U.S. patent application number 15/599314 was filed with the patent office on 2018-04-12 for preparation of 6-aminocaproic acid from 5-formyl valeric acid.
The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Stefaan Marie Andre DE WILDEMAN, Petronella Catharina RAEMAKERS-FRANKEN, Martin SCHURMANN, Axel Christoph TREFZER.
Application Number | 20180100172 15/599314 |
Document ID | / |
Family ID | 39651405 |
Filed Date | 2018-04-12 |
United States Patent
Application |
20180100172 |
Kind Code |
A1 |
RAEMAKERS-FRANKEN; Petronella
Catharina ; et al. |
April 12, 2018 |
PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERIC ACID
Abstract
The invention relates to a method for preparing 6-aminocaproic
acid (hereinafter also referred to as `6-ACA`) using a biocatalyst.
The invention further relates to a method for preparing
-caprolactam (hereafter referred to as `caprolactam`) by cyclising
such 6-ACA. The invention further relates to a host cell, a
micro-organism, or a polynucleotide which may be used in the
preparation of 6-ACA or caprolactam.
Inventors: |
RAEMAKERS-FRANKEN; Petronella
Catharina; (Budel, NL) ; SCHURMANN; Martin;
(Julich, DE) ; TREFZER; Axel Christoph;
(Leidschendam, NL) ; DE WILDEMAN; Stefaan Marie
Andre; (Maasmechelen, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
39651405 |
Appl. No.: |
15/599314 |
Filed: |
May 18, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14105705 |
Dec 13, 2013 |
9663805 |
|
|
15599314 |
|
|
|
|
12921733 |
Dec 21, 2010 |
8673599 |
|
|
PCT/NL2009/050117 |
Mar 11, 2009 |
|
|
|
14105705 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 13/02 20130101;
C07D 223/10 20130101; C12P 13/005 20130101; C07D 201/08 20130101;
C12P 13/001 20130101; C07C 227/06 20130101; C07C 227/06 20130101;
C07C 229/08 20130101 |
International
Class: |
C12P 13/00 20060101
C12P013/00; C12P 13/02 20060101 C12P013/02; C07C 227/06 20060101
C07C227/06; C07D 201/08 20060101 C07D201/08; C07D 223/10 20060101
C07D223/10; C07C 229/08 20060101 C07C229/08 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 2008 |
EP |
08152584.2 |
Claims
1. (canceled)
2. Method for preparing 6-aminocaproic acid, wherein the
6-aminocaproic acid is prepared from 5-formylpentanoate, using at
least one biocatalyst.
3-25. (canceled)
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 14,105,705, filed Dec. 13, 2013, which is a continuation
of U.S. patent application Ser. No. 12/921,733, filed Dec. 21,
2010, now issued U.S. Pat. No. 8,673,599, issued Mar. 18, 2014,
which is a U.S. National Stage Application under 35 U.S.C. .sctn.
371 of International Patent Application No. PCT/NL2009/050117,
filed Mar. 11, 2009, which claims the benefit of European Patent
Application No. 08152584.2, filed Mar. 11, 2008, the entire
contents of which are each incorporated herein by reference.
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 17, 2017, is named Sequence_Listing_12956-416-999.txt and is
249,748 bytes in size.
[0003] The invention relates to a method for preparing
6-aminocaproic acid (hereinafter also referred to as `6-ACA`). The
invention further relates to a method for preparing -caprolactam
(hereafter referred to as `caprolactam`) from 6-ACA. The invention
further relates to a host cell which may be used in the preparation
of 6-ACA or caprolactam.
[0004] Caprolactam is a lactam which may be used for the production
of polyamide, for instance nylon-6 or nylon-6,12 (a copolymer of
caprolactam and laurolactam). Various manners of preparing
caprolactam from bulk chemicals are known in the art and include
the preparation of caprolactam from cyclohexanone, toluene, phenol,
cyclohexanol, benzene or cyclohexane. These intermediate compounds
are generally obtained from mineral oil. In view of a growing
desire to prepare materials using more sustainable technology it
would be desirable to provide a method wherein caprolactam is
prepared from an intermediate compound that can be obtained from a
biologically renewable source or at least from an intermediate
compound that is converted into caprolactam using a biochemical
method. Further, it would be desirable to provide a method that
requires less energy than conventional chemical processes making
use of bulk chemicals from petrochemical origin.
[0005] It is known to prepare caprolactam from 6-ACA, e.g. as
described in U.S. Pat. No. 6,194,572. As disclosed in WO
2005/068643, 6-ACA may be prepared biochemically by converting
6-aminohex-2-enoic acid (6-AHEA) in the presence of an enzyme
having .alpha.,.beta.-enoate reductase activity. The 6-AHEA may be
prepared from lysine, e.g. biochemically or by pure chemical
synthesis. Although the preparation of 6-ACA via the reduction of
6-AHEA is feasible by the methods disclosed in WO 2005/068643, the
inventors have found that--under the reduction reaction
conditions--6-AHEA may spontaneously and substantially irreversibly
cyclise to form an undesired side-product, notably
.beta.-homoproline. This cyclisation may be a bottleneck in the
production of 6-ACA, and may lead to a considerable loss in
yield.
[0006] It is an object of the invention to provide a novel method
for preparing 6-ACA or caprolactam--which may, inter alia, be used
for the preparation of polyamide--or an intermediate compound for
the preparation of 6-ACA or caprolactam, that can serve as an
alternative for known methods.
[0007] It is a further object to provide a novel method that would
overcome one or more of the drawbacks mentioned above.
[0008] One or more further objects which may be solved in
accordance with the invention, will follow from the description,
below.
[0009] It has now been found possible to prepare 6-ACA from a
specific starting compound, namely it has been found possible to
prepare 6-aminocaproic acid (6-ACA), wherein the 6-aminocaproic
acid is prepared from 2-oxo-heptanedioic acid also known as
.alpha.-ketopimelic acid (AKP). In particular, the preparation may
be carried out in two or more reaction steps. For instance, a
method is provided wherein AKP is first converted into
5-formylpentanoate (5-formylvaleric acid, 5-FVA), which 5-FVA is
converted into 6-ACA. Further a method is provided wherein AKP is
first converted into alpha-aminopimelic acid (AAP). Thereafter, AAP
is converted into 6-ACA.
[0010] The inventors realised that in principle, it is possible to
prepare 6-ACA from AKP in an entirely chemical (i.e. without the
use of a biocatalyst) manner. Examples of suitable chemical ways of
carrying out individual reaction steps are given herein below.
However, the inventors also realised that it is possible to prepare
6-ACA biochemically from AKP.
[0011] Accordingly, the present invention in particular relates to
a method for preparing 6-ACA, wherein the 6-ACA is prepared from
AKP, using at least one biocatalyst.
[0012] The invention further relates to a method, wherein 6-ACA is
prepared from 5-formylpentanoate (5-formylvaleric acid, 5-FVA),
using a biocatalyst. As indicated above, the 5-FVA may be obtained
from AKP.
[0013] In an embodiment, 6-ACA prepared in a method of the
invention is used for preparing caprolactam. Such method comprises
cyclising the 6-amino-caproic acid, optionally in the presence of a
biocatalyst.
[0014] When referring herein to carboxylic acids or carboxylates,
e.g. 6-ACA, 2-aminoheptanedioic acid (.alpha.-aminopimelic acid,
herein after abbreviated as `AAP`), another amino acid, 5-FVA or
AKP, these terms are meant to include the protonated carboxylic
acid group (i.e. the neutral group), their corresponding
carboxylate (their conjugated bases) as well as salts thereof. When
referring herein to amino acids, e.g. 6-ACA, this term is meant to
include amino acids in their zwitterionic form (in which the amino
group is in the protonated and the carboxylate group is in the
deprotonated form), the amino acid in which the amino group is
protonated and the carboxylic group is in its neutral form, and the
amino acid in which the amino group is in its neutral form and the
carboxylate group is in the deprotonated form, as well as salts
thereof.
[0015] In accordance with the invention, no problems have been
noticed with respect to an undesired cyclisation of an intermediate
product, when forming 6-ACA and optionally caprolactam, resulting
in a loss of yield.
[0016] It is envisaged that a method of the invention allows a
comparable or even better yield than the method described in WO
2005/68643. It is envisaged that a method of the invention may in
particular be favourable if a use is made of a living organism--in
particular in a method wherein growth and maintenance of the
organism is taken into account.
[0017] It is further envisaged that in an embodiment of the
invention the productivity of 6-ACA (g/l.h formed) in a method of
the invention may be improved.
[0018] The term "or" as used herein is defined as "and/or" unless
specified otherwise.
[0019] The term "a" or "an" as used herein is defined as "at least
one" unless specified otherwise.
[0020] When referring to a noun (e.g. a compound, an additive,
etc.) in the singular, the plural is meant to be included.
[0021] When referring to a compound of which stereoisomers exist,
the compound may be any of such stereoisomers or a combination
thereof. Thus, when referred to, e.g., an amino acid of which
enantiomers exist, the amino acid may be the L-enantiomer, the
D-enantiomer or a combination thereof. In case a natural
stereoisomer exists, the compound is preferably a natural
stereoisomer.
[0022] When an enzyme is mentioned with reference to an enzyme
class (EC) between brackets, the enzyme class is a class wherein
the enzyme is classified or may be classified, on the basis of the
Enzyme Nomenclature provided by the Nomenclature Committee of the
International Union of Biochemistry and Molecular Biology
(NC-IUBMB), which nomenclature may be found at
http://www.chem.qmul.ac.uk/iubmb/enzyme/. Other suitable enzymes
that have not (yet) been classified in a specified class but may be
classified as such, are meant to be included.
[0023] The term "homologue" is used herein in particular for
polynucleotides or polypeptides having a sequence identity of at
least 30%, preferably at least 40%, more preferably at least 60%,
more preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, in
particular at least 85%, more in particular at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98% or at least 99%. The term
homologue is also meant to include nucleic acid sequences
(polynucleotide sequences) which differ from another nucleic acid
sequence due to the degeneracy of the genetic code and encode the
same polypeptide sequence.
[0024] Sequence identity or similarity is herein defined as a
relationship between two or more polypeptide sequences or two or
more nucleic acid sequences, as determined by comparing the
sequences. Usually, sequence identities or similarities are
compared over the whole length of the sequences, but may however
also be compared only for a part of the sequences aligning with
each other. In the art, "identity" or "similarity" also means the
degree of sequence relatedness between polypeptide sequences or
nucleic acid sequences, as the case may be, as determined by the
match between such sequences. Preferred methods to determine
identity or similarity are designed to give the largest match
between the sequences tested. In context of this invention a
preferred computer program method to determine identity and
similarity between two sequences includes BLASTP and BLASTN
(Altschul, S. F. et al., J. Mol. Biol. 1990, 215, 403-410, publicly
available from NCBI and other sources (BLAST Manual, Altschul, S.,
et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred parameters for
polypeptide sequence comparison using BLASTP are gap open 10.0, gap
extend 0.5, Blosum 62 matrix. Preferred parameters for nucleic acid
sequence comparison using BLASTN are gap open 10.0, gap extend 0.5,
DNA full matrix (DNA identity matrix).
[0025] In accordance with the invention, a biocatalyst is used,
i.e. at least one reaction step in the method is catalysed by a
biological material or moiety derived from a biological source, for
instance an organism or a biomolecule derived there from. The
biocatalyst may in particular comprise one or more enzymes. The
biocatalyst may be used in any form. In an embodiment, one or more
enzymes are used isolated from the natural environment (isolated
from the organism it has been produced in), for instance as a
solution, an emulsion, a dispersion, (a suspension of) freeze-dried
cells, as a lysate, or immobilised on a support. In an embodiment,
one or more enzymes form part of a living organism (such as living
whole cells).
[0026] The enzymes may perform a catalytic function inside the
cell. It is also possible that the enzyme may be secreted into a
medium, wherein the cells are present.
[0027] Living cells may be growing cells, resting or dormant cells
(e.g. spores) or cells in a stationary phase. It is also possible
to use an enzyme forming part of a permeabilised cell (i.e. made
permeable to a substrate for the enzyme or a precursor for a
substrate for the enzyme or enzymes).
[0028] A biocatalyst used in a method of the invention may in
principle be any organism, or be obtained or derived from any
organism. The organism may be eukaryotic or prokaryotic. In
particular the organism may be selected from animals (including
humans), plants, bacteria, archaea, yeasts and fungi.
[0029] In an embodiment a biocatalyst originates from an animal, in
particular from a part thereof--e.g. liver, pancreas, brain,
kidney, heart or other organ. The animal may in particular be
selected from the group of mammals, more in particular selected
from the group of Leporidae, Muridae, Suidae and Bovidae.
[0030] Suitable plants in particular include plants selected from
the group of Asplenium; Cucurbitaceae, in particular Curcurbita,
e.g. Curcurbita moschata (squash), or Cucumis; Mercurialis, e.g.
Mercurialis perennis; Hydnocarpus; and Ceratonia.
[0031] Suitable bacteria may in particular be selected amongst the
group of Vibrio, Pseudomonas, Bacillus, Corynebacterium,
Brevibacterium, Enterococcus, Streptococcus, Klebsiella,
Lactococcus, Lactobacillus, Clostridium, Escherichia, Thermus,
Mycobacterium, Zymomonas, Proteus, Agrobacterium, Geobacillus,
Acinetobacter, Ralstonia, Rhodobacter, Paracoccus, Novosphingobium,
Nitrosomonas, Legionella, Neisseria, Rhodopseudomonas,
Staphylococcus, Deinococcus and Salmonella.
[0032] Suitable archaea may in particular be selected amongst the
group of Archaeoglobus, Aeropyrum, Halobacterium, Methanosarcina,
Methanococcus, Thermoplasma, Pyrobaculum, Methanocaldococcus,
Methanobacterium, Methanosphaera, Methanopyrus and
Methanobrevibacter.
[0033] Suitable fungi may in particular be selected amongst the
group of Rhizopus, Neurospora, Penicillium and Aspergillus.
[0034] A suitable yeast may in particular be selected amongst the
group of Candida, Hansenula, Kluyveromyces and Saccharomyces.
[0035] It will be clear to the person skilled in the art that use
can be made of a naturally occurring biocatalyst (wild type) or a
mutant of a naturally occurring biocatalyst with suitable activity
in a method according to the invention. Properties of a naturally
occurring biocatalyst may be improved by biological techniques
known to the skilled person in the art, such as e.g. molecular
evolution or rational design. Mutants of wild-type biocatalysts can
for example be made by modifying the encoding DNA of an organism
capable of acting as a biocatalyst or capable of producing a
biocatalytic moiety (such as an enzyme) using mutagenesis
techniques known to the person skilled in the art (random
mutagenesis, site-directed mutagenesis, directed evolution, gene
recombination, etc.). In particular the DNA may be modified such
that it encodes an enzyme that differs by at least one amino acid
from the wild-type enzyme, so that it encodes an enzyme that
comprises one or more amino acid substitutions, deletions and/or
insertions compared to the wild-type, or such that the mutants
combine sequences of two or more parent enzymes or by effecting the
expression of the thus modified DNA in a suitable (host) cell. The
latter may be achieved by methods known to the skilled person in
the art such as codon optimisation or codon pair optimisation, e.g.
based on a method as described in WO 2008/000632.
[0036] A mutant biocatalyst may have improved properties, for
instance with respect to one or more of the following aspects:
selectivity towards the substrate, activity, stability, solvent
tolerance, pH profile, temperature profile, substrate profile,
susceptibility to inhibition, cofactor utilisation and
substrate-affinity. Mutants with improved properties can be
identified by applying e.g. suitable high through-put screening or
selection methods based on such methods known to the skilled person
in the art.
[0037] When referred to a biocatalyst, in particular an enzyme,
from a particular source, recombinant biocatalysts, in particular
enzymes, originating from a first organism, but actually produced
in a (genetically modified) second organism, are specifically meant
to be included as biocatalysts, in particular enzymes, from that
first organism.
[0038] In a preferred method of the invention, the preparation
comprises a biocatalytic (usually an enzymatic) reaction in the
presence of a biocatalyst capable of catalysing the decarboxylation
of an .alpha.-keto acid or an amino acid (i.e. a compound
comprising at least one carboxylic acid group and at least one
amino group). An enzyme having such catalytic activity may
therefore be referred to as an .alpha.-keto acid decarboxylase
respectively an amino acid decarboxylase.
[0039] Said acid preferably is a diacid, wherein the said
biocatalyst is selective towards the acid group next to the keto-
or amino-group.
[0040] In general, a suitable decarboxylase has
.alpha.-ketopimelate decarboxylase activity, capable of catalysing
the conversion of AKP into 5-FVA or .alpha.-aminopimelate
decarboxylase activity, capable of catalysing the conversion of AAP
to 6-ACA.
[0041] An enzyme capable of decarboxylating an .alpha.-keto acid or
an amino acid may in particular be selected from the group of
decarboxylases (E.C. 4.1.1), preferably from the group of
oxaloacetate decarboxylases (EC 4.1.1.3), diaminopimelate
decarboxylases (EC 4.1.1.20), branched chain .alpha.-keto acid
decarboxylases (EC 4.1.1.72), .alpha.-ketoisovalerate
decarboxylases, .alpha.-ketoglutarate decarboxylases (EC 4.1.1.71),
and pyruvate decarboxylases (EC 4.1.1.1).
[0042] One or more other suitable decarboxylases may be selected
amongst the group of oxalate decarboxylases (EC 4.1.1.2),
acetoacetate decarboxylases (EC 4.1.1.4), valine
decarboxylases/leucine decarboxylases (EC 4.1.1.14), glutamate
decarboxylases (EC 4.1.1.15), aspartate 1-decarboxylases (EC
4.1.1.11), 3-hydroxyglutamate decarboxylases (EC 4.1.1.16),
ornithine decarboxylases (EC 4.1.1.17), lysine decarboxylases (EC
4.1.1.18), arginine decarboxylases (EC 4.1.1.19), 2-oxoglutarate
decarboxylases (EC 4.1.1.71), and diaminobutyrate decarboxylases
(EC 4.1.1.86)
[0043] A decarboxylase may in particular be a decarboxylase of an
organism selected from the group of squashes; cucumbers; yeasts;
fungi, e.g. Saccharomyces cerevisiae, Candida flareri, Hansenula
sp., Kluyveromyces marxianus, Rhizopus javanicus, and Neurospora
crassa; mammals, in particular from mammalian brain; and bacteria,
such as Escherichia coli, Lactococcus lactis, Mycobacterium
tuberculosis, Pseudomonas sp. and Zymomonas mobilis.
[0044] The pyruvate decarboxylase may originate from Saccharomyces
cerevisiae or Zymomonas mobilis. In particular, pyruvate
decarboxylase mutant I472A from Zymomonas mobilis may be used.
[0045] Glutamate decarboxylase, diaminopimelate decarboxylase or
aspartate decarboxylase from Escherichia coli (E. coli) may be
used.
[0046] Glutamate decarboxylase from Neurospora crassa,
Mycobacterium leprae, Clostridium perfringens, Lactobacillus
brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus
may be used. Examples of Lactococcus species from which the
glutamate decarboxylase may originate in particular include
Lactococcus lactis, such as Lactococcus lactis strain B1157,
Lactococcus lactis IFPL730, more in particular Lactococcus lactis
var. maltigenes (formerly named Streptococcus lactis var.
maltigenes).
[0047] An oxaloacetate decarboxylase from Pseudomonas may in
particular be used.
[0048] A branched-chain alpha-keto acid decarboxylase from
Lactococcus lactis may be used. More in particular, an
alpha-ketoisovalerate decarboxylase from Lactococcus lactis may be
used.
[0049] An alpha-ketoglutarate decarboxylase from Mycobacterium
tuberculosis may in particular be used.
[0050] In a preferred method of the invention, the preparation of
6-ACA comprises an enzymatic reaction in the presence of an enzyme
capable of catalysing a transamination reaction in the presence of
an amino donor, selected from the group of aminotransferases (E.C.
2.6.1).
[0051] In general, a suitable aminotransferase has 6-aminocaproic
acid 6-aminotransferase activity, capable of catalysing the
conversion of 5-FVA into 6-ACA or .alpha.-aminopimelate
2-aminotransferase activity, capable of catalysing the conversion
of AKP into AAP.
[0052] The aminotransferase may in particular be selected amongst
the group of .beta.-aminoisobutyrate:.alpha.-ketoglutarate
aminotransferases, .beta.-alanine aminotransferases, aspartate
aminotransferases, 4-amino-butyrate aminotransferases (EC
2.6.1.19), L-lysine 6-aminotransferase (EC 2.6.1.36),
2-aminoadipate aminotransferases (EC 2.6.1.39), 5-aminovalerate
aminotransferases (EC 2.6.1.48), 2-aminohexanoate aminotransferases
(EC 2.6.1.67) and lysine:pyruvate 6-aminotransferases (EC
2.6.1.71).
[0053] In an embodiment an aminotransferase may be selected amongst
the group of alanine aminotransferases (EC 2.6.1.2), leucine
aminotransferases (EC 2.6.1.6), alanine-oxo-acid aminotransferases
(EC 2.6.1.12), .beta.-alanine-pyruvate aminotransferases (EC
2.6.1.18), (S)-3-amino-2-methylpropionate aminotransferases (EC
2.6.1.22), L,L-diaminopimelate aminotransferase (EC 2.6.1.83).
[0054] The aminotransferase may in particular be selected amongst
aminotransferases from a mammal; Mercurialis, in particular
Mercurialis perennis, more in particular shoots of Mercurialis
perennis; Asplenium, more in particular Asplenium unilaterale or
Asplenium septentrionale; Ceratonia, more in particular Ceratonia
siliqua; Rhodobacter, in particular Rhodobacter sphaeroides,
Staphylococcus, in particular Staphylococcus aureus; Vibrio, in
particular Vibrio fluvialis; Pseudomonas, in particular Pseudomonas
aeruginosa; Rhodopseusomonas; Bacillus, in particular Bacillus
weihenstephanensis and Bacillus subtilis; Legionella; Nitrosomas;
Neisseria; or yeast, in particular Saccharomyces cerevisiae.
[0055] In case the enzyme is of a mammal, it may in particular
originate from mammalian kidney, from mammalian liver, from
mammalian heart or from mammalian brain. For instance a suitable
enzyme may be selected amongst the group of
.beta.-aminoisobutyrate:.alpha.-ketoglutarate aminotransferase from
mammalian kidney, in particular
.beta.-aminoisobutyrate:.alpha.-ketoglutarate aminotransferase from
hog kidney; .beta.-alanine aminotransferase from mammalian liver,
in particular .beta.-alanine aminotransferase from rabbit liver;
aspartate aminotransferase from mammalian heart; in particular
aspartate aminotransferase from pig heart; 4-amino-butyrate
aminotransferase from mammalian liver, in particular
4-amino-butyrate aminotransferase from pig liver; 4-amino-butyrate
aminotransferase from mammalian brain, in particular
4-aminobutyrate aminotransferase from human, pig, or rat brain;
.alpha.-ketoadipate-glutamate aminotransferase from Neurospora, in
particular .alpha.-ketoadipate:glutamate aminotransferase from
Neurospora crassa; 4-amino-butyrate aminotransferase from E. coli,
or .alpha.-aminoadipate aminotransferase from Thermus, in
particular .alpha.-aminoadipate aminotransferase from Thermus
thermophilus, and 5-aminovalerate aminotransferase from Clostridium
in particular from Clostridium aminovalericum. A suitable
2-aminoadipate aminotransferase may e.g. be provided by Pyrobaculum
islandicum.
[0056] In particular, the amino donor can be selected from the
group of ammonia, ammonium ions, amines and amino acids. Suitable
amines are primary amines and secondary amines. The amino acid may
have a D- or L-configuration. Examples of amino donors are alanine,
glutamate, isopropylamine, 2-aminobutane, 2-aminoheptane,
phenylmethanamine, 1-phenyl-1-aminoethane, glutamine, tyrosine,
phenylalanine, aspartate, .beta.-aminoisobutyrate, 4-aminobutyrate,
and .alpha.-aminoadipate.
[0057] In a further preferred embodiment, the method for preparing
6-ACA comprises a biocatalytic reaction in the presence of an
enzyme capable of catalysing a reductive amination reaction in the
presence of an ammonia source, selected from the group of
oxidoreductases acting on the CH--NH.sub.2 group of donors (EC
1.4), in particular from the group of amino acid dehydrogenases
(E.C. 1.4.1). In general, a suitable amino acid dehydrogenase has
6-aminocaproic acid 6-dehydrogenase activity, catalysing the
conversion of 5-FVA into 6-ACA or has .alpha.-aminopimelate
2-dehydrogenase activity, catalysing the conversion of AKP into
AAP. In particular a suitable amino acid dehydrogenase be selected
amongst the group of diaminopimelate dehydrogenases (EC 1.4.1.16),
lysine 6-dehydrogenases (EC 1.4.1.18), glutamate dehydrogenases (EC
1.4.1.3; EC 1.4.1.4), and leucine dehydrogenases (EC 1.4.1.9).
[0058] In an embodiment, an amino acid dehydrogenase may be
selected amongst an amino acid dehydrogenases classified as
glutamate dehydrogenases acting with NAD or NADP as acceptor (EC
1.4.1.3), glutamate dehydrogenases acting with NADP as acceptor (EC
1.4.1.4), leucine dehydrogenases (EC 1.4.1.9), diaminopimelate
dehydrogenases (EC 1.4.1.16), and lysine 6-dehydrogenases (EC
1.4.1.18).
[0059] An amino acid dehydrogenase may in particular originate from
an organism selected from the group of Corynebacterium, in
particular Corynebacterium glutamicum; Proteus, in particular
Proteus vulgaris; Agrobacterium, in particular Agrobacterium
tumefaciens; Geobacillus, in particular Geobacillus
stearothermophilus; Acinetobacter, in particular Acinetobacter sp.
ADP1; Ralstonia, in particular Ralstonia solanacearum; Salmonella,
in particular Salmonella typhimurium; Saccharomyces, in particular
Saccharomyces cerevisiae; Brevibacterium, in particular
Brevibacterium flavum; and Bacillus, in particular Bacillus
sphaericus, Bacillus cereus or Bacillus subtilis. For instance a
suitable amino acid dehydrogenase may be selected amongst
diaminopimelate dehydrogenases from Bacillus, in particular
Bacillus sphaericus; diaminopimelate dehydrogenases from
Brevibacterium sp.; diaminopimelate dehydrogenases from
Corynebacterium, in particular diaminopimelate dehydrogenases from
Corynebacterium glutamicum; diaminopimelate dehydrogenases from
Proteus, in particular diaminopimelate dehydrogenase from Proteus
vulgaris; lysine 6-dehydrogenases from Agrobacterium, in particular
Agrobacterium tumefaciens, lysine 6-dehydrogenases from
Geobacillus, in particular from Geobacillus stearothermophilus;
glutamate dehydrogenases acting with NADH or NADPH as cofactor (EC
1.4.1.3) from Acinetobacter, in particular glutamate dehydrogenases
from Acinetobacter sp. ADP1; glutamate dehydrogenases (EC 1.4.1.3)
from Ralstonia, in particular glutamate dehydrogenases from
Ralstonia solanacearum; glutamate dehydrogenases acting with NADPH
as cofactor (EC 1.4.1.4) from Salmonella, in particular glutamate
dehydrogenases from Salmonella typhimurium; glutamate
dehydrogenases (EC 1.4.1.4) from Saccharomyces, in particular
glutamate dehydrogenases from Saccharomyces cerevisiae; glutamate
dehydrogenases (EC 1.4.1.4) from Brevibacterium, in particular
glutamate dehydrogenases from Brevibacterium flavum; and leucine
dehydrogenases from Bacillus, in particular leucine dehydrogenases
from Bacillus cereus or Bacillus subtilis.
[0060] In a specific embodiment, AKP is biocatalytically converted
into 5-formylpentanoate (5-FVA) in the presence of a decarboxylase
or other biocatalyst catalysing such conversion. A decarboxylase
used in accordance with the invention may in particular be selected
from the group of .alpha.-keto acid decarboxylases from Lactococcus
lactis, Lactococcus lactis var. maltigenes or Lactococcus lactis
subsp. cremoris; branched chain .alpha.-keto acid decarboxylases
from Lactococcus lactis strain B1157 or Lactococcus lactis IFPL730;
pyruvate decarboxylases from Saccharomyces cerevisiae, Candida
flareri, Zymomonas mobilis, Hansenula sp., Rhizopus javanicus,
Neurospora crassa, or Kluyveromyces marxianus;
.alpha.-ketoglutarate decarboxylases from Mycobacterium
tuberculosis; glutamate decarboxylases from E. coli, Lactobacillus
brevis, Mycobacterium leprae, Neurospora crassa or Clostridium
perfringens; and aspartate decarboxylases from E. coli.
[0061] In particular, a decarboxylase from Escherichia coli,
Zymomonas mobilis, Saccharomyces cerevisiae, Mycobacterium
tuberculosis, Pseudomonas species, or Lactococcus lactis has been
found suitable to catalyse the conversion of AKP into 5-FVA. More
in particular, a biocatalyst comprising a decarboxylase having a
amino acid sequence as identified by Sequence ID 31, Sequence ID
34, Sequence ID 37, Sequence ID 40, Sequence ID 43, Sequence ID 46
or a homologue thereof may be used. It is also envisaged that such
decarboxylase may be used to prepare 6-ACA from AAP.
[0062] Thereafter 5-FVA is converted into 6-ACA. This can be done
chemically: 6-ACA can be prepared in high yield by reductive
amination of 5-FVA with ammonia over a hydrogenation catalyst, for
example Ni on SiO.sub.2/Al.sub.2O.sub.3 support, as described for
9-aminononanoic acid (9-aminopelargonic acid) and
12-aminododecanoic acid (12-aminolauric acid) in EP-A 628 535 or DE
4 322 065.
[0063] Alternatively, 6-ACA can be obtained by hydrogenation over
PtO.sub.2 of 6-oximocaproic acid, prepared by reaction of 5-FVA and
hydroxylamine. (see e.g. F. O. Ayorinde, E. Y. Nana, P. D. Nicely,
A. S. Woods, E. O. Price, C. P. Nwaonicha J. Am. Oil Chem. Soc.
1997, 74, 531-538 for synthesis of the homologous
12-aminododecanoic acid).
[0064] In an embodiment, the conversion of 5-FVA to 6-ACA is
performed biocatalytically in the presence of (i) an amino donor
and (ii) an aminotransferase, an amino acid dehydrogenase or
another biocatalyst capable of catalysing such conversion. In
particular in such an embodiment the aminotransferase may be
selected from the group of aminotransferases from Vibrio fluvialis,
Pseudomonas aeruginosa, Bacillus subtilis, Bacillus
weihenstephanensis or Escherichia coli;
.beta.-aminoisobutyrate:.alpha.-ketoglutarate aminotransferase from
hog kidney; .beta.-alanine aminotransferase from rabbit liver;
aminotransferase from shoots from Mercurialis perennis;
4-aminobutyrate aminotransferase from pig liver or from human, rat,
or pig brain; .beta.-alanine aminotransferase from rabbit liver;
and L-lysine:.alpha.-ketoglutarate- -aminotransferase. In case an
amino acid dehydrogenase is used, such amino acid dehydrogenase may
in particular be selected from the group of lysine 6-dehydrogenases
from Agrobacterium tumefaciens or Geobacillus stearothermophilus.
Another suitable amino acid dehydrogenase may be selected from the
group of diaminopimelate dehydrogenases from Bacillus sphaericus,
Brevibacterium sp., Corynebacterium glutamicum, or Proteus
vulgaris; from the group of glutamate dehydrogenases acting with
NADH or NADPH as cofactor (EC 1.4.1.3) from Acinetobacter sp. ADP1
or Ralstonia solanacearum; from the group of glutamate
dehydrogenases acting with NADPH as cofactor (EC 1.4.1.4) from
Salmonella typhimurium; from the group of glutamate dehydrogenases
(EC 1.4.1.4) from Saccharomyces cerevisiae or Brevibacterium
flavum; or from the group of leucine dehydrogenases from Bacillus
cereus or Bacillus subtilis.
[0065] In a specific embodiment, the conversion of 5-FVA to 6-ACA
is catalysed by a biocatalyst comprising an aminotransferase
comprising an amino acid sequence according to Sequence ID 2,
Sequence ID 5, Sequence ID 8, Sequence ID 65, Sequence ID 67,
Sequence ID 69 or a homologue of any of these sequences.
[0066] In a specific embodiment, AKP is chemically converted into
5-FVA. Efficient chemical decarboxylation of a 2-keto carboxylic
acid into the corresponding aldehyde can be performed by
intermediate enamine formation using a secondary amine, for
instance morpholine, under azeotropic water removal and
simultaneous loss of CO.sub.2, e.g. based on a method as described
in Tetrahedron Lett. 1982, 23(4), 459-462. The intermediate
terminal enamide is subsequently hydrolysed to the corresponding
aldehyde. 5-FVA may thereafter be biocatalytically converted into
6-ACA by transamination in the presence of an aminotransferase or
by enzymatic reductive amination by an amino acid dehydrogenase or
another biocatalyst able of catalysing such conversion. Such
aminotransferase or amino acid dehydrogenase may in particular be
selected from the biocatalysts mentioned above when describing the
conversion of 5-FVA to 6-ACA.
[0067] Alternatively, the conversion of 5-FVA to 6-ACA may be
performed by a chemical method, e.g. as mentioned above.
[0068] In a specific embodiment, AKP is biocatalytically converted
into AAP in the presence of (i) an aminotransferase, an amino acid
dehydrogenase, or another biocatalyst capable of catalysing such
conversion and (ii) an amino donor. Such aminotransferase used in
accordance with the invention for the conversion of AKP to AAP may
in particular be selected from aminotransferases mentioned above,
more in particular from the group of aspartate aminotransferases
from pig heart; .alpha.-ketoadipate:glutamate aminotransferases
from Neurospora crassa or yeast; aminotransferases from shoots from
Mercurialis perennis; 4-aminobutyrate aminotransferases from E.
coli; .alpha.-aminoadipate aminotransferases from Thermus
thermophilus; aminotransferases from Asplenium septentrionale or
Asplenium unilaterale; and aminotransferases from Ceratonia
siliqua.
[0069] In a preferred embodiment, the aminotransferase for the
conversion of AKP to AAP is selected from the group of
aminotransferases from Vibrio, Pseudomonas, Bacillus, Legionella,
Nitrosomonas, Neisseria, Rhodobacter, Escherichia and
Rhodopseudomonas.
[0070] In particular, aminotransferases from an organism selected
from the group of Bacillus subtilis, Rhodobacter sphaeroides,
Legionella pneumophila, Nitrosomonas europaea, Neisseria
gonorrhoeae, Pseudomonas syringae, Rhodopseudomonas palustris,
Vibrio fluvialis, Escherichia coli and Pseudomonas aeruginosa, have
been found suitable to catalyse the conversion of AKP to AAP.
[0071] In a specific embodiment, for the conversion of AKP to AAP
an aminotransferase is used comprising an amino acid sequence
according to Sequence ID 2, Sequence ID 8, Sequence ID 12, Sequence
ID 15, Sequence ID 17, Sequence ID 19, Sequence ID 21, Sequence ID
23, Sequence ID 25, Sequence ID 27, Sequence ID 29 or a homologue
of any of these sequences.
[0072] In a further embodiment, the method for preparing AAP
comprises a biocatalytic reaction in the presence of an enzyme
capable of catalysing a reductive amination reaction in the
presence of an ammonia source, selected from the group of
oxidoreductases acting on the CH--NH.sub.2 group of donors (EC
1.4), in particular from the group of amino acid dehydrogenases
(E.C. 1.4.1). In general, a suitable amino acid dehydrogenase has
.alpha.-aminopimelate 2-dehydrogenase activity, catalysing the
conversion of AKP into AAP.
[0073] In particular a suitable amino acid dehydrogenase may be
selected from the group of diaminopimelate dehydrogenases (EC
1.4.1.16), glutamate dehydrogenases (EC 1.4.1.3; EC 1.4.1.4), and
leucine dehydrogenases (EC 1.4.1.9).
[0074] In an embodiment, an amino acid dehydrogenase is selected
amongst amino acid dehydrogenases classified as glutamate
dehydrogenases acting with NAD or NADP as acceptor (EC 1.4.1.3),
glutamate dehydrogenases acting with NADP as acceptor (EC 1.4.1.4),
leucine dehydrogenases (EC 1.4.1.9), and diaminopimelate
dehydrogenases (EC 1.4.1.16).
[0075] An amino acid dehydrogenase may in particular originate from
an organism selected from the group of Corynebacterium, in
particular Corynebacterium glutamicum; Proteus, in particular
Proteus vulgaris; Agrobacterium, in particular Agrobacterium
tumefaciens; Geobacillus, in particular Geobacillus
stearothermophilus; Acinetobacter, in particular Acinetobacter sp.
ADP1; Ralstonia, in particular Ralstonia solanacearum; Salmonella,
in particular Salmonella typhimurium; Saccharomyces, in particular
Saccharomyces cerevisiae; Brevibacterium, in particular
Brevibacterium flavum; and Bacillus, in particular Bacillus
sphaericus, Bacillus cereus or Bacillus subtilis.
[0076] For instance a suitable amino acid dehydrogenase may be
selected amongst diaminopimelate dehydrogenases from Bacillus, in
particular Bacillus sphaericus; diaminopimelate dehydrogenases from
Brevibacterium sp.; diaminopimelate dehydrogenases from
Corynebacterium, in particular diaminopimelate dehydrogenases from
Corynebacterium glutamicum; diaminopimelate dehydrogenases from
Proteus, in particular diaminopimelate dehydrogenase from Proteus
vulgaris; glutamate dehydrogenases acting with NADH or NADPH as
cofactor (EC 1.4.1.3) from Acinetobacter, in particular glutamate
dehydrogenases from Acinetobacter sp. ADP1; glutamate
dehydrogenases (EC 1.4.1.3) from Ralstonia, in particular glutamate
dehydrogenases from Ralstonia solanacearum; glutamate
dehydrogenases acting with NADPH as cofactor (EC 1.4.1.4) from
Salmonella, in particular glutamate dehydrogenases from Salmonella
typhimurium; glutamate dehydrogenases (EC 1.4.1.4) from
Saccharomyces, in particular glutamate dehydrogenases from
Saccharomyces cerevisiae; glutamate dehydrogenases (EC 1.4.1.4)
from Brevibacterium, in particular glutamate dehydrogenases from
Brevibacterium flavum; and leucine dehydrogenases from Bacillus, in
particular leucine dehydrogenases from Bacillus cereus or Bacillus
subtilis.
[0077] Another suitable amino acid dehydrogenase may be selected
from the group of lysine 6-dehydrogenases from Agrobacterium
tumefaciens or Geobacillus stearothermophilus; or from the group of
leucine dehydrogenases from Bacillus cereus or Bacillus
subtilis.
[0078] AAP prepared in a method of the invention may further be
used for the preparation of 6-ACA. The inventors have realised that
AAP, prepared from AKP, can be converted into 6-ACA by a
decarboxylation reaction. This can be performed chemically, for
instance by heating in a high boiling solvent in the presence of a
ketone or aldehyde catalyst. For example, amino acids are
decarboxylated in good yields in cyclohexanol at 150-160.degree. C.
with 1-2 v/v % of cyclohexenone as described by M. Hashimoto, Y.
Eda, Y. Osanai, T. Iwai and S. Aoki in Chem. Lett. 1986, 893-896.
Similar methods are described in Eur. Pat. Appl. 1586553, 2005 by
Daiso, and by S. D. Brandt, D. Mansell, S. Freeman, I. A. Fleet, J.
F. Alder J. Pharm. Biomed. Anal. 2006, 41, 872-882.
[0079] Alternatively, the decarboxylation of AAP to 6-ACA may be
performed biocatalytically in the presence of a decarboxylase or
other biocatalyst catalysing such decarboxylation.
[0080] The decarboxylase may be selected amongst decarboxylases
capable of catalysing the decarboxylation of an .alpha.-amino acid.
An enzyme capable of decarboxylating an alpha-amino acid may in
particular be selected from the group of decarboxylases (E.C.
4.1.1), preferably from the group of pyruvate decarboxylases (EC
4.1.1.1), diaminopimelate decarboxylases (EC 4.1.1.20),
diaminopimelate decarboxylases (EC 4.1.1.20), branched chain
alpha-keto acid decarboxylases (EC 4.1.1.72), which include
alpha-ketoisovalerate decarboxylases, and alpha-ketoglutarate
decarboxylases (EC 4.1.1.71).
[0081] One or more other suitable decarboxylases may in particular
be selected amongst the group of oxalate decarboxylases (EC
4.1.1.2), oxaloacetate decarboxylases (EC 4.1.1.3), acetoacetate
decarboxylases (EC 4.1.1.4), aspartate 1-decarboxylases (EC
4.1.1.11), valine decarboxylases/leucine decarboxylases (EC
4.1.1.14), glutamate decarboxylases (EC 4.1.1.15),
3-hydroxyglutamate decarboxylases (EC 4.1.1.16), ornithine
decarboxylases (EC 4.1.1.17), lysine decarboxylases (EC 4.1.1.18),
arginine decarboxylases (EC 4.1.1.19), 2-oxoglutarate
decarboxylases (EC 4.1.1.71), and diaminobutyrate decarboxylases
(EC 4.1.1.86).
[0082] A decarboxylase may in particular be a decarboxylase of an
organism selected from the group of squashes, e.g. Curcurbita
moschata; cucumbers; yeasts; fungi, e.g. Saccharomyces cerevisiae,
Candida flareri, Hansenula sp., Kluyveromyces marxianus, Rhizopus
javanicus, and Neurospora crassa; mammals, in particular from
mammalian brain; and bacteria such as Escherichia coli, Lactococcus
lactis, Mycobacterium tuberculosis, Pseudomonas sp. and Zymomonas
mobilis.
[0083] The pyruvate decarboxylase may originate from Saccharomyces
cerevisiae or Zymomonas mobilis. In particular, pyruvate
decarboxylase mutant I472A from Zymomonas mobilis may be used. An
oxaloacetate decarboxylase from Pseudomonas may in particular be
used. Glutamate decarboxylase or aspartate decarboxylase from
Escherichia coli (E. coli) may be used, or glutamate decarboxylase
from Neurospora crassa, Mycobacterium leprae, Clostridium
perfringens, Lactobacillus brevis, Mycobacterium tuberculosis,
Streptococcus or Lactococcus may be used. Examples of Lactococcus
species from which the glutamate decarboxylase may originate in
particular include Lactococcus lactis, such as Lactococcus lactis
strain B1157, Lactococcus lactis IFPL730, more in particular
Lactococcus lactis var. maltigenes (formerly named Streptococcus
lactis var. maltigenes). A diaminopimelate decarboxylase may, e.g.,
be from an organism capable of synthesising lysine from
diaminopimelate. Such organism may in particular be found amongst
bacteria, archaea and plants. In particular, the diaminopimelate
decarboxylase may be from a gram negative bacterium, for instance
E. coli. Branched-chain alpha-keto acid decarboxylases from
Lactococcus lactis may be used. More in particular, branched chain
alpha-keto acid decarboxylases and alpha-ketoisovalerate
decarboxylases from Lactococcus lactis may be used.
[0084] An alpha-ketoglutarate decarboxylase from Mycobacterium
tuberculosis may in particular be used. The inventors have found
that alpha-ketoglutarate decarboxylase (Kgd) from Mycobacterium
tuberculosis may be used for converting AAP into 6-ACA. In
particular, the inventors have found that such decarboxylase
comprising a sequence as shown in SEQUENCE ID No. 46 or a
functional analogue thereof may be capable of catalysing the
formation of 6-ACA from AAP.
[0085] A glutamate decarboxylase may in particular be selected from
Curcurbita moschata, cucumber, yeast, or calf brain; and
diaminopimelate decarboxylases (EC 4.1.1.20).
[0086] A diaminopimelate decarboxylase may, e.g., be from an
organism capable of synthesising lysine from diaminopimelate. Such
organism may in particular be found amongst bacteria, archaea and
plants.
[0087] In particular, the diaminopimelate decarboxylase may be from
a gram negative bacterium, for instance E. coli.
[0088] In a specific embodiment, AKP is chemically converted into
AAP. AAP can be prepared from 2-oxopimelic acid by catalytic
Leuckart-Wallach reaction as described for similar compounds. This
reaction is performed with ammonium formate in methanol and
[RhCp*Cl.sub.2].sub.2 as homogeneous catalyst (M. Kitamura, D. Lee,
S. Hayashi, S. Tanaka, M. Yoshimura J. Org. Chem. 2002, 67,
8685-8687). Alternatively, the Leuckart-Wallach reaction can be
performed with aqueous ammonium formate using
[Ir.sup.IIICp*(bpy)H.sub.2O]SO.sub.4 as catalyst as described by S.
Ogo, K. Uehara and S. Fukuzumi in J. Am. Chem. Soc. 2004, 126,
3020-3021. Transformation of .alpha.-keto acids into
(enantiomerically enriched) amino acids is also possible by
reaction with (chiral) benzylamines and subsequent hydrogenation of
the intermediate imine over Pd/C or Pd(OH).sub.2/C. See for
example, R. G. Hiskey, R. C. Northrop J. Am. Chem. Soc. 1961, 83,
4798.
[0089] Thereafter AAP is biocatalytically converted into 6-ACA, in
the presence of a decarboxylase or another biocatalyst capable of
performing such decarboxylation. Such decarboxylase may in
particular be selected amongst the biocatalysts referred to above,
when describing biocatalysts for the conversion of AAP to
6-ACA.
[0090] Alternatively, the conversion of AAP to 6-ACA may be
performed by a chemical method, e.g. as mentioned above.
[0091] In a specific embodiment, AKP is biocatalytically converted
into 5-FVA in the presence of a decarboxylase or other biocatalyst
capable of catalysing such conversion and 5-FVA is thereafter
converted into 6-ACA in the presence of an aminotransferase, amino
acid dehydrogenase, or other biocatalyst capable of catalysing such
conversion. Decarboxylases suitable for these reactions may in
particular be selected from the group of decarboxylases mentioned
above, when describing the biocatalytic conversion of AKP into
5-FVA. A suitable aminotransferase or amino acid dehydrogenase for
the conversion of 5-FVA may in particular be selected from those
mentioned above, when describing the biocatalytic conversion of
5-FVA to 6-ACA.
[0092] In a specific embodiment, AKP is biocatalytically converted
into AAP in the presence of an aminotransferase, amino acid
dehydrogenase, or other biocatalyst capable of catalysing such
conversion and AAP is thereafter converted into 6-ACA in the
presence of a decarboxylase or other biocatalyst capable of
catalysing such conversion.
[0093] Enzymes suitable for these reactions may in particular be
selected from the group of aminotransferases, amino acid
dehydrogenases, and decarboxylases which have been described above
when describing the biocatalytic conversion of AKP into AAP and the
biocatalytic conversion of AAP into 6-ACA respectively.
[0094] AKP used to prepare 6-ACA may in principle be obtained in
any way. For instance, AKP may be obtained based on a method as
described by H. Jager et al. Chem. Ber. 1959, 92, 2492-2499. AKP
can be prepared by alkylating cyclopentanone with diethyl oxalate
using sodium ethoxide as a base, refluxing the resultant product in
a strong acid (2 M HCl) and recovering the product, e.g. by
crystallisation from toluene.
[0095] It is also possible to obtain AKP from a natural source,
e.g. from methanogenic Archaea, from Asplenium septentrionale, or
from Hydnocarpus anthelminthica. AKP may for instance be extracted
from such organism, or a part thereof, e.g. from Hydnocarpus
anthelminthica seeds. A suitable extraction method may e.g. be
based on the method described in A. I. Virtanen and A. M. Berg in
Acta Chemica Scandinavica 1954, 6,1085-1086, wherein the extraction
of amino acids and AKP from Asplenium, using 70% ethanol, is
described.
[0096] In a specific embodiment, AKP is prepared in a method
comprising converting alpha-ketoglutaric acid (AKG) into
alpha-ketoadipic acid (AKA) and converting alpha-ketoadipic acid
into alpha-ketopimelic acid. This reaction may be catalysed by a
biocatalyst. AKG may, e.g., be prepared biocatalytically from a
carbon source, such as a carbohydrate, in a manner known in the art
per se.
[0097] A suitable biocatalyst for preparing AKP from AKG may in
particular be selected amongst biocatalysts catalysing
C.sub.1-elongation of alpha-ketoglutaric acid into alpha-ketoadipic
acid and/or C.sub.1-elongation of alpha-ketoadipic acid into
alpha-ketopimelic acid.
[0098] In a specific embodiment, the preparation of AKP is
catalysed by a biocatalyst comprising
[0099] a. an AksA enzyme or an homologue thereof;
[0100] b. at least one enzyme selected from the group of AksD
enzymes, AksE enzymes, homologues of AksD enzymes and homologues of
AksE enzymes; and
[0101] c. an AksF enzyme or a homologue thereof.
[0102] One or more of the AksA, AksD, AksE, AksF enzymes or
homologues thereof may be found in an organism selected from the
group of methanogenic archaea, preferably selected from the group
of Methanococcus, Methanocaldococcus, Methanosarcina,
Methanothermobacter, Methanosphaera, Methanopyrus and
Methanobrevibacter.
[0103] In a specific embodiment, the biocatalyst catalysing the
preparation of AKP from alpha-ketoglutaric acid (AKG) comprises an
enzyme system catalysing the conversion of alpha-ketoglutaric acid
into alpha-ketoadipic acid, wherein said enzyme system forms part
of the alpha-amino adipate pathway for lysine biosynthesis. The
term `enzyme system` is in particular used herein for a single
enzyme or a group of enzymes whereby a specific conversion can be
catalysed.
[0104] The preparation of AKP from AKG may comprise one or more
biocatalytic reactions with known or unknown intermediates e.g. the
conversion of AKG into AKA or the conversion of AKA into AKP. Such
system may be present inside a cell or isolated from a cell. The
enzyme system may in particular be from an organism selected from
the group of yeasts, fungi, archaea and bacteria, in particular
from the group of Penicillium, Cephalosporium, Paelicomyces,
Trichophytum, Aspergillus, Phanerochaete, Emericella, Ustilago,
Schizosaccharomyces, Saccharomyces, Candida, Yarrowia, Pichia,
Kluyveromyces, Thermus, Deinococcus, Pyrococcus, Sulfolobus,
Thermococcus, Methanococcus, Methanocaldococcus, Methanosphaera,
Methanopyrus, Methanobrevibacter, Methanosarcina and
Methanothermobacter.
[0105] In a specific embodiment, the biocatalyst catalysing the
preparation of AKP from alpha-ketoglutaric acid comprises an enzyme
system catalysing the conversion of alpha-ketoglutaric acid into
alpha-ketoadipic acid, wherein at least one of the enzymes of the
enzyme system originates from nitrogen fixing bacteria selected
from the group of cyanobacteria, rhizobiales,
.gamma.-proteobacteria and actinobacteria, in particular from the
group of Anabaena, Microcystis, Synechocystis, Rhizobium,
Bradyrhizobium, Pseudomonas, Azotobacter, Klebsiella and
Frankia.
[0106] Examples of homologues for these Aks enzymes and the genes
encoding these enzymes are given in the Tables 1A and 1B on the
following pages.
TABLE-US-00001 TABLE 1A Enzyme name Organism gene Protein AksA
Methanocaldococcus jannashii MJ0503 NP_247479 Methanothermobacter
thermoautotropicum .DELTA.H MTH1630 NP_276742 Methanococcus
maripaludis S2 MMP0153 NP_987273 Methanococcus maripaludis C5
MmarC5_1522 YP_001098033 Methanococcus maripaludis C7 MmarC7_1153
YP_001330370 Methanosphaera stadtmanae DSM 3091 Msp_0199 YP_447259
Methanopyrus kandleri AV19 MK1209 NP_614492 Methanobrevibacter
smithii ATCC35061 Msm_0722 YP_001273295 Methanococcus vannielii SB
Mevan_1158 YP_001323668 Methanococcus aeolicus Nankai 3 Maeo_0994
YP_001325184 AksD Methanocaldococcus jannashii MJ1003 NP_247997
Methanothermobacter thermoautotropicum .DELTA.H MTH1386 NP_276502
Methanococcus maripaludis S2 Mmp1480 NP_988600 Methanococcus
maripaludis C5 MmarC5_0098 YP_001096630 Methanococcus maripaludis
C7 MmarC7_0724 YP_001329942 Methanosphaera stadtmanae DSM 3091
Msp_1486 YP_448499 Methanopyrus kandleri AV19 MK1440 NP_614723
Methanobrevibacter smithii ATCC35061 Msm_0723 YP_001273296
Methanococcus vannielii SB Mevan_0789 YP_001323307 Methanococcus
aeolicus Nankai 3 Maeo_0311 YP_001324511 References to gene and
protein can be found via www.ncbi.nlm.nih.gov/, (as available on 15
Apr. 2008)
TABLE-US-00002 TABLE 1B Enzyme name Orgamism gene Protein AksE
Methanocaldococcus jannashii MJ1271 NP_248267 Methanothermobacter
thermoautotropicum .DELTA.H MTH1387 NP_276503 Methanococcus
maripaludis S2 MMP0381 NP_987501 Methanococcus maripaludis C5
MmarC5_1257 YP_001097769 Methanococcus maripaludis C7 MmarC7_1379
YP_001330593 Methanosphaera stadtmanae DSM 3091 Msp_1485 YP_448498
Methanopyrus kandleri AV19 MK0781 NP_614065 Methanobrevibacter
smithii ATCC35061 Msm_0847 YP_001273420 Methanococcus vannielii SB
Mevan_1368 YP_001323877 Methanococcus aeolicus Nankai 3 Maeo_0652
YP_001324848 AksF Methanocaldococcus jannashii MJ1596 NP_248605
Methanothermobacter thermoautotropicum .DELTA.H MTH184 NP_275327
Methanococcus maripaludis S2 MMP0880 NP988000 Methanococcus
maripaludis C5 MmarC5_0688 YP001097214 Methanococcus maripaludis C7
MmarC7_0128 YP_001329349 Methanosphaera stadtmanae DSM 3091
Msp_0674 YP_447715 Methanopyrus kandleri AV19 MK0782 NP_614066
Methanobrevibacter smithii ATCC35061 Msm_0373 YP001272946
Methanococcus vannielii SB Mevan_0040 YP_001322567 Methanococcus
aeolicus Nankai 3 Maeo_1484 YP_001325672 References to gene and
protein can be found via www.ncbi.nlm.nih.gov/, (as available on 15
Apr. 2008)
[0107] If desired, 6-ACA obtained in accordance with the invention
can be cyclised to form caprolactam, e.g. as described in U.S. Pat.
No. 6,194,572.
[0108] Reaction conditions for any biocatalytic step in the context
of the present invention may be chosen depending upon known
conditions for the biocatalyst, in particular the enzyme, the
information disclosed herein and optionally some routine
experimentation.
[0109] In principle, the pH of the reaction medium used may be
chosen within wide limits, as long as the biocatalyst is active
under the pH conditions. Alkaline, neutral or acidic conditions may
be used, depending on the biocatalyst and other factors. In case
the method includes the use of a micro-organism, e.g. for
expressing an enzyme catalysing a method of the invention, the pH
is selected such that the micro-organism is capable of performing
its intended function or functions. The pH may in particular be
chosen within the range of four pH units below neutral pH and two
pH units above neutral pH, i.e. between pH 3 and pH 9 in case of an
essentially aqueous system at 25.degree. C. A system is considered
aqueous if water is the only solvent or the predominant solvent
(>50 wt. %, in particular >90 wt. %, based on total liquids),
wherein e.g. a minor amount of alcohol or another solvent (<50
wt. %, in particular <10 wt. %, based on total liquids) may be
dissolved (e.g. as a carbon source) in such a concentration that
micro-organisms which may be present remain active. In particular
in case a yeast and/or a fungus is used, acidic conditions may be
preferred, in particular the pH may be in the range of pH 3 to pH
8, based on an essentially aqueous system at 25.degree. C. If
desired, the pH may be adjusted using an acid and/or a base or
buffered with a suitable combination of an acid and a base.
[0110] In principle, the incubation conditions can be chosen within
wide limits as long as the biocatalyst shows sufficient activity
and/or growth. This includes aerobic, micro-aerobic, oxygen limited
and anaerobic conditions.
[0111] Anaerobic conditions are herein defined as conditions
without any oxygen or in which substantially no oxygen is consumed
by the biocatalyst, in particular a micro-organism, and usually
corresponds to an oxygen consumption of less than 5 mmol/l.h, in
particular to an oxygen consumption of less than 2.5 mmol/l.h, or
less than 1 mmol/l.h.
[0112] Aerobic conditions are conditions in which a sufficient
level of oxygen for unrestricted growth is dissolved in the medium,
able to support a rate of oxygen consumption of at least 10
mmol/l.h, more preferably more than 20 mmol/l.h, even more
preferably more than 50 mmol/l.h, and most preferably more than 100
mmol/l.h.
[0113] Oxygen-limited conditions are defined as conditions in which
the oxygen consumption is limited by the oxygen transfer from the
gas to the liquid. The lower limit for oxygen-limited conditions is
determined by the upper limit for anaerobic conditions, i.e.
usually at least 1 mmol/l.h, and in particular at least 2.5
mmol/l.h, or at least 5 mmol/l.h. The upper limit for
oxygen-limited conditions is determined by the lower limit for
aerobic conditions, i.e. less than 100 mmol/l.h, less than 50
mmol/l.h, less than 20 mmol/l.h, or less than to 10 mmol/l.h.
[0114] Whether conditions are aerobic, anaerobic or oxygen limited
is dependent on the conditions under which the method is carried
out, in particular by the amount and composition of ingoing gas
flow, the actual mixing/mass transfer properties of the equipment
used, the type of micro-organism used and the micro-organism
density.
[0115] In principle, the temperature used is not critical, as long
as the biocatalyst, in particular the enzyme, shows substantial
activity. Generally, the temperature may be at least 0.degree. C.,
in particular at least 15.degree. C., more in particular at least
20.degree. C. A desired maximum temperature depends upon the
biocatalyst. In general such maximum temperature is known in the
art, e.g. indicated in a product data sheet in case of a
commercially available biocatalyst, or can be determined routinely
based on common general knowledge and the information disclosed
herein. The temperature is usually 90.degree. C. or less,
preferably 70.degree. C. or less, in particular 50.degree. C. or
less, more in particular or 40.degree. C. or less.
[0116] In particular if a biocatalytic reaction is performed
outside a host organism, a reaction medium comprising an organic
solvent may be used in a high concentration (e.g. more than 50%, or
more than 90 wt. %), in case an enzyme is used that retains
sufficient activity in such a medium.
[0117] In an advantageous method 6-ACA is prepared making use of a
whole cell biotransformation of the substrate for 6-ACA or an
intermediate for forming 6-ACA (AKP, AAP or 5-FVA), comprising a
micro-organism wherein one or more biocatalysts (usually one or
more enzymes) catalysing the biotransformation are produced, such
as one or more biocatalysts selected from the group of biocatalysts
capable of catalysing the conversion of AKP to AAP, biocatalysts
capable of catalysing the conversion of AAP to 6-ACA, biocatalysts
capable of catalysing the conversion of AKP to 5-FVA and
biocatalysts capable of catalysing the conversion of 5-FVA to
6-ACA. In a preferred embodiment the micro-organism is capable of
producing a decarboxylase and/or at least one enzyme selected from
amino acid dehydrogenases and aminotransferases are produced.
capable of catalysing a reaction step as described above, and a
carbon source for the micro-organism.
[0118] The carbon source may in particular contain at least one
compound selected from the group of monohydric alcohols, polyhydric
alcohols, carboxylic acids, carbon dioxide, fatty acids,
glycerides, including mixtures comprising any of said compounds.
Suitable monohydric alcohols include methanol and ethanol, Suitable
polyols include glycerol and carbohydrates. Suitable fatty acids or
glycerides may in particular be provided in the form of an edible
oil, preferably of plant origin.
[0119] In particular a carbohydrate may be used, because usually
carbohydrates can be obtained in large amounts from a biologically
renewable source, such as an agricultural product, preferably an
agricultural waste-material. Preferably a carbohydrate is used
selected from the group of glucose, fructose, sucrose, lactose,
saccharose, starch, cellulose and hemi-cellulose. Particularly
preferred are glucose, oligosaccharides comprising glucose and
polysaccharides comprising glucose.
[0120] A cell, in particular a recombinant cell, comprising one or
more biocatalysts (usually one or more enzymes) for catalysing a
reaction step in a method of the invention can be constructed using
molecular biological techniques, which are known in the art per se.
For instance, if one or more biocatalysts are to be produced in a
recombinant cell (which may be a heterologous system), such
techniques can be used to provide a vector (such as a recombinant
vector) which comprises one or more genes encoding one or more of
said biocatalysts. One or more vectors may be used, each comprising
one or more of such genes. Such vector can comprise one or more
regulatory elements, e.g. one or more promoters, which may be
operably linked to a gene encoding an biocatalyst.
[0121] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements (or coding sequences or nucleic
acid sequence) in a functional relationship. A nucleic acid
sequence is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a
promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the coding sequence.
[0122] As used herein, the term "promoter" refers to a nucleic acid
fragment that functions to control the transcription of one or more
genes, located upstream with respect to the direction of
transcription of the transcription initiation site of the gene, and
is structurally identified by the presence of a binding site for
DNA-dependent RNA polymerase, transcription initiation sites and
any other DNA sequences, including, but not limited to
transcription factor binding sites, repressor and activator protein
binding sites, and any other sequences of nucleotides known to one
of skilled in the art to act directly or indirectly to regulate the
amount of transcription from the promoter. A "constitutive"
promoter is a promoter that is active under most environmental and
developmental conditions. An "inducible" promoter is a promoter
that is active under environmental or developmental regulation. The
term "homologous" when used to indicate the relation between a
given (recombinant) nucleic acid or polypeptide molecule and a
given host organism or host cell, is understood to mean that in
nature the nucleic acid or polypeptide molecule is produced by a
host cell or organisms of the same species, preferably of the same
variety or strain.
[0123] The promoter that could be used to achieve the expression of
the nucleic acid sequences coding for an enzyme for use in a method
of the invention, in particular an aminotransferase, an amino acid
dehydrogenase or a decarboxylase, such as described herein above
may be native to the nucleic acid sequence coding for the enzyme to
be expressed, or may be heterologous to the nucleic acid sequence
(coding sequence) to which it is operably linked. Preferably, the
promoter is homologous, i.e. endogenous to the host cell.
[0124] If a heterologous promoter (to the nucleic acid sequence
encoding for the enzyme of interest) is used, the heterologous
promoter is preferably capable of producing a higher steady state
level of the transcript comprising the coding sequence (or is
capable of producing more transcript molecules, i.e. mRNA
molecules, per unit of time) than is the promoter that is native to
the coding sequence. Suitable promoters in this context include
both constitutive and inducible natural promoters as well as
engineered promoters, which are well known to the person skilled in
the art.
[0125] A "strong constitutive promoter" is one which causes mRNAs
to be initiated at high frequency compared to a native host cell.
Examples of such strong constitutive promoters in Gram-positive
micro-organisms include SP01-26, SP01-15, veg, pyc (pyruvate
carboxylase promoter), and amyE.
[0126] Examples of inducible promoters in Gram-positive
micro-organisms include, the IPTG inducible Pspac promoter, the
xylose inducible PxylA promoter.
[0127] Examples of constitutive and inducible promoters in
Gram-negative microorganisms include, but are not limited to, tac,
tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara
(P.sub.BAD), SP6, .lamda.-P.sub.R, and .lamda.-P.sub.L.
[0128] Promoters for (filamentous) fungal cells are known in the
art and can be, for example, the glucose-6-phosphate dehydrogenase
gpdA promoters, protease promoters such as pepA, pepB, pepC, the
glucoamylase glaA promoters, amylase amyA, amyB promoters, the
catalase catR or catA promoters, glucose oxidase goxC promoter,
beta-galactosidase lacA promoter, alpha-glucosidase aglA promoter,
translation elongation factor tefA promoter, xylanase promoters
such as xlnA, xlnB, xlnC, xlnD, cellulase promoters such as eglA,
eglB, cbhA, promoters of transcriptional regulators such as areA,
creA, xlnR, pacC, prtT, or another promotor, and can be found among
others at the NCBI website
(http://www.ncbi.nlm.nih.gov/entrez/).
[0129] The term "heterologous" when used with respect to a nucleic
acid (DNA or RNA) or protein refers to a nucleic acid or protein
that does not occur naturally as part of the organism, cell, genome
or DNA or RNA sequence in which it is present, or that is found in
a cell or location or locations in the genome or DNA or RNA
sequence that differ from that in which it is found in nature.
Heterologous nucleic acids or proteins are not endogenous to the
cell into which it is introduced, but has been obtained from
another cell or synthetically or recombinantly produced. Generally,
though not necessarily, such nucleic acids encode proteins that are
not normally produced by the cell in which the DNA is transcribed
or expressed. Similarly exogenous RNA encodes for proteins not
normally expressed in the cell in which the exogenous RNA is
present. Heterologous nucleic acids and proteins may also be
referred to as foreign nucleic acids or proteins. Any nucleic acid
or protein that one of skill in the art would recognize as
heterologous or foreign to the cell in which it is expressed is
herein encompassed by the term heterologous nucleic acid or
protein.
[0130] A method according to the invention may be carried out in a
host organism, which may be novel.
[0131] Accordingly, the invention also relates to a host cell
comprising one or more biocatalysts capable of catalysing at least
one reaction step in a method of the invention, in particular
capable of catalysing at least one reaction step in the conversion
of AKP, AAP or 5-FVA to 6-ACA. The invention also relates to a
novel vector comprising one or more genes encoding for one or more
enzymes capable of catalysing at least one reaction step in a
method of the invention, in particular capable of catalysing at
least one reaction step in the conversion of AKP to 6-ACA and to a
novel host cell comprising one or more genes encoding for one or
more enzymes capable of catalysing at least one reaction step in a
method of the invention, in particular capable of catalysing at
least one reaction step in the conversion of AKP to 6-ACA (which
one or more genes may form part of one or more vectors).
[0132] In a specific embodiment, a host cell according to the
invention is a recombinant cell comprising a nucleic acid sequence
encoding a biocatalyst capable of catalysing a transamination
reaction or a reductive amination reaction to form
alpha-aminopimelic acid from alpha-ketopimelic acid. Said sequence
may be part of a vector or may have been inserted into the
chromosomal DNA.
[0133] In particular, a host cell or vector according to the
invention may comprise at least one nucleic acid sequence, in
particular at least two nucleic acid sequences, selected from the
group of nucleic acid sequences encoding an enzyme with
.alpha.-ketopimelic acid decarboxylase activity, nucleic acid
sequences encoding an enzyme with 5-formylpentanoate
aminotransferase activity, nucleic acid sequences encoding an
enzyme with .alpha.-ketopimelic acid aminotransferase activity,
nucleic acid sequences encoding an enzyme with .alpha.-ketopimelic
acid dehydrogenase activity and nucleic acid sequences encoding an
enzyme with .alpha.-aminopimelic acid decarboxylase activity. Of
these sequences, typically one or more, in particular two or more,
are recombinant sequences.
[0134] In preferred embodiment the host cell, typically a
recombinant host cell, or the vector according to the invention
comprises a nucleic acid sequence encoding at least one biocatalyst
having .alpha.-ketopimelic acid decarboxylase activity, and/or at
least one nucleic acid sequence selected from sequences encoding a
biocatalyst with 5-formylpentanoate aminotransferase activity.
[0135] In such an embodiment, the nucleic acid sequence encoding an
enzyme with .alpha.-ketopimelic acid decarboxylase activity may in
particular comprise an amino acid sequence according to Sequence ID
31, Sequence ID 34, Sequence ID 37, Sequence ID 40, Sequence ID 43
or Sequence ID 46 or a homologue of any of these sequences and/or
the nucleic acid sequence encoding an enzyme with
5-formylpentanoate aminotransferase may in particular comprise an
amino acid sequence according to Sequence ID 2, Sequence ID 5,
Sequence ID 8, Sequence ID 65 Sequence ID 67, Sequence ID 69 or a
homologue thereof. One or more of said nucleic acid sequences may
form part of one or more recombinant vectors.
[0136] In a further preferred embodiment, the vector or host cell
comprises a nucleic acid sequence encoding an enzyme with
.alpha.-ketopimelic acid aminotransferase activity and/or a nucleic
acid sequence encoding an enzyme with .alpha.-aminopimelic acid
decarboxylase activity. The nucleic acid sequence encoding an
enzyme with .alpha.-ketopimelic acid aminotransferase activity may
in particular comprise an amino acid sequence according to Sequence
ID 2, Sequence ID 8, Sequence ID 12, Sequence ID 15, Sequence ID
17, Sequence ID 19, Sequence ID 21, Sequence ID 23, Sequence ID 25,
Sequence ID 27, Sequence ID 29, or a homologue thereof. One or more
of said nucleic acid sequences may form part of one or more
recombinant vectors.
[0137] In a specific preferred embodiment, a host cell according to
the invention comprises a nucleic acid sequence encoding an enzyme
with .alpha.-aminopimelate 2-dehydrogenase activity and a nucleic
acid sequence encoding an enzyme with .alpha.-aminopimelate
decarboxylase activity.
[0138] In a specific preferred embodiment, a host cell according to
the invention comprises a nucleic acid sequence encoding an enzyme
with 6-aminocaproic acid 6-dehydrogenase activity and a nucleic
acid sequence encoding an enzyme with .alpha.-ketopimelic acid
decarboxylase activity.
[0139] One or more suitable genes of a host cell or vectors
according to the invention may in particular be selected amongst
genes encoding an enzyme as mentioned herein above.
[0140] In a specific embodiment, the host cell is a recombinant
cell comprising at least one nucleic acid sequence selected from
the group of sequences as identified in any of Sequence ID 1,
Sequence ID 3, Sequence ID 4, Sequence ID 6, Sequence ID 7,
Sequence ID 11, Sequence ID 13, Sequence ID 14, Sequence ID 16,
Sequence ID 18, Sequence ID 20, Sequence ID 22, Sequence ID 24,
Sequence ID 26, Sequence ID 28, Sequence ID 30, Sequence ID 32,
Sequence ID 33, Sequence ID 35, Sequence ID 36, Sequence ID 38,
Sequence ID 39, Sequence ID 41, Sequence ID 42, Sequence ID 44,
Sequence ID 45, Sequence ID 47, Sequence ID 64, Sequence ID 66,
Sequence ID 68 and functional analogues thereof.
[0141] A nucleic acid sequence encoding an enzyme with 5-FVA
aminotransferase activity, may in particular be a sequence selected
from the group of sequences represented by any of the Sequence ID's
1, 3, 4, 6, 7, 64, 66, 68, and functional analogues of any of these
sequences.
[0142] As used herein, the term "functional analogues" at least
includes other sequences encoding an enzyme having the same amino
acid sequence and other sequences encoding a homologue of such
enzyme.
[0143] A nucleic acid sequence encoding an enzyme with AKP
decarboxylase activity may in particular be a sequence selected
from the group of sequences represented by any of the Sequence ID's
30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47 and functional
analogues of any of these sequences.
[0144] In a preferred embodiment, the host cell comprises a nucleic
acid sequence encoding an enzyme, capable of catalysing the
conversion of AAP to AKP, according to Sequence ID No.: 1, 3, 7,
11, 13, 14, 16, 18, 20, 22, 24, 26, 28, or a functional analogue
thereof, which may be a wild type or non-wild type sequence
[0145] In a specific embodiment, the host cell comprises at least
one nucleic acid sequence encoding a biocatalyst having
alpha-aminopimelic acid decarboxylase activity, which may be
homologous or heterologous to the host cell. In particular such
biocatalyst may be selected from the group of decarboxylases (E.C.
4.1.1), more in particular from the group of glutamate
decarboxylases (EC 4.1.1.15), diaminopimelate decarboxylases (EC
4.1.1.20) aspartate 1-decarboxylases (EC 4.1.1.11), branched chain
alpha-keto acid decarboxylases, alpha-ketoisovalerate
decarboxylases, alpha-ketoglutarate decarboxylases, pyruvate
decarboxylases (EC 4.1.1.1) and oxaloacetate decarboxylases (E.C.
4.1.1.3).
[0146] In a specific embodiment, the host cell comprises one or
more enzymes catalysing the formation of AKP from AKG (see also
above). Use may be made of an enzyme system forming part of the
alpha-amino adipate pathway for lysine biosynthesis. The term
`enzyme system` is in particular used herein for a single enzyme or
a group of enzymes whereby a specific conversion can be catalysed.
Said conversion may comprise one or more chemical reactions with
known or unknown intermediates e.g. the conversion of AKG into AKA
or the conversion of AKA into AKP. Such system may be present
inside a cell or isolated from a cell. It is known that
aminotransferases often have a wide substrate range. If present, it
may be desired to decrease activity of one or more such enzymes in
a host cell such that activity in the conversion of AKA to
alpha-aminoadipate (AAA) is reduced, whilst maintaining relevant
catalytic functions for biosynthesis of other amino acids or
cellular components. Also a host cell devoid of any other enzymatic
activity resulting in the conversion of AKA to an undesired side
product is preferred.
[0147] In a preferred host cell, suitable for preparing AAP making
use of a whole cell biotransformation process, one or more
biocatalysts capable of catalysing at least one reaction step in
the preparation of alpha-ketopimelic acid from alpha-ketoglutaric
acid are encoded for. Suitable biocatalysts are, e.g., as described
above when discussing the preparation of AKP.
[0148] The host cell may for instance be selected from bacteria,
yeasts or fungi. In particular the host cell may be selected from
the genera selected from the group of Aspergillus, Penicillium,
Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus,
Corynebacterium, Pseudomonas, Gluconobacter, Methanococcus,
Methanobacterium, Methanocaldococcus and Methanosarcina and
Escherichia. Herein, usually one or more encoding nucleic acid
sequences as mentioned above have been cloned and expressed.
[0149] In particular, the host strain and, thus, a host cell
suitable for the biochemical synthesis of 6-ACA may be selected
from the group of Escherichia coli, Bacillus subtilis, Bacillus
amyloliquefaciens, Corynebacterium glutamicum, Aspergillus niger,
Penicillium chrysogenum, Saccharomyces cervisiae, Hansenula
polymorpha, Candida albicans, Kluyveromyces lactis, Pichia
stipitis, Pichia pastoris, Methanobacterium thermoautothrophicum
.DELTA.H, Methanococcus maripaludis, Methanococcus voltae,
Methanosarcina acetivorans, Methanosarcina barkeri and
Methanosarcina mazei host cells. In a preferred embodiment, the
host cell is capable of producing lysine (as a precursor).
[0150] The host cell may be in principle a naturally occurring
organism or may be an engineered organism. Such an organism can be
engineered using a mutation screening or metabolic engineering
strategies known in the art. In a specific embodiment, the host
cell naturally comprises (or is capable of producing) one or more
of the enzymes suitable for catalysing a reaction step in a method
of the invention, such as one or more activities selected from the
group of decarboxylases, aminotransferases and amino acid
dehydrogenases capable of catalysing a reaction step in a method of
the invention. For instance E. coli may naturally be capable of
producing an enzyme catalysing a transamination in a method of the
invention. It is also possible to provide a recombinant host cell
with both a recombinant gene encoding an aminotransferase or amino
acid dehydrogenase capable of catalysing a reaction step in a
method of the invention and a recombinant gene encoding a
decarboxylase gene capable of catalysing a reaction step in a
method of the invention.
[0151] For instance a host cell may be selected of the genus
Corynebacterium, in particular C. glutamicum, enteric bacteria, in
particular Escherichia coli, Bacillus, in particular B. subtilis
and B. methanolicus, and Saccharomyces, in particular S.
cerevisiae. Particularly suitable are C. glutamicum or B.
methanolicus strains which have been developed for the industrial
production of lysine.
[0152] The invention further relates to a micro-organism, which may
be a wild-type micro-organism isolated from its natural environment
or a recombinant micro-organism, comprising DNA containing a
nucleic acid sequence as identified in any Sequence ID selected
from the group of Sequence ID 3, Sequence ID 6, Sequence ID 13,
Sequence ID No. 32, Sequence ID No. 35, Sequence ID No. 41,
Sequence ID No. 44, Sequence ID No. 47, and functional analogues
thereof.
[0153] Functional analogues of a nucleotides sequence, as referred
to herein, are in particular nucleotide sequences encoding the same
amino acid sequence as that nucleotide sequence or encoding a
homologue of that nucleotide sequence. In particular, preferred
functional analogues are nucleotide sequence having a similar, the
same or a better level of expression in a host cell of interest as
the nucleotide sequence of which it is referred to as being a
functional analogue of.
[0154] The invention further relates to a polynucleotide or vector
comprising a nucleic acid sequence as identified in any Sequence ID
selected from the group of Sequence ID 3, Sequence ID 6, Sequence
ID 13, Sequence ID No. 32, Sequence ID No. 35, Sequence ID No. 41,
Sequence ID No. 44, Sequence ID No. 47 and non-wild-type functional
analogues thereof. Such polynucleotide or vector is in particular
advantageous for providing a host cell, especially an E. coli host
cell, or another host cell which is capable of catalysing at least
one reaction step in the conversion of AKP to 6-ACA with a high
yield, compared to a corresponding wild-type gene.
[0155] Optionally, the polynucleotide or vector comprises one or
more nucleic acid sequences encoding one or more other biocatalysts
suitable for catalysing a reaction step in a method according to
the invention, in particular such one or more of the biocatalyst
referred to above.
[0156] The invention further relates to a method for preparing
alpha-aminopimelic acid (AAP), comprising converting AKP into AAP,
which conversion is catalysed by a biocatalyst.
[0157] For such method in particular a biocatalyst may be used
having aminotransferase activity or reductive amination activity as
described above.
[0158] As indicated above, the AAP may thereafter be used for the
preparation of 6-ACA. Alternatively, AAP may be used as such, e.g.
as a chemical for biochemical research or as a pH-buffer compound,
e.g. for use in an preparative or analytical separation technique
such as liquid chromatography or capillary electrophoresis.
[0159] Further, AAP prepared in a method of the invention may
further be used in the preparation of another compound, for
instance, AAP may be converted into caprolactam. As described
above, and illustrated in an example, below. AAP can be chemically
converted in caprolactam, e.g. by exposure to a high temperature.
Without being bound by theory, it is contemplated that also in this
reaction 6-ACA may be formed as a short-lived intermediate.
[0160] Next, the invention will be illustrated by the following
examples.
EXAMPLES
[0161] General Methods
[0162] Molecular and Genetic Techniques
[0163] Standard genetic and molecular biology techniques are
generally known in the art and have been previously described
(Maniatis et al. 1982 "Molecular cloning: a laboratory manual".
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Miller
1972 "Experiments in molecular genetics", Cold Spring Harbor
Laboratory, Cold Spring Harbor; Sambrook and Russell 2001
"Molecular cloning: a laboratory manual" (3rd edition), Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press; F. Ausubel
et al, eds., "Current protocols in molecular biology", Green
Publishing and Wiley Interscience, New York 1987).
[0164] Plasmids and Strains
[0165] pBAD/Myc-His C was obtained from Invitrogen (Carlsbad,
Calif., USA). Plasmid pBAD/Myc-His-DEST constructed as described in
WO2005/068643, was used for protein expression. E. coli TOP10
(Invitrogen, Carlsbad, Calif., USA) was used for all cloning
procedures and for expression of target genes.
[0166] Media
[0167] LB medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl)
was used for growth of E. coli. Antibiotics (50 .mu.g/ml
carbenicillin) were supplemented to maintain plasmids. For
induction of gene expression under control of the P.sub.BAD
promoter in pBAD/Myc-His-DEST derived plasmids, L-arabinose was
added to a final concentration of 0.2% (w/v).
[0168] Identification of Plasmids
[0169] Plasmids carrying the different genes were identified by
genetic, biochemical, and/or phenotypic means generally known in
the art, such as resistance of transformants to antibiotics, PCR
diagnostic analysis of transformant or purification of plasmid DNA,
restriction analysis of the purified plasmid DNA or DNA sequence
analysis.
[0170] HPLC-MS Analysis Method for the Determination of 5-FVA
[0171] 5-FVA was detected by selective reaction monitoring
(SRM)-MS, measuring the transition m/z 1294.fwdarw.83.
Concentrations for 5-FVA were calculated by measuring the peak area
of the 5-FVA peak eluting at approximately 6 min. Calibration was
performed by using an external standard procedure. All the LC-MS
experiments were performed on an Agilent 1200 LC system, consisting
of a quaternary pump, autosampler and column oven, coupled with an
Agilent 6410 QQQ triple quadrupole MS.
LC Conditions:
[0172] Column: 50.times.4.6 mm Nucleosil C18, 5 .mu.m (Machery
& Nagel) pre column coupled to a 250.times.4.6 mm id. Prevail
C18, 5 .mu.m (Alltech) [0173] Column temperature: room temperature
[0174] Eluent: A: water containing 0.1% formic acid
[0175] B: acetonitrile containing 0.1% formic acid
TABLE-US-00003 time (min) % eluent B Gradient: 0 10 6 50 6.1 10 11
10
[0176] Flow: 1.2 ml/min, before entering the MS the flow is split
1:3 Injection volume: 2 .mu.l
MS Conditions:
[0176] [0177] Ionisation: negative ion electrospray [0178] source
conditions: ionspray voltage: 5 kV [0179] temperature: 350.degree.
C. [0180] fragmentor voltage and collision energy optimized [0181]
Scan mode: selective reaction mode: transition m/z
129.fwdarw.83
[0182] HPLC-MS Analysis for the Determination of AAP
[0183] AAP was detected by selected ion monitoring (SIM)-MS,
measuring the protonated molecule for AAP with m/z 176.
Concentrations for AAP were calculated by measuring the peak area
of the AAP peak eluting at a retention time of 2.7 minutes in the
samples. Calibration was performed by using an external standard
procedure. All the LC-MS experiments were performed on an Agilent
1100 LC system consisting of a quaternary pump, degasser,
autosampler and column oven, coupled with an API 2000 triple
quadrupole MS (Applied Biosystems).
[0184] LC conditions were as follows: [0185] Column: 50*4 Nucleosil
C18, 5 .mu.m (Macherey-Nagel)+250.times.4.6 Prevail C18, 5 .mu.m
(Alltech), both at room temperature (RT) [0186] Eluent: A=0.1%
(v/v) formic acid in ultrapure water [0187] B=0.1% (v/v) formic
acid in acetonitrile (pa, Merck) [0188] Flow: 1.2 ml/min, before
entering the MS the flow was split 1:3 [0189] Gradient: The
gradient was started at t=0 minutes with 90% (v/v) A and changed
within 6 minutes to 50% (v/v) A. At 6.1 minutes the gradient was
changed to the original condition. [0190] Injection volume: 2 .mu.l
[0191] MS conditions: Positive ion electrospray was used for
ionization [0192] Detection: in SIM mode on m/z 176, with a dwell
time of 100 msec.
[0193] HPLC-MS Analysis for the Determination of 6-ACA
[0194] Calibration:
[0195] The calibration was performed by an external calibration
line of 6-ACA (m/z 132.fwdarw.m/z 114, Rt 7.5 min). All the LC-MS
experiments were performed on an Agilent 1100, equipped with a
quaternary pump, degasser, autosampler, column oven, and a
single-quadrupole MS (Agilent, Waldbronn, Germany). The LC-MS
conditions were: [0196] Column: 50*4 Nucleosil
(Mancherey-Nagel)+250.times.4.6 Prevail C18 (Alltech), both at room
temperature (RT) [0197] Eluent: A=0.1(v/v) formic acid in ultrapure
water [0198] B=Acetonitrile (pa, Merck) [0199] Flow: 1.0 ml/min,
before entering the MS the flow was split 1:3 [0200] Gradient: The
gradient was started at t=0 minutes with 100% (v/v) A, remaining
for 15 minutes and changed within 15 minutes to 80% (v/v) B (t=30
minutes). From 30 to 31 minutes the gradient was kept at constant
at 80% (v/v) B. [0201] Injection volume: 5 .mu.l [0202] MS
detection: ESI(+)-MS [0203] The electrospray ionization (ESI) was
run in the positive scan mode with the following conditions; m/z
50-500, 50 V fragmentor, 0.1 m/z step size, 350.degree. C. drying
gas temperature, 10 L N.sub.2/min drying gas, 50 psig nebuliser
pressure and 2.5 kV capillary voltage.
[0204] Cloning of Target Genes
[0205] Design of Expression Constructs
[0206] attB sites were added to all genes upstream of the ribosomal
binding site and start codon and downstream of the stop codon to
facilitate cloning using the Gateway technology (Invitrogen,
Carlsbad, Calif., USA).
[0207] Gene Synthesis and Construction of Plasmids
[0208] Synthetic genes were obtained from DNA2.0 and codon
optimised for expression in E. coli according to standard
procedures of DNA2.0. The aminotransferase genes from Vibrio
fluvialis JS17 [SEQ ID No. 1] and Bacillus weihenstephanensis KBAB4
[SEQ ID No. 4] encoding the amino acid sequences of the V.
fluvialis JS17 .omega.-aminotransferase [SEQ ID No. 2] and the B.
weihenstephanensis KBAB4 aminotransferase (ZP_01186960) [SEQ ID No.
5], respectively, were codon optimised and the resulting sequences
[SEQ ID No. 3] and [SEQ ID No. 6] were obtained by DNA
synthesis.
[0209] The decarboxylase genes from Escherichia coli [SEQ ID No.
30], Saccharomyces cerevisiae [SEQ ID No. 33], Zymomonas mobilis
[SEQ ID No. 36], Lactococcus lactis [SEQ ID No. 39], [SEQ ID No.
42], and Mycobacterium tuberculosis [SEQ ID No. 45] encoding the
amino acid sequences of the V. fluvialis JS17
.omega.-aminotransferase [SEQ ID No. 3], the B. weihenstephanensis
KBAB4 aminotransferase (ZP_01186960) [SEQ ID No. 6], the
Escherichia coli diaminopimelate decarboxylase LysA [SEQ ID No.
31], the Saccharomyces cerevisiae pyruvate decarboxylase Pdc [SEQ
ID No. 34], the Zymomonas mobilis pyruvate decarboxylase Pdc1472A
[SEQ ID No. 37], the Lactococcus lactis branched chain alpha-keto
acid decarboxylase KdcA [SEQ ID No. 40] and alpha-ketoisovalerate
decarboxylase KivD [SEQ ID No. 43], and the Mycobacterium
tuberculosis alpha-ketoglutarate decarboxylase Kgd [SEQ ID No. 46],
respectively, were also codon optimised and the resulting sequences
[SEQ ID No. 32], [SEQ ID No. 35], [SEQ ID No. 38], [SEQ ID No. 41],
[SEQ ID No. 44], and [SEQ ID No. 47] were obtained by DNA
synthesis, respectively.
[0210] The gene constructs were cloned into pBAD/Myc-His-DEST
expression vectors using the Gateway technology (Invitrogen) via
the introduced attB sites and pDONR201 (Invitrogen) as entry vector
as described in the manufacturer's protocols (www.invitrogen.com).
This way the expression vectors pBAD-Vfl_AT and pBAD-Bwe_AT were
obtained, respectively. The corresponding expression strains were
obtained by transformation of chemically competent E. coli TOP10
(Invitrogen) with the respective pBAD-expression vectors.
[0211] Cloning by PCR
[0212] Various genes encoding a biocatalyst were amplified from
genomic DNA by PCR using PCR Supermix High Fidelity (Invitrogen)
according to the manufacturer's specifications, using primers as
listed in the following table.
TABLE-US-00004 TABLE 2 enzyme primer origin of gene gene Sequence
ID Sequence ID Sequence ID's Pseudomonas 7 8 9&10 aeruginosa
Pseudomonas 26 27 60&61 aeruginosa Pseudomonas 66 67 72&73
aeruginosa Pseudomonas 68 69 74&75 aeruginosa Bacillus subtilis
14 15 48&49 Bacillus subtilis 16 17 50&51 Bacillus subtilis
64 65 70&71 Rhodobacter 18 19 52&53 sphaeroides Legionella
20 21 54&55 pneumophilia Nitrosomas europaea 22 23 56&57
Neisseria 24 25 58&59 gonorrhoeae Rhodopseudomonas 28 29
62&63 palustris
[0213] PCR reactions were analysed by agarose gel electrophoresis
and PCR products of the correct size were eluted from the gel using
the QIAquick PCR purification kit (Qiagen, Hilden, Germany).
Purified PCR products were cloned into pBAD/Myc-His-DEST expression
vectors using the Gateway technology (Invitrogen) via the
introduced attB sites and pDONR-zeo (Invitrogen) as entry vector as
described in the manufacturer's protocols. The sequence of genes
cloned by PCR was verified by DNA sequencing. This way the
expression vectors pBAD-Pae-_gi9946143_AT, pBAD-Bsu_gi16078032_AT,
pBAD-Bsu_gi16080075_AT, pBAD-Bsu_gi16077991_AT, pBAD-Rsp_AT ,
pBAD-Lpn_AT, pBAD-Neu_AT, pBAD-Ngo_AT, pBAD-Pae_gi9951299_AT,
pBAD-Pae_gi9951072_AT, pBAD-Pae_gi9951630_AT and pBAD-Rpa_AT were
obtained. The corresponding expression strains were obtained by
transformation of chemically competent E. coli TOP10 (Invitrogen)
with the pBAD constructs.
[0214] Growth of E. coli for Protein Expression
[0215] Small scale growth was carried out in 96-deep-well plates
with 940 .mu.l media containing 0.02% (w/v) L-arabinose.
Inoculation was performed by transferring cells from frozen stock
cultures with a 96-well stamp (Kuhner, Birsfelden, Switzerland).
Plates were incubated on an orbital shaker (300 rpm, 5 cm
amplitude) at 25.degree. C. for 48 h. Typically an OD.sub.620nm of
2-4 was reached.
[0216] Preparation of Cell Lysates
[0217] Preparation of Lysis Buffer
[0218] The lysis buffer contained the following ingredients:
TABLE-US-00005 TABLE 3 1M MOPS pH 7.5 5 ml DNAse I grade II (Roche)
10 mg Lysozyme 200 mg MgSO.sub.4.cndot.7H.sub.2O 123.2 mg
dithiothreitol (DTT) 154.2 mg H.sub.2O (MilliQ) Balance to 100
ml
[0219] The solution was freshly prepared directly before use.
[0220] Preparation of Cell Free Extract by Lysis
[0221] Cells from small scales growth (see previous paragraph) were
harvested by centrifugation and the supernatant was discarded. The
cell pellets formed during centrifugation were frozen at
-20.degree. C. for at least 16 h and then thawed on ice. 500 .mu.l
of freshly prepared lysis buffer were added to each well and cells
were resuspended by vigorously vortexing the plate for 2-5 min. To
achieve lysis, the plate was incubated at room temperature for 30
min. To remove cell debris, the plate was centrifuged at 4.degree.
C. and 6000 g for 20 min. The supernatant was transferred to a
fresh plate and kept on ice until further use.
[0222] Preparation of Cell Free Extract by Sonification
[0223] Cells from medium scales growth (see previous paragraph)
were harvested by centrifugation and the supernatant was discarded.
1 ml of potassium phosphate buffer pH7 was added to 0.5 g of wet
cell pellet and cells were resuspended by vigorously vortexing. To
achieve lysis, the cells were sonicated for 20 min. To remove cell
debris, the lysates were centrifuged at 4.degree. C. and 6000 g for
20 min. The supernatant was transferred to a fresh tube and frozen
at -20.degree. C. until further use.
[0224] Preparation of 5-formylpentanoic Acid by Chemical Hydrolysis
of Methyl 5-formylpentanoate
[0225] The substrate for the aminotransferase reaction i.e.
5-formylpentanoic acid was prepared by chemical hydrolysis of
methyl 5-formylpentanoate as follows: a 10% (w/v) solution of
methyl 5-formylpentanoate in water was set at pH 14.1 with NaOH.
After 24 h of incubation at 20.degree. C. the pH was set to 7.1
with HCl.
[0226] Enzymatic Reactions for Conversion of 5-formylpentanoic Acid
to 6-ACA
[0227] Unless specified otherwise, a reaction mixture was prepared
comprising 10 mM 5-formylpentanoic acid, 20 mM racemic
.alpha.-methylbenzylamine, and 200 .mu.M.quadrature. pyridoxal
5'-phosphate in 50 mM potassium phosphate buffer, pH 7.0. 100 .mu.l
of the reaction mixture were dispensed into each well of the well
plates. To start the reaction, 20 .mu.l of the cell free extracts
were added, to each of the wells. Reaction mixtures were incubated
on a shaker at 37.degree. C. for 24 h. Furthermore, a chemical
blank mixture (without cell free extract) and a biological blank
(E. coli TOP10 with pBAD/Myc-His C) were incubated under the same
conditions. Samples were analysed by HPLC-MS. The results are
summarised in the following table.
TABLE-US-00006 TABLE 4 6-ACA formation from 5-FVA in the presence
of aminotransferases 6-ACA Biocatalyst concentration [mg/kg] E.
coli TOP10/pBAD-Vfl_AT 43* E. coli TOP10/pBAD-Pae_AT 930 E. coli
TOP10/pBAD-Pae_AT 25* E. coli TOP10/pBAD-Bwe_AT 24* E. coli
TOP10/pBAD-Bsu_gi16077991_AT 288 E. coli
TOP10/pBAD-Pae_gi9951072_AT 1087 E. coli
TOP10/pBAD-Pae_gi9951630_AT 92 E. coli TOP10 with pBAD/Myc-His C
(biological 0.6 blank) None (chemical blank) n.d. n.d.: not
detectable *method differed in that 10 .mu.l cell free extract was
used instead of 20 .mu.l, the pyridoxal-5'-phosphate concentration
was 50 .mu.M instead of 200 .mu.M and the reaction mixture volume
in the wells was 190 .mu.l instead of 100 .mu.l.
[0228] It is shown that 6-ACA is formed from 5-FVA in the presence
of an aminotransferase.
[0229] Enzymatic Reactions for Conversion of AKP to
5-formylpentanoic Acid
[0230] A reaction mixture was prepared comprising 50 mM AKP, 5 mM
magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate
(for LysA) or 1 mM thiamine diphosphate (for all other enzymes) in
100 mM potassium phosphate buffer, pH 6.5. 4 ml of the reaction
mixture were dispensed into a reaction vessel. To start the
reaction, 1 ml of the cell free extracts obtained by sonification
were added, to each of the wells. In case of the commercial
oxaloacetate decarboxylase (Sigma-Aldrich product number 04878), 50
U were used. Reaction mixtures were incubated with a magnetic
stirrer at 37.degree. C. for 48 h. Furthermore, a chemical blank
mixture (without cell free extract) and a biological blank (E. coli
TOP10 with pBAD/Myc-His C) were incubated under the same
conditions. Samples from different time points during the reaction
were analysed by HPLC-MS. The results are summarised in the
following table.
TABLE-US-00007 TABLE 5 5-FVA formation from AKP in the presence of
decarboxylases 5-FVA concentration [mg/kg] Biocatalyst 3 h 18 h 48
h E. coli TOP10/pBAD-LysA 150 590 720 E. coli TOP10/pBAD-Pdc 1600
1700 1300 E. coli TOP10/pBAD-PdcI472A 2000 2000 1600 E. coli
TOP10/pBAD-KdcA 3300 2300 2200 E. coli TOP10/pBAD-KivD 820 1400
1500 Oxaloacetate decarboxylase n.d. 6 10 E. coli TOP10 with
pBAD/Myc- n.d. n.d. n.d. His C (biological blank) None (chemical
blank) n.d. n.d. n.d. n.d.: not detectable
[0231] It is shown that 5-FVA is formed from AKP in the presence of
a decarboxylase.
[0232] Enzymatic Reactions for Conversion of AKP to 6-ACA in
Presence of Recombinant Decarboxylase
[0233] A reaction mixture was prepared comprising 50 mM AKP, 5 mM
magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate
(for LysA) or 1 mM thiamine diphosphate (for all other tested
biocatalysts) in 100 mM potassium phosphate buffer, pH 6.5. 4 ml of
the reaction mixture were dispensed into a reaction vessel. To
start the reaction, 1 ml of the cell free extracts were added, to
each of the wells. Reaction mixtures were incubated with a magnetic
stirrer at 37.degree. C. for 48 h. Furthermore, a chemical blank
mixture (without cell free extract) and a biological blank (E. coli
TOP10 with pBAD/Myc-His C) were incubated under the same
conditions. Samples from different time points during the reaction
were analysed by HPLC-MS. The results are summarised in the
following table.
TABLE-US-00008 TABLE 6 6-ACA formation from AKP in the presence of
decarboxylases 6-ACA concentration [mg/kg] Biocatalyst 3 h 18 h 48
h E. coli TOP10/pBAD-LysA n.a. 0.01 0 E. coli TOP10/pBAD-Pdc 0.1
0.3 n.a. E. coli TOP10/pBAD-PdcI472A 0.03 0.1 0.2 E. coli
TOP10/pBAD-KdcA 0.04 0.1 0.3 E. coli TOP10/pBAD-KivD n.a. 0.3 0.6
E. coli TOP10 with pBAD/Myc- n.d. n.d. n.d. His C (biological
blank) None (chemical blank) n.d. n.d. n.d. n.a. = not analysed
n.d. = not detectable
[0234] It is shown that 6-ACA is formed from AKP in the presence of
a decarboxylase. It is contemplated that the E. coli contained
natural 5-FVA aminotransferase activity.
[0235] Enzymatic Reactions for Conversion of AKP to 6-ACA in
Presence of Recombinant Decarboxylase and Recombinant
Aminotransferase
[0236] A reaction mixture was prepared comprising 50 mM AKP, 5 mM
magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate, 1
mM thiamine diphosphate and 50 mM racemic .alpha.-methylbenzylamine
in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml of the
reaction mixture were dispensed into a reaction vessel. To start
the reaction, 0.2 ml of the decarboxylase containing cell free
extract and 0.2 ml of the aminotransferase containing cell free
extract were added, to each of the reaction vessels. Reaction
mixtures were incubated with a magnetic stirrer at 37.degree. C.
for 48 h. Furthermore, a chemical blank mixture (without cell free
extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C)
were incubated under the same conditions. Samples from different
time points during the reaction were analysed by HPLC-MS. The
results are summarised in the following table.
TABLE-US-00009 TABLE 7 6-ACA formation from AKP in the presence of
a recombinant decarboxylase and a recombinant aminotransferase
6-ACA concentration [mg/kg] after 48 hours AT E. coli TOP10/ E.
coli TOP10/ E. coli TOP10/ pBAD- pBAD- DC pBAD-Vfl-AT Bwe-AT
PAE_gi9946143_AT E. coli TOP10/ 183.4 248.9 117.9 pBAD-Pdc E. coli
TOP10/ 458.5 471.6 170.3 pBAD-PdcI472A E. coli TOP10/ 497.8 497.8
275.1 pBAD-KdcA E. coli TOP10/ 510.9 510.9 314.4 pBAD-KivD AT =
aminotransferase DC = decarboxylase
[0237] In the chemical blank and in the biological blank no 6-ACA
was detectable.
[0238] Further, the results show that compared to the example
wherein a host-cell with only recombinant decarboxylase (and no
recombinant aminotransferase) the conversion to 6-ACA was
improved.
[0239] Construction of Plasmids for Expression of Aminotransferases
and Decarboxylases in S. cerevisiae
[0240] The aminotransferase gene from Vibrio fluvialis JS17
encoding the amino acid sequence of the V. fluvialis JS17
.omega.-aminotransferase [SEQ ID No. 2] was amplified by PCR from
pBAD-Vfl_AT [SEQ ID No. 3] using Phusion DNA polymerase (Finnzymes)
according to the manufacturers specifications and using specific
primers [SEQ ID No. 76 & 77].
[0241] The aminotransferase gene from Pseudomonas aeruginosa [SEQ
ID No. 7] coding for P. aeruginosa aminotransferase [SEQ ID No. 8]
was amplified from pBAD-Pae_AT by PCR using Phusion DNA polymerase
(Finnzymes) according to the manufacturers specifications and using
specific primers [SEQ ID No. 78 & 79].
[0242] The resulting PCR products were cloned into vector pAKP-41
using SpeI and BamHI restriction enzymes resulting in vectors
pAKP-79 and pAKP-80 respectively, which now contain the
aminotransferase gene under the S. cerevisiae gal10 promoter and
the S. cerevisiae adh2 terminator.
[0243] The decarboxylase gene from Saccharamyces cerevisiae [SEQ ID
No. 33] coding for Saccharamyces cerevisiae pyruvate decarboxylase
Pdc [SEQ ID No. 34] was amplified from pBAD-Pdc by PCR using
Phusion DNA polymerase (Finnzymes) according to the manufacturers
specifications and using specific primers [SEQ ID No 80 &
81].
[0244] The decarboxylase gene from Lactococcus lactis [SEQ ID No.
39] coding for Lactococcus lactis branched chain alpha-keto acid
decarboxylase KdcA [SEQ ID No. 40] was amplified from pBAD-KdcA by
PCR using Phusion DNA polymerase (Finnzymes) according to the
manufacturers specifications and using specific primers [SEQ ID No
82 & 83].
[0245] The resulting PCR products were cloned into vector pAKP-44
using AscI and BamHI restriction enzymes resulting in vectors
pAKP-81 and pAKP-82 respectively, which now contain the
decarboxylase gene under the S. cerevisiae gal2 promoter and the S.
cerevisiae pma1 terminator.
[0246] Plasmids pAKP-79 and pAKP-80 were restriction enzyme
digested with SacI and XbaI and plasmids pAKP-81 and pAKP-82 were
restriction enzyme digested with SalI and XbaI. A SacI/XbaI
aminotransferase fragment was combined with a SalI/XbaI
decarboxylase fragment into the S. cerevisiae low copy episomal
vector pRS414, which was restriction enzyme digested with SalI and
SacI.
[0247] The resulting plasmids were obtained: [0248] pAKP-85:
Pgal10-Pae_AT-Tadh2 Pgal2-Pdc_DC-Tpma1 [0249] pAKP-86:
Pgal10-Pae_AT-Tadh2 Pgal2-KdcA_DC-Tpma1 [0250] pAKP-87:
Pgal10-Vfl_AT-Tadh2 Pgal2-Pdc_DC-Tpma1 [0251] pAKP-88:
Pgal10-Vfl_AT-Tadh2 Pgal2-KdcA_DC-Tpma1
[0252] Transformation and Growth of S. cerevisiae
[0253] S. cerevisiae strain CEN.PK113-3C was transformed with 1
.mu.g of plasmid DNA according to the method as described by Gietz
and Woods (Gietz, R. D. and Woods, R. A. (2002). Transformation of
yeast by the Liac/SS carrier DNA/PEG method. Methods in Enzymology
350: 87-96). Cells were plated on agar plates with 1.times. Yeast
Nitrogen Base without amino acids and 2% glucose.
[0254] The resulting strains were grown aerobically at 30.degree.
C. for 48 hour in Verduyn minimal medium containing 0.05% glucose
and 4% galactose.
[0255] Preparation of Cell Free Extract
[0256] 1 ml of potassium phosphate buffer (pH 7) was added to 0.5 g
of the cell pellet. This mixture was added to a 2 ml eppendorf tube
which contained 0.5 g of glassbeads with a diameter of 0.4-0.5 mM.
Samples were vigorously shaken with an eppendorf shaker (IKA
VIBRAX-VXR) for 20 s. The resulting cell free extract was
centrifuged for 5 minutes at 14000 rpm and 4.degree. C. The
supernatant was used for enzyme activity assays.
[0257] Enzymatic Reactions for Conversion of AKP to 6-ACA in
Presence of Decarboxylase and Aminotransferase Co-Expressed in S.
cerevisiae
[0258] A reaction mixture was prepared comprising 50 mM AKP, 5 mM
magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate, 1
mM thiamine diphosphate and 50 mM racemic .alpha.-methylbenzylamine
in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml of the
reaction mixture were dispensed into a reaction vessel. To start
the reaction, 0.4 ml of the cell free extract from S. cerevisiae
containing decarboxylase and aminotransferase were added, to each
of the reaction vessels. Reaction mixtures were incubated with a
magnetic stirrer at 37.degree. C. Furthermore, a chemical blank
mixture (without cell free extract) and a biological blank (S.
cerevisiae) were incubated under the same conditions. Samples,
taken after 19 hours of incubation, were analysed by HPLC-MS. The
results are summarised in the following table.
TABLE-US-00010 TABLE 8 6-ACA formation from AKP using a
micro-organism as a biocatalyst 6-ACA concentration Biocatalyst
[mg/kg] S. cerevisiae pAKP-85 63 S. cerevisiae pAKP-86 226 S.
cerevisiae pAKP-87 1072 S. cerevisiae pAKP-88 4783 S. cerevisiae
3.9 (biological blank) None (chemical blank) 1.3
[0259] Enzymatic Reactions for Conversion of Alpha-Ketopimelic Acid
to Alpha-Aminopimelic Acid
[0260] A reaction mixture was prepared comprising 10 mM
alpha-ketopimelic acid, 20 mM L-alanine, and 50 .mu.M.quadrature.
pyridoxal 5'-phosphate in 50 mM potassium phosphate buffer, pH 7.0.
800 .mu.l of the reaction mixture were dispensed into each well of
the well plates. To start the reaction, 200 .mu.l of the cell
lysates were added, to each of the wells. Reaction mixtures were
incubated on a shaker at 37.degree. C. for 24 h. Furthermore, a
chemical blank mixture (without cell free extract) and a biological
blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the
same conditions. Samples were analysed by HPLC-MS. The results are
summarised in the following table.
TABLE-US-00011 TABLE 9 AAP formation from AKP in the presence of
aminotransferases AAP concentration [mg/kg] Biocatalyst (after 24
hrs) E. coli TOP10/pBAD-Vfl_AT 3.7 E. coli TOP10/pBAD-Psy_AT 15.8
E. coli TOP10/pBAD-Bsu_gi16078032_AT 11.2 E. coli TOP10/pBAD-Rsp_AT
9.8 E. coli TOP10/pBAD-Bsu_gi16080075_AT 4.6 E. coli
TOP10/pBAD-Lpn_AT 5.4 E. coli TOP10/pBAD-Neu_AT 7.7 E. coli
TOP10/pBAD-Ngo_AT 5.1 E. coli TOP10/pBAD-Pae_gi9951299_AT 5.6 E.
coli TOP10/pBAD-Rpa_AT 5.4 E. coli TOP10 with pBAD/Myc-His C 1.4
(biological blank) None (chemical blank) 0
[0261] It is shown that the formation of AAP from AKP is catalysed
by the biocatalyst.
[0262] Chemical Conversion of AAP to Caprolactam
[0263] To a suspension of 1.5 grams of D,L-2-aminopimelic acid in
21 ml cyclohexanone, 0.5 ml of cyclohexenone was added. The mixture
was heated on an oil bath for 20 h at reflux (approximately
160.degree. C.). After cooling to room temperature the reaction
mixture was decanted and the clear solution was evaporated under
reduced pressure. The remaining 2 grams of brownish oil were
analyzed by .sup.1H-NMR and HPLC and contained 0.8 wt % caprolactam
and 6 wt % of cyclic oligomers of caprolactam.
Sequence CWU 1
1
8311362DNAVibrio fluvialisCDS(1)..(1362) 1atg aac aaa ccg caa agc
tgg gaa gcc cgg gcc gag acc tat tcg ctc 48Met Asn Lys Pro Gln Ser
Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu 1 5 10 15 tat ggt ttc acc
gac atg cct tcg ctg cat cag cgc ggc acg gtc gtc 96Tyr Gly Phe Thr
Asp Met Pro Ser Leu His Gln Arg Gly Thr Val Val 20 25 30 gtg acc
cat ggc gag gga ccc tat atc gtc gat gtg aat ggc cgg cgt 144Val Thr
His Gly Glu Gly Pro Tyr Ile Val Asp Val Asn Gly Arg Arg 35 40 45
tat ctg gac gcc aac tcg ggc ctg tgg aac atg gtc gcg ggc ttt gac
192Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp
50 55 60 cac aag ggg ctg atc gac gcc gcc aag gcc caa tac gag cgt
ttt ccc 240His Lys Gly Leu Ile Asp Ala Ala Lys Ala Gln Tyr Glu Arg
Phe Pro 65 70 75 80 ggt tat cac gcc ttt ttc ggc cgc atg tcc gat cag
acg gta atg ctg 288Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln
Thr Val Met Leu 85 90 95 tcg gaa aag ctg gtc gag gtg tcg ccc ttt
gat tcg ggc cgg gtg ttc 336Ser Glu Lys Leu Val Glu Val Ser Pro Phe
Asp Ser Gly Arg Val Phe 100 105 110 tat aca aac tcg ggg tcc gag gcg
aat gac acc atg gtc aag atg cta 384Tyr Thr Asn Ser Gly Ser Glu Ala
Asn Asp Thr Met Val Lys Met Leu 115 120 125 tgg ttc ctg cat gca gcc
gag ggc aaa ccg caa aag cgc aag atc ctg 432Trp Phe Leu His Ala Ala
Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu 130 135 140 acc cgc tgg aac
gcc tat cac ggc gtg acc gcc gtt tcg gcc agc atg 480Thr Arg Trp Asn
Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met 145 150 155 160 acc
ggc aag ccc tat aat tcg gtc ttt ggc ctg ccg ctg ccg ggc ttt 528Thr
Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe 165 170
175 gtg cat ctg acc tgc ccg cat tac tgg cgc tat ggc gaa gag ggc gaa
576Val His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu
180 185 190 acc gaa gag cag ttc gtc gcc cgc ctc gcc cgc gag ctg gag
gaa acg 624Thr Glu Glu Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu
Glu Thr 195 200 205 atc cag cgc gag ggc gcc gac acc atc gcc ggt ttc
ttt gcc gaa ccg 672Ile Gln Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe
Phe Ala Glu Pro 210 215 220 gtg atg ggc gcg ggc ggc gtg att ccc ccg
gcc aag ggc tat ttc cag 720Val Met Gly Ala Gly Gly Val Ile Pro Pro
Ala Lys Gly Tyr Phe Gln 225 230 235 240 gcg atc ctg cca atc ctg cgc
aaa tat gac atc ccg gtc atc tcg gac 768Ala Ile Leu Pro Ile Leu Arg
Lys Tyr Asp Ile Pro Val Ile Ser Asp 245 250 255 gag gtg atc tgc ggt
ttc gga cgc acc ggt aac acc tgg ggc tgc gtg 816Glu Val Ile Cys Gly
Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val 260 265 270 acc tat gac
ttt aca ccc gat gca atc atc tcg tcc aag aat ctt aca 864Thr Tyr Asp
Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr 275 280 285 gcg
ggc ttt ttc ccc atg ggg gcg gtg atc ctt ggc ccg gaa ctt tcc 912Ala
Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu Leu Ser 290 295
300 aaa cgg ctg gaa acc gca atc gag gcg atc gag gaa ttc ccc cat ggc
960Lys Arg Leu Glu Thr Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly
305 310 315 320 ttt acc gcc tcg ggc cat ccg gtc ggc tgt gct att gcg
ctg aaa gca 1008Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala
Leu Lys Ala 325 330 335 atc gac gtg gtg atg aat gaa ggg ctg gct gag
aac gtc cgc cgc ctt 1056Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu
Asn Val Arg Arg Leu 340 345 350 gcc ccc cgt ttc gag gaa agg ctg aaa
cat atc gcc gag cgc ccg aac 1104Ala Pro Arg Phe Glu Glu Arg Leu Lys
His Ile Ala Glu Arg Pro Asn 355 360 365 atc ggt gaa tat cgc ggc atc
ggc ttc atg tgg gcg ctg gag gct gtc 1152Ile Gly Glu Tyr Arg Gly Ile
Gly Phe Met Trp Ala Leu Glu Ala Val 370 375 380 aag gac aag gca agc
aag acg ccg ttc gac ggc aac ctg tcg gtc agc 1200Lys Asp Lys Ala Ser
Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser 385 390 395 400 gag cgt
atc gcc aat acc tgc acc gat ctg ggg ctg att tgc cgg ccg 1248Glu Arg
Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro 405 410 415
ctt ggt cag tcc gtc gtc ctt tgt ccg ccc ttt atc ctg acc gag gcg
1296Leu Gly Gln Ser Val Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala
420 425 430 cag atg gat gag atg ttc gat aaa ctc gaa aaa gcc ctt gat
aag gtc 1344Gln Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp
Lys Val 435 440 445 ttt gcc gag gtt gcc tga 1362Phe Ala Glu Val Ala
450 2453PRTVibrio fluvialis 2Met Asn Lys Pro Gln Ser Trp Glu Ala
Arg Ala Glu Thr Tyr Ser Leu 1 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 Pro 65 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 Met 145 150 155 160 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 Gln 225 230 235 240 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 Gly 305 310 315
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 Ser 385 390 395 400 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 31362DNAArtificialVibrio fluvialis JS17
omega-aminotransferase codon optimised gene 3atgaataaac cacagtcttg
ggaagctcgt gctgaaacct atagcctgta cggctttacc 60gatatgccgt ctctgcacca
gcgtggtact gtagtggtaa cgcacggtga gggcccgtac 120atcgtggacg
ttaatggccg ccgttacctg gatgcaaaca gcggcctgtg gaacatggtt
180gcgggcttcg accacaaagg cctgatcgat gccgcaaaag cgcagtacga
acgcttcccg 240ggttatcacg cgttctttgg ccgtatgagc gaccagactg
tgatgctgag cgaaaaactg 300gttgaagtgt ccccgttcga tagcggtcgt
gtcttttaca ctaactctgg cagcgaggct 360aacgatacca tggttaagat
gctgtggttc ctgcacgcag cggaaggcaa acctcagaaa 420cgtaaaattc
tgacccgttg gaacgcttat cacggtgtga ctgctgtttc cgcatctatg
480accggtaaac cgtataacag cgtgttcggt ctgccgctgc ctggcttcgt
gcatctgacc 540tgcccgcact actggcgtta tggtgaggaa ggcgaaactg
aggaacagtt cgtggcgcgt 600ctggctcgtg aactggaaga aaccattcaa
cgcgaaggtg cagatactat cgcgggcttc 660tttgcggagc ctgttatggg
tgccggcggt gtgattccgc cggcgaaggg ctatttccag 720gcaatcctgc
cgatcctgcg caagtacgac attccggtta tttctgacga agtgatctgc
780ggcttcggcc gcaccggtaa cacctggggc tgcgtgacgt atgacttcac
tccggacgca 840atcattagct ctaaaaacct gactgcgggt ttcttcccta
tgggcgccgt aatcctgggc 900ccagaactgt ctaagcgcct ggaaaccgcc
atcgaggcaa tcgaagagtt cccgcacggt 960ttcactgcta gcggccatcc
ggtaggctgc gcaatcgcgc tgaaggcgat cgatgttgtc 1020atgaacgagg
gcctggcgga aaacgtgcgc cgcctggcgc cgcgttttga agaacgtctg
1080aaacacattg ctgagcgccc gaacattggc gaatatcgcg gcatcggttt
catgtgggcc 1140ctggaagcag ttaaagataa agctagcaag accccgttcg
acggcaacct gtccgtgagc 1200gaacgtatcg ctaatacctg tacggacctg
ggtctgatct gccgtccgct gggtcagtcc 1260gtagttctgt gcccaccatt
tatcctgacc gaagcgcaga tggatgaaat gttcgataaa 1320ctggagaaag
ctctggataa agtgttcgct gaagtcgcgt aa 136241350DNABacillus
weihenstephanensisCDS(1)..(1350) 4gtg caa gcg acg gag caa aca caa
agt ttg aaa aaa aca gat gaa aag 48Val Gln Ala Thr Glu Gln Thr Gln
Ser Leu Lys Lys Thr Asp Glu Lys 1 5 10 15 tac ctt tgg cat gcg atg
aga gga gca gcc cct agt cca acg aat tta 96Tyr Leu Trp His Ala Met
Arg Gly Ala Ala Pro Ser Pro Thr Asn Leu 20 25 30 att atc aca aaa
gca gaa ggg gca tgg gtg acg gat att gat gga aac 144Ile Ile Thr Lys
Ala Glu Gly Ala Trp Val Thr Asp Ile Asp Gly Asn 35 40 45 cgt tat
tta gac ggt atg tcc ggt ctt tgg tgc gtg aat gtt ggg tat 192Arg Tyr
Leu Asp Gly Met Ser Gly Leu Trp Cys Val Asn Val Gly Tyr 50 55 60
ggt cga aaa gaa ctt gca aga gcg gcg ttt gaa cag ctt gaa gaa atg
240Gly Arg Lys Glu Leu Ala Arg Ala Ala Phe Glu Gln Leu Glu Glu Met
65 70 75 80 ccg tat ttc cct ctg act caa agt cat gtt cct gct att aaa
tta gca 288Pro Tyr Phe Pro Leu Thr Gln Ser His Val Pro Ala Ile Lys
Leu Ala 85 90 95 gaa aaa ttg aat gaa tgg ctt gat gat gaa tac gtc
att ttc ttt tct 336Glu Lys Leu Asn Glu Trp Leu Asp Asp Glu Tyr Val
Ile Phe Phe Ser 100 105 110 aac agt gga tcg gaa gcg aat gaa aca gca
ttt aaa att gct cgt caa 384Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala
Phe Lys Ile Ala Arg Gln 115 120 125 tat cat caa caa aaa ggt gat cat
gga cgc tat aag ttt att tcc cgc 432Tyr His Gln Gln Lys Gly Asp His
Gly Arg Tyr Lys Phe Ile Ser Arg 130 135 140 tac cgc gct tat cac ggt
aac tca atg gga gct ctt gca gca aca ggt 480Tyr Arg Ala Tyr His Gly
Asn Ser Met Gly Ala Leu Ala Ala Thr Gly 145 150 155 160 caa gca cag
cga aag tat aaa tat gaa cca ctc ggg caa gga ttc ctg 528Gln Ala Gln
Arg Lys Tyr Lys Tyr Glu Pro Leu Gly Gln Gly Phe Leu 165 170 175 cat
gta gca ccg cct gat acg tat cga aat cca gag gat gtt cat aca 576His
Val Ala Pro Pro Asp Thr Tyr Arg Asn Pro Glu Asp Val His Thr 180 185
190 ctg gca agt gct gag gaa atc gat cgt gtc atg aca tgg gag tta agc
624Leu Ala Ser Ala Glu Glu Ile Asp Arg Val Met Thr Trp Glu Leu Ser
195 200 205 caa aca gta gcc ggt gtg att atg gag cca atc att act ggg
ggc gga 672Gln Thr Val Ala Gly Val Ile Met Glu Pro Ile Ile Thr Gly
Gly Gly 210 215 220 att tta atg cct cct gat gga tat atg gga aaa gta
aaa gaa att tgc 720Ile Leu Met Pro Pro Asp Gly Tyr Met Gly Lys Val
Lys Glu Ile Cys 225 230 235 240 gag aag cac ggt gcg ttg ctc att tgt
gat gaa gtt ata tgt gga ttt 768Glu Lys His Gly Ala Leu Leu Ile Cys
Asp Glu Val Ile Cys Gly Phe 245 250 255 ggc cgg aca ggg aag cca ttt
gga ttt atg aat tat ggc gtc aaa cca 816Gly Arg Thr Gly Lys Pro Phe
Gly Phe Met Asn Tyr Gly Val Lys Pro 260 265 270 gat atc att aca atg
gca aaa ggt att aca agt gcg tat ctt cct ttg 864Asp Ile Ile Thr Met
Ala Lys Gly Ile Thr Ser Ala Tyr Leu Pro Leu 275 280 285 tca gca aca
gca gtt aga cga gag gtt tat gag gca ttc gta ggt agt 912Ser Ala Thr
Ala Val Arg Arg Glu Val Tyr Glu Ala Phe Val Gly Ser 290 295 300 gat
gat tat gat cgc ttc cgc cat gta aat acg ttc gga ggg aat cct 960Asp
Asp Tyr Asp Arg Phe Arg His Val Asn Thr Phe Gly Gly Asn Pro 305 310
315 320 gct gct tgc gct tta gct ttg aag aat tta gaa att atg gag aat
gag 1008Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu Ile Met Glu Asn
Glu 325 330 335 aaa ctc att gaa cgt tcc aaa gaa ttg ggt gaa cga ctg
tta tat gag 1056Lys Leu Ile Glu Arg Ser Lys Glu Leu Gly Glu Arg Leu
Leu Tyr Glu 340 345 350 cta gag gat gta aaa gag cat cca aac gta ggg
gat gtt cgc gga aag 1104Leu Glu Asp Val Lys Glu His Pro Asn Val Gly
Asp Val Arg Gly Lys 355 360 365 ggc ctt ctt tta ggc att gaa cta gtg
gaa gat aag caa aca aaa gaa 1152Gly Leu Leu Leu Gly Ile Glu Leu Val
Glu Asp Lys Gln Thr Lys Glu 370 375 380 ccg gct tcc att gaa aag atg
aac aaa gtc atc aat gct tgt aaa gaa 1200Pro Ala Ser Ile Glu Lys Met
Asn Lys Val Ile Asn Ala Cys Lys Glu 385 390 395 400 aaa ggt cta att
att ggt aaa aat ggt gac act gtc gca ggt tac aat 1248Lys Gly Leu Ile
Ile Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn 405 410 415 aat att
ttg cag ctt gca cct cca tta agc atc aca gag gaa gac ttt 1296Asn Ile
Leu Gln Leu Ala Pro Pro Leu Ser Ile Thr Glu Glu Asp Phe 420 425 430
act ttt atc gtt aaa aca atg aaa gaa tgt tta tcc cgc att aac ggg
1344Thr Phe Ile Val Lys Thr Met Lys Glu Cys Leu Ser Arg Ile Asn Gly
435 440 445 cag taa 1350Gln 5449PRTBacillus weihenstephanensis 5Val
Gln Ala Thr Glu Gln Thr Gln Ser Leu Lys Lys Thr Asp Glu Lys 1 5 10
15 Tyr Leu Trp His Ala Met Arg Gly Ala Ala Pro Ser Pro Thr Asn Leu
20 25 30
Ile Ile Thr Lys Ala Glu Gly Ala Trp Val Thr Asp Ile Asp Gly Asn 35
40 45 Arg Tyr Leu Asp Gly Met Ser Gly Leu Trp Cys Val Asn Val Gly
Tyr 50 55 60 Gly Arg Lys Glu Leu Ala Arg Ala Ala Phe Glu Gln Leu
Glu Glu Met 65 70 75 80 Pro Tyr Phe Pro Leu Thr Gln Ser His Val Pro
Ala Ile Lys Leu Ala 85 90 95 Glu Lys Leu Asn Glu Trp Leu Asp Asp
Glu Tyr Val Ile Phe Phe Ser 100 105 110 Asn Ser Gly Ser Glu Ala Asn
Glu Thr Ala Phe Lys Ile Ala Arg Gln 115 120 125 Tyr His Gln Gln Lys
Gly Asp His Gly Arg Tyr Lys Phe Ile Ser Arg 130 135 140 Tyr Arg Ala
Tyr His Gly Asn Ser Met Gly Ala Leu Ala Ala Thr Gly 145 150 155 160
Gln Ala Gln Arg Lys Tyr Lys Tyr Glu Pro Leu Gly Gln Gly Phe Leu 165
170 175 His Val Ala Pro Pro Asp Thr Tyr Arg Asn Pro Glu Asp Val His
Thr 180 185 190 Leu Ala Ser Ala Glu Glu Ile Asp Arg Val Met Thr Trp
Glu Leu Ser 195 200 205 Gln Thr Val Ala Gly Val Ile Met Glu Pro Ile
Ile Thr Gly Gly Gly 210 215 220 Ile Leu Met Pro Pro Asp Gly Tyr Met
Gly Lys Val Lys Glu Ile Cys 225 230 235 240 Glu Lys His Gly Ala Leu
Leu Ile Cys Asp Glu Val Ile Cys Gly Phe 245 250 255 Gly Arg Thr Gly
Lys Pro Phe Gly Phe Met Asn Tyr Gly Val Lys Pro 260 265 270 Asp Ile
Ile Thr Met Ala Lys Gly Ile Thr Ser Ala Tyr Leu Pro Leu 275 280 285
Ser Ala Thr Ala Val Arg Arg Glu Val Tyr Glu Ala Phe Val Gly Ser 290
295 300 Asp Asp Tyr Asp Arg Phe Arg His Val Asn Thr Phe Gly Gly Asn
Pro 305 310 315 320 Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu Ile
Met Glu Asn Glu 325 330 335 Lys Leu Ile Glu Arg Ser Lys Glu Leu Gly
Glu Arg Leu Leu Tyr Glu 340 345 350 Leu Glu Asp Val Lys Glu His Pro
Asn Val Gly Asp Val Arg Gly Lys 355 360 365 Gly Leu Leu Leu Gly Ile
Glu Leu Val Glu Asp Lys Gln Thr Lys Glu 370 375 380 Pro Ala Ser Ile
Glu Lys Met Asn Lys Val Ile Asn Ala Cys Lys Glu 385 390 395 400 Lys
Gly Leu Ile Ile Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn 405 410
415 Asn Ile Leu Gln Leu Ala Pro Pro Leu Ser Ile Thr Glu Glu Asp Phe
420 425 430 Thr Phe Ile Val Lys Thr Met Lys Glu Cys Leu Ser Arg Ile
Asn Gly 435 440 445 Gln 61350DNAArtificialB. weihenstephanensis
KBAB4 aminotransferase codon-optimised gene 6atgcaggcta ccgaacaaac
ccaatctctg aaaaagactg acgaaaaata tctgtggcac 60gcgatgcgcg gtgcagctcc
gtctccgacc aacctgatta ttaccaaagc tgaaggcgcg 120tgggtgaccg
acattgacgg taaccgttat ctggatggca tgagcggcct gtggtgtgtt
180aatgtcggtt atggccgtaa ggagctggcg cgcgcggcat ttgaacaact
ggaagaaatg 240ccgtacttcc cgctgactca aagccatgtg ccggctatca
aactggcgga aaaactgaac 300gaatggctgg acgacgaata cgtgattttc
ttctctaatt ctggctccga agcaaacgaa 360accgcattca aaatcgcccg
tcaatatcac cagcagaaag gtgaccacgg ccgctataaa 420ttcatcagcc
gttatcgtgc ataccatggt aattctatgg gtgcgctggc tgctaccggt
480caggctcagc gcaaatacaa gtacgaaccg ctgggtcagg gttttctgca
cgttgcacca 540ccggatacct accgtaaccc ggaagacgtc cacaccctgg
cttctgccga agaaatcgat 600cgtgttatga cctgggagct gtcccagact
gttgcgggtg ttatcatgga acctattatt 660accggtggtg gcattctgat
gccgccggac ggttatatgg gtaaagtcaa ggaaatctgc 720gaaaaacacg
gcgcgctgct gatctgcgat gaagttatct gtggcttcgg tcgcaccggc
780aaaccatttg gcttcatgaa ttatggcgta aaacctgaca ttattaccat
ggctaaaggc 840attacttccg cttatctgcc gctgagcgcg accgcagttc
gccgcgaagt ttatgaagcg 900tttgttggtt ctgatgatta cgaccgtttc
cgtcatgtaa acacgtttgg cggtaaccca 960gcggcatgtg cgctggcgct
gaaaaacctg gaaatcatgg aaaacgaaaa gctgatcgaa 1020cgtagcaaag
aactgggtga acgtctgctg tacgaactgg aagatgtcaa agaacacccg
1080aacgtgggcg atgttcgcgg taaaggcctg ctgctgggta ttgaactggt
tgaagacaaa 1140cagaccaagg aaccggcttc cattgaaaag atgaacaaag
tgattaacgc gtgcaaagag 1200aaaggcctga tcattggtaa gaacggtgat
accgtggcag gttataacaa cattctgcag 1260ctggcgccgc ctctgagcat
cactgaagaa gatttcacct tcatcgtcaa aactatgaag 1320gagtgcctga
gccgcatcaa tggtcagtaa 135071371DNAPseudomonas
aeruginosaCDS(1)..(1371) 7atg aac agc caa atc acc aac gcc aag acc
cgt gag tgg cag gcg ttg 48Met Asn Ser Gln Ile Thr Asn Ala Lys Thr
Arg Glu Trp Gln Ala Leu 1 5 10 15 agc cgc gac cac cat ctg ccg ccg
ttc acc gac tac aag cag ttg aac 96Ser Arg Asp His His Leu Pro Pro
Phe Thr Asp Tyr Lys Gln Leu Asn 20 25 30 gag aag ggc gcg cgg atc
atc acc aag gcc gaa ggc gtc tat atc tgg 144Glu Lys Gly Ala Arg Ile
Ile Thr Lys Ala Glu Gly Val Tyr Ile Trp 35 40 45 gac agc gag ggc
aac aag atc ctc gat gcg atg gcc ggc ctc tgg tgc 192Asp Ser Glu Gly
Asn Lys Ile Leu Asp Ala Met Ala Gly Leu Trp Cys 50 55 60 gtc aac
gtc ggc tac ggc cgc gag gag ctg gtc cag gcc gcc acc cgg 240Val Asn
Val Gly Tyr Gly Arg Glu Glu Leu Val Gln Ala Ala Thr Arg 65 70 75 80
cag atg cgc gag ttg ccg ttc tac aac ctg ttc ttc cag acc gcc cac
288Gln Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gln Thr Ala His
85 90 95 ccg ccg gtg gtc gag ctg gcc aag gcg atc gcc gac gtc gct
ccg gaa 336Pro Pro Val Val Glu Leu Ala Lys Ala Ile Ala Asp Val Ala
Pro Glu 100 105 110 ggc atg aac cac gtg ttc ttc acc ggc tcc ggc tcc
gag gcc aac gac 384Gly Met Asn His Val Phe Phe Thr Gly Ser Gly Ser
Glu Ala Asn Asp 115 120 125 acc gtg ctg cgt atg gtc cgc cac tat tgg
gcg acc aag ggc cag ccg 432Thr Val Leu Arg Met Val Arg His Tyr Trp
Ala Thr Lys Gly Gln Pro 130 135 140 cag aag aaa gtg gtg atc ggc cgc
tgg aac ggc tac cac ggc tcc acc 480Gln Lys Lys Val Val Ile Gly Arg
Trp Asn Gly Tyr His Gly Ser Thr 145 150 155 160 gtc gcc ggc gtc agc
ctg ggc ggc atg aag gcg ttg cat gag cag ggt 528Val Ala Gly Val Ser
Leu Gly Gly Met Lys Ala Leu His Glu Gln Gly 165 170 175 gat ttc ccc
atc ccg ggc atc gtc cac atc gcc cag ccc tac tgg tac 576Asp Phe Pro
Ile Pro Gly Ile Val His Ile Ala Gln Pro Tyr Trp Tyr 180 185 190 ggc
gag ggc ggc gac atg tcg ccg gac gag ttc ggc gtc tgg gcc gcc 624Gly
Glu Gly Gly Asp Met Ser Pro Asp Glu Phe Gly Val Trp Ala Ala 195 200
205 gag cag ttg gag aag aag att ctc gaa gtg ggc gag gaa aac gtc gcc
672Glu Gln Leu Glu Lys Lys Ile Leu Glu Val Gly Glu Glu Asn Val Ala
210 215 220 gcc ttc atc gcc gag ccg atc cag ggc gcc ggc ggc gtg atc
gtc ccg 720Ala Phe Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile
Val Pro 225 230 235 240 ccg gac acc tac tgg ccg aag atc cgc gag atc
ctc gcc aag tac gac 768Pro Asp Thr Tyr Trp Pro Lys Ile Arg Glu Ile
Leu Ala Lys Tyr Asp 245 250 255 atc ctg ttc atc gcc gac gaa gtg atc
tgc ggc ttc ggc cgt acc ggc 816Ile Leu Phe Ile Ala Asp Glu Val Ile
Cys Gly Phe Gly Arg Thr Gly 260 265 270 gag tgg ttc ggc agc cag tac
tac ggc aac gcc ccg gac ctg atg ccg 864Glu Trp Phe Gly Ser Gln Tyr
Tyr Gly Asn Ala Pro Asp Leu Met Pro 275 280 285 atc gcc aag ggc ctc
acc tcc ggc tac atc ccc atg ggc ggg gtg gtg 912Ile Ala Lys Gly Leu
Thr Ser Gly Tyr Ile Pro Met Gly Gly Val Val 290 295 300 gtg cgc gac
gag atc gtc gaa gtg ctc aac cag ggc ggc gag ttc tac 960Val Arg Asp
Glu Ile Val Glu Val Leu Asn Gln Gly Gly Glu Phe Tyr 305 310 315 320
cac ggc ttc acc tat tcc ggt cac ccg gtg gcg gcc gcc gtg gcc ctg
1008His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335 gag aac atc cgc atc ctg cgc gaa gag aag atc atc gag aag
gtg aag 1056Glu Asn Ile Arg Ile Leu Arg Glu Glu Lys Ile Ile Glu Lys
Val Lys 340 345 350 gcg gaa acg gca ccg tat ttg cag aaa cgc tgg cag
gag ctg gcc gac 1104Ala Glu Thr Ala Pro Tyr Leu Gln Lys Arg Trp Gln
Glu Leu Ala Asp 355 360 365 cac ccg ttg gtg ggc gaa gcg cgc ggg gtc
ggc atg gtc gcc gcc ctg 1152His Pro Leu Val Gly Glu Ala Arg Gly Val
Gly Met Val Ala Ala Leu 370 375 380 gag ctg gtc aag aac aag aag acc
cgc gag cgt ttc acc gac aag ggc 1200Glu Leu Val Lys Asn Lys Lys Thr
Arg Glu Arg Phe Thr Asp Lys Gly 385 390 395 400 gtc ggg atg ctg tgc
cgg gaa cat tgt ttc cgc aac ggt ttg atc atg 1248Val Gly Met Leu Cys
Arg Glu His Cys Phe Arg Asn Gly Leu Ile Met 405 410 415 cgc gcg gtg
ggc gac act atg att atc tcg ccg ccg ctg gtg atc gat 1296Arg Ala Val
Gly Asp Thr Met Ile Ile Ser Pro Pro Leu Val Ile Asp 420 425 430 ccg
tcg cag atc gat gag ttg atc acc ctg gcg cgc aag tgc ctc gat 1344Pro
Ser Gln Ile Asp Glu Leu Ile Thr Leu Ala Arg Lys Cys Leu Asp 435 440
445 cag acc gcc gcc gcc gtc ctg gct tga 1371Gln Thr Ala Ala Ala Val
Leu Ala 450 455 8456PRTPseudomonas aeruginosa 8Met Asn Ser Gln Ile
Thr Asn Ala Lys Thr Arg Glu Trp Gln Ala Leu 1 5 10 15 Ser Arg Asp
His His Leu Pro Pro Phe Thr Asp Tyr Lys Gln Leu Asn 20 25 30 Glu
Lys Gly Ala Arg Ile Ile Thr Lys Ala Glu Gly Val Tyr Ile Trp 35 40
45 Asp Ser Glu Gly Asn Lys Ile Leu Asp Ala Met Ala Gly Leu Trp Cys
50 55 60 Val Asn Val Gly Tyr Gly Arg Glu Glu Leu Val Gln Ala Ala
Thr Arg 65 70 75 80 Gln Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe
Gln Thr Ala His 85 90 95 Pro Pro Val Val Glu Leu Ala Lys Ala Ile
Ala Asp Val Ala Pro Glu 100 105 110 Gly Met Asn His Val Phe Phe Thr
Gly Ser Gly Ser Glu Ala Asn Asp 115 120 125 Thr Val Leu Arg Met Val
Arg His Tyr Trp Ala Thr Lys Gly Gln Pro 130 135 140 Gln Lys Lys Val
Val Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr 145 150 155 160 Val
Ala Gly Val Ser Leu Gly Gly Met Lys Ala Leu His Glu Gln Gly 165 170
175 Asp Phe Pro Ile Pro Gly Ile Val His Ile Ala Gln Pro Tyr Trp Tyr
180 185 190 Gly Glu Gly Gly Asp Met Ser Pro Asp Glu Phe Gly Val Trp
Ala Ala 195 200 205 Glu Gln Leu Glu Lys Lys Ile Leu Glu Val Gly Glu
Glu Asn Val Ala 210 215 220 Ala Phe Ile Ala Glu Pro Ile Gln Gly Ala
Gly Gly Val Ile Val Pro 225 230 235 240 Pro Asp Thr Tyr Trp Pro Lys
Ile Arg Glu Ile Leu Ala Lys Tyr Asp 245 250 255 Ile Leu Phe Ile Ala
Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly 260 265 270 Glu Trp Phe
Gly Ser Gln Tyr Tyr Gly Asn Ala Pro Asp Leu Met Pro 275 280 285 Ile
Ala Lys Gly Leu Thr Ser Gly Tyr Ile Pro Met Gly Gly Val Val 290 295
300 Val Arg Asp Glu Ile Val Glu Val Leu Asn Gln Gly Gly Glu Phe Tyr
305 310 315 320 His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala
Val Ala Leu 325 330 335 Glu Asn Ile Arg Ile Leu Arg Glu Glu Lys Ile
Ile Glu Lys Val Lys 340 345 350 Ala Glu Thr Ala Pro Tyr Leu Gln Lys
Arg Trp Gln Glu Leu Ala Asp 355 360 365 His Pro Leu Val Gly Glu Ala
Arg Gly Val Gly Met Val Ala Ala Leu 370 375 380 Glu Leu Val Lys Asn
Lys Lys Thr Arg Glu Arg Phe Thr Asp Lys Gly 385 390 395 400 Val Gly
Met Leu Cys Arg Glu His Cys Phe Arg Asn Gly Leu Ile Met 405 410 415
Arg Ala Val Gly Asp Thr Met Ile Ile Ser Pro Pro Leu Val Ile Asp 420
425 430 Pro Ser Gln Ile Asp Glu Leu Ile Thr Leu Ala Arg Lys Cys Leu
Asp 435 440 445 Gln Thr Ala Ala Ala Val Leu Ala 450 455
970DNAArtificialprimer 9ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta
accatgaaca gccaaatcac 60caacgccaag 701049DNAArtificialprimer
10ggggaccact ttgtacaaga aagctgggtt caagccagga cggcggcgg
49111365DNAPseudomonas syringaeCDS(1)..(1365) 11atg agt gcc aac aac
ccg caa acc ctc gaa tgg cag gcc ctg agc agc 48Met Ser Ala Asn Asn
Pro Gln Thr Leu Glu Trp Gln Ala Leu Ser Ser 1 5 10 15 gag cat cac
ctg gca ccg ttc agc gac tac aaa caa ctg aaa gag aaa 96Glu His His
Leu Ala Pro Phe Ser Asp Tyr Lys Gln Leu Lys Glu Lys 20 25 30 ggc
ccg cgc atc atc acc cgt gcc gag ggc gtt tat ctg tgg gac agc 144Gly
Pro Arg Ile Ile Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser 35 40
45 gag ggc aac aag atc ctc gat ggc atg tcc ggc ctg tgg tgc gtg gcc
192Glu Gly Asn Lys Ile Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala
50 55 60 atc ggt tat ggc cgc gaa gaa ctg gcc gac gca gcc agc aaa
cag atg 240Ile Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys
Gln Met 65 70 75 80 cgc gag ctg ccg tac tac aac ctg ttc ttc cag acc
gcc cac ccg ccg 288Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gln Thr
Ala His Pro Pro 85 90 95 gtg ctg gaa ctg gcc aag gcc atc tcc gac
atc gct ccc gag ggc atg 336Val Leu Glu Leu Ala Lys Ala Ile Ser Asp
Ile Ala Pro Glu Gly Met 100 105 110 aac cat gtg ttc ttc acc ggt tca
ggc tct gaa ggc aat gac acg atg 384Asn His Val Phe Phe Thr Gly Ser
Gly Ser Glu Gly Asn Asp Thr Met 115 120 125 ctg cgc atg gtt cgt cat
tac tgg gcg ctg aaa ggc cag ccg aac aag 432Leu Arg Met Val Arg His
Tyr Trp Ala Leu Lys Gly Gln Pro Asn Lys 130 135 140 aaa acc atc atc
agc cgc gtc aat ggc tac cac ggc tcc acc gtc gcc 480Lys Thr Ile Ile
Ser Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala 145 150 155 160 ggt
gcc agc ctg ggt ggc atg acc tac atg cac gaa cag ggc gac ctg 528Gly
Ala Ser Leu Gly Gly Met Thr Tyr Met His Glu Gln Gly Asp Leu 165 170
175 ccg atc ccg ggg gtg gtg cac att cca cag cct tac tgg ttc ggc gaa
576Pro Ile Pro Gly Val Val His Ile Pro Gln Pro Tyr Trp Phe Gly Glu
180 185 190 ggc ggc gac atg acg ccg gac gag ttc ggc atc tgg gcg gcc
gag caa 624Gly Gly Asp Met Thr Pro Asp Glu Phe Gly Ile Trp Ala Ala
Glu Gln 195 200 205 ctg gaa aag aaa att ctc gag ctg ggc gtc gag aac
gtc ggt gcg ttc
672Leu Glu Lys Lys Ile Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe
210 215 220 att gcc gag cca atc cag ggc gcg ggc ggt gtg att gtc ccg
cct gat 720Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro
Pro Asp 225 230 235 240 tcc tac tgg ccg aag atc aag gaa atc ctt tcc
cgc tac gac atc ctg 768Ser Tyr Trp Pro Lys Ile Lys Glu Ile Leu Ser
Arg Tyr Asp Ile Leu 245 250 255 ttc gcc gcc gat gag gtg att tgt ggc
ttc ggg cgt acc agt gag tgg 816Phe Ala Ala Asp Glu Val Ile Cys Gly
Phe Gly Arg Thr Ser Glu Trp 260 265 270 ttc ggt agc gat ttc tat ggc
ctc agg ccg gac atg atg acc atc gcc 864Phe Gly Ser Asp Phe Tyr Gly
Leu Arg Pro Asp Met Met Thr Ile Ala 275 280 285 aaa ggc ctg acc tcc
ggt tac gta ccg atg ggc ggc ctg atc gtg cgc 912Lys Gly Leu Thr Ser
Gly Tyr Val Pro Met Gly Gly Leu Ile Val Arg 290 295 300 gat gaa atc
gtt gcg gtg ctc aat gag ggt ggc gat ttc aat cac ggc 960Asp Glu Ile
Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly 305 310 315 320
ttt acc tac tcc ggg cac ccg gtg gcg gcc gcg gtt gcg ctg gag aac
1008Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn
325 330 335 atc cgt atc ctg cgc gaa gaa aag atc gtc gaa cgg gtc agg
tcg gaa 1056Ile Arg Ile Leu Arg Glu Glu Lys Ile Val Glu Arg Val Arg
Ser Glu 340 345 350 acg gca ccg tat ttg caa aag cgt ttg cgt gag ttg
agc gat cat ccg 1104Thr Ala Pro Tyr Leu Gln Lys Arg Leu Arg Glu Leu
Ser Asp His Pro 355 360 365 ctg gtg ggc gaa gtc cgg ggt gtc ggg ctg
ctc ggg gcc att gag ctg 1152Leu Val Gly Glu Val Arg Gly Val Gly Leu
Leu Gly Ala Ile Glu Leu 370 375 380 gtg aag gac aag acc acc cgc gag
cgc tat acc gac aag ggc gcg gga 1200Val Lys Asp Lys Thr Thr Arg Glu
Arg Tyr Thr Asp Lys Gly Ala Gly 385 390 395 400 atg atc tgt cga acc
ttc tgc ttc gac aat ggc ctg atc atg cgg gct 1248Met Ile Cys Arg Thr
Phe Cys Phe Asp Asn Gly Leu Ile Met Arg Ala 405 410 415 gtg ggc gat
acc atg atc att gcg ccg cca ctg gtg atc agt ttt gcg 1296Val Gly Asp
Thr Met Ile Ile Ala Pro Pro Leu Val Ile Ser Phe Ala 420 425 430 caa
atc gat gag ctg gta gag aag gcg cgc acg tgt ctg gat ctg acg 1344Gln
Ile Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr 435 440
445 ctg gcg gtg ttg cag ggc tga 1365Leu Ala Val Leu Gln Gly 450
12454PRTPseudomonas syringae 12Met Ser Ala Asn Asn Pro Gln Thr Leu
Glu Trp Gln Ala Leu Ser Ser 1 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 Met 65 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 Ala 145 150 155 160 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 Asp 225 230 235 240 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 Gly 305 310 315
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 Gly 385 390 395 400 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 131365DNAArtificialPseudomonas
syringae codon optimised aminotransferase gene 13atgtctgcta
acaatccaca aactctggaa tggcaggcac tgagctccga acatcacctg 60gctccgttct
ccgactacaa acaactgaaa gagaaaggcc cgcgtatcat tacccgcgct
120gaaggtgtgt acctgtggga ttctgaaggc aacaaaattc tggacggtat
gagcggcctg 180tggtgcgtag caatcggtta tggccgtgaa gaactggctg
acgcggcgag caaacagatg 240cgtgaactgc cgtattataa cctgttcttc
caaaccgcac acccgccggt tctggaactg 300gctaaagcta tcagcgatat
cgcaccggag ggcatgaatc acgtcttctt cactggttcc 360ggtagcgaag
gcaacgacac gatgctgcgc atggtacgtc actattgggc gctgaagggc
420cagccgaaca agaaaacgat tatcagccgt gtaaacggtt atcacggcag
caccgttgcg 480ggtgcgagcc tgggcggtat gacctacatg cacgaacagg
gtgacctgcc gatcccgggt 540gtagtgcaca ttccgcagcc gtattggttc
ggtgaaggcg gtgacatgac gccggacgaa 600ttcggcatct gggcggcaga
gcagctggaa aagaaaatcc tggaactggg cgtggaaaac 660gtcggcgcgt
tcatcgcgga accgattcag ggcgcgggcg gcgtaattgt tccgccggac
720agctactggc caaaaatcaa agagatcctg tctcgttacg acatcctgtt
cgccgcagac 780gaagtgatct gcggttttgg ccgcacctct gaatggttcg
gctccgactt ctacggtctg 840cgtccggaca tgatgaccat cgccaaaggc
ctgacctccg gttatgttcc tatgggtggc 900ctgatcgtgc gcgacgaaat
tgttgcggtt ctgaacgaag gcggcgattt caaccacggc 960ttcacctatt
ccggtcaccc agttgctgct gctgtagcac tggaaaacat ccgcatcctg
1020cgtgaagaaa agatcgtaga acgcgtacgt tccgaaaccg caccttacct
gcagaagcgc 1080ctgcgcgaac tgagcgacca ccctctggta ggtgaagttc
gcggcgtggg cctgctgggc 1140gcgatcgagc tggtgaaaga caaaactacc
cgtgaacgtt acaccgacaa aggcgcaggc 1200atgatctgcc gtaccttttg
cttcgataac ggtctgatca tgcgcgcagt cggtgatacc 1260atgatcattg
ctccgcctct ggttatttct tttgcccaga ttgatgagct ggtcgaaaaa
1320gcgcgcactt gtctggatct gactctggct gttctgcagg gttaa
136514849DNABacillus subtilisCDS(1)..(849) 14atg aag gtt tta gtc
aat ggc cgg ctg att ggg cgc agt gaa gca tca 48Met Lys Val Leu Val
Asn Gly Arg Leu Ile Gly Arg Ser Glu Ala Ser 1 5 10 15 atc gat ttg
gaa gat cgc ggt tat cag ttt ggt gac ggc atc tat gaa 96Ile Asp Leu
Glu Asp Arg Gly Tyr Gln Phe Gly Asp Gly Ile Tyr Glu 20 25 30 gtg
atc agg gtg tac aaa gga gta ttg ttc ggc tta cgt gag cat gca 144Val
Ile Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala 35 40
45 gag cgt ttt ttc aga agt gct gct gaa atc gga att tca ctg cca ttc
192Glu Arg Phe Phe Arg Ser Ala Ala Glu Ile Gly Ile Ser Leu Pro Phe
50 55 60 agt ata gaa gat ctc gag tgg gac ctg caa aag ctt gta cag
gaa aat 240Ser Ile Glu Asp Leu Glu Trp Asp Leu Gln Lys Leu Val Gln
Glu Asn 65 70 75 80 gcg gtc agt gag gga gcg gta tac att cag aca aca
aga ggt gtg gcc 288Ala Val Ser Glu Gly Ala Val Tyr Ile Gln Thr Thr
Arg Gly Val Ala 85 90 95 ccg cga aaa cac cag tat gaa gcc ggc ctc
gag ccg cag act act gcc 336Pro Arg Lys His Gln Tyr Glu Ala Gly Leu
Glu Pro Gln Thr Thr Ala 100 105 110 tat acg ttt acg gtg aaa aaa ccg
gag caa gag cag gca tac gga gtg 384Tyr Thr Phe Thr Val Lys Lys Pro
Glu Gln Glu Gln Ala Tyr Gly Val 115 120 125 gcg gcc att aca gat gag
gat ctt cgc tgg tta aga tgt gat atc aaa 432Ala Ala Ile Thr Asp Glu
Asp Leu Arg Trp Leu Arg Cys Asp Ile Lys 130 135 140 agt ctg aat tta
ctg tat aat gtc atg acg aag caa agg gcc tat gaa 480Ser Leu Asn Leu
Leu Tyr Asn Val Met Thr Lys Gln Arg Ala Tyr Glu 145 150 155 160 gcc
gga gca ttt gaa gcc att tta ctt agg gac ggc gtt gtt acg gag 528Ala
Gly Ala Phe Glu Ala Ile Leu Leu Arg Asp Gly Val Val Thr Glu 165 170
175 ggt aca tcc tct aac gtt tat gcc gtt atc aac ggc aca gtg cga aca
576Gly Thr Ser Ser Asn Val Tyr Ala Val Ile Asn Gly Thr Val Arg Thr
180 185 190 cat ccg gct aat cgg ctc att ctc aat gga att aca cgg atg
aat att 624His Pro Ala Asn Arg Leu Ile Leu Asn Gly Ile Thr Arg Met
Asn Ile 195 200 205 tta gga ctg att gag aag aat ggg atc aaa ctg gat
gag act cct gtc 672Leu Gly Leu Ile Glu Lys Asn Gly Ile Lys Leu Asp
Glu Thr Pro Val 210 215 220 agt gaa gaa gag ttg aaa cag gcg gaa gag
atc ttt att tcg tca acg 720Ser Glu Glu Glu Leu Lys Gln Ala Glu Glu
Ile Phe Ile Ser Ser Thr 225 230 235 240 acg gca gaa att att ccg gtc
gtg acg ctc gat gga caa tcg atc gga 768Thr Ala Glu Ile Ile Pro Val
Val Thr Leu Asp Gly Gln Ser Ile Gly 245 250 255 agc ggg aaa ccc gga
ccg gtg acc aaa cag ctt cag gct gct ttt caa 816Ser Gly Lys Pro Gly
Pro Val Thr Lys Gln Leu Gln Ala Ala Phe Gln 260 265 270 gaa agc att
caa cag gct gct agc att tca taa 849Glu Ser Ile Gln Gln Ala Ala Ser
Ile Ser 275 280 15282PRTBacillus subtilis 15Met Lys Val Leu Val Asn
Gly Arg Leu Ile Gly Arg Ser Glu Ala Ser 1 5 10 15 Ile Asp Leu Glu
Asp Arg Gly Tyr Gln Phe Gly Asp Gly Ile Tyr Glu 20 25 30 Val Ile
Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala 35 40 45
Glu Arg Phe Phe Arg Ser Ala Ala Glu Ile Gly Ile Ser Leu Pro Phe 50
55 60 Ser Ile Glu Asp Leu Glu Trp Asp Leu Gln Lys Leu Val Gln Glu
Asn 65 70 75 80 Ala Val Ser Glu Gly Ala Val Tyr Ile Gln Thr Thr Arg
Gly Val Ala 85 90 95 Pro Arg Lys His Gln Tyr Glu Ala Gly Leu Glu
Pro Gln Thr Thr Ala 100 105 110 Tyr Thr Phe Thr Val Lys Lys Pro Glu
Gln Glu Gln Ala Tyr Gly Val 115 120 125 Ala Ala Ile Thr Asp Glu Asp
Leu Arg Trp Leu Arg Cys Asp Ile Lys 130 135 140 Ser Leu Asn Leu Leu
Tyr Asn Val Met Thr Lys Gln Arg Ala Tyr Glu 145 150 155 160 Ala Gly
Ala Phe Glu Ala Ile Leu Leu Arg Asp Gly Val Val Thr Glu 165 170 175
Gly Thr Ser Ser Asn Val Tyr Ala Val Ile Asn Gly Thr Val Arg Thr 180
185 190 His Pro Ala Asn Arg Leu Ile Leu Asn Gly Ile Thr Arg Met Asn
Ile 195 200 205 Leu Gly Leu Ile Glu Lys Asn Gly Ile Lys Leu Asp Glu
Thr Pro Val 210 215 220 Ser Glu Glu Glu Leu Lys Gln Ala Glu Glu Ile
Phe Ile Ser Ser Thr 225 230 235 240 Thr Ala Glu Ile Ile Pro Val Val
Thr Leu Asp Gly Gln Ser Ile Gly 245 250 255 Ser Gly Lys Pro Gly Pro
Val Thr Lys Gln Leu Gln Ala Ala Phe Gln 260 265 270 Glu Ser Ile Gln
Gln Ala Ala Ser Ile Ser 275 280 161347DNABacillus
subtilisCDS(1)..(1347) 16atg act cat gat ttg ata gaa aaa agt aaa
aag cac ctc tgg ctg cca 48Met Thr His Asp Leu Ile Glu Lys Ser Lys
Lys His Leu Trp Leu Pro 1 5 10 15 ttt acc caa atg aaa gat tat gat
gaa aac ccc tta atc atc gaa agc 96Phe Thr Gln Met Lys Asp Tyr Asp
Glu Asn Pro Leu Ile Ile Glu Ser 20 25 30 ggg act gga atc aaa gtc
aaa gac ata aac ggc aag gaa tac tat gac 144Gly Thr Gly Ile Lys Val
Lys Asp Ile Asn Gly Lys Glu Tyr Tyr Asp 35 40 45 ggt ttt tca tcg
gtt tgg ctt aat gtc cac gga cac cgc aaa aaa gaa 192Gly Phe Ser Ser
Val Trp Leu Asn Val His Gly His Arg Lys Lys Glu 50 55 60 cta gat
gac gcc ata aaa aaa cag ctc gga aaa att gcg cac tcc acg 240Leu Asp
Asp Ala Ile Lys Lys Gln Leu Gly Lys Ile Ala His Ser Thr 65 70 75 80
tta ttg ggc atg acc aat gtt cca gca acc cag ctt gcc gaa aca tta
288Leu Leu Gly Met Thr Asn Val Pro Ala Thr Gln Leu Ala Glu Thr Leu
85 90 95 atc gac atc agc cca aaa aag ctc acg cgg gtc ttt tat tca
gac agc 336Ile Asp Ile Ser Pro Lys Lys Leu Thr Arg Val Phe Tyr Ser
Asp Ser 100 105 110 ggc gca gag gcg atg gaa ata gcc cta aaa atg gcg
ttt cag tat tgg 384Gly Ala Glu Ala Met Glu Ile Ala Leu Lys Met Ala
Phe Gln Tyr Trp 115 120 125 aag aac atc ggg aag ccc gag aaa caa aaa
ttc atc gca atg aaa aac 432Lys Asn Ile Gly Lys Pro Glu Lys Gln Lys
Phe Ile Ala Met Lys Asn 130 135 140 ggg tat cac ggt gat acg att ggc
gcc gtc agt gtc ggt tca att gag 480Gly Tyr His Gly Asp Thr Ile Gly
Ala Val Ser Val Gly Ser Ile Glu 145 150 155 160 ctt ttt cac cac gta
tac ggc ccg ttg atg ttc gag agt tac aag gcc 528Leu Phe His His Val
Tyr Gly Pro Leu Met Phe Glu Ser Tyr Lys Ala 165 170 175 ccg att cct
tat gtg tat cgt tct gaa agc ggt gat cct gat gag tgc 576Pro Ile Pro
Tyr Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys 180 185 190 cgt
gat cag tgc ctc cga gag ctt gca cag ctg ctt gag gaa cat cat 624Arg
Asp Gln Cys Leu Arg Glu Leu Ala Gln Leu Leu Glu Glu His His 195 200
205 gag gaa att gcc gcg ctt tcc att gaa tca atg gta caa ggc gcg tcc
672Glu Glu Ile Ala Ala Leu Ser Ile Glu Ser Met Val Gln Gly Ala Ser
210 215 220 ggt atg atc gtg atg ccg gaa gga tat ttg gca ggc gtg cgc
gag cta 720Gly Met Ile Val Met Pro Glu Gly Tyr Leu Ala Gly Val Arg
Glu Leu 225 230 235
240 tgt aca aca tac gat gtc tta atg atc gtt gat gaa gtc gct aca ggc
768Cys Thr Thr Tyr Asp Val Leu Met Ile Val Asp Glu Val Ala Thr Gly
245 250 255 ttt ggc cgt aca gga aaa atg ttt gcg tgc gag cac gag aat
gtc cag 816Phe Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His Glu Asn
Val Gln 260 265 270 cct gat ctg atg gct gcc ggt aaa ggc att aca gga
ggc tat ttg cca 864Pro Asp Leu Met Ala Ala Gly Lys Gly Ile Thr Gly
Gly Tyr Leu Pro 275 280 285 att gcc gtt acg ttt gcc act gaa gac atc
tat aag gca ttc tat gat 912Ile Ala Val Thr Phe Ala Thr Glu Asp Ile
Tyr Lys Ala Phe Tyr Asp 290 295 300 gat tat gaa aac cta aaa acc ttt
ttc cat ggc cat tcc tat aca ggc 960Asp Tyr Glu Asn Leu Lys Thr Phe
Phe His Gly His Ser Tyr Thr Gly 305 310 315 320 aat cag ctt ggc tgt
gcg gtt gcg ctt gaa aat ctg gca tta ttt gaa 1008Asn Gln Leu Gly Cys
Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu 325 330 335 tct gaa aac
att gtg gaa caa gta gcg gaa aaa agt aaa aag ctc cat 1056Ser Glu Asn
Ile Val Glu Gln Val Ala Glu Lys Ser Lys Lys Leu His 340 345 350 ttt
ctt ctt caa gat ctg cac gct ctt cct cat gtt ggg gat att cgg 1104Phe
Leu Leu Gln Asp Leu His Ala Leu Pro His Val Gly Asp Ile Arg 355 360
365 cag ctt ggc ttt atg tgc ggt gca gag ctt gta cga tca aag gaa act
1152Gln Leu Gly Phe Met Cys Gly Ala Glu Leu Val Arg Ser Lys Glu Thr
370 375 380 aaa gaa cct tac ccg gct gat cgg cgg att gga tac aaa gtt
tcc tta 1200Lys Glu Pro Tyr Pro Ala Asp Arg Arg Ile Gly Tyr Lys Val
Ser Leu 385 390 395 400 aaa atg aga gag tta gga atg ctg aca aga ccg
ctt ggg gac gtg att 1248Lys Met Arg Glu Leu Gly Met Leu Thr Arg Pro
Leu Gly Asp Val Ile 405 410 415 gca ttt ctt cct cct ctt gcc agc aca
gct gaa gag ctc tcg gaa atg 1296Ala Phe Leu Pro Pro Leu Ala Ser Thr
Ala Glu Glu Leu Ser Glu Met 420 425 430 gtt gcc att atg aaa caa gcg
atc cac gag gtt acg agc ctt gaa gat 1344Val Ala Ile Met Lys Gln Ala
Ile His Glu Val Thr Ser Leu Glu Asp 435 440 445 tga 1347
17448PRTBacillus subtilis 17Met Thr His Asp Leu Ile Glu Lys Ser Lys
Lys His Leu Trp Leu Pro 1 5 10 15 Phe Thr Gln Met Lys Asp Tyr Asp
Glu Asn Pro Leu Ile Ile Glu Ser 20 25 30 Gly Thr Gly Ile Lys Val
Lys Asp Ile Asn Gly Lys Glu Tyr Tyr Asp 35 40 45 Gly Phe Ser Ser
Val Trp Leu Asn Val His Gly His Arg Lys Lys Glu 50 55 60 Leu Asp
Asp Ala Ile Lys Lys Gln Leu Gly Lys Ile Ala His Ser Thr 65 70 75 80
Leu Leu Gly Met Thr Asn Val Pro Ala Thr Gln Leu Ala Glu Thr Leu 85
90 95 Ile Asp Ile Ser Pro Lys Lys Leu Thr Arg Val Phe Tyr Ser Asp
Ser 100 105 110 Gly Ala Glu Ala Met Glu Ile Ala Leu Lys Met Ala Phe
Gln Tyr Trp 115 120 125 Lys Asn Ile Gly Lys Pro Glu Lys Gln Lys Phe
Ile Ala Met Lys Asn 130 135 140 Gly Tyr His Gly Asp Thr Ile Gly Ala
Val Ser Val Gly Ser Ile Glu 145 150 155 160 Leu Phe His His Val Tyr
Gly Pro Leu Met Phe Glu Ser Tyr Lys Ala 165 170 175 Pro Ile Pro Tyr
Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys 180 185 190 Arg Asp
Gln Cys Leu Arg Glu Leu Ala Gln Leu Leu Glu Glu His His 195 200 205
Glu Glu Ile Ala Ala Leu Ser Ile Glu Ser Met Val Gln Gly Ala Ser 210
215 220 Gly Met Ile Val Met Pro Glu Gly Tyr Leu Ala Gly Val Arg Glu
Leu 225 230 235 240 Cys Thr Thr Tyr Asp Val Leu Met Ile Val Asp Glu
Val Ala Thr Gly 245 250 255 Phe Gly Arg Thr Gly Lys Met Phe Ala Cys
Glu His Glu Asn Val Gln 260 265 270 Pro Asp Leu Met Ala Ala Gly Lys
Gly Ile Thr Gly Gly Tyr Leu Pro 275 280 285 Ile Ala Val Thr Phe Ala
Thr Glu Asp Ile Tyr Lys Ala Phe Tyr Asp 290 295 300 Asp Tyr Glu Asn
Leu Lys Thr Phe Phe His Gly His Ser Tyr Thr Gly 305 310 315 320 Asn
Gln Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu 325 330
335 Ser Glu Asn Ile Val Glu Gln Val Ala Glu Lys Ser Lys Lys Leu His
340 345 350 Phe Leu Leu Gln Asp Leu His Ala Leu Pro His Val Gly Asp
Ile Arg 355 360 365 Gln Leu Gly Phe Met Cys Gly Ala Glu Leu Val Arg
Ser Lys Glu Thr 370 375 380 Lys Glu Pro Tyr Pro Ala Asp Arg Arg Ile
Gly Tyr Lys Val Ser Leu 385 390 395 400 Lys Met Arg Glu Leu Gly Met
Leu Thr Arg Pro Leu Gly Asp Val Ile 405 410 415 Ala Phe Leu Pro Pro
Leu Ala Ser Thr Ala Glu Glu Leu Ser Glu Met 420 425 430 Val Ala Ile
Met Lys Gln Ala Ile His Glu Val Thr Ser Leu Glu Asp 435 440 445
181467DNARhodobacter sphaeroidesCDS(1)..(1467) 18atg ccc ggt tgc
ggg ggc ttg ccc ggg aat gaa ccg aaa tgc gga cga 48Met Pro Gly Cys
Gly Gly Leu Pro Gly Asn Glu Pro Lys Cys Gly Arg 1 5 10 15 gag ggg
agg tcg gcg atg acg cgg aat gac gcg acg aat gct gcc gga 96Glu Gly
Arg Ser Ala Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly 20 25 30
gcg gtg ggc gcg gcg atg cgg gat cac atc ctc ttg cct gca cag gaa
144Ala Val Gly Ala Ala Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu
35 40 45 atg gcg aag ctc ggc aag tcc gcg cag ccg gtg ctg act cat
gcc gag 192Met Ala Lys Leu Gly Lys Ser Ala Gln Pro Val Leu Thr His
Ala Glu 50 55 60 ggc atc tat gtc cat acc gag gac ggc cgc cgc ctg
atc gac ggg ccg 240Gly Ile Tyr Val His Thr Glu Asp Gly Arg Arg Leu
Ile Asp Gly Pro 65 70 75 80 gcg ggc atg tgg tgc gcg cag gtg ggc tac
ggc cgc cgc gag atc gtc 288Ala Gly Met Trp Cys Ala Gln Val Gly Tyr
Gly Arg Arg Glu Ile Val 85 90 95 gat gcc atg gcg cat cag gcg atg
gtg ctg ccc tat gcc tcg ccc tgg 336Asp Ala Met Ala His Gln Ala Met
Val Leu Pro Tyr Ala Ser Pro Trp 100 105 110 tat atg gcc acg agc ccc
gcg gcg cgg ctg gcg gag aag atc gcc acg 384Tyr Met Ala Thr Ser Pro
Ala Ala Arg Leu Ala Glu Lys Ile Ala Thr 115 120 125 ctg acg ccg ggc
gat ctc aac cgg atc ttt ttc acc acg ggc ggg tcg 432Leu Thr Pro Gly
Asp Leu Asn Arg Ile Phe Phe Thr Thr Gly Gly Ser 130 135 140 acc gcg
gtg gac agc gcg ctg cgc ttc tcg gaa ttc tac aac aac gtg 480Thr Ala
Val Asp Ser Ala Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val 145 150 155
160 ctg ggc cgg ccg cag aag aag cgc atc atc gtg cgc tac gac ggc tat
528Leu Gly Arg Pro Gln Lys Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr
165 170 175 cac ggc tcg acg gcg ctc acc gcc gcc tgc acc ggc cgc acc
ggc aac 576His Gly Ser Thr Ala Leu Thr Ala Ala Cys Thr Gly Arg Thr
Gly Asn 180 185 190 tgg ccg aac ttc gac atc gcg cag gac cgg atc tcg
ttc ctc tcg agc 624Trp Pro Asn Phe Asp Ile Ala Gln Asp Arg Ile Ser
Phe Leu Ser Ser 195 200 205 ccc aat ccg cgc cac gcc ggc aac cgc agc
cag gag gcg ttc ctc gac 672Pro Asn Pro Arg His Ala Gly Asn Arg Ser
Gln Glu Ala Phe Leu Asp 210 215 220 gat ctg gtg cag gaa ttc gag gac
cgg atc gag agc ctc ggc ccc gac 720Asp Leu Val Gln Glu Phe Glu Asp
Arg Ile Glu Ser Leu Gly Pro Asp 225 230 235 240 acg atc gcg gcc ttc
ctg gcc gag ccg atc ctc gcc tcg ggc ggc gtc 768Thr Ile Ala Ala Phe
Leu Ala Glu Pro Ile Leu Ala Ser Gly Gly Val 245 250 255 att att ccg
ccc gca ggc tat cat gcg cgc ttc aag gcg atc tgc gag 816Ile Ile Pro
Pro Ala Gly Tyr His Ala Arg Phe Lys Ala Ile Cys Glu 260 265 270 aag
cac gac atc ctc tat atc tcg gac gag gtg gtg acg ggc ttc ggc 864Lys
His Asp Ile Leu Tyr Ile Ser Asp Glu Val Val Thr Gly Phe Gly 275 280
285 cgt tgc ggc gag tgg ttc gcc tcg gag aag gtg ttc ggg gtg gtg ccg
912Arg Cys Gly Glu Trp Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro
290 295 300 gac atc atc acc ttc gcc aag ggc gtg acc tcg ggc tat gtg
ccg ctc 960Asp Ile Ile Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val
Pro Leu 305 310 315 320 ggc ggc ctt gcg atc tcc gag gcg gtg ctg gcg
cgg atc tcg ggc gag 1008Gly Gly Leu Ala Ile Ser Glu Ala Val Leu Ala
Arg Ile Ser Gly Glu 325 330 335 aat gcc aag gga agc tgg ttc acc aac
ggc tat acc tac agc aat cag 1056Asn Ala Lys Gly Ser Trp Phe Thr Asn
Gly Tyr Thr Tyr Ser Asn Gln 340 345 350 ccg gtg gcc tgc gcc gcg gcg
ctt gcc aac atc gag ctg atg gag cgc 1104Pro Val Ala Cys Ala Ala Ala
Leu Ala Asn Ile Glu Leu Met Glu Arg 355 360 365 gag ggc atc gtc gat
cag gcg cgc gag atg gcg gac tat ttc gcc gcg 1152Glu Gly Ile Val Asp
Gln Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala 370 375 380 gcg ctg gct
tcg ctg cgc gat ctg ccg ggc gtg gcg gaa acc cgg tcg 1200Ala Leu Ala
Ser Leu Arg Asp Leu Pro Gly Val Ala Glu Thr Arg Ser 385 390 395 400
gtg ggc ctc gtg ggt tgc gtg caa tgc ctg ctc gac ccg acc cgg gcg
1248Val Gly Leu Val Gly Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala
405 410 415 gac ggc acg gcc gag gac aag gcc ttc acc ctg aag atc gac
gag cgc 1296Asp Gly Thr Ala Glu Asp Lys Ala Phe Thr Leu Lys Ile Asp
Glu Arg 420 425 430 tgc ttc gag ctc ggg ctg atc gtg cgc ccg ctg ggc
gat ctc tgc gtg 1344Cys Phe Glu Leu Gly Leu Ile Val Arg Pro Leu Gly
Asp Leu Cys Val 435 440 445 atc tcg ccg ccg ctc atc atc tcg cgc gcg
cag atc gac gag atg gtc 1392Ile Ser Pro Pro Leu Ile Ile Ser Arg Ala
Gln Ile Asp Glu Met Val 450 455 460 gcg atc atg cgg cag gcc atc acc
gaa gtg agc gcc gcc cac ggt ctg 1440Ala Ile Met Arg Gln Ala Ile Thr
Glu Val Ser Ala Ala His Gly Leu 465 470 475 480 acc gcg aaa gaa ccg
gcc gcc gtc tga 1467Thr Ala Lys Glu Pro Ala Ala Val 485
19488PRTRhodobacter sphaeroides 19Met Pro Gly Cys Gly Gly Leu Pro
Gly Asn Glu Pro Lys Cys Gly Arg 1 5 10 15 Glu Gly Arg Ser Ala Met
Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly 20 25 30 Ala Val Gly Ala
Ala Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu 35 40 45 Met Ala
Lys Leu Gly Lys Ser Ala Gln Pro Val Leu Thr His Ala Glu 50 55 60
Gly Ile Tyr Val His Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro 65
70 75 80 Ala Gly Met Trp Cys Ala Gln Val Gly Tyr Gly Arg Arg Glu
Ile Val 85 90 95 Asp Ala Met Ala His Gln Ala Met Val Leu Pro Tyr
Ala Ser Pro Trp 100 105 110 Tyr Met Ala Thr Ser Pro Ala Ala Arg Leu
Ala Glu Lys Ile Ala Thr 115 120 125 Leu Thr Pro Gly Asp Leu Asn Arg
Ile Phe Phe Thr Thr Gly Gly Ser 130 135 140 Thr Ala Val Asp Ser Ala
Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val 145 150 155 160 Leu Gly Arg
Pro Gln Lys Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr 165 170 175 His
Gly Ser Thr Ala Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn 180 185
190 Trp Pro Asn Phe Asp Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser
195 200 205 Pro Asn Pro Arg His Ala Gly Asn Arg Ser Gln Glu Ala Phe
Leu Asp 210 215 220 Asp Leu Val Gln Glu Phe Glu Asp Arg Ile Glu Ser
Leu Gly Pro Asp 225 230 235 240 Thr Ile Ala Ala Phe Leu Ala Glu Pro
Ile Leu Ala Ser Gly Gly Val 245 250 255 Ile Ile Pro Pro Ala Gly Tyr
His Ala Arg Phe Lys Ala Ile Cys Glu 260 265 270 Lys His Asp Ile Leu
Tyr Ile Ser Asp Glu Val Val Thr Gly Phe Gly 275 280 285 Arg Cys Gly
Glu Trp Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro 290 295 300 Asp
Ile Ile Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu 305 310
315 320 Gly Gly Leu Ala Ile Ser Glu Ala Val Leu Ala Arg Ile Ser Gly
Glu 325 330 335 Asn Ala Lys Gly Ser Trp Phe Thr Asn Gly Tyr Thr Tyr
Ser Asn Gln 340 345 350 Pro Val Ala Cys Ala Ala Ala Leu Ala Asn Ile
Glu Leu Met Glu Arg 355 360 365 Glu Gly Ile Val Asp Gln Ala Arg Glu
Met Ala Asp Tyr Phe Ala Ala 370 375 380 Ala Leu Ala Ser Leu Arg Asp
Leu Pro Gly Val Ala Glu Thr Arg Ser 385 390 395 400 Val Gly Leu Val
Gly Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala 405 410 415 Asp Gly
Thr Ala Glu Asp Lys Ala Phe Thr Leu Lys Ile Asp Glu Arg 420 425 430
Cys Phe Glu Leu Gly Leu Ile Val Arg Pro Leu Gly Asp Leu Cys Val 435
440 445 Ile Ser Pro Pro Leu Ile Ile Ser Arg Ala Gln Ile Asp Glu Met
Val 450 455 460 Ala Ile Met Arg Gln Ala Ile Thr Glu Val Ser Ala Ala
His Gly Leu 465 470 475 480 Thr Ala Lys Glu Pro Ala Ala Val 485
20837DNALegionella pneumophilaCDS(1)..(837) 20atg agt atc gca ttt
gtt aac ggc aag tat tgt tgt caa tct gaa gca 48Met Ser Ile Ala Phe
Val Asn Gly Lys Tyr Cys Cys Gln Ser Glu Ala 1 5 10 15 aaa att tca
ata ttt gat cga ggg ttt ctt ttt ggt gac tcg gtt tat 96Lys Ile Ser
Ile Phe Asp Arg Gly Phe Leu Phe Gly Asp Ser Val Tyr 20 25 30 gaa
gtg ctg cct gtt tac cat ggg cag cct tac ttt gta gac caa cat 144Glu
Val Leu Pro Val Tyr His Gly Gln Pro Tyr Phe Val Asp Gln His 35 40
45 ctt gac cga tta ttc tca aat atg aaa aaa att aag atg att ata cca
192Leu Asp Arg Leu Phe Ser Asn Met Lys Lys Ile Lys Met Ile Ile Pro
50 55 60 aat tat gat tgg cat ggt tta att cat aga cta ata tca gaa
aat aat 240Asn Tyr Asp Trp His Gly Leu Ile His Arg Leu Ile Ser Glu
Asn Asn 65 70 75
80 ggc ggt aat tta caa gta tat atc caa gtc aca cga ggg aat caa ggg
288Gly Gly Asn Leu Gln Val Tyr Ile Gln Val Thr Arg Gly Asn Gln Gly
85 90 95 gtg cgc aag cat gat atc cct act tcc atc aca cct tct gtt
atc gca 336Val Arg Lys His Asp Ile Pro Thr Ser Ile Thr Pro Ser Val
Ile Ala 100 105 110 ttc act atg cat aat cca ttt ccc acc ctc gaa gat
aag gaa cag gga 384Phe Thr Met His Asn Pro Phe Pro Thr Leu Glu Asp
Lys Glu Gln Gly 115 120 125 atg tca gca aaa ctg gtt gaa gat ttt cgg
tgg atg aga tgt gat ata 432Met Ser Ala Lys Leu Val Glu Asp Phe Arg
Trp Met Arg Cys Asp Ile 130 135 140 aaa act act tct tta att gcc aat
ata tta ctg aat gat gag gct gta 480Lys Thr Thr Ser Leu Ile Ala Asn
Ile Leu Leu Asn Asp Glu Ala Val 145 150 155 160 tct gca gga ttc cac
act gca att ctt gcc cgg aac ggt cta att aca 528Ser Ala Gly Phe His
Thr Ala Ile Leu Ala Arg Asn Gly Leu Ile Thr 165 170 175 gag gga agt
agt acc aac gta ttt att gtc gca cag gat ggt gtt att 576Glu Gly Ser
Ser Thr Asn Val Phe Ile Val Ala Gln Asp Gly Val Ile 180 185 190 aag
aca cca ccc atg aat aat ttc tgt tta cca gga att act cgg caa 624Lys
Thr Pro Pro Met Asn Asn Phe Cys Leu Pro Gly Ile Thr Arg Gln 195 200
205 gtt gtt att gaa ata att aaa aaa tta gat tta aag ttc aga gaa ata
672Val Val Ile Glu Ile Ile Lys Lys Leu Asp Leu Lys Phe Arg Glu Ile
210 215 220 gaa att agc att tca gag ctt ttt tct gct cag gaa gtt tgg
ata aca 720Glu Ile Ser Ile Ser Glu Leu Phe Ser Ala Gln Glu Val Trp
Ile Thr 225 230 235 240 agt acg aca aaa gaa gta ttc cct att aca aag
att aat gac tct ttg 768Ser Thr Thr Lys Glu Val Phe Pro Ile Thr Lys
Ile Asn Asp Ser Leu 245 250 255 att aat ggc gga aaa gtt ggc gaa tat
tgg cgg ata att aat gat tcc 816Ile Asn Gly Gly Lys Val Gly Glu Tyr
Trp Arg Ile Ile Asn Asp Ser 260 265 270 tac caa caa cta gta aac taa
837Tyr Gln Gln Leu Val Asn 275 21278PRTLegionella pneumophila 21Met
Ser Ile Ala Phe Val Asn Gly Lys Tyr Cys Cys Gln Ser Glu Ala 1 5 10
15 Lys Ile Ser Ile Phe Asp Arg Gly Phe Leu Phe Gly Asp Ser Val Tyr
20 25 30 Glu Val Leu Pro Val Tyr His Gly Gln Pro Tyr Phe Val Asp
Gln His 35 40 45 Leu Asp Arg Leu Phe Ser Asn Met Lys Lys Ile Lys
Met Ile Ile Pro 50 55 60 Asn Tyr Asp Trp His Gly Leu Ile His Arg
Leu Ile Ser Glu Asn Asn 65 70 75 80 Gly Gly Asn Leu Gln Val Tyr Ile
Gln Val Thr Arg Gly Asn Gln Gly 85 90 95 Val Arg Lys His Asp Ile
Pro Thr Ser Ile Thr Pro Ser Val Ile Ala 100 105 110 Phe Thr Met His
Asn Pro Phe Pro Thr Leu Glu Asp Lys Glu Gln Gly 115 120 125 Met Ser
Ala Lys Leu Val Glu Asp Phe Arg Trp Met Arg Cys Asp Ile 130 135 140
Lys Thr Thr Ser Leu Ile Ala Asn Ile Leu Leu Asn Asp Glu Ala Val 145
150 155 160 Ser Ala Gly Phe His Thr Ala Ile Leu Ala Arg Asn Gly Leu
Ile Thr 165 170 175 Glu Gly Ser Ser Thr Asn Val Phe Ile Val Ala Gln
Asp Gly Val Ile 180 185 190 Lys Thr Pro Pro Met Asn Asn Phe Cys Leu
Pro Gly Ile Thr Arg Gln 195 200 205 Val Val Ile Glu Ile Ile Lys Lys
Leu Asp Leu Lys Phe Arg Glu Ile 210 215 220 Glu Ile Ser Ile Ser Glu
Leu Phe Ser Ala Gln Glu Val Trp Ile Thr 225 230 235 240 Ser Thr Thr
Lys Glu Val Phe Pro Ile Thr Lys Ile Asn Asp Ser Leu 245 250 255 Ile
Asn Gly Gly Lys Val Gly Glu Tyr Trp Arg Ile Ile Asn Asp Ser 260 265
270 Tyr Gln Gln Leu Val Asn 275 22861DNANitrosomonas
europaeaCDS(1)..(861) 22atg att tac ctc aat ggc aaa ttt ctg ccg atg
gaa cag gct acc gtt 48Met Ile Tyr Leu Asn Gly Lys Phe Leu Pro Met
Glu Gln Ala Thr Val 1 5 10 15 cca gtg ctg gat aga ggc ttc atc ttc
ggt gat ggt gtc tat gaa gtc 96Pro Val Leu Asp Arg Gly Phe Ile Phe
Gly Asp Gly Val Tyr Glu Val 20 25 30 ata ccg gtt tat tca cgt aaa
ccg ttc cgg ctg ggc gaa cat ctt tcc 144Ile Pro Val Tyr Ser Arg Lys
Pro Phe Arg Leu Gly Glu His Leu Ser 35 40 45 cgg ctg cag cac agt
ctg gat ggc ata cgt ctc cag aat ccg cac act 192Arg Leu Gln His Ser
Leu Asp Gly Ile Arg Leu Gln Asn Pro His Thr 50 55 60 gaa gaa caa
tgg gct ggt ctg atc gaa cgc atc atc gag ctg aat gaa 240Glu Glu Gln
Trp Ala Gly Leu Ile Glu Arg Ile Ile Glu Leu Asn Glu 65 70 75 80 ggt
gat gat cag tac ctt tac ctg cac att aca cgc ggg gtg gca aaa 288Gly
Asp Asp Gln Tyr Leu Tyr Leu His Ile Thr Arg Gly Val Ala Lys 85 90
95 cgt gac cat gcc ttt cct cgc gaa gta acg ccc act gtc ttc atc atg
336Arg Asp His Ala Phe Pro Arg Glu Val Thr Pro Thr Val Phe Ile Met
100 105 110 agc aac ccg ctt ccg gct cca cct gca aaa ttg ctc gtt tcc
gga gtt 384Ser Asn Pro Leu Pro Ala Pro Pro Ala Lys Leu Leu Val Ser
Gly Val 115 120 125 tca gcg att acc gcc agg gat aat cgc tgg ggg cgc
tgt gat atc aaa 432Ser Ala Ile Thr Ala Arg Asp Asn Arg Trp Gly Arg
Cys Asp Ile Lys 130 135 140 gcc att tca ctg ttg cca aat atc tta ttg
cgc cag ctt gcc gtg gac 480Ala Ile Ser Leu Leu Pro Asn Ile Leu Leu
Arg Gln Leu Ala Val Asp 145 150 155 160 gca caa gcc atg gaa acg atc
ctg tta cgc gat ggt ctg ttg acc gaa 528Ala Gln Ala Met Glu Thr Ile
Leu Leu Arg Asp Gly Leu Leu Thr Glu 165 170 175 ggg gcc gcc agc aat
att ttc atc gta aaa gac gac ctg ctg ctg acc 576Gly Ala Ala Ser Asn
Ile Phe Ile Val Lys Asp Asp Leu Leu Leu Thr 180 185 190 ccc ccc aaa
gat cac cgt ata ttg cct ggc att act tat gat gta gta 624Pro Pro Lys
Asp His Arg Ile Leu Pro Gly Ile Thr Tyr Asp Val Val 195 200 205 ctg
gaa ctg gct gaa aca cat ggt gtt cca cat gcg aca aga gaa ata 672Leu
Glu Leu Ala Glu Thr His Gly Val Pro His Ala Thr Arg Glu Ile 210 215
220 tca gag ctt gag tta cgt act gca cgg gaa atc atg ctg act tct tcc
720Ser Glu Leu Glu Leu Arg Thr Ala Arg Glu Ile Met Leu Thr Ser Ser
225 230 235 240 acc aaa gaa att ctc ccg atc aca cag ctg gat gga caa
ccg atc ggt 768Thr Lys Glu Ile Leu Pro Ile Thr Gln Leu Asp Gly Gln
Pro Ile Gly 245 250 255 aat ggc acc cca ggg cca gta ttt cag caa ctg
gat cgg ctc tat cag 816Asn Gly Thr Pro Gly Pro Val Phe Gln Gln Leu
Asp Arg Leu Tyr Gln 260 265 270 gca tat aag ctg gaa gtc atg cgc ggg
cat gct cca cgc cag taa 861Ala Tyr Lys Leu Glu Val Met Arg Gly His
Ala Pro Arg Gln 275 280 285 23286PRTNitrosomonas europaea 23Met Ile
Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gln Ala Thr Val 1 5 10 15
Pro Val Leu Asp Arg Gly Phe Ile Phe Gly Asp Gly Val Tyr Glu Val 20
25 30 Ile Pro Val Tyr Ser Arg Lys Pro Phe Arg Leu Gly Glu His Leu
Ser 35 40 45 Arg Leu Gln His Ser Leu Asp Gly Ile Arg Leu Gln Asn
Pro His Thr 50 55 60 Glu Glu Gln Trp Ala Gly Leu Ile Glu Arg Ile
Ile Glu Leu Asn Glu 65 70 75 80 Gly Asp Asp Gln Tyr Leu Tyr Leu His
Ile Thr Arg Gly Val Ala Lys 85 90 95 Arg Asp His Ala Phe Pro Arg
Glu Val Thr Pro Thr Val Phe Ile Met 100 105 110 Ser Asn Pro Leu Pro
Ala Pro Pro Ala Lys Leu Leu Val Ser Gly Val 115 120 125 Ser Ala Ile
Thr Ala Arg Asp Asn Arg Trp Gly Arg Cys Asp Ile Lys 130 135 140 Ala
Ile Ser Leu Leu Pro Asn Ile Leu Leu Arg Gln Leu Ala Val Asp 145 150
155 160 Ala Gln Ala Met Glu Thr Ile Leu Leu Arg Asp Gly Leu Leu Thr
Glu 165 170 175 Gly Ala Ala Ser Asn Ile Phe Ile Val Lys Asp Asp Leu
Leu Leu Thr 180 185 190 Pro Pro Lys Asp His Arg Ile Leu Pro Gly Ile
Thr Tyr Asp Val Val 195 200 205 Leu Glu Leu Ala Glu Thr His Gly Val
Pro His Ala Thr Arg Glu Ile 210 215 220 Ser Glu Leu Glu Leu Arg Thr
Ala Arg Glu Ile Met Leu Thr Ser Ser 225 230 235 240 Thr Lys Glu Ile
Leu Pro Ile Thr Gln Leu Asp Gly Gln Pro Ile Gly 245 250 255 Asn Gly
Thr Pro Gly Pro Val Phe Gln Gln Leu Asp Arg Leu Tyr Gln 260 265 270
Ala Tyr Lys Leu Glu Val Met Arg Gly His Ala Pro Arg Gln 275 280 285
241293DNANeisseria gonorrhoeaeCDS(1)..(1293) 24atg agg ata aat atg
aac cgt aac gaa att tta ttc gac cgc gcc aag 48Met Arg Ile Asn Met
Asn Arg Asn Glu Ile Leu Phe Asp Arg Ala Lys 1 5 10 15 gcc atc atc
ccc ggc ggc gtg aat tcg ccc gtg cgc gca ttc ggc agc 96Ala Ile Ile
Pro Gly Gly Val Asn Ser Pro Val Arg Ala Phe Gly Ser 20 25 30 gtc
ggc ggc gtg ccg cgc ttc atc aaa aaa gcc gaa ggc gcg tat gtt 144Val
Gly Gly Val Pro Arg Phe Ile Lys Lys Ala Glu Gly Ala Tyr Val 35 40
45 tgg gac gaa aac ggc acg cgc tac acc gat tat gtc ggc tct tgg ggg
192Trp Asp Glu Asn Gly Thr Arg Tyr Thr Asp Tyr Val Gly Ser Trp Gly
50 55 60 cct gcg att gtc gga cac gcg cat ccc gaa gtc gtc gaa gcc
gtg cgc 240Pro Ala Ile Val Gly His Ala His Pro Glu Val Val Glu Ala
Val Arg 65 70 75 80 gaa gct gcg ttg ggc ggt ttg tcg ttc ggc gcg ccc
acc gaa ggc gaa 288Glu Ala Ala Leu Gly Gly Leu Ser Phe Gly Ala Pro
Thr Glu Gly Glu 85 90 95 atc gcc att gcc gaa caa att gcc gaa att
atg ccg tct gtc gaa cgg 336Ile Ala Ile Ala Glu Gln Ile Ala Glu Ile
Met Pro Ser Val Glu Arg 100 105 110 ctg cgc ctc gtc agc tcc ggc acg
gaa gcg acg atg act gcc atc cgt 384Leu Arg Leu Val Ser Ser Gly Thr
Glu Ala Thr Met Thr Ala Ile Arg 115 120 125 ctg gca cgc ggt ttt acc
ggc cgc gac aaa atc atc aaa ttt gaa ggc 432Leu Ala Arg Gly Phe Thr
Gly Arg Asp Lys Ile Ile Lys Phe Glu Gly 130 135 140 tgc tac cac ggc
cat tcc gac agc ctg ttg gtg aaa gca ggc agc ggt 480Cys Tyr His Gly
His Ser Asp Ser Leu Leu Val Lys Ala Gly Ser Gly 145 150 155 160 ctg
ctt acc ttc ggc aat cct tct tcc gcc ggt gtg cct gcc gac ttt 528Leu
Leu Thr Phe Gly Asn Pro Ser Ser Ala Gly Val Pro Ala Asp Phe 165 170
175 acc aaa cat act ttg gta ctc gaa tac aac aac atc gcc caa ctc gaa
576Thr Lys His Thr Leu Val Leu Glu Tyr Asn Asn Ile Ala Gln Leu Glu
180 185 190 gaa gcc ttt gcc caa agc ggc gac gaa atc gcc tgc gtg att
gtc gaa 624Glu Ala Phe Ala Gln Ser Gly Asp Glu Ile Ala Cys Val Ile
Val Glu 195 200 205 ccc ttc gtc ggc aat atg aac ctc gtc cgc ccg acc
gaa gcc ttt gtc 672Pro Phe Val Gly Asn Met Asn Leu Val Arg Pro Thr
Glu Ala Phe Val 210 215 220 aaa gcc ttg cgc gga ttg acc gaa aaa cac
ggc gcg gtg ttg att tac 720Lys Ala Leu Arg Gly Leu Thr Glu Lys His
Gly Ala Val Leu Ile Tyr 225 230 235 240 gac gaa gtg atg acc ggt ttc
cgc gtc gcg ctc ggc ggc gcg cag tcg 768Asp Glu Val Met Thr Gly Phe
Arg Val Ala Leu Gly Gly Ala Gln Ser 245 250 255 ctg cac ggc atc acg
ccc gac ctg acc acg atg ggc aaa gtc atc ggc 816Leu His Gly Ile Thr
Pro Asp Leu Thr Thr Met Gly Lys Val Ile Gly 260 265 270 ggc ggt atg
ccg ctt gcc gcg ttc ggc gga cgc aaa gac atc atg gaa 864Gly Gly Met
Pro Leu Ala Ala Phe Gly Gly Arg Lys Asp Ile Met Glu 275 280 285 tgt
att tcc ccg ttg ggc ggc gtg tat cag gca ggt aca tta tca ggc 912Cys
Ile Ser Pro Leu Gly Gly Val Tyr Gln Ala Gly Thr Leu Ser Gly 290 295
300 aac ccg att gcc gtc gcc gcc ggc ttg aaa acg ctg gaa atc atc cag
960Asn Pro Ile Ala Val Ala Ala Gly Leu Lys Thr Leu Glu Ile Ile Gln
305 310 315 320 cgc gaa ggc ttc tat gaa aac ctg acc gcc ttg aca caa
cgc ctt gcc 1008Arg Glu Gly Phe Tyr Glu Asn Leu Thr Ala Leu Thr Gln
Arg Leu Ala 325 330 335 aac ggt att gcc gcc gcc aaa gcg cac ggt atc
gag ttt gcc gcc gac 1056Asn Gly Ile Ala Ala Ala Lys Ala His Gly Ile
Glu Phe Ala Ala Asp 340 345 350 agc gtg ggc ggt atg ttc ggt ctg tat
ttc gcc gca cac gtg ccg cga 1104Ser Val Gly Gly Met Phe Gly Leu Tyr
Phe Ala Ala His Val Pro Arg 355 360 365 aac tat gcc gat atg gcg cgc
tcc aat atc gac gct ttc aaa cgc ttc 1152Asn Tyr Ala Asp Met Ala Arg
Ser Asn Ile Asp Ala Phe Lys Arg Phe 370 375 380 ttc cac ggc atg ctc
gac cgc ggc att gcc ttc ggc ccg tcc gct tat 1200Phe His Gly Met Leu
Asp Arg Gly Ile Ala Phe Gly Pro Ser Ala Tyr 385 390 395 400 gaa gcg
ggt ttc gtt tcc gcc gcg cat acg ccc gag ctg att gac gaa 1248Glu Ala
Gly Phe Val Ser Ala Ala His Thr Pro Glu Leu Ile Asp Glu 405 410 415
acg gtt gcg gtt gcg gtt gaa gtg ttc aag gcg atg gct gca tga 1293Thr
Val Ala Val Ala Val Glu Val Phe Lys Ala Met Ala Ala 420 425 430
25430PRTNeisseria gonorrhoeae 25Met Arg Ile Asn Met Asn Arg Asn Glu
Ile Leu Phe Asp Arg Ala Lys 1 5 10 15 Ala Ile Ile Pro Gly Gly Val
Asn Ser Pro Val Arg Ala Phe Gly Ser 20 25 30 Val Gly Gly Val Pro
Arg Phe Ile Lys Lys Ala Glu Gly Ala Tyr Val 35 40 45 Trp Asp Glu
Asn Gly Thr Arg Tyr Thr Asp Tyr Val Gly Ser Trp Gly 50 55 60 Pro
Ala Ile Val Gly His Ala His Pro Glu Val Val Glu Ala Val Arg 65 70
75 80 Glu Ala Ala Leu Gly Gly Leu Ser Phe Gly Ala Pro Thr Glu Gly
Glu 85 90 95 Ile Ala Ile Ala Glu Gln Ile Ala Glu Ile Met Pro Ser
Val Glu Arg 100 105 110 Leu Arg Leu Val Ser Ser Gly Thr Glu Ala Thr
Met Thr Ala Ile Arg 115 120
125 Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys Ile Ile Lys Phe Glu Gly
130 135 140 Cys Tyr His Gly His Ser Asp Ser Leu Leu Val Lys Ala Gly
Ser Gly 145 150 155 160 Leu Leu Thr Phe Gly Asn Pro Ser Ser Ala Gly
Val Pro Ala Asp Phe 165 170 175 Thr Lys His Thr Leu Val Leu Glu Tyr
Asn Asn Ile Ala Gln Leu Glu 180 185 190 Glu Ala Phe Ala Gln Ser Gly
Asp Glu Ile Ala Cys Val Ile Val Glu 195 200 205 Pro Phe Val Gly Asn
Met Asn Leu Val Arg Pro Thr Glu Ala Phe Val 210 215 220 Lys Ala Leu
Arg Gly Leu Thr Glu Lys His Gly Ala Val Leu Ile Tyr 225 230 235 240
Asp Glu Val Met Thr Gly Phe Arg Val Ala Leu Gly Gly Ala Gln Ser 245
250 255 Leu His Gly Ile Thr Pro Asp Leu Thr Thr Met Gly Lys Val Ile
Gly 260 265 270 Gly Gly Met Pro Leu Ala Ala Phe Gly Gly Arg Lys Asp
Ile Met Glu 275 280 285 Cys Ile Ser Pro Leu Gly Gly Val Tyr Gln Ala
Gly Thr Leu Ser Gly 290 295 300 Asn Pro Ile Ala Val Ala Ala Gly Leu
Lys Thr Leu Glu Ile Ile Gln 305 310 315 320 Arg Glu Gly Phe Tyr Glu
Asn Leu Thr Ala Leu Thr Gln Arg Leu Ala 325 330 335 Asn Gly Ile Ala
Ala Ala Lys Ala His Gly Ile Glu Phe Ala Ala Asp 340 345 350 Ser Val
Gly Gly Met Phe Gly Leu Tyr Phe Ala Ala His Val Pro Arg 355 360 365
Asn Tyr Ala Asp Met Ala Arg Ser Asn Ile Asp Ala Phe Lys Arg Phe 370
375 380 Phe His Gly Met Leu Asp Arg Gly Ile Ala Phe Gly Pro Ser Ala
Tyr 385 390 395 400 Glu Ala Gly Phe Val Ser Ala Ala His Thr Pro Glu
Leu Ile Asp Glu 405 410 415 Thr Val Ala Val Ala Val Glu Val Phe Lys
Ala Met Ala Ala 420 425 430 26924DNAPseudomonas
aeruginosaCDS(1)..(924) 26atg tcg atg gcc gat cgt gat ggc gtg atc
tgg tat gac ggt gaa ctg 48Met Ser Met Ala Asp Arg Asp Gly Val Ile
Trp Tyr Asp Gly Glu Leu 1 5 10 15 gtg cag tgg cgc gac gcg acc acg
cac gtg ctg acc cat acc ctg cac 96Val Gln Trp Arg Asp Ala Thr Thr
His Val Leu Thr His Thr Leu His 20 25 30 tat gga atg ggc gtg ttc
gag ggc gtg cgc gcc tac gac acc ccg cag 144Tyr Gly Met Gly Val Phe
Glu Gly Val Arg Ala Tyr Asp Thr Pro Gln 35 40 45 ggc acg gcg atc
ttc cgc ctg cag gcg cat acc gac cgg ctg ttc gac 192Gly Thr Ala Ile
Phe Arg Leu Gln Ala His Thr Asp Arg Leu Phe Asp 50 55 60 tcc gcg
cac atc atg aac atg cag atc ccg tac agc cgc gac gag atc 240Ser Ala
His Ile Met Asn Met Gln Ile Pro Tyr Ser Arg Asp Glu Ile 65 70 75 80
aac gag gcg acc cgc gcc gcc gtg cgc gag aac aac ctg gaa agc gcc
288Asn Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala
85 90 95 tat atc cgc ccg atg gtg ttc tac gga agc gaa ggc atg ggc
ctg cgc 336Tyr Ile Arg Pro Met Val Phe Tyr Gly Ser Glu Gly Met Gly
Leu Arg 100 105 110 gcc agc ggc ctg aag gtc cat gtg atc atc gcc gcc
tgg agc tgg ggc 384Ala Ser Gly Leu Lys Val His Val Ile Ile Ala Ala
Trp Ser Trp Gly 115 120 125 gcc tac atg ggc gag gaa gcc ctg cag caa
ggc atc aag gtg cgc acc 432Ala Tyr Met Gly Glu Glu Ala Leu Gln Gln
Gly Ile Lys Val Arg Thr 130 135 140 agt tcc ttc acc cgc cac cac gtc
aac atc tcg atg acc cgc gcc aag 480Ser Ser Phe Thr Arg His His Val
Asn Ile Ser Met Thr Arg Ala Lys 145 150 155 160 tcc aac ggc gcc tac
atc aac tcg atg ctg gcc ctc cag gaa gcg atc 528Ser Asn Gly Ala Tyr
Ile Asn Ser Met Leu Ala Leu Gln Glu Ala Ile 165 170 175 tcc ggc ggc
gcc gac gag gcc atg atg ctc gat ccg gaa ggc tac gtg 576Ser Gly Gly
Ala Asp Glu Ala Met Met Leu Asp Pro Glu Gly Tyr Val 180 185 190 gcc
gaa ggc tcc ggc gag aac atc ttc atc atc aag gat ggc gtg atc 624Ala
Glu Gly Ser Gly Glu Asn Ile Phe Ile Ile Lys Asp Gly Val Ile 195 200
205 tac acc ccg gaa gtc acc gcc tgc ctg aac ggc atc act cgt aac act
672Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn Gly Ile Thr Arg Asn Thr
210 215 220 atc ctg acc ctg gcc gcc gaa cac ggt ttt aaa ctg gtc gag
aag cgc 720Ile Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu
Lys Arg 225 230 235 240 atc acc cgc gac gag gtg tac atc gcc gac gag
gcc ttc ttc act ggc 768Ile Thr Arg Asp Glu Val Tyr Ile Ala Asp Glu
Ala Phe Phe Thr Gly 245 250 255 act gcc gcg gaa gtc acg ccg atc cgc
gaa gtg gac ggt cgc aag atc 816Thr Ala Ala Glu Val Thr Pro Ile Arg
Glu Val Asp Gly Arg Lys Ile 260 265 270 ggc gcc ggc cgc cgt ggc ccg
gtc acc gaa aag ctg cag aaa gcc tat 864Gly Ala Gly Arg Arg Gly Pro
Val Thr Glu Lys Leu Gln Lys Ala Tyr 275 280 285 ttc gac ctg gtc agc
ggc aag acc gag gcc cac gcc gag tgg cgt acc 912Phe Asp Leu Val Ser
Gly Lys Thr Glu Ala His Ala Glu Trp Arg Thr 290 295 300 ctg gtc aag
taa 924Leu Val Lys 305 27307PRTPseudomonas aeruginosa 27Met Ser Met
Ala Asp Arg Asp Gly Val Ile Trp Tyr Asp Gly Glu Leu 1 5 10 15 Val
Gln Trp Arg Asp Ala Thr Thr His Val Leu Thr His Thr Leu His 20 25
30 Tyr Gly Met Gly Val Phe Glu Gly Val Arg Ala Tyr Asp Thr Pro Gln
35 40 45 Gly Thr Ala Ile Phe Arg Leu Gln Ala His Thr Asp Arg Leu
Phe Asp 50 55 60 Ser Ala His Ile Met Asn Met Gln Ile Pro Tyr Ser
Arg Asp Glu Ile 65 70 75 80 Asn Glu Ala Thr Arg Ala Ala Val Arg Glu
Asn Asn Leu Glu Ser Ala 85 90 95 Tyr Ile Arg Pro Met Val Phe Tyr
Gly Ser Glu Gly Met Gly Leu Arg 100 105 110 Ala Ser Gly Leu Lys Val
His Val Ile Ile Ala Ala Trp Ser Trp Gly 115 120 125 Ala Tyr Met Gly
Glu Glu Ala Leu Gln Gln Gly Ile Lys Val Arg Thr 130 135 140 Ser Ser
Phe Thr Arg His His Val Asn Ile Ser Met Thr Arg Ala Lys 145 150 155
160 Ser Asn Gly Ala Tyr Ile Asn Ser Met Leu Ala Leu Gln Glu Ala Ile
165 170 175 Ser Gly Gly Ala Asp Glu Ala Met Met Leu Asp Pro Glu Gly
Tyr Val 180 185 190 Ala Glu Gly Ser Gly Glu Asn Ile Phe Ile Ile Lys
Asp Gly Val Ile 195 200 205 Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn
Gly Ile Thr Arg Asn Thr 210 215 220 Ile Leu Thr Leu Ala Ala Glu His
Gly Phe Lys Leu Val Glu Lys Arg 225 230 235 240 Ile Thr Arg Asp Glu
Val Tyr Ile Ala Asp Glu Ala Phe Phe Thr Gly 245 250 255 Thr Ala Ala
Glu Val Thr Pro Ile Arg Glu Val Asp Gly Arg Lys Ile 260 265 270 Gly
Ala Gly Arg Arg Gly Pro Val Thr Glu Lys Leu Gln Lys Ala Tyr 275 280
285 Phe Asp Leu Val Ser Gly Lys Thr Glu Ala His Ala Glu Trp Arg Thr
290 295 300 Leu Val Lys 305 281407DNARhodopseudomonas
palustrisCDS(1)..(1407) 28atg aag ctg ata ccg tgc cgc gcc ttt cac
ccc ccg gcc gcg cag tgc 48Met Lys Leu Ile Pro Cys Arg Ala Phe His
Pro Pro Ala Ala Gln Cys 1 5 10 15 atg agg agc gcc atg tta gac aag
atc aag ccc acg tcc gcc gtc aac 96Met Arg Ser Ala Met Leu Asp Lys
Ile Lys Pro Thr Ser Ala Val Asn 20 25 30 gcg ccg aac gat ctc aac
gcg ttc tgg atg ccg ttc acc gcg aac cgg 144Ala Pro Asn Asp Leu Asn
Ala Phe Trp Met Pro Phe Thr Ala Asn Arg 35 40 45 gcc ttc aag cgc
gcg ccg aag atg gtc gtg ggt gcc gaa ggc atg cac 192Ala Phe Lys Arg
Ala Pro Lys Met Val Val Gly Ala Glu Gly Met His 50 55 60 tac atc
acc gcc gat ggt cgc aag atc atc gac gcc gcc tcg ggc atg 240Tyr Ile
Thr Ala Asp Gly Arg Lys Ile Ile Asp Ala Ala Ser Gly Met 65 70 75 80
tgg tgc acc aat gcg ggc cat ggc cgc aag gaa atc gcc gag gcg atc
288Trp Cys Thr Asn Ala Gly His Gly Arg Lys Glu Ile Ala Glu Ala Ile
85 90 95 aag gcg cag gcc gat gaa ctc gac ttc tcg ccg ccg ttc cag
ttc ggc 336Lys Ala Gln Ala Asp Glu Leu Asp Phe Ser Pro Pro Phe Gln
Phe Gly 100 105 110 cag ccg aag gcg ttc gaa ctc gcc agc cgg atc gcc
gat ctg gcg ccg 384Gln Pro Lys Ala Phe Glu Leu Ala Ser Arg Ile Ala
Asp Leu Ala Pro 115 120 125 gaa ggc ctc gat cac gtg ttc ttc tgc aat
tcg ggc tcg gaa gcc ggc 432Glu Gly Leu Asp His Val Phe Phe Cys Asn
Ser Gly Ser Glu Ala Gly 130 135 140 gac acc gcg ctg aag atc gcg gtc
gcc tat cag cag atc aag ggc cag 480Asp Thr Ala Leu Lys Ile Ala Val
Ala Tyr Gln Gln Ile Lys Gly Gln 145 150 155 160 ggc tca cgc acc cgc
ctg atc ggc cgc gag cgc ggc tat cac ggc gtc 528Gly Ser Arg Thr Arg
Leu Ile Gly Arg Glu Arg Gly Tyr His Gly Val 165 170 175 ggc ttc ggc
ggc acc gcg gtc ggc ggc atc ggc aac aac cgc aag atg 576Gly Phe Gly
Gly Thr Ala Val Gly Gly Ile Gly Asn Asn Arg Lys Met 180 185 190 ttc
ggt ccg ctg ctc aac ggc gtc gat cat ctg cct gcg act tat gat 624Phe
Gly Pro Leu Leu Asn Gly Val Asp His Leu Pro Ala Thr Tyr Asp 195 200
205 cgc gac aag cag gct ttc acc atc ggc gag ccg gaa tac ggc gcg cac
672Arg Asp Lys Gln Ala Phe Thr Ile Gly Glu Pro Glu Tyr Gly Ala His
210 215 220 ttc gcc gaa gcg ctt gaa ggc ctc gtc aat ctg cac ggc gcc
aac acc 720Phe Ala Glu Ala Leu Glu Gly Leu Val Asn Leu His Gly Ala
Asn Thr 225 230 235 240 atc gcg gcg gtg atc gtc gag ccg atg gcc ggc
tcc acc ggc gtg ctg 768Ile Ala Ala Val Ile Val Glu Pro Met Ala Gly
Ser Thr Gly Val Leu 245 250 255 ccg gcg ccg aag ggc tat ctc aag aag
ctg cgc gag atc acc aag aag 816Pro Ala Pro Lys Gly Tyr Leu Lys Lys
Leu Arg Glu Ile Thr Lys Lys 260 265 270 cac ggc atc ctg ctg atc ttc
gac gag gtc atc acc ggc tac ggc cgt 864His Gly Ile Leu Leu Ile Phe
Asp Glu Val Ile Thr Gly Tyr Gly Arg 275 280 285 ctc ggc tat gcc ttc
gcg tcc gaa cgt tac ggc gtc acc ccg gac atg 912Leu Gly Tyr Ala Phe
Ala Ser Glu Arg Tyr Gly Val Thr Pro Asp Met 290 295 300 atc acc ttc
gcc aag ggc gtc acc aat ggt gcg gtg ccg atg ggc ggc 960Ile Thr Phe
Ala Lys Gly Val Thr Asn Gly Ala Val Pro Met Gly Gly 305 310 315 320
gtg atc acc tcg gcg gag atc cac gat gcg ttc atg acc ggc ccc gag
1008Val Ile Thr Ser Ala Glu Ile His Asp Ala Phe Met Thr Gly Pro Glu
325 330 335 cac gcg gtc gag ctg gcg cac ggc tac acc tat tcg gcg cat
ccg ctc 1056His Ala Val Glu Leu Ala His Gly Tyr Thr Tyr Ser Ala His
Pro Leu 340 345 350 gcc tgc gcg gcc ggc atc gcc acc ctc gac atc tac
cgc gac gag aag 1104Ala Cys Ala Ala Gly Ile Ala Thr Leu Asp Ile Tyr
Arg Asp Glu Lys 355 360 365 ctg ttc gag cgc gcc aag gcg ctg gag ccg
aag ttt gcc gag gcg gtg 1152Leu Phe Glu Arg Ala Lys Ala Leu Glu Pro
Lys Phe Ala Glu Ala Val 370 375 380 atg tcg ctg aag tcg gcc ccg aac
gtg gtc gac atc cgc acc gtc ggc 1200Met Ser Leu Lys Ser Ala Pro Asn
Val Val Asp Ile Arg Thr Val Gly 385 390 395 400 ctg acg gcg ggt atc
gac ctc gct tcg atc gcc gat gcg gtc ggc aag 1248Leu Thr Ala Gly Ile
Asp Leu Ala Ser Ile Ala Asp Ala Val Gly Lys 405 410 415 cgt ggc ttc
gaa gcg atg aat gcc ggc ttc cac gac cac gag ctg atg 1296Arg Gly Phe
Glu Ala Met Asn Ala Gly Phe His Asp His Glu Leu Met 420 425 430 ctg
cgg atc gcc ggc gac acc ctg gcg ctg acc ccg ccg ctg atc ctc 1344Leu
Arg Ile Ala Gly Asp Thr Leu Ala Leu Thr Pro Pro Leu Ile Leu 435 440
445 agc gag gac cac atc ggt gag atc gtc gac aag gtc ggc aag gtg atc
1392Ser Glu Asp His Ile Gly Glu Ile Val Asp Lys Val Gly Lys Val Ile
450 455 460 cgc gcg gtc gcc tga 1407Arg Ala Val Ala 465
29468PRTRhodopseudomonas palustris 29Met Lys Leu Ile Pro Cys Arg
Ala Phe His Pro Pro Ala Ala Gln Cys 1 5 10 15 Met Arg Ser Ala Met
Leu Asp Lys Ile Lys Pro Thr Ser Ala Val Asn 20 25 30 Ala Pro Asn
Asp Leu Asn Ala Phe Trp Met Pro Phe Thr Ala Asn Arg 35 40 45 Ala
Phe Lys Arg Ala Pro Lys Met Val Val Gly Ala Glu Gly Met His 50 55
60 Tyr Ile Thr Ala Asp Gly Arg Lys Ile Ile Asp Ala Ala Ser Gly Met
65 70 75 80 Trp Cys Thr Asn Ala Gly His Gly Arg Lys Glu Ile Ala Glu
Ala Ile 85 90 95 Lys Ala Gln Ala Asp Glu Leu Asp Phe Ser Pro Pro
Phe Gln Phe Gly 100 105 110 Gln Pro Lys Ala Phe Glu Leu Ala Ser Arg
Ile Ala Asp Leu Ala Pro 115 120 125 Glu Gly Leu Asp His Val Phe Phe
Cys Asn Ser Gly Ser Glu Ala Gly 130 135 140 Asp Thr Ala Leu Lys Ile
Ala Val Ala Tyr Gln Gln Ile Lys Gly Gln 145 150 155 160 Gly Ser Arg
Thr Arg Leu Ile Gly Arg Glu Arg Gly Tyr His Gly Val 165 170 175 Gly
Phe Gly Gly Thr Ala Val Gly Gly Ile Gly Asn Asn Arg Lys Met 180 185
190 Phe Gly Pro Leu Leu Asn Gly Val Asp His Leu Pro Ala Thr Tyr Asp
195 200 205 Arg Asp Lys Gln Ala Phe Thr Ile Gly Glu Pro Glu Tyr Gly
Ala His 210 215 220 Phe Ala Glu Ala Leu Glu Gly Leu Val Asn Leu His
Gly Ala Asn Thr 225 230 235 240 Ile Ala Ala Val Ile Val Glu Pro Met
Ala Gly Ser Thr Gly Val Leu 245 250 255 Pro Ala Pro Lys Gly Tyr Leu
Lys Lys Leu Arg Glu Ile Thr Lys Lys 260 265 270 His Gly Ile Leu Leu
Ile Phe Asp Glu Val Ile Thr Gly Tyr Gly Arg 275 280 285 Leu Gly Tyr
Ala Phe Ala Ser Glu Arg Tyr Gly Val Thr Pro Asp Met 290 295 300 Ile
Thr Phe Ala Lys Gly
Val Thr Asn Gly Ala Val Pro Met Gly Gly 305 310 315 320 Val Ile Thr
Ser Ala Glu Ile His Asp Ala Phe Met Thr Gly Pro Glu 325 330 335 His
Ala Val Glu Leu Ala His Gly Tyr Thr Tyr Ser Ala His Pro Leu 340 345
350 Ala Cys Ala Ala Gly Ile Ala Thr Leu Asp Ile Tyr Arg Asp Glu Lys
355 360 365 Leu Phe Glu Arg Ala Lys Ala Leu Glu Pro Lys Phe Ala Glu
Ala Val 370 375 380 Met Ser Leu Lys Ser Ala Pro Asn Val Val Asp Ile
Arg Thr Val Gly 385 390 395 400 Leu Thr Ala Gly Ile Asp Leu Ala Ser
Ile Ala Asp Ala Val Gly Lys 405 410 415 Arg Gly Phe Glu Ala Met Asn
Ala Gly Phe His Asp His Glu Leu Met 420 425 430 Leu Arg Ile Ala Gly
Asp Thr Leu Ala Leu Thr Pro Pro Leu Ile Leu 435 440 445 Ser Glu Asp
His Ile Gly Glu Ile Val Asp Lys Val Gly Lys Val Ile 450 455 460 Arg
Ala Val Ala 465 301263DNAEscherichia coliCDS(1)..(1263) 30atg cca
cat tca ctg ttc agc acc gat acc gat ctc acc gcc gaa aat 48Met Pro
His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn 1 5 10 15
ctg ctg cgt ttg ccc gct gaa ttt ggc tgc ccg gtg tgg gtc tac gat
96Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp
20 25 30 gcg caa att att cgt cgg cag att gca gcg ctg aaa cag ttt
gat gtg 144Ala Gln Ile Ile Arg Arg Gln Ile Ala Ala Leu Lys Gln Phe
Asp Val 35 40 45 gtg cgc ttt gca cag aaa gcc tgt tcc aat att cat
att ttg cgc tta 192Val Arg Phe Ala Gln Lys Ala Cys Ser Asn Ile His
Ile Leu Arg Leu 50 55 60 atg cgt gag cag ggc gtg aaa gtg gat tcc
gtc tcg tta ggc gaa ata 240Met Arg Glu Gln Gly Val Lys Val Asp Ser
Val Ser Leu Gly Glu Ile 65 70 75 80 gag cgt gcg ttg gcg gcg ggt tac
aat ccg caa acg cac ccc gat gat 288Glu Arg Ala Leu Ala Ala Gly Tyr
Asn Pro Gln Thr His Pro Asp Asp 85 90 95 att gtt ttt acg gca gat
gtt atc gat cag gcg acg ctt gaa cgc gtc 336Ile Val Phe Thr Ala Asp
Val Ile Asp Gln Ala Thr Leu Glu Arg Val 100 105 110 agt gaa ttg caa
att ccg gtg aat gcg ggt tct gtt gat atg ctc gac 384Ser Glu Leu Gln
Ile Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp 115 120 125 caa ctg
ggc cag gtt tcg cca ggg cat cgg gta tgg ctg cgc gtt aat 432Gln Leu
Gly Gln Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn 130 135 140
ccg ggg ttt ggt cac gga cat agc caa aaa acc aat acc ggt ggc gaa
480Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu
145 150 155 160 aac agc aag cac ggt atc tgg tac acc gat ctg ccc gcc
gca ctg gac 528Asn Ser Lys His Gly Ile Trp Tyr Thr Asp Leu Pro Ala
Ala Leu Asp 165 170 175 gtg ata caa cgt cat cat ctg cag ctg gtc ggc
att cac atg cac att 576Val Ile Gln Arg His His Leu Gln Leu Val Gly
Ile His Met His Ile 180 185 190 ggt tct ggc gtt gat tat gcc cat ctg
gaa cag gtg tgt ggt gct atg 624Gly Ser Gly Val Asp Tyr Ala His Leu
Glu Gln Val Cys Gly Ala Met 195 200 205 gtg cgt cag gtc atc gaa ttc
ggt cag gat tta cag gct att tct gcg 672Val Arg Gln Val Ile Glu Phe
Gly Gln Asp Leu Gln Ala Ile Ser Ala 210 215 220 ggc ggt ggg ctt tct
gtt cct tat caa cag ggt gaa gag gcg gtt gat 720Gly Gly Gly Leu Ser
Val Pro Tyr Gln Gln Gly Glu Glu Ala Val Asp 225 230 235 240 acc gaa
cat tat tat ggt ctg tgg aat gcc gcg cgt gag caa atc gcc 768Thr Glu
His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg Glu Gln Ile Ala 245 250 255
cgc cat ttg ggc cac cct gtg aaa ctg gaa att gaa ccg ggt cgc ttc
816Arg His Leu Gly His Pro Val Lys Leu Glu Ile Glu Pro Gly Arg Phe
260 265 270 ctg gta gcg cag tct ggc gta tta att act cag gtg cgg agc
gtc aaa 864Leu Val Ala Gln Ser Gly Val Leu Ile Thr Gln Val Arg Ser
Val Lys 275 280 285 caa atg ggg agc cgc cac ttt gtg ctg gtt gat gcc
ggg ttc aac gat 912Gln Met Gly Ser Arg His Phe Val Leu Val Asp Ala
Gly Phe Asn Asp 290 295 300 ctg atg cgc ccg gca atg tac ggt agt tac
cac cat atc agt gcc ctg 960Leu Met Arg Pro Ala Met Tyr Gly Ser Tyr
His His Ile Ser Ala Leu 305 310 315 320 gca gct gat ggt cgt tct ctg
gaa cac gcg cca acg gtg gaa acc gtc 1008Ala Ala Asp Gly Arg Ser Leu
Glu His Ala Pro Thr Val Glu Thr Val 325 330 335 gtc gcc gga ccg tta
tgt gaa tcg ggc gat gtc ttt acc cag cag gaa 1056Val Ala Gly Pro Leu
Cys Glu Ser Gly Asp Val Phe Thr Gln Gln Glu 340 345 350 ggg gga aat
gtt gaa acc cgc gcc ttg ccg gaa gtg aag gca ggt gat 1104Gly Gly Asn
Val Glu Thr Arg Ala Leu Pro Glu Val Lys Ala Gly Asp 355 360 365 tat
ctg gta ctg cat gat aca ggg gca tat ggc gca tca atg tca tcc 1152Tyr
Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser 370 375
380 aac tac aat agc cgt ccg ctg tta cca gaa gtt ctg ttt gat aat ggt
1200Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu Phe Asp Asn Gly
385 390 395 400 cag gcg cgg ttg att cgc cgt cgc cag acc atc gaa gaa
tta ctg gcg 1248Gln Ala Arg Leu Ile Arg Arg Arg Gln Thr Ile Glu Glu
Leu Leu Ala 405 410 415 ctg gaa ttg ctt taa 1263Leu Glu Leu Leu 420
31420PRTEscherichia coli 31Met Pro His Ser Leu Phe Ser Thr Asp Thr
Asp Leu Thr Ala Glu Asn 1 5 10 15 Leu Leu Arg Leu Pro Ala Glu Phe
Gly Cys Pro Val Trp Val Tyr Asp 20 25 30 Ala Gln Ile Ile Arg Arg
Gln Ile Ala Ala Leu Lys Gln Phe Asp Val 35 40 45 Val Arg Phe Ala
Gln Lys Ala Cys Ser Asn Ile His Ile Leu Arg Leu 50 55 60 Met Arg
Glu Gln Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu Ile 65 70 75 80
Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gln Thr His Pro Asp Asp 85
90 95 Ile Val Phe Thr Ala Asp Val Ile Asp Gln Ala Thr Leu Glu Arg
Val 100 105 110 Ser Glu Leu Gln Ile Pro Val Asn Ala Gly Ser Val Asp
Met Leu Asp 115 120 125 Gln Leu Gly Gln Val Ser Pro Gly His Arg Val
Trp Leu Arg Val Asn 130 135 140 Pro Gly Phe Gly His Gly His Ser Gln
Lys Thr Asn Thr Gly Gly Glu 145 150 155 160 Asn Ser Lys His Gly Ile
Trp Tyr Thr Asp Leu Pro Ala Ala Leu Asp 165 170 175 Val Ile Gln Arg
His His Leu Gln Leu Val Gly Ile His Met His Ile 180 185 190 Gly Ser
Gly Val Asp Tyr Ala His Leu Glu Gln Val Cys Gly Ala Met 195 200 205
Val Arg Gln Val Ile Glu Phe Gly Gln Asp Leu Gln Ala Ile Ser Ala 210
215 220 Gly Gly Gly Leu Ser Val Pro Tyr Gln Gln Gly Glu Glu Ala Val
Asp 225 230 235 240 Thr Glu His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg
Glu Gln Ile Ala 245 250 255 Arg His Leu Gly His Pro Val Lys Leu Glu
Ile Glu Pro Gly Arg Phe 260 265 270 Leu Val Ala Gln Ser Gly Val Leu
Ile Thr Gln Val Arg Ser Val Lys 275 280 285 Gln Met Gly Ser Arg His
Phe Val Leu Val Asp Ala Gly Phe Asn Asp 290 295 300 Leu Met Arg Pro
Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu 305 310 315 320 Ala
Ala Asp Gly Arg Ser Leu Glu His Ala Pro Thr Val Glu Thr Val 325 330
335 Val Ala Gly Pro Leu Cys Glu Ser Gly Asp Val Phe Thr Gln Gln Glu
340 345 350 Gly Gly Asn Val Glu Thr Arg Ala Leu Pro Glu Val Lys Ala
Gly Asp 355 360 365 Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala
Ser Met Ser Ser 370 375 380 Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu
Val Leu Phe Asp Asn Gly 385 390 395 400 Gln Ala Arg Leu Ile Arg Arg
Arg Gln Thr Ile Glu Glu Leu Leu Ala 405 410 415 Leu Glu Leu Leu 420
321265DNAArtificialEscherichia.coli diaminopimelate decarboxylase
LysA codon optimised gene 32atatgccaca ctctctgttt tctactgata
ctgatctgac tgcggaaaac ctgctgcgtc 60tgccggctga attcggttgt ccggtatggg
tgtacgacgc tcagattatt cgtcgccaga 120tcgcagcact gaagcagttc
gatgtagtgc gttttgcaca gaaggcgtgc tccaacatcc 180atatcctgcg
cctgatgcgt gagcagggcg ttaaagttga ctccgtctct ctgggtgaga
240ttgagcgcgc cctggcagcc ggctataacc cacagaccca tcctgacgac
attgtattta 300ctgccgacgt gatcgaccag gctactctgg aacgcgtttc
tgaactgcag atcccggtta 360atgctggttc tgtggacatg ctggaccagc
tgggccaggt atccccaggt catcgtgtgt 420ggctgcgtgt caacccaggt
ttcggccacg gccactctca gaaaactaac actggtggtg 480agaactccaa
gcatggcatt tggtataccg atctgccggc tgcactggac gtaatccagc
540gtcaccacct gcagctggtg ggcatccaca tgcacattgg ctccggcgta
gactacgccc 600acctggagca agtctgcggt gctatggtac gtcaggtaat
cgagttcggc caagatctgc 660aggcaatcag cgctggtggc ggcctgtctg
taccttatca gcagggcgag gaggcggttg 720acactgagca ctactacggt
ctgtggaacg ccgctcgtga gcaaattgca cgtcacctgg 780gccacccggt
gaaactggag atcgagccgg gccgcttcct ggtagcacag tccggcgtac
840tgattaccca ggtacgctct gttaaacaga tgggctcccg tcactttgtg
ctggtagacg 900caggcttcaa cgacctgatg cgtccggcta tgtatggttc
ctatcatcac atctctgcgc 960tggccgccga cggccgctct ctggaacacg
cgccgacggt tgaaacggtg gtggctggtc 1020cgctgtgcga gtccggcgac
gttttcactc agcaggaggg cggcaatgta gagacgcgtg 1080cgctgccgga
agtgaaagcc ggtgattatc tggtgctgca tgataccggc gcctatggtg
1140cgagcatgag cagcaactac aactctcgcc cgctgctgcc ggaggtcctg
ttcgataacg 1200gccaagcccg cctgatccgt cgtcgtcaga ccatcgagga
actgctggca ctggagctgc 1260tgtaa 1265331692DNASaccharomyces
cerevisiaeCDS(1)..(1692) 33atg tct gaa att act ttg ggt aaa tat ttg
ttc gaa aga tta aag caa 48Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu
Phe Glu Arg Leu Lys Gln 1 5 10 15 gtc aac gtt aac acc gtt ttc ggt
ttg cca ggt gac ttc aac ttg tcc 96Val Asn Val Asn Thr Val Phe Gly
Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 ttg ttg gac aag atc tac
gaa gtt gaa ggt atg aga tgg gct ggt aac 144Leu Leu Asp Lys Ile Tyr
Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 gcc aac gaa ttg
aac gct gct tac gcc gct gat ggt tac gct cgt atc 192Ala Asn Glu Leu
Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 aag ggt
atg tct tgt atc atc acc acc ttc ggt gtc ggt gaa ttg tct 240Lys Gly
Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80
gct ttg aac ggt att gcc ggt tct tac gct gaa cac gtc ggt gtt ttg
288Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu
85 90 95 cac gtt gtt ggt gtc cca tcc atc tct gct caa gct aag caa
ttg ttg 336His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln
Leu Leu 100 105 110 ttg cac cac acc ttg ggt aac ggt gac ttc act gtt
ttc cac aga atg 384Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val
Phe His Arg Met 115 120 125 tct gcc aac att tct gaa acc act gct atg
atc act gac att gct acc 432Ser Ala Asn Ile Ser Glu Thr Thr Ala Met
Ile Thr Asp Ile Ala Thr 130 135 140 gcc cca gct gaa att gac aga tgt
atc aga acc act tac gtc acc caa 480Ala Pro Ala Glu Ile Asp Arg Cys
Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 aga cca gtc tac tta
ggt ttg cca gct aac ttg gtc gac ttg aac gtc 528Arg Pro Val Tyr Leu
Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175 cca gct aag
ttg ttg caa act cca att gac atg tct ttg aag cca aac 576Pro Ala Lys
Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190 gat
gct gaa tcc gaa aag gaa gtc att gac acc atc ttg gct ttg gtc 624Asp
Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val 195 200
205 aag gat gct aag aac cca gtt atc ttg gct gat gct tgt tgt tcc aga
672Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg
210 215 220 cac gac gtc aag gct gaa act aag aag ttg att gac ttg act
caa ttc 720His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr
Gln Phe 225 230 235 240 cca gct ttc gtc acc cca atg ggt aag ggt tcc
att gac gaa caa cac 768Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser
Ile Asp Glu Gln His 245 250 255 cca aga tac ggt ggt gtt tac gtc ggt
acc ttg tcc aag cca gaa gtt 816Pro Arg Tyr Gly Gly Val Tyr Val Gly
Thr Leu Ser Lys Pro Glu Val 260 265 270 aag gaa gcc gtt gaa tct gct
gac ttg att ttg tct gtc ggt gct ttg 864Lys Glu Ala Val Glu Ser Ala
Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 ttg tct gat ttc aac
acc ggt tct ttc tct tac tct tac aag acc aag 912Leu Ser Asp Phe Asn
Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 aac att gtc
gaa ttc cac tcc gac cac atg aag atc aga aac gcc act 960Asn Ile Val
Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320
ttc cca ggt gtc caa atg aaa ttc gtt ttg caa aag ttg ttg acc act
1008Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr
325 330 335 att gct gac gcc gct aag ggt tac aag cca gtt gct gtc cca
gct aga 1056Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro
Ala Arg 340 345 350 act cca gct aac gct gct gtc cca gct tct acc cca
ttg aag caa gaa 1104Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro
Leu Lys Gln Glu 355 360 365 tgg atg tgg aac caa ttg ggt aac ttc ttg
caa gaa ggt gat gtt gtc 1152Trp Met Trp Asn Gln Leu Gly Asn Phe Leu
Gln Glu Gly Asp Val Val 370 375 380 att gct gaa acc ggt acc tcc gct
ttc ggt atc aac caa acc act ttc 1200Ile Ala Glu Thr Gly Thr Ser Ala
Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 cca aac aac acc tac
ggt atc tct caa gtc tta tgg ggt tcc att ggt 1248Pro Asn Asn Thr Tyr
Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 ttc acc act
ggt gct acc ttg ggt gct gct ttc gct gct gaa gaa att 1296Phe Thr Thr
Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 gat
cca aag aag aga gtt atc tta ttc att ggt gac ggt tct ttg caa
1344Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln
435 440 445 ttg act gtt caa gaa atc tcc acc atg atc aga tgg ggc ttg
aag cca 1392Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu
Lys Pro 450 455 460 tac ttg ttc gtc ttg aac aac gat ggt tac acc att
gaa aag ttg att 1440Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile
Glu Lys Leu Ile 465 470 475 480 cac ggt cca aag gct caa tac aac gaa
att caa ggt tgg gac cac cta 1488His Gly Pro Lys Ala Gln Tyr Asn Glu
Ile Gln Gly Trp Asp His Leu 485 490 495 tcc ttg ttg cca act ttc ggt
gct aag gac tat gaa acc cac aga gtc 1536Ser Leu Leu Pro Thr Phe Gly
Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 gct acc acc ggt gaa
tgg gac aag ttg acc caa gac aag tct ttc aac 1584Ala Thr Thr Gly Glu
Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525 gac aac tct
aag atc aga atg att gaa atc atg ttg cca gtc ttc gat 1632Asp Asn Ser
Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp 530 535 540 gct
cca caa aac ttg gtt gaa caa gct aag ttg act gct gct acc aac 1680Ala
Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550
555 560 gct aag caa taa 1692Ala Lys Gln 34563PRTSaccharomyces
cerevisiae 34Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg
Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly
Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu
Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala
Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys
Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn
Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His
Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100 105
110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met
115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile
Ala Thr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr
Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala
Asn Leu Val Asp Leu Asn Val 165 170 175 Pro Ala Lys Leu Leu Gln Thr
Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190 Asp Ala Glu Ser Glu
Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val 195 200 205 Lys Asp Ala
Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 His
Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230
235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln
His 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys
Pro Glu Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu
Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe
Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His Ser
Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val
Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr 325 330 335 Ile Ala
Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350
Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355
360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val
Val 370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln
Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val
Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly
Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val
Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln
Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu
Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475
480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu
485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His
Arg Val 500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp
Lys Ser Phe Asn 515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Ile
Met Leu Pro Val Phe Asp 530 535 540 Ala Pro Gln Asn Leu Val Glu Gln
Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln
351692DNAArtificialSaccharomyces cerevisiae pyruvate decarboxylase
Pdc codon optimised gene 35atgtccgaga tcactctggg caaatacctg
tttgaacgtc tgaaacaggt gaacgttaat 60accgtattcg gcctgccggg tgatttcaac
ctgtccctgc tggacaaaat ctatgaagtt 120gaaggtatgc gttgggctgg
caacgctaac gagctgaacg cagcgtacgc ggcagatggt 180tacgctcgta
tcaaaggtat gtcttgtatc atcaccacct tcggtgttgg tgagctgagc
240gccctgaacg gcatcgccgg ctcctatgca gagcacgtgg gcgtgctgca
cgttgtgggt 300gtaccgtcca tcagcgccca ggcaaaacag ctgctgctgc
accacaccct gggtaacggc 360gactttaccg ttttccatcg tatgtctgcg
aacatcagcg aaactactgc aatgattact 420gacatcgcta cggcaccggc
agaaatcgac cgttgcattc gtaccacgta cgttactcag 480cgcccggttt
atctgggcct gccagccaac ctggtggatc tgaacgtccc ggctaaactg
540ctgcagactc cgatcgatat gtctctgaaa cctaacgacg cagaatctga
gaaagaagtt 600atcgatacta ttctggctct ggtgaaagat gcaaagaacc
cagttatcct ggctgacgca 660tgttgctctc gtcatgatgt aaaggcagaa
accaaaaagc tgatcgacct gacgcagttc 720ccggcgttcg ttaccccgat
gggcaagggt tccatcgatg agcagcaccc gcgttatggt 780ggtgtatacg
ttggcacgct gtccaaaccg gaggtaaaag aagcggttga aagcgcagat
840ctgatcctgt ctgttggtgc actgctgagc gacttcaaca ccggttcttt
ctcctatagc 900tacaagacca aaaacattgt ggagtttcac tccgatcaca
tgaaaatccg caacgcgacc 960tttcctggtg tgcagatgaa attcgtactg
cagaaactgc tgaccaccat cgccgacgct 1020gcgaaaggtt ataaaccggt
agctgtgccg gcacgtaccc cggcgaacgc cgcggttcct 1080gcatccactc
cactgaagca ggaatggatg tggaatcagc tgggtaattt cctgcaagaa
1140ggcgacgttg taatcgcaga aaccggcact agcgcgtttg gcattaacca
gacgaccttc 1200ccaaacaaca cctacggtat cagccaagtc ctgtggggct
ctatcggctt caccaccggt 1260gcaaccctgg gtgcggcttt cgctgctgag
gagatcgacc cgaagaaacg tgttatcctg 1320ttcatcggtg acggctccct
gcagctgacc gtccaggaga tttctaccat gatccgctgg 1380ggcctgaaac
cgtacctgtt tgtgctgaac aacgacggct acactattga gaaactgatc
1440cacggtccga aagcacagta taatgagatc cagggttggg atcatctgtc
tctgctgccg 1500acctttggcg ctaaagacta cgagacccac cgcgtggcta
ccaccggcga gtgggataaa 1560ctgacgcagg ataaatcctt caatgacaat
agcaagattc gtatgatcga aatcatgctg 1620ccggtctttg atgctccgca
gaacctggta gagcaagcaa aactgaccgc ggcaactaac 1680gctaaacagt aa
1692361707DNAZymomonas mobilisCDS(1)..(1707) 36atg agt tat act gtc
ggt acc tat tta gcg gag cgg ctt gtc cag att 48Met Ser Tyr Thr Val
Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile 1 5 10 15 ggt ctc aag
cat cac ttc gca gtc gcg ggc gac tac aac ctc gtc ctt 96Gly Leu Lys
His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu 20 25 30 ctt
gac aac ctg ctt ttg aac aaa aac atg gag cag gtt tat tgc tgt 144Leu
Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys 35 40
45 aac gaa ctg aac tgc ggt ttc agt gca gaa ggt tat gct cgt gcc aaa
192Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys
50 55 60 ggc gca gca gca gcc gtc gtt acc tac agc gtc ggt gcg ctt
tcc gca 240Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu
Ser Ala 65 70 75 80 ttt gat gct atc ggt ggc gcc tat gca gaa aac ctt
ccg gtt atc ctg 288Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu
Pro Val Ile Leu 85 90 95 atc tcc ggt gct ccg aac aac aat gat cac
gct gct ggt cac gtg ttg 336Ile Ser Gly Ala Pro Asn Asn Asn Asp His
Ala Ala Gly His Val Leu 100 105 110 cat cac gct ctt ggc aaa acc gac
tat cac tat cag ttg gaa atg gcc 384His His Ala Leu Gly Lys Thr Asp
Tyr His Tyr Gln Leu Glu Met Ala 115 120 125 aag aac atc acg gcc gcc
gct gaa gcg att tac acc ccg gaa gaa gct 432Lys Asn Ile Thr Ala Ala
Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala 130 135 140 ccg gct aaa atc
gat cac gtg att aaa act gct ctt cgt gag aag aag 480Pro Ala Lys Ile
Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys 145 150 155 160 ccg
gtt tat ctc gaa atc gct tgc aac att gct tcc atg ccc tgc gcc 528Pro
Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys Ala 165 170
175 gct cct gga ccg gca agc gca ttg ttc aat gac gaa gcc agc gac gaa
576Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu
180 185 190 gct tct ttg aat gca gcg gtt gaa gaa acc ctg aaa ttc atc
gcc aac 624Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe Ile
Ala Asn 195 200 205 cgc gac aaa gtt gcc gtc ctc gtc ggc agc aag ctg
cgc gca gct ggt 672Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu
Arg Ala Ala Gly 210 215 220 gct gaa gaa gct gct gtc aaa ttt gct gat
gct ctc ggt ggc gca gtt 720Ala Glu Glu Ala Ala Val Lys Phe Ala Asp
Ala Leu Gly Gly Ala Val 225 230 235 240 gct acc atg gct gct gca aaa
agc ttc ttc cca gaa gaa aac ccg cat 768Ala Thr Met Ala Ala Ala Lys
Ser Phe Phe Pro Glu Glu Asn Pro His 245 250 255 tac atc ggc acc tca
tgg ggt gaa gtc agc tat ccg ggc gtt gaa aag 816Tyr Ile Gly Thr Ser
Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys 260 265 270 acg atg aaa
gaa gcc gat gcg gtt atc gct ctg gct cct gtc ttc aac 864Thr Met Lys
Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn 275 280 285 gac
tac tcc acc act ggt tgg acg gat att cct gat cct aag aaa ctg 912Asp
Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu 290 295
300 gtt ctc gct gaa ccg cgt tct gtc gtc gtt aac ggc att cgc ttc ccc
960Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro
305 310 315 320 agc gtc cat ctg aaa gac tat ctg acc cgt ttg gct cag
aaa gtt tcc 1008Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln
Lys Val Ser 325 330 335 aag aaa acc ggt gca ttg gac ttc ttc aaa tcc
ctc aat gca ggt gaa 1056Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys Ser
Leu Asn Ala Gly Glu 340 345 350 ctg aag aaa gcc gct ccg gct gat ccg
agt gct ccg ttg gtc aac gca 1104Leu Lys Lys Ala Ala Pro Ala Asp Pro
Ser Ala Pro Leu Val Asn Ala 355 360 365 gaa atc gcc cgt cag gtc gaa
gct ctt ctg acc ccg aac acg acg gtt 1152Glu Ile Ala Arg Gln Val Glu
Ala Leu Leu Thr Pro Asn Thr Thr Val 370 375 380 att gct gaa acc ggt
gac tct tgg ttc aat gct cag cgc atg aag ctc 1200Ile Ala Glu Thr Gly
Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu 385 390 395 400 ccg aac
ggt gct cgc gtt gaa tat gaa atg cag tgg ggt cac att ggt 1248Pro Asn
Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly 405 410 415
tgg tcc gtt cct gcc gcc ttc ggt tat gcc gtc ggt gct ccg gaa cgt
1296Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg
420 425 430 cgc aac atc ctc atg gtt ggt gat ggt tcc ttc cag ctg acg
gct cag 1344Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr
Ala Gln 435 440 445 gaa gtc gct cag atg gtt cgc ctg aaa ctg ccg gtt
atc atc ttc ttg 1392Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val
Ile Ile Phe Leu 450 455 460 atc aat aac tat ggt tac acc gcc gaa gtt
atg atc cat gat ggt ccg 1440Ile Asn Asn Tyr Gly Tyr Thr Ala Glu Val
Met Ile His Asp Gly Pro 465 470 475 480 tac aac aac atc aag aac tgg
gat tat gcc ggt ctg atg gaa gtg ttc 1488Tyr Asn Asn Ile Lys Asn Trp
Asp Tyr Ala Gly Leu Met Glu Val Phe 485 490 495 aac ggt aac ggt ggt
tat gac agc ggt gct ggt aaa ggc ctg aag gct 1536Asn Gly Asn Gly Gly
Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys Ala 500 505 510 aaa acc ggt
ggc gaa ctg gca gaa gct atc aag gtt gct ctg gca aac 1584Lys Thr Gly
Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn 515 520 525 acc
gac ggc cca acc ctg atc gaa tgc ttc atc ggt cgt gaa gac tgc 1632Thr
Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys 530 535
540 act gaa gaa ttg gtc aaa tgg ggt aag cgc gtt gct gcc gcc aac agc
1680Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser
545 550 555 560 cgt aag cct gtt aac aag ctc ctc tag 1707Arg Lys Pro
Val Asn Lys Leu Leu 565 37568PRTZymomonas mobilis 37Met Ser Tyr Thr
Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile 1 5 10 15 Gly Leu
Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu 20 25 30
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys 35
40 45 Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala
Lys 50 55 60 Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala
Leu Ser Ala 65 70 75 80 Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn
Leu Pro Val Ile Leu 85 90 95 Ile Ser Gly Ala Pro Asn Asn Asn Asp
His Ala Ala Gly His Val Leu 100 105 110 His His Ala Leu Gly Lys Thr
Asp Tyr His Tyr Gln Leu Glu Met Ala 115 120 125 Lys Asn Ile Thr Ala
Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala 130 135 140 Pro Ala Lys
Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys 145 150 155 160
Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys Ala 165
170 175 Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp
Glu 180 185 190 Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe
Ile Ala Asn 195 200 205 Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys
Leu Arg Ala Ala Gly 210 215 220 Ala Glu Glu Ala Ala Val Lys Phe Ala
Asp Ala Leu Gly Gly Ala Val 225 230
235 240 Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro
His 245 250 255 Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly
Val Glu Lys 260 265 270 Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu
Ala Pro Val Phe Asn 275 280 285 Asp Tyr Ser Thr Thr Gly Trp Thr Asp
Ile Pro Asp Pro Lys Lys Leu 290 295 300 Val Leu Ala Glu Pro Arg Ser
Val Val Val Asn Gly Ile Arg Phe Pro 305 310 315 320 Ser Val His Leu
Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser 325 330 335 Lys Lys
Thr Gly Ala Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu 340 345 350
Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala 355
360 365 Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr
Val 370 375 380 Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg
Met Lys Leu 385 390 395 400 Pro Asn Gly Ala Arg Val Glu Tyr Glu Met
Gln Trp Gly His Ile Gly 405 410 415 Trp Ser Val Pro Ala Ala Phe Gly
Tyr Ala Val Gly Ala Pro Glu Arg 420 425 430 Arg Asn Ile Leu Met Val
Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln 435 440 445 Glu Val Ala Gln
Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu 450 455 460 Ile Asn
Asn Tyr Gly Tyr Thr Ala Glu Val Met Ile His Asp Gly Pro 465 470 475
480 Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe
485 490 495 Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Gly Lys Gly Leu
Lys Ala 500 505 510 Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val
Ala Leu Ala Asn 515 520 525 Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe
Ile Gly Arg Glu Asp Cys 530 535 540 Thr Glu Glu Leu Val Lys Trp Gly
Lys Arg Val Ala Ala Ala Asn Ser 545 550 555 560 Arg Lys Pro Val Asn
Lys Leu Leu 565 381707DNAArtificialZymomonas mobilis pyruvate
decarboxylase PdcI472A codon optimised gene 38atgtcttata ctgttggtac
ttatctggct gagcgtctgg tgcaaatcgg cctgaaacac 60cactttgcag ttgctggcga
ctacaacctg gttctgctgg ataacctgct gctgaacaaa 120aacatggagc
aagtttattg ctgtaacgag ctgaactgcg gcttctctgc ggagggttat
180gcgcgtgcga aaggtgccgc tgcagcagtc gtaacctact ctgtgggcgc
tctgtccgcg 240ttcgacgcaa tcggtggcgc ttacgctgaa aacctgccgg
tgatcctgat tagcggtgcg 300ccgaataata acgaccatgc tgctggccac
gttctgcacc acgccctggg taaaactgat 360taccattacc agctggagat
ggctaaaaac atcactgcag cagcagaagc gatctacacc 420ccggaagagg
ctccggcaaa aatcgaccac gtgattaaaa ccgctctgcg tgagaaaaag
480ccggtatacc tggaaatcgc gtgcaacatc gcgtctatgc cgtgcgccgc
accgggtccg 540gcttctgccc tgttcaacga tgaggcgagc gatgaggcat
ctctgaacgc agcagtagaa 600gaaaccctga aatttatcgc aaaccgtgac
aaagtagcag tcctggtagg ttctaaactg 660cgtgcggctg gtgcggaaga
ggctgcggta aagttcgcgg atgctctggg cggtgcagtg 720gcgaccatgg
cagcggctaa atccttcttc ccagaggaga acccgcatta cattggtacc
780tcctggggcg aagtttccta ccctggtgtg gagaaaacca tgaaagaagc
cgatgctgtg 840attgccctgg cgcctgtatt caacgattat tccaccaccg
gttggaccga tatcccggac 900ccgaagaaac tggtcctggc tgaaccgcgc
tccgtagtag tgaatggcat tcgtttcccg 960tccgtacacc tgaaggatta
cctgacgcgt ctggcacaga aagtatccaa gaaaactggc 1020gcgctggact
tctttaaatc cctgaacgct ggtgagctga aaaaggcggc tccggccgat
1080ccgtccgcac cgctggtgaa cgcagagatt gcacgtcagg ttgaggcact
gctgacgccg 1140aacaccaccg taatcgcgga aacgggcgac tcttggttca
acgcacagcg catgaaactg 1200ccgaacggtg cccgcgttga atatgaaatg
cagtggggtc acatcggctg gtctgtccca 1260gcagcgtttg gttacgcggt
tggtgcaccg gagcgtcgca acatcctgat ggtgggtgac 1320ggctccttcc
agctgactgc tcaggaggtg gcgcagatgg tgcgcctgaa gctgccggtt
1380atcattttcc tgatcaacaa ctacggctac accgccgagg taatgatcca
cgatggtccg 1440tacaacaaca tcaaaaactg ggactacgcc ggtctgatgg
aggtttttaa cggtaacggc 1500ggttacgaca gcggtgctgg taagggtctg
aaagccaaaa ccggtggcga actggcagag 1560gcgattaaag ttgcgctggc
aaacaccgat ggcccgaccc tgatcgagtg cttcatcggc 1620cgtgaggact
gcaccgagga gctggtcaaa tggggcaaac gtgtggcggc tgctaactct
1680cgcaagccgg taaacaaact gctgtaa 1707391644DNALactococcus
lactisCDS(1)..(1644) 39atg tat aca gta gga gat tac ctg tta gac cga
tta cac gag ttg gga 48Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg
Leu His Glu Leu Gly 1 5 10 15 att gaa gaa att ttt gga gtt cct ggt
gac tat aac tta caa ttt tta 96Ile Glu Glu Ile Phe Gly Val Pro Gly
Asp Tyr Asn Leu Gln Phe Leu 20 25 30 gat caa att att tca cgc gaa
gat atg aaa tgg att gga aat gct aat 144Asp Gln Ile Ile Ser Arg Glu
Asp Met Lys Trp Ile Gly Asn Ala Asn 35 40 45 gaa tta aat gct tct
tat atg gct gat ggt tat gct cgt act aaa aaa 192Glu Leu Asn Ala Ser
Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 gct gcc gca
ttt ctc acc aca ttt gga gtc ggc gaa ttg agt gcg atc 240Ala Ala Ala
Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Ile 65 70 75 80 aat
gga ctg gca gga agt tat gcc gaa aat tta cca gta gta gaa att 288Asn
Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90
95 gtt ggt tca cca act tca aaa gta caa aat gac gga aaa ttt gtc cat
336Val Gly Ser Pro Thr Ser Lys Val Gln Asn Asp Gly Lys Phe Val His
100 105 110 cat aca cta gca gat ggt gat ttt aaa cac ttt atg aag atg
cat gaa 384His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met
His Glu 115 120 125 cct gtt aca gca gcg cgg act tta ctg aca gca gaa
aat gcc aca tat 432Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu
Asn Ala Thr Tyr 130 135 140 gaa att gac cga gta ctt tct caa tta cta
aaa gaa aga aaa cca gtc 480Glu Ile Asp Arg Val Leu Ser Gln Leu Leu
Lys Glu Arg Lys Pro Val 145 150 155 160 tat att aac tta cca gtc gat
gtt gct gca gca aaa gca gag aag cct 528Tyr Ile Asn Leu Pro Val Asp
Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 gca tta tct tta gaa
aaa gaa agc tct aca aca aat aca act gaa caa 576Ala Leu Ser Leu Glu
Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln 180 185 190 gtg att ttg
agt aag att gaa gaa agt ttg aaa aat gcc caa aaa cca 624Val Ile Leu
Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala Gln Lys Pro 195 200 205 gta
gtg att gca gga cac gaa gta att agt ttt ggt tta gaa aaa acg 672Val
Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Leu Glu Lys Thr 210 215
220 gta act cag ttt gtt tca gaa aca aaa cta ccg att acg aca cta aat
720Val Thr Gln Phe Val Ser Glu Thr Lys Leu Pro Ile Thr Thr Leu Asn
225 230 235 240 ttt ggt aaa agt gct gtt gat gaa tct ttg ccc tca ttt
tta gga ata 768Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe
Leu Gly Ile 245 250 255 tat aac ggg aaa ctt tca gaa atc agt ctt aaa
aat ttt gtg gag tcc 816Tyr Asn Gly Lys Leu Ser Glu Ile Ser Leu Lys
Asn Phe Val Glu Ser 260 265 270 gca gac ttt atc cta atg ctt gga gtg
aag ctt acg gac tcc tca aca 864Ala Asp Phe Ile Leu Met Leu Gly Val
Lys Leu Thr Asp Ser Ser Thr 275 280 285 ggt gca ttc aca cat cat tta
gat gaa aat aaa atg att tca cta aac 912Gly Ala Phe Thr His His Leu
Asp Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 ata gat gaa gga ata
att ttc aat aaa gtg gta gaa gat ttt gat ttt 960Ile Asp Glu Gly Ile
Ile Phe Asn Lys Val Val Glu Asp Phe Asp Phe 305 310 315 320 aga gca
gtg gtt tct tct tta tca gaa tta aaa gga ata gaa tat gaa 1008Arg Ala
Val Val Ser Ser Leu Ser Glu Leu Lys Gly Ile Glu Tyr Glu 325 330 335
gga caa tat att gat aag caa tat gaa gaa ttt att cca tca agt gct
1056Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu Phe Ile Pro Ser Ser Ala
340 345 350 ccc tta tca caa gac cgt cta tgg cag gca gtt gaa agt ttg
act caa 1104Pro Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Ser Leu
Thr Gln 355 360 365 agc aat gaa aca atc gtt gct gaa caa gga acc tca
ttt ttt gga gct 1152Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser
Phe Phe Gly Ala 370 375 380 tca aca att ttc tta aaa tca aat agt cgt
ttt att gga caa cct tta 1200Ser Thr Ile Phe Leu Lys Ser Asn Ser Arg
Phe Ile Gly Gln Pro Leu 385 390 395 400 tgg ggt tct att gga tat act
ttt cca gcg gct tta gga agc caa att 1248Trp Gly Ser Ile Gly Tyr Thr
Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 gcg gat aaa gag agc
aga cac ctt tta ttt att ggt gat ggt tca ctt 1296Ala Asp Lys Glu Ser
Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 caa ctt acc
gta caa gaa tta gga cta tca atc aga gaa aaa ctc aat 1344Gln Leu Thr
Val Gln Glu Leu Gly Leu Ser Ile Arg Glu Lys Leu Asn 435 440 445 cca
att tgt ttt atc ata aat aat gat ggt tat aca gtt gaa aga gaa 1392Pro
Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455
460 atc cac gga cct act caa agt tat aac gac att cca atg tgg aat tac
1440Ile His Gly Pro Thr Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr
465 470 475 480 tcg aaa tta cca gaa aca ttt gga gca aca gaa gat cgt
gta gta tca 1488Ser Lys Leu Pro Glu Thr Phe Gly Ala Thr Glu Asp Arg
Val Val Ser 485 490 495 aaa att gtt aga aca gag aat gaa ttt gtg tct
gtc atg aaa gaa gcc 1536Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser
Val Met Lys Glu Ala 500 505 510 caa gca gat gtc aat aga atg tat tgg
ata gaa cta gtt ttg gaa aaa 1584Gln Ala Asp Val Asn Arg Met Tyr Trp
Ile Glu Leu Val Leu Glu Lys 515 520 525 gaa gat gcg cca aaa tta ctg
aaa aaa atg ggt aaa tta ttt gct gag 1632Glu Asp Ala Pro Lys Leu Leu
Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 caa aat aaa tag
1644Gln Asn Lys 545 40547PRTLactococcus lactis 40Met Tyr Thr Val
Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu
Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30
Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile Gly Asn Ala Asn 35
40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys
Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu
Ser Ala Ile 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu
Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln
Asn Asp Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp
Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala
Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr 130 135 140 Glu Ile Asp
Arg Val Leu Ser Gln Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160
Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165
170 175 Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu
Gln 180 185 190 Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala
Gln Lys Pro 195 200 205 Val Val Ile Ala Gly His Glu Val Ile Ser Phe
Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Val Ser Glu Thr Lys
Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ala Val
Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Lys
Leu Ser Glu Ile Ser Leu Lys Asn Phe Val Glu Ser 260 265 270 Ala Asp
Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285
Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met Ile Ser Leu Asn 290
295 300 Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu Asp Phe Asp
Phe 305 310 315 320 Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly
Ile Glu Tyr Glu 325 330 335 Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu
Phe Ile Pro Ser Ser Ala 340 345 350 Pro Leu Ser Gln Asp Arg Leu Trp
Gln Ala Val Glu Ser Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val
Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Thr Ile Phe
Leu Lys Ser Asn Ser Arg Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp
Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410
415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu
420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ser Ile Arg Glu Lys
Leu Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr
Val Glu Arg Glu 450 455 460 Ile His Gly Pro Thr Gln Ser Tyr Asn Asp
Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Thr Phe
Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr
Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp
Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys 515 520 525 Glu
Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535
540 Gln Asn Lys 545 411644DNAArtificialLactococcus lactis branched
chain alpha-ketoacid decarboxylase KdcA codon optimised gene
41atgtatactg ttggtgatta tctgctggac cgtctgcatg aactgggcat tgaagaaatc
60ttcggtgtcc caggcgacta caacctgcag ttcctggacc agatcatctc ccgcgaagat
120atgaaatgga tcggtaacgc aaacgagctg aacgcgtctt atatggctga
tggttatgct 180cgcaccaaaa aggctgcggc ctttctgacc acctttggtg
tgggcgagct gagcgcgatc 240aacggcctgg caggttccta cgctgagaac
ctgccggtag tagaaatcgt tggttccccg 300acctctaagg ttcagaacga
cggcaaattc gtacatcaca ccctggcgga cggcgatttt 360aagcacttta
tgaaaatgca cgaaccggtc accgccgctc gcactctgct gaccgcggaa
420aacgcaacgt acgagatcga tcgtgtactg tcccagctgc tgaaagaacg
taaaccggtg 480tatatcaatc tgccggttga tgtcgctgcg gccaaagcag
agaaaccggc actgtccctg 540gagaaggaga gctccactac taacaccacc
gaacaggtta tcctgtccaa aattgaagaa 600tctctgaaaa acgcacagaa
accggtggtt atcgcaggtc acgaggttat ctccttcggc 660ctggagaaaa
ctgttactca attcgtctct gaaacgaaac tgccgatcac gaccctgaac
720tttggcaagt ccgcagttga cgaatctctg ccttctttcc tgggcattta
caacggcaaa 780ctgtccgaga tctccctgaa gaacttcgta gaatccgctg
actttatcct gatgctgggt 840gtgaaactga ccgactcctc taccggtgcg
ttcacgcacc atctggatga aaacaaaatg 900atcagcctga acatcgacga
gggtatcatc ttcaacaagg tagttgaaga tttcgacttc 960cgtgctgttg
tcagcagcct gtccgagctg aaaggcattg agtacgaggg tcaatacatc
1020gataaacagt acgaagagtt
tattccgtct tctgcaccgc tgagccagga ccgcctgtgg 1080caggcagttg
agtccctgac gcagtccaac gaaactatcg tagcggaaca aggtacctct
1140ttcttcggtg cttctaccat ctttctgaag tccaactctc gctttatcgg
tcagccgctg 1200tggggttcta tcggttacac gttcccggct gcgctgggta
gccagatcgc tgataaagag 1260tctcgtcatc tgctgttcat cggtgatggt
tccctgcagc tgactgtaca ggaactgggt 1320ctgtctatcc gtgaaaaact
gaacccgatt tgttttatca tcaataacga tggctacact 1380gttgagcgtg
aaattcatgg tccgactcag tcttacaacg atattccgat gtggaactac
1440tctaaactgc cggaaacctt cggtgcaact gaggatcgcg tcgtgagcaa
gattgtgcgt 1500actgagaacg agttcgtatc tgttatgaaa gaggcgcagg
cagatgtgaa ccgcatgtac 1560tggatcgaac tggttctgga aaaagaggat
gcaccgaaac tgctgaagaa aatgggtaaa 1620ctgtttgcgg agcagaacaa gtaa
1644421647DNALactococcus lactisCDS(1)..(1647) 42atg tat aca gta gga
gat tac cta tta gac cga tta cac gag tta gga 48Met Tyr Thr Val Gly
Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 att gaa gaa
att ttt gga gtc cct gga gac tat aac tta caa ttt tta 96Ile Glu Glu
Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 gat
caa att att tcc cac aag gat atg aaa tgg gtc gga aat gct aat 144Asp
Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40
45 gaa tta aat gct tca tat atg gct gat ggc tat gct cgt act aaa aaa
192Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys
50 55 60 gct gcc gca ttt ctt aca acc ttt gga gta ggt gaa ttg agt
gca gtt 240Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser
Ala Val 65 70 75 80 aat gga tta gca gga agt tac gcc gaa aat tta cca
gta gta gaa ata 288Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro
Val Val Glu Ile 85 90 95 gtg gga tca cct aca tca aaa gtt caa aat
gaa gga aaa ttt gtt cat 336Val Gly Ser Pro Thr Ser Lys Val Gln Asn
Glu Gly Lys Phe Val His 100 105 110 cat acg ctg gct gac ggt gat ttt
aaa cac ttt atg aaa atg cac gaa 384His Thr Leu Ala Asp Gly Asp Phe
Lys His Phe Met Lys Met His Glu 115 120 125 cct gtt aca gca gct cga
act tta ctg aca gca gaa aat gca acc gtt 432Pro Val Thr Ala Ala Arg
Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 gaa att gac cga
gta ctt tct gca cta tta aaa gaa aga aaa cct gtc 480Glu Ile Asp Arg
Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 tat
atc aac tta cca gtt gat gtt gct gct gca aaa gca gag aaa ccc 528Tyr
Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170
175 tca ctc cct ttg aaa aag gaa aac tca act tca aat aca agt gac caa
576Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln
180 185 190 gaa att ttg aac aaa att caa gaa agc ttg aaa aat gcc aaa
aaa cca 624Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys
Lys Pro 195 200 205 atc gtg att aca gga cat gaa ata att agt ttt ggc
tta gaa aaa aca 672Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly
Leu Glu Lys Thr 210 215 220 gtc act caa ttt att tca aag aca aaa cta
cct att acg aca tta aac 720Val Thr Gln Phe Ile Ser Lys Thr Lys Leu
Pro Ile Thr Thr Leu Asn 225 230 235 240 ttt ggt aaa agt tca gtt gat
gaa gcc ctc cct tca ttt tta gga atc 768Phe Gly Lys Ser Ser Val Asp
Glu Ala Leu Pro Ser Phe Leu Gly Ile 245 250 255 tat aat ggt aca ctc
tca gag cct aat ctt aaa gaa ttc gtg gaa tca 816Tyr Asn Gly Thr Leu
Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 gcc gac ttc
atc ttg atg ctt gga gtt aaa ctc aca gac tct tca aca 864Ala Asp Phe
Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 gga
gcc ttc act cat cat tta aat gaa aat aaa atg att tca ctg aat 912Gly
Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295
300 ata gat gaa gga aaa ata ttt aac gaa aga atc caa aat ttt gat ttt
960Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe
305 310 315 320 gaa tcc ctc atc tcc tct ctc tta gac cta agc gaa ata
gaa tac aaa 1008Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile
Glu Tyr Lys 325 330 335 gga aaa tat atc gat aaa aag caa gaa gac ttt
gtt cca tca aat gcg 1056Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe
Val Pro Ser Asn Ala 340 345 350 ctt tta tca caa gac cgc cta tgg caa
gca gtt gaa aac cta act caa 1104Leu Leu Ser Gln Asp Arg Leu Trp Gln
Ala Val Glu Asn Leu Thr Gln 355 360 365 agc aat gaa aca atc gtt gct
gaa caa ggg aca tca ttc ttt ggc gct 1152Ser Asn Glu Thr Ile Val Ala
Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 tca tca att ttc tta
aaa tca aag agt cat ttt att ggt caa ccc tta 1200Ser Ser Ile Phe Leu
Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 tgg gga
tca att gga tat aca ttc cca gca gca tta gga agc caa att 1248Trp Gly
Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415
gca gat aaa gaa agc aga cac ctt tta ttt att ggt gat ggt tca ctt
1296Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu
420 425 430 caa ctt aca gtg caa gaa tta gga tta gca atc aga gaa aaa
att aat 1344Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys
Ile Asn 435 440 445 cca att tgc ttt att atc aat aat gat ggt tat aca
gtc gaa aga gaa 1392Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr
Val Glu Arg Glu 450 455 460 att cat gga cca aat caa agc tac aat gat
att cca atg tgg aat tac 1440Ile His Gly Pro Asn Gln Ser Tyr Asn Asp
Ile Pro Met Trp Asn Tyr 465 470 475 480 tca aaa tta cca gaa tcg ttt
gga gca aca gaa gat cga gta gtc tca 1488Ser Lys Leu Pro Glu Ser Phe
Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 aaa atc gtt aga act
gaa aat gaa ttt gtg tct gtc atg aaa gaa gct 1536Lys Ile Val Arg Thr
Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 caa gca gat
cca aat aga atg tac tgg att gag tta att ttg gca aaa 1584Gln Ala Asp
Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys 515 520 525 gaa
ggt gca cca aaa gta ctg aaa aaa atg ggc aaa cta ttt gct gaa 1632Glu
Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535
540 caa aat aaa tca taa 1647Gln Asn Lys Ser 545 43548PRTLactococcus
lactis 43Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu
Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn
Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys
Trp Val Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala
Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr
Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala
Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly
Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His 100 105 110
His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115
120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr
Val 130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg
Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala
Ala Lys Ala Glu Lys Pro 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn
Ser Thr Ser Asn Thr Ser Asp Gln 180 185 190 Glu Ile Leu Asn Lys Ile
Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 Ile Val Ile Thr
Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr
Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235
240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile
245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val
Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr
Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn
Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn
Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser
Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys 325 330 335 Gly Lys Tyr
Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala 340 345 350 Leu
Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln 355 360
365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala
370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln
Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala
Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu
Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu
Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445 Pro Ile Cys Phe Ile
Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly
Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480
Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485
490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu
Ala 500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile
Leu Ala Lys 515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly
Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys Ser 545
441647DNAArtificialLactococcus lactis -ketoisovalerate
decarboxylase KivD codon optimised gene 44atgtatactg ttggtgatta
cctgctggat cgtctgcatg aactgggcat cgaggaaatt 60ttcggcgtac ctggtgacta
taacctgcag ttcctggatc agatcatttc ccacaaagat 120atgaaatggg
ttggtaacgc gaacgagctg aatgcaagct acatggctga cggttatgca
180cgcaccaaga aagctgcggc gttcctgact acttttggcg tcggcgagct
gtctgcggta 240aacggtctgg ccggctccta cgcggaaaac ctgccggtag
tagaaatcgt cggttccccg 300acctctaaag ttcagaacga gggtaaattc
gtgcaccata ctctggccga tggtgacttc 360aaacacttca tgaagatgca
cgaaccggtc actgctgctc gtacgctgct gaccgcggaa 420aatgcgactg
tcgagattga tcgtgtactg agcgcactgc tgaaagaacg caagcctgta
480tacatcaacc tgccggttga tgtcgcggcc gccaaagcgg aaaaaccatc
tctgccgctg 540aaaaaggaga acagcacctc taacaccagc gaccaggaaa
tcctgaacaa gatccaggag 600tctctgaaga acgctaaaaa gccgatcgta
atcaccggcc atgagattat ctctttcggt 660ctggagaaaa ctgtcaccca
gttcatcagc aaaaccaaac tgccgatcac caccctgaac 720ttcggtaaat
cctccgttga cgaagcgctg ccgtcctttc tgggtattta caacggcact
780ctgtctgagc cgaacctgaa agagttcgtg gagtctgcgg attttatcct
gatgctgggc 840gtgaaactga cggattcctc caccggtgca ttcacccacc
acctgaatga gaataaaatg 900atctctctga acattgatga gggcaaaatc
ttcaacgagc gtattcagaa cttcgatttc 960gaatccctga tctcctccct
gctggatctg tccgagattg aatataaagg caaatacatt 1020gataagaagc
aagaggactt cgtaccgtct aacgcgctgc tgagccagga ccgtctgtgg
1080caagctgtgg aaaacctgac ccagtccaac gaaaccatcg tggcggaaca
gggtacctcc 1140ttcttcggtg ctagctctat cttcctgaaa tctaaaagcc
acttcatcgg tcagccactg 1200tggggctcta ttggctacac cttcccggca
gcgctgggtt cccaaatcgc agacaaagaa 1260tcccgccacc tgctgttcat
tggtgacggc tctctgcaac tgaccgtaca ggagctgggt 1320ctggcgattc
gtgagaaaat caacccgatt tgtttcatca tcaacaacga tggctacact
1380gttgagcgtg agatccacgg cccgaaccag tcctacaacg acattccgat
gtggaactac 1440tctaaactgc cggaatcctt cggtgcgact gaagaccgtg
tcgtaagcaa gatcgtccgt 1500accgaaaacg aattcgtgtc tgtcatgaaa
gaagcacagg cggacccgaa ccgcatgtac 1560tggatcgagc tgattctggc
taaagagggc gcgccaaaag tactgaaaaa gatgggtaaa 1620ctgttcgcag
aacagaacaa atcctaa 1647453696DNAMycobacterium
tuberculosisCDS(1)..(3696) 45gtg gcc aac ata agt tca cca ttc ggg
caa aac gaa tgg ctg gtc gaa 48Val Ala Asn Ile Ser Ser Pro Phe Gly
Gln Asn Glu Trp Leu Val Glu 1 5 10 15 gag atg tac cgc aag ttc cgc
gac gac ccc tcc tcg gtc gat ccc agc 96Glu Met Tyr Arg Lys Phe Arg
Asp Asp Pro Ser Ser Val Asp Pro Ser 20 25 30 tgg cac gag ttc ctg
gtt gac tac agc ccc gaa ccc acc tcc caa cca 144Trp His Glu Phe Leu
Val Asp Tyr Ser Pro Glu Pro Thr Ser Gln Pro 35 40 45 gct gcc gaa
cca acc cgg gtt acc tcg cca ctc gtt gcc gag cgg gcc 192Ala Ala Glu
Pro Thr Arg Val Thr Ser Pro Leu Val Ala Glu Arg Ala 50 55 60 gct
gcg gcc gcc ccg cag gca ccc ccc aag ccg gcc gac acc gcg gcc 240Ala
Ala Ala Ala Pro Gln Ala Pro Pro Lys Pro Ala Asp Thr Ala Ala 65 70
75 80 gcg ggc aac ggc gtg gtc gcc gca ctg gcc gcc aaa act gcc gtt
ccc 288Ala Gly Asn Gly Val Val Ala Ala Leu Ala Ala Lys Thr Ala Val
Pro 85 90 95 ccg cca gcc gaa ggt gac gag gta gcg gtg ctg cgc ggc
gcc gcc gcg 336Pro Pro Ala Glu Gly Asp Glu Val Ala Val Leu Arg Gly
Ala Ala Ala 100 105 110 gcc gtc gtc aag aac atg tcc gcg tcg ttg gag
gtg ccg acg gcg acc 384Ala Val Val Lys Asn Met Ser Ala Ser Leu Glu
Val Pro Thr Ala Thr 115 120 125 agc gtc cgg gcg gtc ccg gcc aag cta
ctg atc gac aac cgg atc gtc 432Ser Val Arg Ala Val Pro Ala Lys Leu
Leu Ile Asp Asn Arg Ile Val 130 135 140 atc aac aac cag ttg aag cgg
acc cgc ggc ggc aag atc tcg ttc acg 480Ile Asn Asn Gln Leu Lys Arg
Thr Arg Gly Gly Lys Ile Ser Phe Thr 145 150 155 160 cat ttg ctg ggc
tac gcc ctg gtg cag gcg gtg aag aaa ttc ccg aac 528His Leu Leu Gly
Tyr Ala Leu Val Gln Ala Val Lys Lys Phe Pro Asn 165 170 175 atg aac
cgg cac tac acc gaa gtc gac ggc aag ccc acc gcg gtc acg 576Met Asn
Arg His Tyr Thr Glu Val Asp Gly Lys Pro Thr Ala Val Thr 180 185 190
ccg gcg cac acc aat ctc ggc ctg gcg atc gac ctg caa ggc aag gac
624Pro Ala His Thr Asn Leu Gly Leu Ala Ile Asp Leu Gln Gly Lys Asp
195 200 205 ggg aag cgt tcc ctg gtg gtg gcc ggc atc aag cgg tgc gag
acc atg 672Gly Lys Arg Ser Leu Val Val Ala Gly Ile Lys Arg Cys Glu
Thr Met 210 215 220 cga ttc gcg cag ttc gtc acg gcc tac gaa gac atc
gta cgc cgg gcc 720Arg Phe Ala Gln Phe Val Thr Ala Tyr Glu Asp Ile
Val Arg Arg Ala 225 230 235 240
cgc gac ggc aag ctg acc act gaa gac ttt gcc ggc gtg acg att tcg
768Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala Gly Val Thr Ile Ser
245 250 255 ctg acc aat ccc gga acc atc ggc acc gtg cat tcg gtg ccg
cgg ctg 816Leu Thr Asn Pro Gly Thr Ile Gly Thr Val His Ser Val Pro
Arg Leu 260 265 270 atg ccc ggc cag ggc gcc atc atc ggc gtg ggc gcc
atg gaa tac ccc 864Met Pro Gly Gln Gly Ala Ile Ile Gly Val Gly Ala
Met Glu Tyr Pro 275 280 285 gcc gag ttt caa ggc gcc agc gag gaa cgc
atc gcc gag ctg ggc atc 912Ala Glu Phe Gln Gly Ala Ser Glu Glu Arg
Ile Ala Glu Leu Gly Ile 290 295 300 ggc aaa ttg atc act ttg acc tcc
acc tac gac cac cgc atc atc cag 960Gly Lys Leu Ile Thr Leu Thr Ser
Thr Tyr Asp His Arg Ile Ile Gln 305 310 315 320 ggc gcg gaa tcg ggc
gac ttc ctg cgc acc atc cac gag ttg ctg ctc 1008Gly Ala Glu Ser Gly
Asp Phe Leu Arg Thr Ile His Glu Leu Leu Leu 325 330 335 tcg gat ggc
ttc tgg gac gag gtc ttc cgc gaa ctg agc atc cca tat 1056Ser Asp Gly
Phe Trp Asp Glu Val Phe Arg Glu Leu Ser Ile Pro Tyr 340 345 350 ctg
ccg gtg cgc tgg agc acc gac aac ccc gac tcg atc gtc gac aag 1104Leu
Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser Ile Val Asp Lys 355 360
365 aac gct cgc gtc atg aac ttg atc gcg gcc tac cgc aac cgc ggc cat
1152Asn Ala Arg Val Met Asn Leu Ile Ala Ala Tyr Arg Asn Arg Gly His
370 375 380 ctg atg gcc gat acc gac ccg ctg cgg ttg gac aaa gct cgg
ttc cgc 1200Leu Met Ala Asp Thr Asp Pro Leu Arg Leu Asp Lys Ala Arg
Phe Arg 385 390 395 400 agt cac ccc gac ctc gaa gtg ctg acc cac ggc
ctg acg ctg tgg gat 1248Ser His Pro Asp Leu Glu Val Leu Thr His Gly
Leu Thr Leu Trp Asp 405 410 415 ctc gat cgg gtg ttc aag gtc gac ggc
ttt gcc ggt gcg cag tac aag 1296Leu Asp Arg Val Phe Lys Val Asp Gly
Phe Ala Gly Ala Gln Tyr Lys 420 425 430 aaa ctg cgc gac gtg ctg ggc
ttg ctg cgc gat gcc tac tgc cgc cac 1344Lys Leu Arg Asp Val Leu Gly
Leu Leu Arg Asp Ala Tyr Cys Arg His 435 440 445 atc ggc gtg gag tac
gcc cat atc ctc gac ccc gaa caa aag gag tgg 1392Ile Gly Val Glu Tyr
Ala His Ile Leu Asp Pro Glu Gln Lys Glu Trp 450 455 460 ctc gaa caa
cgg gtc gag acc aag cac gtc aaa ccc act gtg gcc caa 1440Leu Glu Gln
Arg Val Glu Thr Lys His Val Lys Pro Thr Val Ala Gln 465 470 475 480
cag aaa tac atc ctc agc aag ctc aac gcc gcc gag gcc ttt gaa acg
1488Gln Lys Tyr Ile Leu Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu Thr
485 490 495 ttc cta cag acc aag tac gtc ggc cag aag cgg ttc tcg ctg
gaa ggc 1536Phe Leu Gln Thr Lys Tyr Val Gly Gln Lys Arg Phe Ser Leu
Glu Gly 500 505 510 gcc gaa agc gtg atc ccg atg atg gac gcg gcg atc
gac cag tgc gct 1584Ala Glu Ser Val Ile Pro Met Met Asp Ala Ala Ile
Asp Gln Cys Ala 515 520 525 gag cac ggc ctc gac gag gtg gtc atc ggg
atg ccg cac cgg ggc cgg 1632Glu His Gly Leu Asp Glu Val Val Ile Gly
Met Pro His Arg Gly Arg 530 535 540 ctc aac gtg ctg gcc aac atc gtc
ggc aag ccg tac tcg cag atc ttc 1680Leu Asn Val Leu Ala Asn Ile Val
Gly Lys Pro Tyr Ser Gln Ile Phe 545 550 555 560 acc gag ttc gag ggc
aac ctg aat ccg tcg cag gcg cac ggc tcc ggt 1728Thr Glu Phe Glu Gly
Asn Leu Asn Pro Ser Gln Ala His Gly Ser Gly 565 570 575 gac gtc aag
tac cac ctg ggc gcc acc ggg ctg tac ctg cag atg ttc 1776Asp Val Lys
Tyr His Leu Gly Ala Thr Gly Leu Tyr Leu Gln Met Phe 580 585 590 ggc
gac aac gac att cag gtg tcg ctg acc gcc aac ccg tcg cat ctg 1824Gly
Asp Asn Asp Ile Gln Val Ser Leu Thr Ala Asn Pro Ser His Leu 595 600
605 gag gcc gtc gac ccg gtg ctg gag gga ttg gtg cgg gcc aag cag gat
1872Glu Ala Val Asp Pro Val Leu Glu Gly Leu Val Arg Ala Lys Gln Asp
610 615 620 ctg ctc gac cac gga agc atc gac agc gac ggc caa cgg gcg
ttc tcg 1920Leu Leu Asp His Gly Ser Ile Asp Ser Asp Gly Gln Arg Ala
Phe Ser 625 630 635 640 gtg gtg ccg ctg atg ttg cat ggc gat gcc gcg
ttc gcc ggt cag ggt 1968Val Val Pro Leu Met Leu His Gly Asp Ala Ala
Phe Ala Gly Gln Gly 645 650 655 gtg gtc gcc gag acg ctg aac ctg gcg
aat ctg ccg ggc tac cgc gtc 2016Val Val Ala Glu Thr Leu Asn Leu Ala
Asn Leu Pro Gly Tyr Arg Val 660 665 670 ggc ggc acc atc cac atc atc
gtc aac aac cag atc ggc ttc acc acc 2064Gly Gly Thr Ile His Ile Ile
Val Asn Asn Gln Ile Gly Phe Thr Thr 675 680 685 gcg ccc gag tat tcc
agg tcc agc gag tac tgc acc gac gtc gca aag 2112Ala Pro Glu Tyr Ser
Arg Ser Ser Glu Tyr Cys Thr Asp Val Ala Lys 690 695 700 atg atc ggg
gca ccg atc ttt cac gtc aac ggc gac gac ccg gag gcg 2160Met Ile Gly
Ala Pro Ile Phe His Val Asn Gly Asp Asp Pro Glu Ala 705 710 715 720
tgt gtc tgg gtg gcg cgg ttg gcg gtg gac ttc cga caa cgg ttc aag
2208Cys Val Trp Val Ala Arg Leu Ala Val Asp Phe Arg Gln Arg Phe Lys
725 730 735 aag gac gtc gtc atc gac atg ctg tgc tac cgc cgc cgc ggg
cac aac 2256Lys Asp Val Val Ile Asp Met Leu Cys Tyr Arg Arg Arg Gly
His Asn 740 745 750 gag ggt gac gac ccg tcg atg acc aac ccc tac gtg
tac gac gtc gtc 2304Glu Gly Asp Asp Pro Ser Met Thr Asn Pro Tyr Val
Tyr Asp Val Val 755 760 765 gac acc aag cgc ggg gcc cgc aaa agc tac
acc gaa gcc ctg atc gga 2352Asp Thr Lys Arg Gly Ala Arg Lys Ser Tyr
Thr Glu Ala Leu Ile Gly 770 775 780 cgt ggc gac atc tcg atg aag gag
gcc gag gac gcg ctg cgc gac tac 2400Arg Gly Asp Ile Ser Met Lys Glu
Ala Glu Asp Ala Leu Arg Asp Tyr 785 790 795 800 cag ggc cag ctg gaa
cgg gtg ttc aac gaa gtg cgc gag ctg gag aag 2448Gln Gly Gln Leu Glu
Arg Val Phe Asn Glu Val Arg Glu Leu Glu Lys 805 810 815 cac ggt gtg
cag ccg agc gag tcg gtc gag tcc gac cag atg att ccc 2496His Gly Val
Gln Pro Ser Glu Ser Val Glu Ser Asp Gln Met Ile Pro 820 825 830 gcg
ggg ctg gcc act gcg gtg gac aag tcg ctg ctg gcc cgg atc ggc 2544Ala
Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu Ala Arg Ile Gly 835 840
845 gat gcg ttc ctc gcc ttg ccg aac ggc ttc acc gcg cac ccg cga gtc
2592Asp Ala Phe Leu Ala Leu Pro Asn Gly Phe Thr Ala His Pro Arg Val
850 855 860 caa ccg gtg ctg gag aag cgc cgg gag atg gcc tat gaa ggc
aag atc 2640Gln Pro Val Leu Glu Lys Arg Arg Glu Met Ala Tyr Glu Gly
Lys Ile 865 870 875 880 gac tgg gcc ttt ggc gag ctg ctg gcg ctg ggc
tcg ctg gtg gcc gaa 2688Asp Trp Ala Phe Gly Glu Leu Leu Ala Leu Gly
Ser Leu Val Ala Glu 885 890 895 ggc aag ctg gtg cgc ttg tcg ggg cag
gac agc cgc cgc ggc acc ttc 2736Gly Lys Leu Val Arg Leu Ser Gly Gln
Asp Ser Arg Arg Gly Thr Phe 900 905 910 tcc cag cgg cat tcg gtt ctc
atc gac cgc cac act ggc gag gag ttc 2784Ser Gln Arg His Ser Val Leu
Ile Asp Arg His Thr Gly Glu Glu Phe 915 920 925 aca cca ctg cag ctg
ctg gcg acc aac tcc gac ggc agc ccg acc ggc 2832Thr Pro Leu Gln Leu
Leu Ala Thr Asn Ser Asp Gly Ser Pro Thr Gly 930 935 940 gga aag ttc
ctg gtc tac gac tcg cca ctg tcg gag tac gcc gcc gtc 2880Gly Lys Phe
Leu Val Tyr Asp Ser Pro Leu Ser Glu Tyr Ala Ala Val 945 950 955 960
ggc ttc gag tac ggc tac act gtg ggc aat ccg gac gcc gtg gtg ctc
2928Gly Phe Glu Tyr Gly Tyr Thr Val Gly Asn Pro Asp Ala Val Val Leu
965 970 975 tgg gag gcg cag ttc ggc gac ttc gtc aac ggc gcg cag tcg
atc atc 2976Trp Glu Ala Gln Phe Gly Asp Phe Val Asn Gly Ala Gln Ser
Ile Ile 980 985 990 gac gag ttc atc agc tcc ggt gag gcc aag tgg ggc
caa ttg tcc aac 3024Asp Glu Phe Ile Ser Ser Gly Glu Ala Lys Trp Gly
Gln Leu Ser Asn 995 1000 1005 gtc gtg ctg ctg tta ccg cac ggg cac
gag ggg cag gga ccc gac 3069Val Val Leu Leu Leu Pro His Gly His Glu
Gly Gln Gly Pro Asp 1010 1015 1020 cac act tct gcc cgg atc gaa cgc
ttc ttg cag ttg tgg gcg gaa 3114His Thr Ser Ala Arg Ile Glu Arg Phe
Leu Gln Leu Trp Ala Glu 1025 1030 1035 ggt tcg atg acc atc gcg atg
ccg tcg act ccg tcg aac tac ttc 3159Gly Ser Met Thr Ile Ala Met Pro
Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050 cac ctg cta cgc cgg cat
gcc ctg gac ggc atc caa cgc ccg ctg 3204His Leu Leu Arg Arg His Ala
Leu Asp Gly Ile Gln Arg Pro Leu 1055 1060 1065 atc gtg ttc acg ccc
aag tcg atg ttg cgt cac aag gcc gcc gtc 3249Ile Val Phe Thr Pro Lys
Ser Met Leu Arg His Lys Ala Ala Val 1070 1075 1080 agc gaa atc aag
gac ttc acc gag atc aag ttc cgc tca gtg ctg 3294Ser Glu Ile Lys Asp
Phe Thr Glu Ile Lys Phe Arg Ser Val Leu 1085 1090 1095 gag gaa ccc
acc tat gag gac ggc atc gga gac cgc aac aag gtc 3339Glu Glu Pro Thr
Tyr Glu Asp Gly Ile Gly Asp Arg Asn Lys Val 1100 1105 1110 agc cgg
atc ctg ctg acc agt ggc aag ctg tat tac gag ctg gcc 3384Ser Arg Ile
Leu Leu Thr Ser Gly Lys Leu Tyr Tyr Glu Leu Ala 1115 1120 1125 gcc
cgc aag gcc aag gac aac cgc aat gac ctc gcg atc gtg cgg 3429Ala Arg
Lys Ala Lys Asp Asn Arg Asn Asp Leu Ala Ile Val Arg 1130 1135 1140
ctt gaa cag ctc gcc ccg ctg ccc agg cgt cga ctg cgt gaa acg 3474Leu
Glu Gln Leu Ala Pro Leu Pro Arg Arg Arg Leu Arg Glu Thr 1145 1150
1155 ctg gac cgc tac gag aac gtc aag gag ttc ttc tgg gtc caa gag
3519Leu Asp Arg Tyr Glu Asn Val Lys Glu Phe Phe Trp Val Gln Glu
1160 1165 1170 gaa ccg gcc aac cag ggt gcg tgg ccg cga ttc ggg ctc
gaa cta 3564Glu Pro Ala Asn Gln Gly Ala Trp Pro Arg Phe Gly Leu Glu
Leu 1175 1180 1185 ccc gag ctg ctg cct gac aag ttg gcc ggg atc aag
cga atc tcg 3609Pro Glu Leu Leu Pro Asp Lys Leu Ala Gly Ile Lys Arg
Ile Ser 1190 1195 1200 cgc cgg gcg atg tca gcc ccg tcg tca ggc tcg
tcg aag gtg cac 3654Arg Arg Ala Met Ser Ala Pro Ser Ser Gly Ser Ser
Lys Val His 1205 1210 1215 gcc gtc gaa cag cag gag atc ctc gac gag
gcg ttc ggc tga 3696Ala Val Glu Gln Gln Glu Ile Leu Asp Glu Ala Phe
Gly 1220 1225 1230 461231PRTMycobacterium tuberculosis 46Val Ala
Asn Ile Ser Ser Pro Phe Gly Gln Asn Glu Trp Leu Val Glu 1 5 10 15
Glu Met Tyr Arg Lys Phe Arg Asp Asp Pro Ser Ser Val Asp Pro Ser 20
25 30 Trp His Glu Phe Leu Val Asp Tyr Ser Pro Glu Pro Thr Ser Gln
Pro 35 40 45 Ala Ala Glu Pro Thr Arg Val Thr Ser Pro Leu Val Ala
Glu Arg Ala 50 55 60 Ala Ala Ala Ala Pro Gln Ala Pro Pro Lys Pro
Ala Asp Thr Ala Ala 65 70 75 80 Ala Gly Asn Gly Val Val Ala Ala Leu
Ala Ala Lys Thr Ala Val Pro 85 90 95 Pro Pro Ala Glu Gly Asp Glu
Val Ala Val Leu Arg Gly Ala Ala Ala 100 105 110 Ala Val Val Lys Asn
Met Ser Ala Ser Leu Glu Val Pro Thr Ala Thr 115 120 125 Ser Val Arg
Ala Val Pro Ala Lys Leu Leu Ile Asp Asn Arg Ile Val 130 135 140 Ile
Asn Asn Gln Leu Lys Arg Thr Arg Gly Gly Lys Ile Ser Phe Thr 145 150
155 160 His Leu Leu Gly Tyr Ala Leu Val Gln Ala Val Lys Lys Phe Pro
Asn 165 170 175 Met Asn Arg His Tyr Thr Glu Val Asp Gly Lys Pro Thr
Ala Val Thr 180 185 190 Pro Ala His Thr Asn Leu Gly Leu Ala Ile Asp
Leu Gln Gly Lys Asp 195 200 205 Gly Lys Arg Ser Leu Val Val Ala Gly
Ile Lys Arg Cys Glu Thr Met 210 215 220 Arg Phe Ala Gln Phe Val Thr
Ala Tyr Glu Asp Ile Val Arg Arg Ala 225 230 235 240 Arg Asp Gly Lys
Leu Thr Thr Glu Asp Phe Ala Gly Val Thr Ile Ser 245 250 255 Leu Thr
Asn Pro Gly Thr Ile Gly Thr Val His Ser Val Pro Arg Leu 260 265 270
Met Pro Gly Gln Gly Ala Ile Ile Gly Val Gly Ala Met Glu Tyr Pro 275
280 285 Ala Glu Phe Gln Gly Ala Ser Glu Glu Arg Ile Ala Glu Leu Gly
Ile 290 295 300 Gly Lys Leu Ile Thr Leu Thr Ser Thr Tyr Asp His Arg
Ile Ile Gln 305 310 315 320 Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr
Ile His Glu Leu Leu Leu 325 330 335 Ser Asp Gly Phe Trp Asp Glu Val
Phe Arg Glu Leu Ser Ile Pro Tyr 340 345 350 Leu Pro Val Arg Trp Ser
Thr Asp Asn Pro Asp Ser Ile Val Asp Lys 355 360 365 Asn Ala Arg Val
Met Asn Leu Ile Ala Ala Tyr Arg Asn Arg Gly His 370 375 380 Leu Met
Ala Asp Thr Asp Pro Leu Arg Leu Asp Lys Ala Arg Phe Arg 385 390 395
400 Ser His Pro Asp Leu Glu Val Leu Thr His Gly Leu Thr Leu Trp Asp
405 410 415 Leu Asp Arg Val Phe Lys Val Asp Gly Phe Ala Gly Ala Gln
Tyr Lys 420 425 430 Lys Leu Arg Asp Val Leu Gly Leu Leu Arg Asp Ala
Tyr Cys Arg His 435 440 445 Ile Gly Val Glu Tyr Ala His Ile Leu Asp
Pro Glu Gln Lys Glu Trp 450 455 460 Leu Glu Gln Arg Val Glu Thr Lys
His Val Lys Pro Thr Val Ala Gln 465 470 475 480 Gln Lys Tyr Ile Leu
Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu Thr 485 490 495 Phe Leu Gln
Thr Lys Tyr Val Gly Gln Lys Arg Phe Ser Leu Glu Gly 500 505 510 Ala
Glu Ser Val Ile Pro Met Met Asp Ala Ala Ile Asp Gln Cys Ala 515 520
525 Glu His Gly Leu Asp Glu Val Val Ile Gly Met Pro His Arg Gly Arg
530 535 540 Leu Asn Val Leu Ala Asn Ile Val Gly Lys Pro Tyr Ser Gln
Ile Phe 545 550 555 560 Thr Glu Phe Glu Gly Asn Leu Asn Pro Ser
Gln Ala His Gly Ser Gly 565 570 575 Asp Val Lys Tyr His Leu Gly Ala
Thr Gly Leu Tyr Leu Gln Met Phe 580 585 590 Gly Asp Asn Asp Ile Gln
Val Ser Leu Thr Ala Asn Pro Ser His Leu 595 600 605 Glu Ala Val Asp
Pro Val Leu Glu Gly Leu Val Arg Ala Lys Gln Asp 610 615 620 Leu Leu
Asp His Gly Ser Ile Asp Ser Asp Gly Gln Arg Ala Phe Ser 625 630 635
640 Val Val Pro Leu Met Leu His Gly Asp Ala Ala Phe Ala Gly Gln Gly
645 650 655 Val Val Ala Glu Thr Leu Asn Leu Ala Asn Leu Pro Gly Tyr
Arg Val 660 665 670 Gly Gly Thr Ile His Ile Ile Val Asn Asn Gln Ile
Gly Phe Thr Thr 675 680 685 Ala Pro Glu Tyr Ser Arg Ser Ser Glu Tyr
Cys Thr Asp Val Ala Lys 690 695 700 Met Ile Gly Ala Pro Ile Phe His
Val Asn Gly Asp Asp Pro Glu Ala 705 710 715 720 Cys Val Trp Val Ala
Arg Leu Ala Val Asp Phe Arg Gln Arg Phe Lys 725 730 735 Lys Asp Val
Val Ile Asp Met Leu Cys Tyr Arg Arg Arg Gly His Asn 740 745 750 Glu
Gly Asp Asp Pro Ser Met Thr Asn Pro Tyr Val Tyr Asp Val Val 755 760
765 Asp Thr Lys Arg Gly Ala Arg Lys Ser Tyr Thr Glu Ala Leu Ile Gly
770 775 780 Arg Gly Asp Ile Ser Met Lys Glu Ala Glu Asp Ala Leu Arg
Asp Tyr 785 790 795 800 Gln Gly Gln Leu Glu Arg Val Phe Asn Glu Val
Arg Glu Leu Glu Lys 805 810 815 His Gly Val Gln Pro Ser Glu Ser Val
Glu Ser Asp Gln Met Ile Pro 820 825 830 Ala Gly Leu Ala Thr Ala Val
Asp Lys Ser Leu Leu Ala Arg Ile Gly 835 840 845 Asp Ala Phe Leu Ala
Leu Pro Asn Gly Phe Thr Ala His Pro Arg Val 850 855 860 Gln Pro Val
Leu Glu Lys Arg Arg Glu Met Ala Tyr Glu Gly Lys Ile 865 870 875 880
Asp Trp Ala Phe Gly Glu Leu Leu Ala Leu Gly Ser Leu Val Ala Glu 885
890 895 Gly Lys Leu Val Arg Leu Ser Gly Gln Asp Ser Arg Arg Gly Thr
Phe 900 905 910 Ser Gln Arg His Ser Val Leu Ile Asp Arg His Thr Gly
Glu Glu Phe 915 920 925 Thr Pro Leu Gln Leu Leu Ala Thr Asn Ser Asp
Gly Ser Pro Thr Gly 930 935 940 Gly Lys Phe Leu Val Tyr Asp Ser Pro
Leu Ser Glu Tyr Ala Ala Val 945 950 955 960 Gly Phe Glu Tyr Gly Tyr
Thr Val Gly Asn Pro Asp Ala Val Val Leu 965 970 975 Trp Glu Ala Gln
Phe Gly Asp Phe Val Asn Gly Ala Gln Ser Ile Ile 980 985 990 Asp Glu
Phe Ile Ser Ser Gly Glu Ala Lys Trp Gly Gln Leu Ser Asn 995 1000
1005 Val Val Leu Leu Leu Pro His Gly His Glu Gly Gln Gly Pro Asp
1010 1015 1020 His Thr Ser Ala Arg Ile Glu Arg Phe Leu Gln Leu Trp
Ala Glu 1025 1030 1035 Gly Ser Met Thr Ile Ala Met Pro Ser Thr Pro
Ser Asn Tyr Phe 1040 1045 1050 His Leu Leu Arg Arg His Ala Leu Asp
Gly Ile Gln Arg Pro Leu 1055 1060 1065 Ile Val Phe Thr Pro Lys Ser
Met Leu Arg His Lys Ala Ala Val 1070 1075 1080 Ser Glu Ile Lys Asp
Phe Thr Glu Ile Lys Phe Arg Ser Val Leu 1085 1090 1095 Glu Glu Pro
Thr Tyr Glu Asp Gly Ile Gly Asp Arg Asn Lys Val 1100 1105 1110 Ser
Arg Ile Leu Leu Thr Ser Gly Lys Leu Tyr Tyr Glu Leu Ala 1115 1120
1125 Ala Arg Lys Ala Lys Asp Asn Arg Asn Asp Leu Ala Ile Val Arg
1130 1135 1140 Leu Glu Gln Leu Ala Pro Leu Pro Arg Arg Arg Leu Arg
Glu Thr 1145 1150 1155 Leu Asp Arg Tyr Glu Asn Val Lys Glu Phe Phe
Trp Val Gln Glu 1160 1165 1170 Glu Pro Ala Asn Gln Gly Ala Trp Pro
Arg Phe Gly Leu Glu Leu 1175 1180 1185 Pro Glu Leu Leu Pro Asp Lys
Leu Ala Gly Ile Lys Arg Ile Ser 1190 1195 1200 Arg Arg Ala Met Ser
Ala Pro Ser Ser Gly Ser Ser Lys Val His 1205 1210 1215 Ala Val Glu
Gln Gln Glu Ile Leu Asp Glu Ala Phe Gly 1220 1225 1230
473696DNAArtificialMycobacterium tuberculosis -ketoglutarate
decarboxylase Kgd codon optimised gene 47atggctaata tctcctctcc
gtttggtcag aatgaatggc tggtagaaga aatgtaccgt 60aaattccgcg atgacccgtc
ctctgtggac ccgtcctggc atgaattcct ggtagactac 120agcccggagc
cgaccagcca accggcagcg gaaccaaccc gcgttacttc tccgctggta
180gcggaacgtg cagctgctgc cgcgcctcag gcgccgccta aaccggcgga
tactgccgca 240gccggtaacg gtgtggtggc cgcactggct gctaagactg
cggttccgcc gccagcagaa 300ggcgatgaag ttgcagtcct gcgcggtgcg
gcggctgcag tggtgaaaaa catgagcgcg 360tccctggagg taccgaccgc
cacgagcgtg cgcgcggtcc ctgctaaact gctgattgat 420aaccgtattg
tgatcaacaa ccagctgaaa cgtacccgtg gtggcaagat ctccttcact
480catctgctgg gttatgcact ggtacaagcg gttaagaaat tccctaacat
gaaccgtcat 540tacactgagg tcgacggtaa accgacggct gttactccgg
cacacacgaa cctgggcctg 600gcgatcgacc tgcaaggtaa agatggtaag
cgctccctgg tagttgcggg tattaaacgt 660tgcgaaacca tgcgtttcgc
acaattcgta accgcctacg aggacattgt ccgccgtgct 720cgtgatggca
aactgaccac cgaagatttt gcgggcgtta ctattagcct gaccaaccca
780ggcaccatcg gcaccgtgca cagcgtacct cgtctgatgc cgggccaagg
tgcgattatc 840ggtgtgggtg ccatggagta cccggcagaa tttcagggtg
cttctgaaga gcgcatcgcc 900gagctgggta ttggtaaact gatcaccctg
acttctacct atgaccaccg catcattcag 960ggcgcagaat ccggtgactt
cctgcgcact attcacgaac tgctgctgtc cgacggtttc 1020tgggatgaag
tttttcgtga actgagcatc ccatatctgc cagttcgctg gtccaccgac
1080aatccggact ctatcgttga caaaaacgct cgcgtaatga acctgatcgc
tgcttatcgt 1140aatcgtggtc acctgatggc tgatacggat ccgctgcgcc
tggataaagc tcgtttccgt 1200tcccacccgg acctggaagt gctgacccat
ggtctgactc tgtgggatct ggaccgcgtg 1260ttcaaagtag atggtttcgc
gggtgctcag tacaagaagc tgcgtgacgt gctgggtctg 1320ctgcgtgatg
cgtactgtcg tcacattggt gtggagtacg cccacattct ggatccggaa
1380cagaaagaat ggctggagca gcgtgtcgag accaaacacg taaaaccgac
cgtagcgcag 1440cagaaatata tcctgtccaa actgaacgcc gccgaggctt
tcgaaacttt cctgcagacc 1500aagtacgtgg gccagaaacg cttcagcctg
gagggtgcgg aaagcgttat tccgatgatg 1560gatgcagcta tcgatcagtg
cgcggaacat ggtctggatg aagtcgttat cggtatgccg 1620caccgtggtc
gcctgaacgt actggcaaac atcgtcggta aaccatattc tcagatcttc
1680acggaattcg agggcaacct gaacccgtcc caagcccacg gctccggcga
cgtaaaatat 1740catctgggtg ctaccggcct gtatctgcag atgttcggtg
ataacgacat ccaggtatct 1800ctgactgcta acccgagcca cctggaggcg
gttgatcctg ttctggaagg tctggttcgc 1860gccaaacagg atctgctgga
ccacggctct atcgacagcg atggccagcg tgcattcagc 1920gttgtaccgc
tgatgctgca tggcgacgcg gcgttcgccg gtcagggtgt cgtagcagaa
1980actctgaacc tggcgaacct gcctggctat cgcgtgggtg gcaccattca
catcatcgtt 2040aacaaccaaa tcggtttcac cacggcaccg gagtatagcc
gttctagcga atattgcacc 2100gacgtagcca aaatgatcgg tgcgccgatc
ttccatgtaa acggtgacga tccagaggcc 2160tgcgtgtggg tggctcgtct
ggccgtagac ttccgccagc gttttaagaa agatgtggtt 2220atcgacatgc
tgtgctaccg ccgtcgtggt cacaacgaag gtgatgatcc gtctatgact
2280aacccgtatg tctatgacgt ggtggacacc aagcgtggtg cacgcaaatc
ttacacggag 2340gccctgatcg gtcgtggcga catctctatg aaagaagcgg
aagacgctct gcgtgattac 2400cagggtcagc tggaacgtgt gttcaatgag
gtgcgtgagc tggaaaagca cggcgtacaa 2460ccgtccgaat ccgtagagtc
cgatcagatg atccctgctg gtctggcaac tgctgttgat 2520aaaagcctgc
tggcgcgtat cggcgacgca ttcctggcgc tgccgaatgg ctttaccgcg
2580cacccgcgcg tacagccggt actggaaaaa cgtcgtgaaa tggcctacga
aggtaaaatc 2640gattgggcct tcggtgagct gctggccctg ggctctctgg
tggctgaggg caagctggta 2700cgcctgagcg gccaggactc ccgtcgcggc
actttttctc agcgtcacag cgtcctgatc 2760gatcgtcaca ccggcgaaga
attcacgccg ctgcaactgc tggctactaa ctccgatggt 2820agcccgaccg
gtggtaagtt cctggtgtac gattccccgc tgtccgaata tgctgcagtt
2880ggtttcgagt atggttacac cgttggcaac ccggacgcag tggttctgtg
ggaagcgcag 2940ttcggcgatt tcgttaacgg tgcccagtcc attatcgatg
agtttattag cagcggcgag 3000gccaaatggg gccagctgtc taacgttgtg
ctgctgctgc ctcacggcca cgagggtcaa 3060ggcccggacc acacctccgc
ccgtatcgaa cgcttcctgc agctgtgggc tgaaggctct 3120atgaccatcg
cgatgccgtc taccccaagc aactacttcc acctgctgcg tcgccacgca
3180ctggacggca ttcagcgccc gctgatcgtt ttcaccccaa aatccatgct
gcgccacaaa 3240gcagctgttt ctgaaatcaa agattttacg gaaattaaat
tccgttctgt gctggaagaa 3300ccaacctacg aagacggtat tggcgaccgc
aacaaggtaa gccgtatcct gctgacctcc 3360ggcaaactgt actacgagct
ggcagcacgt aaggcaaaag ataaccgcaa cgacctggcc 3420atcgtccgcc
tggaacagct ggcgccactg ccacgccgtc gcctgcgtga aaccctggat
3480cgctacgaaa acgtaaaaga attcttctgg gtgcaggaag aaccggcaaa
ccagggtgcg 3540tggccgcgct ttggtctgga actgccggaa ctgctgccgg
ataaactggc aggtatcaag 3600cgcatcagcc gtcgcgctat gagcgccccg
tcttctggta gctctaaagt acacgctgta 3660gaacagcaag agatcctgga
tgaggccttc ggctaa 36964874DNAArtificial sequenceForward primer for
amplification of Bacillus subtilis aminotransferase x 48ggggacaagt
ttgtacaaaa aagcaggcta ggaggaatta accatgaagg ttttagtcaa 60tggccggctg
attg 744962DNAArtificial sequenceReverse primer for amplification
of Bacillus subtilis aminotransferase x 49ggggaccact ttgtacaaga
aagctgggtt tatgaaatgc tagcagcctg ttgaatgctt 60tc
625082DNAArtificial sequenceForward primer for amplification of
Bacillus subtilis aminotransferase y 50ggggacaagt ttgtacaaaa
aagcaggcta ggaggaatta accatgactc atgatttgat 60agaaaaaagt aaaaagcacc
tc 825157DNAArtificial sequenceReverse primer for amplification of
Bacillus subtilis aminotransferase y 51ggggaccact ttgtacaaga
aagctgggtt caatcttcaa ggctcgtaac ctcgtgg 575264DNAArtificial
sequenceForward primer for amplification of Rhodobacter sphaeroides
aminotransferase 52ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta
accatgcccg gttgcggggg 60cttg 645351DNAArtificial sequenceReverse
primer for amplification of Rhodobacter sphaeroides
aminotransferase 53ggggaccact ttgtacaaga aagctgggtt cagacggcgg
ccggttcttt c 515478DNAArtificial sequenceForward primer for
amplification of Legionella pneumophila aminotransferase
54ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgagta tcgcatttgt
60taacggcaag tattgttg 785567DNAArtificial sequenceReverse primer
for amplification of Legionella pneumophila aminotransferase
55ggggaccact ttgtacaaga aagctgggtt tagtttacta gttgttggta ggaatcatta
60attatcc 675676DNAArtificial sequenceForward primer for
amplification of Nitrosomonas europaea aminotransferase
56ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgattt acctcaatgg
60caaatttctg ccgatg 765750DNAArtificial sequenceReverse primer for
amplification of Nitrosomonas europaea aminotransferase
57ggggaccact ttgtacaaga aagctgggtt tactggcgtg gagcatgccc
505879DNAArtificial sequenceForward primer for amplification of
Neisseria gonorrhoeae aminotransferase 58ggggacaagt ttgtacaaaa
aagcaggcta ggaggaatta accatgagga taaatatgaa 60ccgtaacgaa attttattc
795956DNAArtificial sequenceReverse primer for amplification of
Neisseria gonorrhoeae aminotransferase 59ggggaccact ttgtacaaga
aagctgggtt catgcagcca tcgccttgaa cacttc 566066DNAArtificial
sequenceForward primer for amplification of Pseudomonas aeruginosa
aminotransferase 60ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta
accatgtcga tggccgatcg 60tgatgg 666153DNAArtificial sequenceReverse
primer for amplification of Pseudomonas aeruginosa aminotransferase
61ggggaccact ttgtacaaga aagctgggtt tacttgacca gggtacgcca ctc
536267DNAArtificial sequenceForward primer for amplification of
Rhodopseudomonas palustris aminotransferase 62ggggacaagt ttgtacaaaa
aagcaggcta ggaggaatta accatgaagc tgataccgtg 60ccgcgcc
676351DNAArtificial sequenceReverse primer for amplification of
Rhodopseudomonas palustris aminotransferase 63ggggaccact ttgtacaaga
aagctgggtt caggcgaccg cgcggatcac c 51641353DNABacillus subtilis
64atggagatga tggggatgga aaacattcag caaaatcagg gattaaagca aaaagatgag
60caatttgtgt ggcatgccat gaagggagcg catcaagcgg acagcctgat agcccagaag
120gccgaagggg cctgggtaac cgacacagac ggacgccgct atttggatgc
gatgtccggt 180ttgtggtgcg tcaacattgg ttacggcaga aaggagcttg
cggaggctgc ctatgagcaa 240ctaaaggagc tgccttacta cccgttaacg
caaagtcacg cacccgcaat tcaactggcg 300gaaaagctga atgaatggct
tggcggcgat tatgttattt ttttttccaa cagcggatcg 360gaagcaaacg
aaactgcttt taaaattgcc cgccagtacc atctgcaaaa cggcgaccac
420agccgttata aattcatctc aagatatcgg gcataccacg gcaatacatt
gggagcgctc 480tcagctaccg gacaggcgca gcggaaatat aaatacgagc
ctttgagcca agggttcctg 540catgcagctc cgccagatat ataccggaat
cctgatgatg cagacacgct tgaaagcgca 600aatgaaatcg accgcatcat
gacatgggaa ttaagcgaaa cgattgccgg ggtcattatg 660gagcccatca
ttacaggcgg aggcatccta atgccgccgg acggatatat gaagaaggtg
720gaggacattt gccggcgcca cggagccctt ttgatttgcg atgaagtgat
ctgcgggttt 780ggacggacag gtgagccgtt cgggtttatg cactacggtg
tgaagcctga tatcattacg 840atggcaaagg gaatcacaag cgcgtatctg
ccattgtcag cgactgctgt gaaacgggac 900attttcgaag cgtatcaggg
ggaagctcct tatgaccgtt tccgccacgt gaacacgttc 960ggcggaagcc
cggctgcctg tgctttggcg ttgaaaaacc tgcaaattat ggaggacgaa
1020cagctgattc agcgatcccg tgatcttgga gcaaagcttt taggtgagct
tcaagctctg 1080agagaacacc cggcagtcgg ggatgttaga ggaaaagggc
tgctgatcgg aatcgaactc 1140gtcaaagaca aattgactaa agagccggct
gatgccgcca aagtaaacca agtggttgcg 1200gcgtgcaaag aaaaagggct
gatcatcggc aaaaacggcg atacagtcgc cggctacaac 1260aatgtcatcc
acgttgcgcc gccattttgc ctgacagaag aggacctttc ctttatcgtg
1320aaaacggtga aagaaagctt tcaaacgata taa 135365450PRTBacillus
subtilis 65Met Glu Met Met Gly Met Glu Asn Ile Gln Gln Asn Gln Gly
Leu Lys 1 5 10 15 Gln Lys Asp Glu Gln Phe Val Trp His Ala Met Lys
Gly Ala His Gln 20 25 30 Ala Asp Ser Leu Ile Ala Gln Lys Ala Glu
Gly Ala Trp Val Thr Asp 35 40 45 Thr Asp Gly Arg Arg Tyr Leu Asp
Ala Met Ser Gly Leu Trp Cys Val 50 55 60 Asn Ile Gly Tyr Gly Arg
Lys Glu Leu Ala Glu Ala Ala Tyr Glu Gln 65 70 75 80 Leu Lys Glu Leu
Pro Tyr Tyr Pro Leu Thr Gln Ser His Ala Pro Ala 85 90 95 Ile Gln
Leu Ala Glu Lys Leu Asn Glu Trp Leu Gly Gly Asp Tyr Val 100 105 110
Ile Phe Phe Ser Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys 115
120 125 Ile Ala Arg Gln Tyr His Leu Gln Asn Gly Asp His Ser Arg Tyr
Lys 130 135 140 Phe Ile Ser Arg Tyr Arg Ala Tyr His Gly Asn Thr Leu
Gly Ala Leu 145 150 155 160 Ser Ala Thr Gly Gln Ala Gln Arg Lys Tyr
Lys Tyr Glu Pro Leu Ser 165 170 175 Gln Gly Phe Leu His Ala Ala Pro
Pro Asp Ile Tyr Arg Asn Pro Asp 180 185 190 Asp Ala Asp Thr Leu Glu
Ser Ala Asn Glu Ile Asp Arg Ile Met Thr 195 200 205 Trp Glu Leu Ser
Glu Thr Ile Ala Gly Val Ile Met Glu Pro Ile Ile 210 215 220 Thr Gly
Gly Gly Ile Leu Met Pro Pro Asp Gly Tyr Met Lys Lys Val 225 230 235
240 Glu Asp Ile Cys Arg Arg His Gly Ala Leu Leu Ile Cys Asp Glu Val
245 250 255 Ile Cys Gly Phe Gly Arg Thr Gly Glu Pro Phe Gly Phe Met
His Tyr 260 265 270 Gly Val Lys Pro Asp Ile Ile Thr Met Ala Lys Gly
Ile Thr Ser Ala 275 280 285 Tyr Leu Pro Leu Ser Ala Thr Ala Val Lys
Arg Asp Ile Phe Glu Ala 290 295 300 Tyr Gln Gly Glu Ala Pro Tyr Asp
Arg Phe Arg His Val Asn Thr Phe 305 310 315 320 Gly Gly Ser Pro Ala
Ala Cys Ala Leu Ala Leu Lys Asn Leu Gln Ile 325 330 335 Met Glu Asp
Glu Gln Leu Ile Gln Arg Ser Arg Asp Leu Gly Ala Lys 340 345 350 Leu
Leu Gly Glu Leu Gln Ala Leu Arg Glu His Pro Ala Val
Gly Asp 355 360 365 Val Arg Gly Lys Gly Leu Leu Ile Gly Ile Glu Leu
Val Lys Asp Lys 370 375 380 Leu Thr Lys Glu Pro Ala Asp Ala Ala Lys
Val Asn Gln Val Val Ala 385 390 395 400 Ala Cys Lys Glu Lys Gly Leu
Ile Ile Gly Lys Asn Gly Asp Thr Val 405 410 415 Ala Gly Tyr Asn Asn
Val Ile His Val Ala Pro Pro Phe Cys Leu Thr 420 425 430 Glu Glu Asp
Leu Ser Phe Ile Val Lys Thr Val Lys Glu Ser Phe Gln 435 440 445 Thr
Ile 450 661407DNAPseudomonas aeruginosa 66atgaacgcaa gactgcacgc
cacgtccccc ctcggcgacg ccgacctggt ccgtgccgac 60caggcccact acatgcacgg
ctaccacgtg ttcgacgacc accgcgtcaa cggctcgctg 120aacatcgccg
ccggcgacgg cgcctatatc tacgacaccg ccggcaaccg ctacctcgac
180gcggtgggcg gcatgtggtg caccaacatc ggcctggggc gcgaggaaat
ggctcgcacc 240gtggccgagc agacccgcct gctggcctat tccaatccct
tctgcgacat ggccaacccg 300cgcgccatcg aactctgccg caagctcgcc
gagctggccc ccggcgacct cgaccacgtg 360ttcctcacca ccggcggttc
caccgccgtg gacaccgcga tccgcctcat gcactactac 420cagaactgcc
gcggcaagcg cgccaagaag cacgtcatca cgcggatcaa cgcctaccac
480ggctcgacct tcctcggcat gtcgctgggc ggcaagagcg ccgaccggcc
ggccgagttc 540gacttcctcg acgagcgcat ccaccacctc gcctgtccct
attactaccg cgctccggaa 600gggctgggcg aagccgagtt cctcgatggc
ctggtggacg agttcgaacg caagatcctc 660gaactgggcg ccgaccgggt
gggggcgttc atctccgagc cggtgttcgg ctccggcggc 720gtgatcgtcc
cgcccgcggg ctaccacagg cggatgtggg agctgtgcca gcgctacgac
780gtgctgtaca tctccgacga agtggtgacc tccttcggcc gcctcggcca
cttcttcgcc 840agccaggcgg tgttcggcgt acagccggac atcatcctca
ccgccaaggg cctcacctcc 900ggctaccagc cgctgggcgc gtgcatcttc
tcccggcgca tctgggaggt gatcgccgag 960ccggacaagg gccgctgctt
cagccatggt ttcacctact ccggccaccc ggtggcctgc 1020gcggcggcgc
tgaagaacat cgagatcatc gagcgcgagg gcttgctcgc ccacgccgac
1080gaggtcggcc gctacttcga ggagcgcctg caaagcctcc gcgacctgcc
catcgtcggc 1140gacgtgcgcg ggatgcgctt catggcctgt gtcgagttcg
tcgccgacaa ggcgagcaag 1200gcgctgtttc cggaaagcct gaacatcggc
gagtgggtcc acctgcgggc gcagaagcgc 1260ggcctgctgg ttcgtccgat
cgtccacctg aacgtgatgt cgccgccgct gatcctcacc 1320cgcgaacagg
tcgataccgt ggtccgggtg ctgcgcgaga gcatcgagga aaccgtggag
1380gatcttgtcc gcgccggtca ccggtaa 140767468PRTPseudomonas
aeruginosa 67Met Asn Ala Arg Leu His Ala Thr Ser Pro Leu Gly Asp
Ala Asp Leu 1 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 Thr 65 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 His 145 150 155 160 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 Gly 225 230
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 Glu 305 310 315 320 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 Lys 385 390 395 400 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 Arg 465 681335DNAPseudomonas aeruginosa 68atgacaatga atgacgagcc
gcagtcgagc agcctcgaca acttctggat gcccttcacc 60gccaaccgcc agttcaaggc
gcggccgcgc ctgctggaaa gcgccgaagg catccactat 120atcgcccagg
gcgggcgccg catcctcgac ggcaccgccg gcctctggtg ctgcaatgcc
180ggccacggcc ggcgcgagat cagcgaagcg gtggcccggc agatcgccac
cctcgactac 240gccccgccgt tccagatggg tcacccgctg ccgttcgaac
tcgccgcgcg gctgacggaa 300atcgccccgc cgagcctgaa caaagtattc
ttcaccaact ccggctcgga atcggcggac 360accgcgctga agatcgccct
tgcctaccag cgcgccatcg gccagggcac ccgcacccgc 420ctgatcggcc
gcgaactggg ctaccacggg gtcggcttcg gcggcctgtc ggtaggcggt
480atggtcaaca accgcaaggc cttctccgcc aacctgctgc cgggggtcga
ccacctgccg 540cacaccctgg acgtcgcccg caacgccttc accgtcggcc
tgcccgagca tggcgtggaa 600aaggccgagg agctggaacg cctggtgacc
ctgcacggcg ccgagaatat cgccgcggtg 660atcgtcgagc cgatgtccgg
ctcggccggc gtggtgctgc cgcccaaggg ctaccttcag 720cggctgcgcg
agataacccg caagcatggc atcctgctga tcttcgacga agtgatcacc
780ggtttcggcc gcgtcggcga agccttcgcc gcgcagcgct ggggcgtcgt
cccggacctg 840ctgacctgcg ccaaggggct gaccaacggc agcatcccga
tgggcgccgt attcgtcgac 900gagaagatcc atgctgcctt catgcaaggc
ccgcagggcg ccatcgagtt cttccacggc 960tatacctatt ccggccatcc
ggtagcctgc gccgccgccc tggcgaccct ggacatctac 1020cgtcgcgacg
acctgttcca gcgggccgtc gaactggaag gctactggca ggacgcgctg
1080ttcagcctgc gcgacctgcc caacgtggtc gacatccgcg ccgtaggcct
ggtcggcggc 1140gtgcaactgg cgccgcacgc ggacggcccc ggcaagcgcg
gctacgacgt cttcgagcgc 1200tgcttctggg agcacgacct gatggtccgg
gtgaccggcg acatcatcgc catgtcgccg 1260ccgctgatca tcgacaagcc
ccacatcgac cagatcgtcg agcgcctggc ccaggccatc 1320cgcgccagcg tctga
133569444PRTPseudomonas aeruginosa 69Met Thr Met Asn Asp Glu Pro
Gln Ser Ser Ser Leu Asp Asn Phe Trp 1 5 10 15 Met Pro Phe Thr Ala
Asn Arg Gln Phe Lys Ala Arg Pro Arg Leu Leu 20 25 30 Glu Ser Ala
Glu Gly Ile His Tyr Ile Ala Gln Gly Gly Arg Arg Ile 35 40 45 Leu
Asp Gly Thr Ala Gly Leu Trp Cys Cys Asn Ala Gly His Gly Arg 50 55
60 Arg Glu Ile Ser Glu Ala Val Ala Arg Gln Ile Ala Thr Leu Asp Tyr
65 70 75 80 Ala Pro Pro Phe Gln Met Gly His Pro Leu Pro Phe Glu Leu
Ala Ala 85 90 95 Arg Leu Thr Glu Ile Ala Pro Pro Ser Leu Asn Lys
Val Phe Phe Thr 100 105 110 Asn Ser Gly Ser Glu Ser Ala Asp Thr Ala
Leu Lys Ile Ala Leu Ala 115 120 125 Tyr Gln Arg Ala Ile Gly Gln Gly
Thr Arg Thr Arg Leu Ile Gly Arg 130 135 140 Glu Leu Gly Tyr His Gly
Val Gly Phe Gly Gly Leu Ser Val Gly Gly 145 150 155 160 Met Val Asn
Asn Arg Lys Ala Phe Ser Ala Asn Leu Leu Pro Gly Val 165 170 175 Asp
His Leu Pro His Thr Leu Asp Val Ala Arg Asn Ala Phe Thr Val 180 185
190 Gly Leu Pro Glu His Gly Val Glu Lys Ala Glu Glu Leu Glu Arg Leu
195 200 205 Val Thr Leu His Gly Ala Glu Asn Ile Ala Ala Val Ile Val
Glu Pro 210 215 220 Met Ser Gly Ser Ala Gly Val Val Leu Pro Pro Lys
Gly Tyr Leu Gln 225 230 235 240 Arg Leu Arg Glu Ile Thr Arg Lys His
Gly Ile Leu Leu Ile Phe Asp 245 250 255 Glu Val Ile Thr Gly Phe Gly
Arg Val Gly Glu Ala Phe Ala Ala Gln 260 265 270 Arg Trp Gly Val Val
Pro Asp Leu Leu Thr Cys Ala Lys Gly Leu Thr 275 280 285 Asn Gly Ser
Ile Pro Met Gly Ala Val Phe Val Asp Glu Lys Ile His 290 295 300 Ala
Ala Phe Met Gln Gly Pro Gln Gly Ala Ile Glu Phe Phe His Gly 305 310
315 320 Tyr Thr Tyr Ser Gly His Pro Val Ala Cys Ala Ala Ala Leu Ala
Thr 325 330 335 Leu Asp Ile Tyr Arg Arg Asp Asp Leu Phe Gln Arg Ala
Val Glu Leu 340 345 350 Glu Gly Tyr Trp Gln Asp Ala Leu Phe Ser Leu
Arg Asp Leu Pro Asn 355 360 365 Val Val Asp Ile Arg Ala Val Gly Leu
Val Gly Gly Val Gln Leu Ala 370 375 380 Pro His Ala Asp Gly Pro Gly
Lys Arg Gly Tyr Asp Val Phe Glu Arg 385 390 395 400 Cys Phe Trp Glu
His Asp Leu Met Val Arg Val Thr Gly Asp Ile Ile 405 410 415 Ala Met
Ser Pro Pro Leu Ile Ile Asp Lys Pro His Ile Asp Gln Ile 420 425 430
Val Glu Arg Leu Ala Gln Ala Ile Arg Ala Ser Val 435 440
7071DNAArtificial sequenceForward primer for amplification of
Bacillus subtilis aminotransferase (gi16077991) 70ggggacaagt
ttgtacaaaa aagcaggcta ggaggaatta accatggaga tgatggggat 60ggaaaacatt
c 717165DNAArtificial sequenceReverse primer for amplification of
Bacillus subtilis aminotransferase (gi16077991) 71ggggaccact
ttgtacaaga aagctgggtt tatatcgttt gaaagctttc tttcaccgtt 60ttcac
657266DNAArtificial sequenceForward primer for amplification of
Pseudomonas aeruginosa aminotransferase (gi9951072) 72ggggacaagt
ttgtacaaaa aagcaggcta ggaggaatta accatgaacg caagactgca 60cgccac
667348DNAArtificial sequenceReverse primer for amplification of
Pseudomonas aeruginosa aminotransferase (gi9951072) 73ggggaccact
ttgtacaaga aagctgggtt taccggtgac cggcgcgg 487469DNAArtificial
sequenceForward primer for amplification of Pseudomonas aeruginosa
aminotransferase (gi9951630) 74ggggacaagt ttgtacaaaa aagcaggcta
ggaggaatta accatgacaa tgaatgacga 60gccgcagtc 697549DNAArtificial
sequenceReverse primer for amplification of Pseudomonas aeruginosa
aminotransferase (gi9951630) 75ggggaccact ttgtacaaga aagctgggtt
cagacgctgg cgcggatgg 497657DNAArtificial sequenceForward primer
76aaatttacta gtaagaattt ttgaggaggc aatataaatg aataaaccac agtcttg
577732DNAArtificial sequenceReverse primer 77aaatttggat cctacaagaa
agctgggttt ac 327858DNAArtificial sequenceForward primer
78aaatttacta gtaagaattt ttgaggaggc aatataaatg aacagccaaa tcaccaac
587937DNAArtificial sequenceReverse primer 79aaatttggat ccactttgta
caagaaagct gggttca 378057DNAArtificial sequenceForward primer
80aaatttggat ccgttgagga ggcctcaaaa atgtccgaga tcactctggg caaatac
578135DNAArtificial sequenceReverse primer 81aaatttggcg cgccattact
gtttagcgtt agttg 358252DNAArtificial sequenceForward primer
82aaatttggat ccgttgagga ggcctcaaaa atgtatactg ttggtgatta tc
528337DNAArtificial sequenceReverse primer 83aaatttggcg cgccattact
tgttctgctc cgcaaac 37
* * * * *
References