example 3
[0221] Enzyme Activity of Carboxylate Reductase Using Pimelate as
Substrate and Forming Pimelate Semialdehyde
[0222] A sequence encoding a HIS-tag was added to the genes from
Segniliparus rugosus and Segniliparus rotundus that encode the
carboxylate reductases of SEQ ID NOs: 4 and 7, respectively (see
FIG. 7), such that N-terminal HIS tagged carboxylate reductases
could be produced. Each of the modified genes was cloned into a pET
Duet expression vector along with a sfp gene encoding a HIS-tagged
phosphopantetheine transferase from Bacillus subtilis, both under
the T7 promoter. Each expression vector was transformed into a
BL21[DE3] E. coli host and the resulting recombinant E. coli
strains were 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.
[0223] The pellet from each induced shake flask culture was
harvested via centrifugation. Each pellet was resuspended and lysed
via sonication, and the cell debris was separated from the
supernatant via centrifugation. The carboxylate reductases and
phosphopantetheine transferases were purified from the supernatant
using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES
buffer (pH=7.5), and concentrated via ultrafiltration.
[0224] Enzyme activity assays (i.e., from pimelate to pimelate
semialdehyde) were performed in triplicate in a buffer composed of
a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM
pimelate, 10 mM MgCl.sub.2, 1 mM ATP and 1 mM NADPH. Each enzyme
activity assay reaction was initiated by adding purified
carboxylate reductase and phosphopantetheine transferase gene
products or the empty vector control to the assay buffer containing
the pimelate and then incubated at room temperature for 20 min. The
consumption of NADPH was monitored by absorbance at 340 nm. Each
enzyme only control without pimelate demonstrated low base line
consumption of NADPH. See FIG. 9.
[0225] The gene products of SEQ ID NO 4 and SEQ ID NO 7, enhanced
by the gene product of sfp, accepted pimelate as substrate, as
confirmed against the empty vector control (see FIG. 10), and
synthesized pimelate semialdehyde.
Example 4
[0226] Enzyme Activity of Carboxylate Reductase Using
7-hydroxyheptanoate as Substrate and Forming 7-hydroxyheptanal
[0227] A sequence encoding a His-tag was added to the genes from
Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus
rugosus, Mycobacterium smegmatis, Mycobacterium massiliense, and
Segniliparus rotundus that encode the carboxylate reductases of SEQ
ID NOs: 2-7--respectively (see FIG. 7) 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.
[0228] Each expression vector was transformed into a BL21[DE3] E.
coli host and the resulting recombinant E. coli strains were
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.
[0229] 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.
[0230] Enzyme activity (i.e., 7-hydroxyheptanoate to
7-hydroxyheptanal) assays were performed in triplicate in a buffer
composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2
mM 7-hydroxyheptanal, 10 mM MgCl.sub.2, 1 mM ATP, and 1 mM NADPH.
Each 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
7-hydroxyheptanoate and then incubated at room temperature for 20
min. The consumption of NADPH was monitored by absorbance at 340
nm. Each enzyme only control without 7-hydroxyheptanoate
demonstrated low base line consumption of NADPH. See FIG. 9.
[0231] The gene products of SEQ ID NO 2-7, enhanced by the gene
product of sfp, accepted 7-hydroxyheptanoate as substrate as
confirmed against the empty vector control (see FIG. 11), and
synthesized 7-hydroxyheptanal.
Example 5
[0232] Enzyme Activity of .omega.-transaminase for 7-aminoheptanol,
Forming 7-oxoheptanol
[0233] A nucleotide sequence encoding an N-terminal His-tag was
added to the Chromobacterium violaceum, Pseudomonas syringae and
Rhodobacter sphaeroides genes encoding the .omega.-transaminases of
SEQ ID NOs: 8, 10 and 11, respectively (see FIG. 7) such that
N-terminal HIS tagged .omega.-transaminases could be produced. The
modified genes were cloned into a pET21a expression vector under
the T7 promoter. Each expression vector was transformed into a
BL21[DE3] E. coli host. Each resulting recombinant E. coli strain
were 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
16.degree. C. using 1 mM IPTG.
[0234] 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 and the cell free extract was used immediately
in enzyme activity assays.
[0235] Enzyme activity assays in the reverse direction (i.e.,
7-aminoheptanol to 7-oxoheptanol) were performed in a buffer
composed of a final concentration of 50 mM HEPES buffer (pH=7.5),
10 mM 7-aminoheptanol, 10 mM pyruvate, and 100 .mu.M pyridoxyl 5'
phosphate. Each enzyme activity assay reaction was initiated by
adding cell free extract of the .omega.-transaminase gene product
or the empty vector control to the assay buffer containing the
7-aminoheptanol and then incubated at 25.degree. C. for 4 h, with
shaking at 250 rpm. The formation of L-alanine was quantified via
RP-HPLC.
[0236] Each enzyme only control without 7-aminoheptanol had low
base line conversion of pyruvate to L-alanine. See FIG. 14.
[0237] The gene products of SEQ ID NO 8, 10 & 11 accepted
7-aminoheptanol as substrate as confirmed against the empty vector
control (see FIG. 19) and synthesized 7-oxoheptanol as reaction
product. Given the reversibility of the .omega.-transaminase
activity (see Example 2), it can be concluded that the gene
products of SEQ ID 8, 10 & 11 accept 7-oxoheptanol as substrate
and form 7-aminoheptanol.
Example 6
[0238] Enzyme Activity of .omega.-transaminase Using
Heptamethylenediamine as Substrate and Forming 7-aminoheptanal
[0239] A sequence encoding an N-terminal His-tag was added to the
Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas
syringae, Rhodobacter sphaeroides, Escherichia coli, and Vibrio
fluvialis genes encoding the .omega.-transaminases of SEQ ID NOs:
8-13, respectively (see FIG. 7) such that N-terminal HIS tagged
.omega.-transaminases could be produced. The modified genes were
cloned into a pET21a expression vector under the T7 promoter. Each
expression vector was transformed into a BL21[DE3] E. coli host.
Each resulting recombinant E. coli strain were 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 16.degree. C. using 1 mM
IPTG.
[0240] 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 and the cell free extract was used immediately
in enzyme activity assays.
[0241] Enzyme activity assays in the reverse direction (i.e.,
heptamethylenediamine to 7-aminoheptanal) were performed in a
buffer composed of a final concentration of 50 mM HEPES buffer
(pH=7.5), 10 mM heptamethylenediamine, 10 mM pyruvate, and 100
.mu.M pyridoxyl 5' phosphate. Each enzyme activity assay reaction
was initiated by adding cell free extract of the
.omega.-transaminase gene product or the empty vector control to
the assay buffer containing the heptamethylenediamine and then
incubated at 25.degree. C. for 4 h, with shaking at 250 rpm. The
formation of L-alanine was quantified via RP-HPLC.
[0242] Each enzyme only control without heptamethylenediamine had
low base line conversion of pyruvate to L-alanine. See FIG. 14.
[0243] The gene products of SEQ ID NO 8-13 accepted
heptamethylenediamine as substrate as confirmed against the empty
vector control (see FIG. 17) and synthesized 7-aminoheptanal as
reaction product. Given the reversibility of the
.omega.-transaminase activity (see Example 2), it can be concluded
that the gene products of SEQ ID 8-13 accept 7-aminoheptanal as
substrate and form heptamethylenediamine.
Example 7
[0244] Enzyme Activity of Carboxylate Reductase for
N7-acetyl-7-aminoheptanoate, Forming N7-acetyl-7-aminoheptanal
[0245] The activity of each of the N-terminal His-tagged
carboxylate reductases of SEQ ID NOs: 3, 6, and 7 (see Examples 4,
and FIG. 7) for converting N7-acetyl-7-aminoheptanoate to
N7-acetyl-7-aminoheptanal was assayed in triplicate in a buffer
composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2
mM N7-acetyl-7-aminoheptanoate, 10 mM MgCl.sub.2, 1 mM ATP, and 1
mM NADPH. The assays were initiated by adding purified carboxylate
reductase and phosphopantetheine transferase or the empty vector
control to the assay buffer containing the
N7-acetyl-7-aminoheptanoate then incubated at room temperature for
20 min. The consumption of NADPH was monitored by absorbance at 340
nm. Each enzyme only control without N7-acetyl-7-aminoheptanoate
demonstrated low base line consumption of NADPH. See FIG. 9.
[0246] The gene products of SEQ ID NO 3, 6, and 7, enhanced by the
gene product of sfp, accepted N7-acetyl-7-aminoheptanoate as
substrate as confirmed against the empty vector control (see FIG.
12), and synthesized N7-acetyl-7-aminoheptanal.
Example 8
[0247] Enzyme Activity of .omega.-transaminase Using
N7-acetyl-1,7-diaminoheptane, and Forming
N7-acetyl-7-aminoheptanal
[0248] The activity of the N-terminal His-tagged
.omega.-transaminases of SEQ ID NOs: 8-13 (see Example 6, and FIG.
7) for converting N7-acetyl-1,7-diaminoheptane to
N7-acetyl-7-aminoheptanal was assayed using a buffer composed of a
final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM
N7-acetyl-1,7-diaminoheptane, 10 mM pyruvate and 100 .mu.M
pyridoxyl 5' phosphate. Each enzyme activity assay reaction was
initiated by adding a cell free extract of the .omega.-transaminase
or the empty vector control to the assay buffer containing the
N7-acetyl-1,7-diaminoheptane then incubated at 25.degree. C. for 4
h, with shaking at 250 rpm. The formation of L-alanine was
quantified via RP-HPLC.
[0249] Each enzyme only control without
N7-acetyl-1,7-diaminoheptane demonstrated low base line conversion
of pyruvate to L-alanine. See FIG. 14.
[0250] The gene product of SEQ ID NOs: 8-13 accepted
N7-acetyl-1,7-diaminoheptane as substrate as confirmed against the
empty vector control (see FIG. 18) and synthesized
N7-acetyl-7-aminoheptanal as reaction product.
[0251] Given the reversibility of the .omega.-transaminase activity
(see example 2), the gene products of SEQ ID NOs: 8-13 accept
N7-acetyl-7-aminoheptanal as substrate forming
N7-acetyl-1,7-diaminoheptane.
Example 9
Enzyme Activity of Carboxylate Reductase Using Pimelate
Semialdehyde as Substrate and Forming Heptanedial
[0252] The N-terminal His-tagged carboxylate reductase of SEQ ID NO
7 (see Example 4 and FIG. 7) was assayed using pimelate
semialdehyde as substrate. 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 pimelate semialdehyde, 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 pimelate semialdehyde 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 pimelate semialdehyde demonstrated low base line
consumption of NADPH. See FIG. 9.
[0253] The gene product of SEQ ID NO 7, enhanced by the gene
product of sfp, accepted pimelate semialdehyde as substrate as
confirmed against the empty vector control (see FIG. 13) and
synthesized heptanedial.
OTHER EMBODIMENTS
[0254] 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
151286PRTEscherichia coli 1Met 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 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 8459PRTChromobacterium violaceum 8Met
Gln Lys Gln Arg Thr Thr Ser Gln Trp Arg Glu Leu Asp Ala Ala1 5 10
15 His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30 Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp
Ser Glu 35 40 45 Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp
Cys Val Asn Val 50 55 60 Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala
Ala Arg Arg Gln Met Glu65 70 75 80 Glu Leu Pro Phe Tyr Asn Thr Phe
Phe Lys Thr Thr His Pro Ala Val 85 90 95 Val Glu Leu Ser Ser Leu
Leu Ala Glu Val Thr Pro Ala Gly Phe Asp 100 105 110 Arg Val Phe Tyr
Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile 115 120 125 Arg Met
Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys 130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly145
150 155 160 Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp
Leu Pro 165 170 175 Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp
Tyr Lys His Gly 180 185 190 Lys Asp Met Thr Pro Asp Glu Phe Gly Val
Val Ala Ala Arg Trp Leu 195 200 205 Glu Glu Lys Ile Leu Glu Ile Gly
Ala Asp Lys Val Ala Ala Phe Val 210 215 220 Gly Glu Pro Ile Gln Gly
Ala Gly Gly Val Ile Val Pro Pro Ala Thr225 230 235 240 Tyr Trp Pro
Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu 245 250 255 Val
Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe 260 265
270 Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285 Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val
Gly Lys 290 295 300 Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe
Asn His Gly Phe305 310 315 320 Thr Tyr Ser Gly His Pro Val Cys Ala
Ala Val Ala His Ala Asn Val 325 330 335 Ala Ala Leu Arg Asp Glu Gly
Ile Val Gln Arg Val Lys Asp Asp Ile 340 345 350 Gly Pro Tyr Met Gln
Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu 355 360 365 His Val Asp
Asp Val Arg Gly Val Gly Met Val Gln Ala Phe Thr Leu 370 375 380 Val
Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile385 390
395 400 Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met
Arg 405 410 415 Ala Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val
Met Thr Arg 420 425 430 Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu
Arg Cys Leu Glu Glu 435 440 445 Phe Glu Gln Thr Leu Lys Ala Arg Gly
Leu Ala 450 455 9468PRTPseudomonas aeruginosa 9Met Asn Ala Arg Leu
His Ala Thr Ser Pro Leu Gly Asp Ala Asp Leu1 5 10 15 Val Arg Ala
Asp Gln Ala His Tyr Met His Gly Tyr His Val Phe Asp 20 25 30 Asp
His Arg Val Asn Gly Ser Leu Asn Ile Ala Ala Gly Asp Gly Ala 35 40
45 Tyr Ile Tyr Asp Thr Ala Gly Asn Arg Tyr Leu Asp Ala Val Gly Gly
50 55 60 Met Trp Cys Thr Asn Ile Gly Leu Gly Arg Glu Glu Met Ala
Arg Thr65 70 75 80 Val Ala Glu Gln Thr Arg Leu Leu Ala Tyr Ser Asn
Pro Phe Cys Asp 85 90 95 Met Ala Asn Pro Arg Ala Ile Glu Leu Cys
Arg Lys Leu Ala Glu Leu 100 105 110 Ala Pro Gly Asp Leu Asp His Val
Phe Leu Thr Thr Gly Gly Ser Thr 115 120 125 Ala Val Asp Thr Ala Ile
Arg Leu Met His Tyr Tyr Gln Asn Cys Arg 130 135 140 Gly Lys Arg Ala
Lys Lys His Val Ile Thr Arg Ile Asn Ala Tyr His145 150 155 160 Gly
Ser Thr Phe Leu Gly Met Ser Leu Gly Gly Lys Ser Ala Asp Arg 165 170
175 Pro Ala Glu Phe Asp Phe Leu Asp Glu Arg Ile His His Leu Ala Cys
180 185 190 Pro Tyr Tyr Tyr Arg Ala Pro Glu Gly Leu Gly Glu Ala Glu
Phe Leu 195 200 205 Asp Gly Leu Val Asp Glu Phe Glu Arg Lys Ile Leu
Glu Leu Gly Ala 210 215 220 Asp Arg Val Gly Ala Phe Ile Ser Glu Pro
Val Phe Gly Ser Gly Gly225 230 235 240 Val Ile Val Pro Pro Ala Gly
Tyr His Arg Arg Met Trp Glu Leu Cys 245 250 255 Gln Arg Tyr Asp Val
Leu Tyr Ile Ser Asp Glu Val Val Thr Ser Phe 260 265 270 Gly Arg Leu
Gly His Phe Phe Ala Ser Gln Ala Val Phe Gly Val Gln 275 280 285 Pro
Asp Ile Ile Leu Thr Ala Lys Gly Leu Thr Ser Gly Tyr Gln Pro 290 295
300 Leu Gly Ala Cys Ile Phe Ser Arg Arg Ile Trp Glu Val Ile Ala
Glu305 310 315 320 Pro Asp Lys Gly Arg Cys Phe Ser His Gly Phe Thr
Tyr Ser Gly His 325 330 335 Pro Val Ala Cys Ala Ala Ala Leu Lys Asn
Ile Glu Ile Ile Glu Arg 340 345 350 Glu Gly Leu Leu Ala His Ala Asp
Glu Val Gly Arg Tyr Phe Glu Glu 355 360 365 Arg Leu Gln Ser Leu Arg
Asp Leu Pro Ile Val Gly Asp Val Arg Gly 370 375 380 Met Arg Phe Met
Ala Cys Val Glu Phe Val Ala Asp Lys Ala Ser Lys385 390 395 400 Ala
Leu Phe Pro Glu Ser Leu Asn Ile Gly Glu Trp Val His Leu Arg 405 410
415 Ala Gln Lys Arg Gly Leu Leu Val Arg Pro Ile Val His Leu Asn Val
420 425 430 Met Ser Pro Pro Leu Ile Leu Thr Arg Glu Gln Val Asp Thr
Val Val 435 440 445 Arg Val Leu Arg Glu Ser Ile Glu Glu Thr Val Glu
Asp Leu Val Arg 450 455 460 Ala Gly His Arg465 10454PRTPseudomonas
syringae 10Met Ser Ala Asn Asn Pro Gln Thr Leu Glu Trp Gln Ala Leu
Ser Ser1 5 10 15 Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gln
Leu Lys Glu Lys 20 25 30 Gly Pro Arg Ile Ile Thr Arg Ala Glu Gly
Val Tyr Leu Trp Asp Ser 35 40 45 Glu Gly Asn Lys Ile Leu Asp Gly
Met Ser Gly Leu Trp Cys Val Ala 50 55 60 Ile Gly Tyr Gly Arg Glu
Glu Leu Ala Asp Ala Ala Ser Lys Gln Met65 70 75 80 Arg Glu Leu Pro
Tyr Tyr Asn Leu Phe Phe Gln Thr Ala His Pro Pro 85 90 95 Val Leu
Glu Leu Ala Lys Ala Ile Ser Asp Ile Ala Pro Glu Gly Met 100 105 110
Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met 115
120 125 Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gln Pro Asn
Lys 130 135 140 Lys Thr Ile Ile Ser Arg Val Asn Gly Tyr His Gly Ser
Thr Val Ala145 150 155 160 Gly Ala Ser Leu Gly Gly Met Thr Tyr Met
His Glu Gln Gly Asp Leu 165 170 175 Pro Ile Pro Gly Val Val His Ile
Pro Gln Pro Tyr Trp Phe Gly Glu 180 185 190 Gly Gly Asp Met Thr Pro
Asp Glu Phe Gly Ile Trp Ala Ala Glu Gln 195 200 205 Leu Glu Lys Lys
Ile Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe 210 215 220 Ile Ala
Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Asp225 230 235
240 Ser Tyr Trp Pro Lys Ile Lys Glu Ile Leu Ser Arg Tyr Asp Ile Leu
245 250 255 Phe Ala Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Ser
Glu Trp 260 265 270 Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met
Met Thr Ile Ala 275 280 285 Lys Gly Leu Thr Ser Gly Tyr Val Pro Met
Gly Gly Leu Ile Val Arg 290 295 300 Asp Glu Ile Val Ala Val Leu Asn
Glu Gly Gly Asp Phe Asn His Gly305 310 315 320 Phe Thr Tyr Ser Gly
His Pro Val Ala Ala Ala Val Ala Leu Glu Asn 325 330 335 Ile Arg Ile
Leu Arg Glu Glu Lys Ile Val Glu Arg Val Arg Ser Glu 340 345 350 Thr
Ala Pro Tyr Leu Gln Lys Arg Leu Arg Glu Leu Ser Asp His Pro 355 360
365 Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala Ile Glu Leu
370 375 380 Val Lys Asp Lys Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly
Ala Gly385 390 395 400 Met Ile Cys Arg Thr Phe Cys Phe Asp Asn Gly
Leu Ile Met Arg Ala 405 410 415 Val Gly Asp Thr Met Ile Ile Ala Pro
Pro Leu Val Ile Ser Phe Ala 420 425 430 Gln Ile Asp Glu Leu Val Glu
Lys Ala Arg Thr Cys Leu Asp Leu Thr 435 440 445 Leu Ala Val Leu Gln
Gly 450 11467PRTRhodobacter sphaeroides 11Met Thr Arg Asn Asp Ala
Thr Asn Ala Ala Gly Ala Val Gly Ala Ala1 5 10 15 Met Arg Asp His
Ile Leu Leu Pro Ala Gln Glu Met Ala Lys Leu Gly 20 25 30 Lys Ser
Ala Gln Pro Val Leu Thr His Ala Glu Gly Ile Tyr Val His 35 40 45
Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro Ala Gly Met Trp Cys 50
55 60 Ala Gln Val Gly Tyr Gly Arg Arg Glu Ile Val Asp Ala Met Ala
His65 70 75 80 Gln Ala Met Val Leu Pro Tyr Ala Ser Pro Trp Tyr Met
Ala Thr Ser 85 90 95 Pro Ala Ala Arg Leu Ala Glu Lys Ile Ala Thr
Leu Thr Pro Gly Asp 100 105 110 Leu Asn Arg Ile Phe Phe Thr Thr Gly
Gly Ser Thr Ala Val Asp Ser 115 120 125 Ala Leu Arg Phe Ser Glu Phe
Tyr Asn Asn Val Leu Gly Arg Pro Gln 130 135 140 Lys Lys Arg Ile Ile
Val Arg Tyr Asp Gly Tyr His Gly Ser Thr Ala145 150 155 160 Leu Thr
Ala Ala Cys Thr Gly Arg Thr Gly Asn Trp Pro Asn Phe Asp 165 170 175
Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser Pro Asn Pro Arg His 180
185 190 Ala Gly Asn Arg Ser Gln Glu Ala Phe Leu Asp Asp Leu Val Gln
Glu 195 200 205 Phe Glu Asp Arg Ile Glu Ser Leu Gly Pro Asp Thr Ile
Ala Ala Phe 210 215 220 Leu Ala Glu Pro Ile Leu Ala Ser Gly Gly Val
Ile Ile Pro Pro Ala225 230 235 240 Gly Tyr His Ala Arg Phe Lys Ala
Ile Cys Glu Lys His Asp Ile Leu 245 250 255 Tyr Ile Ser Asp Glu Val
Val Thr Gly Phe Gly Arg Cys Gly Glu Trp 260 265 270 Phe Ala Ser Glu
Lys Val Phe Gly Val Val Pro Asp Ile Ile Thr Phe 275 280 285 Ala Lys
Gly Val Thr Ser Gly Tyr Val Pro Leu Gly Gly Leu Ala Ile 290 295 300
Ser Glu Ala Val Leu Ala Arg Ile Ser Gly Glu Asn Ala Lys Gly Ser305
310 315 320 Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gln Pro Val Ala
Cys Ala 325 330 335 Ala Ala Leu Ala Asn Ile Glu Leu Met Glu Arg Glu
Gly Ile Val Asp 340 345 350 Gln Ala Arg Glu Met Ala Asp Tyr Phe Ala
Ala Ala Leu Ala Ser Leu 355 360 365 Arg Asp Leu Pro Gly Val Ala Glu
Thr Arg Ser Val Gly Leu Val Gly 370 375 380 Cys Val Gln Cys Leu Leu
Asp Pro Thr Arg Ala Asp Gly Thr Ala Glu385 390 395 400 Asp Lys Ala
Phe Thr Leu Lys Ile Asp Glu Arg Cys Phe Glu Leu Gly 405 410 415 Leu
Ile Val Arg Pro Leu Gly Asp Leu Cys Val Ile Ser Pro Pro Leu 420 425
430 Ile Ile Ser Arg Ala Gln Ile Asp Glu Met Val Ala Ile Met Arg Gln
435 440 445 Ala Ile Thr Glu Val Ser Ala Ala His Gly Leu Thr Ala Lys
Glu Pro 450 455 460 Ala Ala Val465 12459PRTEscherichia coli 12Met
Asn Arg Leu Pro Ser Ser Ala Ser Ala Leu Ala Cys Ser Ala His1 5 10
15 Ala Leu Asn Leu Ile Glu Lys Arg Thr Leu Asp His Glu Glu Met Lys
20 25 30 Ala Leu Asn Arg Glu Val Ile Glu Tyr Phe Lys Glu His Val
Asn Pro 35
40 45 Gly Phe Leu Glu Tyr Arg Lys Ser Val Thr Ala Gly Gly Asp Tyr
Gly 50 55 60 Ala Val Glu Trp Gln Ala Gly Ser Leu Asn Thr Leu Val
Asp Thr Gln65 70 75 80 Gly Gln Glu Phe Ile Asp Cys Leu Gly Gly Phe
Gly Ile Phe Asn Val 85 90 95 Gly His Arg Asn Pro Val Val Val Ser
Ala Val Gln Asn Gln Leu Ala 100 105 110 Lys Gln Pro Leu His Ser Gln
Glu Leu Leu Asp Pro Leu Arg Ala Met 115 120 125 Leu Ala Lys Thr Leu
Ala Ala Leu Thr Pro Gly Lys Leu Lys Tyr Ser 130 135 140 Phe Phe Cys
Asn Ser Gly Thr Glu Ser Val Glu Ala Ala Leu Lys Leu145 150 155 160
Ala Lys Ala Tyr Gln Ser Pro Arg Gly Lys Phe Thr Phe Ile Ala Thr 165
170 175 Ser Gly Ala Phe His Gly Lys Ser Leu Gly Ala Leu Ser Ala Thr
Ala 180 185 190 Lys Ser Thr Phe Arg Lys Pro Phe Met Pro Leu Leu Pro
Gly Phe Arg 195 200 205 His Val Pro Phe Gly Asn Ile Glu Ala Met Arg
Thr Ala Leu Asn Glu 210 215 220 Cys Lys Lys Thr Gly Asp Asp Val Ala
Ala Val Ile Leu Glu Pro Ile225 230 235 240 Gln Gly Glu Gly Gly Val
Ile Leu Pro Pro Pro Gly Tyr Leu Thr Ala 245 250 255 Val Arg Lys Leu
Cys Asp Glu Phe Gly Ala Leu Met Ile Leu Asp Glu 260 265 270 Val Gln
Thr Gly Met Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His 275 280 285
Glu Asn Val Gln Pro Asp Ile Leu Cys Leu Ala Lys Ala Leu Gly Gly 290
295 300 Gly Val Met Pro Ile Gly Ala Thr Ile Ala Thr Glu Glu Val Phe
Ser305 310 315 320 Val Leu Phe Asp Asn Pro Phe Leu His Thr Thr Thr
Phe Gly Gly Asn 325 330 335 Pro Leu Ala Cys Ala Ala Ala Leu Ala Thr
Ile Asn Val Leu Leu Glu 340 345 350 Gln Asn Leu Pro Ala Gln Ala Glu
Gln Lys Gly Asp Met Leu Leu Asp 355 360 365 Gly Phe Arg Gln Leu Ala
Arg Glu Tyr Pro Asp Leu Val Gln Glu Ala 370 375 380 Arg Gly Lys Gly
Met Leu Met Ala Ile Glu Phe Val Asp Asn Glu Ile385 390 395 400 Gly
Tyr Asn Phe Ala Ser Glu Met Phe Arg Gln Arg Val Leu Val Ala 405 410
415 Gly Thr Leu Asn Asn Ala Lys Thr Ile Arg Ile Glu Pro Pro Leu Thr
420 425 430 Leu Thr Ile Glu Gln Cys Glu Leu Val Ile Lys Ala Ala Arg
Lys Ala 435 440 445 Leu Ala Ala Met Arg Val Ser Val Glu Glu Ala 450
455 13453PRTVibrio fluvialis 13Met Asn Lys Pro Gln Ser Trp Glu Ala
Arg Ala Glu Thr Tyr Ser Leu1 5 10 15 Tyr Gly Phe Thr Asp Met Pro
Ser Leu His Gln Arg Gly Thr Val Val 20 25 30 Val Thr His Gly Glu
Gly Pro Tyr Ile Val Asp Val Asn Gly Arg Arg 35 40 45 Tyr Leu Asp
Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp 50 55 60 His
Lys Gly Leu Ile Asp Ala Ala Lys Ala Gln Tyr Glu Arg Phe Pro65 70 75
80 Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln Thr Val Met Leu
85 90 95 Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg
Val Phe 100 105 110 Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met
Val Lys Met Leu 115 120 125 Trp Phe Leu His Ala Ala Glu Gly Lys Pro
Gln Lys Arg Lys Ile Leu 130 135 140 Thr Arg Trp Asn Ala Tyr His Gly
Val Thr Ala Val Ser Ala Ser Met145 150 155 160 Thr Gly Lys Pro Tyr
Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe 165 170 175 Val His Leu
Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu 180 185 190 Thr
Glu Glu Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr 195 200
205 Ile Gln Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe Phe Ala Glu Pro
210 215 220 Val Met Gly Ala Gly Gly Val Ile Pro Pro Ala Lys Gly Tyr
Phe Gln225 230 235 240 Ala Ile Leu Pro Ile Leu Arg Lys Tyr Asp Ile
Pro Val Ile Ser Asp 245 250 255 Glu Val Ile Cys Gly Phe Gly Arg Thr
Gly Asn Thr Trp Gly Cys Val 260 265 270 Thr Tyr Asp Phe Thr Pro Asp
Ala Ile Ile Ser Ser Lys Asn Leu Thr 275 280 285 Ala Gly Phe Phe Pro
Met Gly Ala Val Ile Leu Gly Pro Glu Leu Ser 290 295 300 Lys Arg Leu
Glu Thr Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly305 310 315 320
Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala Leu Lys Ala 325
330 335 Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg
Leu 340 345 350 Ala Pro Arg Phe Glu Glu Arg Leu Lys His Ile Ala Glu
Arg Pro Asn 355 360 365 Ile Gly Glu Tyr Arg Gly Ile Gly Phe Met Trp
Ala Leu Glu Ala Val 370 375 380 Lys Asp Lys Ala Ser Lys Thr Pro Phe
Asp Gly Asn Leu Ser Val Ser385 390 395 400 Glu Arg Ile Ala Asn Thr
Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro 405 410 415 Leu Gly Gln Ser
Val Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala 420 425 430 Gln Met
Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val 435 440 445
Phe Ala Glu Val Ala 450 14224PRTBacillus subtilis 14Met 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 15222PRTNocardia sp. NRRL 5646
15Met 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