U.S. patent application number 16/105845 was filed with the patent office on 2019-07-18 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 | 20190218580 16/105845 |
Document ID | / |
Family ID | 39651405 |
Filed Date | 2019-07-18 |
United States Patent
Application |
20190218580 |
Kind Code |
A1 |
Raemakers-Franken; Petronella
Catharina ; et al. |
July 18, 2019 |
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
E-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.: |
16/105845 |
Filed: |
August 20, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15599314 |
May 18, 2017 |
|
|
|
16105845 |
|
|
|
|
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 13/005 20130101;
C07D 201/08 20130101; C07D 223/10 20130101; C12P 13/001 20130101;
C12P 13/02 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; C07D 201/08 20060101
C07D201/08; C07C 227/06 20060101 C07C227/06; C07D 223/10 20060101
C07D223/10 |
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. 15/599,314, filed May 18, 2017, which is a divisional of
U.S. patent application Ser. No. 14/105,705, filed Dec. 13, 2013,
now issued U.S. Pat. No. 9,663,805, issued Jun. 8, 2017, 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 Aug. 20, 2018, is named 12956-454-999_Sequence_Listing and is
249,788 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
.epsilon.-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
[0025] 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).
[0026] 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).
[0027] 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.
[0028] 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).
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] Suitable archaea may in particular be selected amongst the
group of Archaeoglobus, Aeropyrum, Halobacterium, Methanosarcina,
Methanococcus, Thermoplasma, Pyrobaculum, Methanocaldococcus,
Methanobacterium, Methanosphaera, Methanopyrus and
Methanobrevibacter.
[0034] Suitable fungi may in particular be selected amongst the
group of Rhizopus, Neurospora, Penicillium and Aspergillus.
[0035] A suitable yeast may in particular be selected amongst the
group of Candida, Hansenula, Kluyveromyces and Saccharomyces.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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 a-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.
[0040] Said acid preferably is a diacid, wherein the said
biocatalyst is selective towards the acid group next to the keto-
or amino-group.
[0041] 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.
[0042] 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).
[0043] 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)
[0044] 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.
[0045] The pyruvate decarboxylase may originate from Saccharomyces
cerevisiae or Zymomonas mobilis. In particular, pyruvate
decarboxylase mutant 1472A from Zymomonas mobilis may be used.
[0046] Glutamate decarboxylase, diaminopimelate decarboxylase or
aspartate decarboxylase from Escherichia coli (E. coli) may be
used.
[0047] 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).
[0048] An oxaloacetate decarboxylase from Pseudomonas may in
particular be used.
[0049] 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.
[0050] An alpha-ketoglutarate decarboxylase from Mycobacterium
tuberculosis may in particular be used.
[0051] 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).
[0052] 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.
[0053] 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).
[0054] 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).
[0055] 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.
[0056] 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.
[0057] 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, .beta.-alanine,
4-aminobutyrate, and .alpha.-aminoadipate.
[0058] 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 a-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).
[0059] 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).
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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).
[0065] 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-.epsilon.-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.
[0066] 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.
[0067] 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.
[0068] Alternatively, the conversion of 5-FVA to 6-ACA may be
performed by a chemical method, e.g. as mentioned above.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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).
[0075] 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).
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] Alternatively, the decarboxylation of AAP to 6-ACA may be
performed biocatalytically in the presence of a decarboxylase or
other biocatalyst catalysing such decarboxylation.
[0081] 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).
[0082] 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).
[0083] 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.
[0084] The pyruvate decarboxylase may originate from Saccharomyces
cerevisiae or Zymomonas mobilis. In particular, pyruvate
decarboxylase mutant 1472A 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.
[0085] 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.
[0086] A glutamate decarboxylase may in particular be selected from
Curcurbita moschata, cucumber, yeast, or calf brain; and
diaminopimelate decarboxylases (EC 4.1.1.20).
[0087] 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.
[0088] In particular, the diaminopimelate decarboxylase may be from
a gram negative bacterium, for instance E. coli.
[0089] 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.
[0090] 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.
[0091] Alternatively, the conversion of AAP to 6-ACA may be
performed by a chemical method, e.g. as mentioned above.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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. 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 PxyIA 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 eg/A,
eg/B, cbhA, promoters of transcriptional regulators such as areA,
creA, xlnR, pacC, prfT, 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
General Methods
Molecular and Genetic Techniques
[0161] 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 Wley Interscience, New York 1987).
Plasmids and Strains
[0162] 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.
Media
[0163] 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).
Identification of Plasmids
[0164] 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.
HPLC-MS Analysis Method for the Determination of 5-FVA
[0165] 5-FVA was detected by selective reaction monitoring
(SRM)-MS, measuring the transition m/z 129.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.
TABLE-US-00003 LC conditions: 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) Column room
temperature temperature: Eluent: A: water containing 0.1% formic
acid B: acetonitrile containing 0.1% formic acid Gradient: time
(min) % eluent B 0 10 6 50 6.1 10 11 10 Flow: 1.2 ml/min, before
entering the MS the flow is split 1:3 Injection volume: 2 .mu.l MS
conditions: Ionisation: negative ion electrospray source
conditions: ionspray voltage: 5 kV temperature: 350.degree. C.
fragmentor voltage and collision energy optimized Scan mode:
selective reaction mode: transition m/z 129 .fwdarw. 83
HPLC-MS Analysis for the Determination of AAP
[0166] 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).
[0167] LC conditions were as follows: [0168] 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) [0169] Eluent: A=0.1%
(v/v) formic acid in ultrapure water
[0170] B=0.1% (v/v) formic acid in acetonitrile (pa, Merck) [0171]
Flow: 1.2 ml/min, before entering the MS the flow was split 1:3
[0172] 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. [0173]
Injection volume: 2 .mu.l [0174] MS conditions: Positive ion
electrospray was used for ionization [0175] Detection: in SIM mode
on m/z 176, with a dwell time of 100 msec.
HPLC-MS Analysis for the Determination of 6-ACA
[0176] Calibration:
[0177] 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: [0178] Column: 50*4 Nucleosil
(Mancherey-Nagel)+250.times.4.6 Prevail C18 (Alltech), both at room
temperature (RT) [0179] Eluent: A=0.1(v/v) formic acid in ultrapure
water
[0180] B=Acetonitrile (pa, Merck) [0181] Flow: 1.0 ml/min, before
entering the MS the flow was split 1:3
[0182] 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. [0183] Injection volume: 5 .mu.l
[0184] MS detection: ESI(+)-MS
[0185] 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.
Cloning of Target Genes
Design of Expression Constructs
[0186] 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
[0187] Gateway technology (Invitrogen, Carlsbad, Calif., USA).
Gene Synthesis and Construction of Plasmids
[0188] 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.
[0189] 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.
[0190] 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.
Cloning by PCR
[0191] 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 gene enzyme primer origin of 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
[0192] PCR reactions were analysed by agarose gel electrophoresis
and PCR products of the correct size were eluted from the gel using
the QlAquick 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.
Growth of E. coli for Protein Expression
[0193] 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.
Preparation of Cell Lysates
Preparation of Lysis Buffer
[0194] 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
[0195] The solution was freshly prepared directly before use.
Preparation of Cell Free Extract by Lysis
[0196] 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.
Preparation of Cell Free Extract by Sonification
[0197] 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 .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.
Preparation of 5-Formylpentanoic Acid by Chemical Hydrolysis of
Methyl 5-Formylpentanoate
[0198] 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.
[0199] Enzymatic Reactions for Conversion of 5-Formylpentanoic Acid
to 6-ACA
[0200] Unless specified otherwise, a reaction mixture was prepared
comprising 10 mM 5-formylpentanoic acid, 20 mM racemic
a-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 concentration Biocatalyst [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
0.6 (biological 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.
[0201] It is shown that 6-ACA is formed from 5-FVA in the presence
of an aminotransferase.
Enzymatic Reactions for Conversion of AKP to 5-Formylpentanoic
Acid
[0202] 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-Pdcl472A 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
[0203] It is shown that 5-FVA is formed from AKP in the presence of
a decarboxylase.
Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of
Recombinant Decarboxylase
[0204] 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-Pdcl472A 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
[0205] 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.
Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of
Recombinant Decarboxylase and Recombinant Aminotransferase
[0206] 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/ DC pBAD-Vfl-AT pBAD-Bwe-AT
pBAD-PAE_gi9946143_AT E. coli TOP10/pBAD-Pdc 183.4 248.9 117.9 E.
coli TOP10/pBAD-Pdcl472A 458.5 471.6 170.3 E. coli TOP10/pBAD-KdcA
497.8 497.8 275.1 E. coli TOP10/pBAD-KivD 510.9 510.9 314.4 AT =
aminotransferase DC = decarboxylase
[0207] In the chemical blank and in the biological blank no 6-ACA
was detectable.
[0208] 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.
Construction of Plasmids for Expression of Aminotransferases and
Decarboxylases in S. cerevisiae
[0209] 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].
[0210] 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].
[0211] 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.
[0212] 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].
[0213] 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].
[0214] 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.
[0215] 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.
[0216] The resulting plasmids were obtained: [0217] pAKP-85:
PgaI10-Pae_AT-Tadh2 PgaI2-Pdc_DC-Tpma1 [0218] pAKP-86:
PgaI10-Pae_AT-Tadh2 PgaI2-KdcA_DC-Tpma1 [0219] pAKP-87:
PgaI10-Vfl_AT-Tadh2 PgaI2-Pdc_DC-Tpma1 [0220] pAKP-88:
PgaI10-Vfl_AT-Tadh2 PgaI2-KdcA_DC-Tpma1
Transformation and Growth of S. cerevisiae
[0221] 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 lx Yeast
Nitrogen Base without amino acids and 2% glucose.
[0222] The resulting strains were grown aerobically at 30.degree.
C. for 48 hour in Verduyn minimal medium containing 0.05% glucose
and 4% galactose.
Preparation of Cell Free Extract
[0223] 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.
Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of
Decarboxylase and Aminotransferase Co-Expressed in S.
Cerevisiae
[0224] 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 Biocatalyst 6-ACA concentration
[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
Enzymatic Reactions for Conversion of Alpha-Ketopimelic Acid to
Alpha-Aminopimelic Acid
[0225] 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
[0226] It is shown that the formation of AAP from AKP is catalysed
by the biocatalyst.
Chemical Conversion of AAP to Caprolactam
[0227] 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 Leu1 5 10 15tat 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 30gtg 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 45tat 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 60cac 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 Pro65 70 75
80ggt 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 95tcg 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 110tat 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 125tgg 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 140acc 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 Met145 150 155 160acc 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 175gtg 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 190acc 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
205atc 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 220gtg 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 Gln225 230 235 240gcg 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 255gag 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 270acc 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 285gcg 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 300aaa 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 Gly305 310 315
320ttt 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 335atc 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 350gcc 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 365atc 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 380aag 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 Ser385 390 395 400gag 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 415ctt 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 430cag
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
445ttt gcc gag gtt gcc tga 1362Phe Ala Glu Val Ala 4502453PRTVibrio
fluvialis 2Met Asn Lys Pro Gln Ser Trp Glu Ala Arg Ala Glu Thr Tyr
Ser Leu1 5 10 15Tyr Gly Phe Thr Asp Met Pro Ser Leu His Gln Arg Gly
Thr Val Val 20 25 30Val Thr His Gly Glu Gly Pro Tyr Ile Val Asp Val
Asn Gly Arg Arg 35 40 45Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met
Val Ala Gly Phe Asp 50 55 60His Lys Gly Leu Ile Asp Ala Ala Lys Ala
Gln Tyr Glu Arg Phe Pro65 70 75 80Gly Tyr His Ala Phe Phe Gly Arg
Met Ser Asp Gln Thr Val Met Leu 85 90 95Ser Glu Lys Leu Val Glu Val
Ser Pro Phe Asp Ser Gly Arg Val Phe 100 105 110Tyr Thr Asn Ser Gly
Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu 115 120 125Trp Phe Leu
His Ala Ala Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu 130 135 140Thr
Arg Trp Asn Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met145 150
155 160Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly
Phe 165 170 175Val His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu
Glu Gly Glu 180 185 190Thr Glu Glu Gln Phe Val Ala Arg Leu Ala Arg
Glu Leu Glu Glu Thr 195 200 205Ile Gln Arg Glu Gly Ala Asp Thr Ile
Ala Gly Phe Phe Ala Glu Pro 210 215 220Val Met Gly Ala Gly Gly Val
Ile Pro Pro Ala Lys Gly Tyr Phe Gln225 230 235 240Ala Ile Leu Pro
Ile Leu Arg Lys Tyr Asp Ile Pro Val Ile Ser Asp 245 250 255Glu Val
Ile Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val 260 265
270Thr Tyr Asp Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr
275 280 285Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu
Leu Ser 290 295 300Lys Arg Leu Glu Thr Ala Ile Glu Ala Ile Glu Glu
Phe Pro His Gly305 310 315 320Phe Thr Ala Ser Gly His Pro Val Gly
Cys Ala Ile Ala Leu Lys Ala 325 330 335Ile Asp Val Val Met Asn Glu
Gly Leu Ala Glu Asn Val Arg Arg Leu 340 345 350Ala Pro Arg Phe Glu
Glu Arg Leu Lys His Ile Ala Glu Arg Pro Asn 355 360 365Ile Gly Glu
Tyr Arg Gly Ile Gly Phe Met Trp Ala Leu Glu Ala Val 370 375 380Lys
Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser385 390
395 400Glu Arg Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg
Pro 405 410 415Leu Gly Gln Ser Val Val Leu Cys Pro Pro Phe Ile Leu
Thr Glu Ala 420 425 430Gln Met Asp Glu Met Phe Asp Lys Leu Glu Lys
Ala Leu Asp Lys Val 435 440 445Phe Ala Glu Val Ala
45031362DNAArtificialVibrio 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 Lys1
5 10 15tac 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 30att 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 45cgt 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 60ggt 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 Met65 70 75 80ccg 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 95gaa 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 110aac 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 125tat 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 140tac 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 Gly145 150 155
160caa 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 175cat 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 190ctg 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 205caa 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 220att 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 Cys225 230 235 240gag 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 255ggc 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 270gat
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
285tca 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 300gat 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 Pro305 310 315 320gct 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 335aaa 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 350cta 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 365ggc 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 380ccg 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 Glu385 390 395
400aaa 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 415aat 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 430act 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 445cag taa 1350Gln5449PRTBacillus
weihenstephanensis 5Val Gln Ala Thr Glu Gln Thr Gln Ser Leu Lys Lys
Thr Asp Glu Lys1 5 10 15Tyr Leu Trp His Ala Met Arg Gly Ala Ala Pro
Ser Pro Thr Asn Leu 20 25 30Ile Ile Thr Lys Ala Glu Gly Ala Trp Val
Thr Asp Ile Asp Gly Asn 35 40 45Arg Tyr Leu Asp Gly Met Ser Gly Leu
Trp Cys Val Asn Val Gly Tyr 50 55 60Gly Arg Lys Glu Leu Ala Arg Ala
Ala Phe Glu Gln Leu Glu Glu Met65 70 75 80Pro Tyr Phe Pro Leu Thr
Gln Ser His Val Pro Ala Ile Lys Leu Ala 85 90 95Glu Lys Leu Asn Glu
Trp Leu Asp Asp Glu Tyr Val Ile Phe Phe Ser 100 105 110Asn Ser Gly
Ser Glu Ala Asn Glu Thr Ala Phe Lys Ile Ala Arg Gln 115 120 125Tyr
His Gln Gln Lys Gly Asp His Gly Arg Tyr Lys Phe Ile Ser Arg 130 135
140Tyr Arg Ala Tyr His Gly Asn Ser Met Gly Ala Leu Ala Ala Thr
Gly145 150 155 160Gln Ala Gln Arg Lys Tyr Lys Tyr Glu Pro Leu Gly
Gln Gly Phe Leu 165 170 175His Val Ala Pro Pro Asp Thr Tyr Arg Asn
Pro Glu Asp Val His Thr 180 185 190Leu Ala Ser Ala Glu Glu Ile Asp
Arg Val Met Thr Trp Glu Leu Ser 195 200 205Gln Thr Val Ala Gly Val
Ile Met Glu Pro Ile Ile Thr Gly Gly Gly 210 215 220Ile Leu Met Pro
Pro Asp Gly Tyr Met Gly Lys Val Lys Glu Ile Cys225 230 235 240Glu
Lys His Gly Ala Leu Leu Ile Cys Asp Glu Val Ile Cys Gly Phe 245 250
255Gly Arg Thr Gly Lys Pro Phe Gly Phe Met Asn Tyr Gly Val Lys Pro
260 265 270Asp Ile Ile Thr Met Ala Lys Gly Ile Thr Ser Ala Tyr Leu
Pro Leu 275 280 285Ser Ala Thr Ala Val Arg Arg Glu Val Tyr Glu Ala
Phe Val Gly Ser 290
295 300Asp Asp Tyr Asp Arg Phe Arg His Val Asn Thr Phe Gly Gly Asn
Pro305 310 315 320Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu Ile
Met Glu Asn Glu 325 330 335Lys Leu Ile Glu Arg Ser Lys Glu Leu Gly
Glu Arg Leu Leu Tyr Glu 340 345 350Leu Glu Asp Val Lys Glu His Pro
Asn Val Gly Asp Val Arg Gly Lys 355 360 365Gly Leu Leu Leu Gly Ile
Glu Leu Val Glu Asp Lys Gln Thr Lys Glu 370 375 380Pro Ala Ser Ile
Glu Lys Met Asn Lys Val Ile Asn Ala Cys Lys Glu385 390 395 400Lys
Gly Leu Ile Ile Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn 405 410
415Asn Ile Leu Gln Leu Ala Pro Pro Leu Ser Ile Thr Glu Glu Asp Phe
420 425 430Thr Phe Ile Val Lys Thr Met Lys Glu Cys Leu Ser Arg Ile
Asn Gly 435 440 445Gln61350DNAArtificialB. 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 Leu1 5 10 15agc 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 30gag 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 45gac 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 60gtc 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 Arg65 70 75 80cag 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 95ccg 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 110ggc
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
125acc 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 140cag 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 Thr145 150 155 160gtc 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 175gat 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 190ggc 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 205gag 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 220gcc 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 Pro225 230 235
240ccg 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 255atc 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 270gag 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 285atc 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 300gtg 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 Tyr305 310 315 320cac 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 335gag 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 350gcg
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
365cac 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 380gag 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 Gly385 390 395 400gtc 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 415cgc 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 430ccg 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 445cag acc gcc gcc gcc
gtc ctg gct tga 1371Gln Thr Ala Ala Ala Val Leu Ala 450
4558456PRTPseudomonas aeruginosa 8Met Asn Ser Gln Ile Thr Asn Ala
Lys Thr Arg Glu Trp Gln Ala Leu1 5 10 15Ser Arg Asp His His Leu Pro
Pro Phe Thr Asp Tyr Lys Gln Leu Asn 20 25 30Glu Lys Gly Ala Arg Ile
Ile Thr Lys Ala Glu Gly Val Tyr Ile Trp 35 40 45Asp Ser Glu Gly Asn
Lys Ile Leu Asp Ala Met Ala Gly Leu Trp Cys 50 55 60Val Asn Val Gly
Tyr Gly Arg Glu Glu Leu Val Gln Ala Ala Thr Arg65 70 75 80Gln Met
Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gln Thr Ala His 85 90 95Pro
Pro Val Val Glu Leu Ala Lys Ala Ile Ala Asp Val Ala Pro Glu 100 105
110Gly Met Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Ala Asn Asp
115 120 125Thr Val Leu Arg Met Val Arg His Tyr Trp Ala Thr Lys Gly
Gln Pro 130 135 140Gln Lys Lys Val Val Ile Gly Arg Trp Asn Gly Tyr
His Gly Ser Thr145 150 155 160Val Ala Gly Val Ser Leu Gly Gly Met
Lys Ala Leu His Glu Gln Gly 165 170 175Asp Phe Pro Ile Pro Gly Ile
Val His Ile Ala Gln Pro Tyr Trp Tyr 180 185 190Gly Glu Gly Gly Asp
Met Ser Pro Asp Glu Phe Gly Val Trp Ala Ala 195 200 205Glu Gln Leu
Glu Lys Lys Ile Leu Glu Val Gly Glu Glu Asn Val Ala 210 215 220Ala
Phe Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro225 230
235 240Pro Asp Thr Tyr Trp Pro Lys Ile Arg Glu Ile Leu Ala Lys Tyr
Asp 245 250 255Ile Leu Phe Ile Ala Asp Glu Val Ile Cys Gly Phe Gly
Arg Thr Gly 260 265 270Glu Trp Phe Gly Ser Gln Tyr Tyr Gly Asn Ala
Pro Asp Leu Met Pro 275 280 285Ile Ala Lys Gly Leu Thr Ser Gly Tyr
Ile Pro Met Gly Gly Val Val 290 295 300Val Arg Asp Glu Ile Val Glu
Val Leu Asn Gln Gly Gly Glu Phe Tyr305 310 315 320His Gly Phe Thr
Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335Glu Asn
Ile Arg Ile Leu Arg Glu Glu Lys Ile Ile Glu Lys Val Lys 340 345
350Ala Glu Thr Ala Pro Tyr Leu Gln Lys Arg Trp Gln Glu Leu Ala Asp
355 360 365His Pro Leu Val Gly Glu Ala Arg Gly Val Gly Met Val Ala
Ala Leu 370 375 380Glu Leu Val Lys Asn Lys Lys Thr Arg Glu Arg Phe
Thr Asp Lys Gly385 390 395 400Val Gly Met Leu Cys Arg Glu His Cys
Phe Arg Asn Gly Leu Ile Met 405 410 415Arg Ala Val Gly Asp Thr Met
Ile Ile Ser Pro Pro Leu Val Ile Asp 420 425 430Pro Ser Gln Ile Asp
Glu Leu Ile Thr Leu Ala Arg Lys Cys Leu Asp 435 440 445Gln Thr Ala
Ala Ala Val Leu Ala 450 455970DNAArtificialprimer 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 Ser1
5 10 15gag 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 30ggc 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 45gag 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 60atc 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 Met65 70 75 80cgc 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 95gtg 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 110aac 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 125ctg 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 140aaa 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 Ala145 150 155
160ggt 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 175ccg 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 190ggc 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 205ctg 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 220att 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 Asp225 230 235 240tcc 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 255ttc 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 270ttc
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
285aaa 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 300gat 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 Gly305 310 315 320ttt 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 335atc 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 350acg 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 365ctg 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 380gtg 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 Gly385 390 395
400atg 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 415gtg 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 430caa 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 445ctg gcg gtg ttg cag ggc tga 1365Leu Ala
Val Leu Gln Gly 45012454PRTPseudomonas syringae 12Met Ser Ala Asn
Asn Pro Gln Thr Leu Glu Trp Gln Ala Leu Ser Ser1 5 10 15Glu His His
Leu Ala Pro Phe Ser Asp Tyr Lys Gln Leu Lys Glu Lys 20 25 30Gly Pro
Arg Ile Ile Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser 35 40 45Glu
Gly Asn Lys Ile Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala 50 55
60Ile Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gln Met65
70 75 80Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gln Thr Ala His Pro
Pro 85 90 95Val Leu Glu Leu Ala Lys Ala Ile Ser Asp Ile
Ala Pro Glu Gly Met 100 105 110Asn His Val Phe Phe Thr Gly Ser Gly
Ser Glu Gly Asn Asp Thr Met 115 120 125Leu Arg Met Val Arg His Tyr
Trp Ala Leu Lys Gly Gln Pro Asn Lys 130 135 140Lys Thr Ile Ile Ser
Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala145 150 155 160Gly Ala
Ser Leu Gly Gly Met Thr Tyr Met His Glu Gln Gly Asp Leu 165 170
175Pro Ile Pro Gly Val Val His Ile Pro Gln Pro Tyr Trp Phe Gly Glu
180 185 190Gly Gly Asp Met Thr Pro Asp Glu Phe Gly Ile Trp Ala Ala
Glu Gln 195 200 205Leu Glu Lys Lys Ile Leu Glu Leu Gly Val Glu Asn
Val Gly Ala Phe 210 215 220Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly
Val Ile Val Pro Pro Asp225 230 235 240Ser Tyr Trp Pro Lys Ile Lys
Glu Ile Leu Ser Arg Tyr Asp Ile Leu 245 250 255Phe Ala Ala Asp Glu
Val Ile Cys Gly Phe Gly Arg Thr Ser Glu Trp 260 265 270Phe Gly Ser
Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr Ile Ala 275 280 285Lys
Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu Ile Val Arg 290 295
300Asp Glu Ile Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His
Gly305 310 315 320Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val
Ala Leu Glu Asn 325 330 335Ile Arg Ile Leu Arg Glu Glu Lys Ile Val
Glu Arg Val Arg Ser Glu 340 345 350Thr Ala Pro Tyr Leu Gln Lys Arg
Leu Arg Glu Leu Ser Asp His Pro 355 360 365Leu Val Gly Glu Val Arg
Gly Val Gly Leu Leu Gly Ala Ile Glu Leu 370 375 380Val Lys Asp Lys
Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly Ala Gly385 390 395 400Met
Ile Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu Ile Met Arg Ala 405 410
415Val Gly Asp Thr Met Ile Ile Ala Pro Pro Leu Val Ile Ser Phe Ala
420 425 430Gln Ile Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp
Leu Thr 435 440 445Leu Ala Val Leu Gln Gly
450131365DNAArtificialPseudomonas 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 Ser1 5 10
15atc 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 30gtg 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 45gag 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 60agt 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 Asn65 70 75 80gcg 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 95ccg 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 110tat 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 125gcg 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 140agt 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 Glu145 150 155 160gcc
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
175ggt 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 190cat 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 205tta 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 220agt 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 Thr225 230 235 240acg 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 255agc 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 270gaa agc att
caa cag gct gct agc att tca taa 849Glu Ser Ile Gln Gln Ala Ala Ser
Ile Ser 275 28015282PRTBacillus subtilis 15Met Lys Val Leu Val Asn
Gly Arg Leu Ile Gly Arg Ser Glu Ala Ser1 5 10 15Ile Asp Leu Glu Asp
Arg Gly Tyr Gln Phe Gly Asp Gly Ile Tyr Glu 20 25 30Val Ile Arg Val
Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala 35 40 45Glu Arg Phe
Phe Arg Ser Ala Ala Glu Ile Gly Ile Ser Leu Pro Phe 50 55 60Ser Ile
Glu Asp Leu Glu Trp Asp Leu Gln Lys Leu Val Gln Glu Asn65 70 75
80Ala Val Ser Glu Gly Ala Val Tyr Ile Gln Thr Thr Arg Gly Val Ala
85 90 95Pro Arg Lys His Gln Tyr Glu Ala Gly Leu Glu Pro Gln Thr Thr
Ala 100 105 110Tyr Thr Phe Thr Val Lys Lys Pro Glu Gln Glu Gln Ala
Tyr Gly Val 115 120 125Ala Ala Ile Thr Asp Glu Asp Leu Arg Trp Leu
Arg Cys Asp Ile Lys 130 135 140Ser Leu Asn Leu Leu Tyr Asn Val Met
Thr Lys Gln Arg Ala Tyr Glu145 150 155 160Ala Gly Ala Phe Glu Ala
Ile Leu Leu Arg Asp Gly Val Val Thr Glu 165 170 175Gly Thr Ser Ser
Asn Val Tyr Ala Val Ile Asn Gly Thr Val Arg Thr 180 185 190His Pro
Ala Asn Arg Leu Ile Leu Asn Gly Ile Thr Arg Met Asn Ile 195 200
205Leu Gly Leu Ile Glu Lys Asn Gly Ile Lys Leu Asp Glu Thr Pro Val
210 215 220Ser Glu Glu Glu Leu Lys Gln Ala Glu Glu Ile Phe Ile Ser
Ser Thr225 230 235 240Thr Ala Glu Ile Ile Pro Val Val Thr Leu Asp
Gly Gln Ser Ile Gly 245 250 255Ser Gly Lys Pro Gly Pro Val Thr Lys
Gln Leu Gln Ala Ala Phe Gln 260 265 270Glu Ser Ile Gln Gln Ala Ala
Ser Ile Ser 275 280161347DNABacillus 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 Pro1 5 10
15ttt 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 30ggg 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 45ggt 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 60cta 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 Thr65 70 75 80tta 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 95atc 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 110ggc 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 125aag 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 140ggg 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 Glu145 150 155 160ctt
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
175ccg 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 190cgt 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 205gag 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 220ggt 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 Leu225 230 235 240tgt 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 255ttt 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 270cct 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 285att
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
300gat 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
Gly305 310 315 320aat 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 335tct 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 350ttt 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 365cag 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 380aaa 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 Leu385 390 395 400aaa
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
415gca 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 430gtt 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 445tga 134717448PRTBacillus subtilis 17Met Thr His
Asp Leu Ile Glu Lys Ser Lys Lys His Leu Trp Leu Pro1 5 10 15Phe Thr
Gln Met Lys Asp Tyr Asp Glu Asn Pro Leu Ile Ile Glu Ser 20 25 30Gly
Thr Gly Ile Lys Val Lys Asp Ile Asn Gly Lys Glu Tyr Tyr Asp 35 40
45Gly Phe Ser Ser Val Trp Leu Asn Val His Gly His Arg Lys Lys Glu
50 55 60Leu Asp Asp Ala Ile Lys Lys Gln Leu Gly Lys Ile Ala His Ser
Thr65 70 75 80Leu Leu Gly Met Thr Asn Val Pro Ala Thr Gln Leu Ala
Glu Thr Leu 85 90 95Ile Asp Ile Ser Pro Lys Lys Leu Thr Arg Val Phe
Tyr Ser Asp Ser 100 105 110Gly Ala Glu Ala Met Glu Ile Ala Leu Lys
Met Ala Phe Gln Tyr Trp 115 120 125Lys Asn Ile Gly Lys Pro Glu Lys
Gln Lys Phe Ile Ala Met Lys Asn 130 135 140Gly Tyr His Gly Asp Thr
Ile Gly Ala Val Ser Val Gly Ser Ile Glu145 150 155 160Leu Phe His
His Val Tyr Gly Pro Leu Met Phe Glu Ser Tyr Lys Ala 165 170 175Pro
Ile Pro Tyr Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys 180 185
190Arg Asp Gln Cys Leu Arg Glu Leu Ala Gln Leu Leu Glu Glu His His
195 200 205Glu Glu Ile Ala Ala Leu Ser Ile Glu Ser Met Val Gln Gly
Ala Ser 210 215 220Gly Met Ile Val Met Pro Glu Gly Tyr Leu Ala Gly
Val Arg Glu Leu225 230 235 240Cys Thr Thr Tyr Asp Val Leu Met Ile
Val Asp Glu Val Ala Thr Gly 245 250 255Phe Gly Arg Thr Gly Lys Met
Phe Ala Cys Glu His Glu Asn Val Gln 260 265 270Pro Asp Leu Met Ala
Ala Gly Lys Gly Ile Thr Gly Gly Tyr Leu Pro 275 280 285Ile Ala Val
Thr Phe Ala Thr Glu Asp Ile Tyr Lys Ala Phe Tyr Asp 290 295 300Asp
Tyr Glu Asn Leu Lys Thr Phe Phe His Gly His Ser Tyr Thr Gly305 310
315 320Asn Gln Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe
Glu 325 330 335Ser Glu Asn Ile Val Glu Gln Val Ala Glu Lys Ser Lys
Lys Leu His 340 345 350Phe Leu Leu Gln Asp Leu His Ala Leu Pro His
Val Gly Asp Ile Arg 355 360 365Gln Leu Gly Phe Met Cys Gly Ala Glu
Leu Val Arg Ser Lys Glu Thr 370 375 380Lys Glu Pro Tyr Pro Ala Asp
Arg Arg Ile Gly Tyr Lys Val Ser Leu385 390 395
400Lys Met Arg Glu Leu Gly Met Leu Thr Arg Pro Leu Gly Asp Val Ile
405 410 415Ala Phe Leu Pro Pro Leu Ala Ser Thr Ala Glu Glu Leu Ser
Glu Met 420 425 430Val Ala Ile Met Lys Gln Ala Ile His Glu Val Thr
Ser Leu Glu Asp 435 440 445181467DNARhodobacter
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 Arg1 5 10 15gag 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 30gcg 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 45atg 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 60ggc 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 Pro65 70 75 80gcg 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 95gat 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 110tat
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
125ctg 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 140acc 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 Val145 150 155 160ctg 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 175cac 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 190tgg 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 205ccc 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 220gat 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 Asp225 230 235
240acg 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 255att 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 270aag 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 285cgt 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 300gac 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 Leu305 310 315 320ggc 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 335aat 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 350ccg
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
365gag 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 380gcg 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 Ser385 390 395 400gtg 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 415gac 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 430tgc 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 445atc 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 460gcg 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 Leu465 470 475
480acc gcg aaa gaa ccg gcc gcc gtc tga 1467Thr Ala Lys Glu Pro Ala
Ala Val 48519488PRTRhodobacter sphaeroides 19Met Pro Gly Cys Gly
Gly Leu Pro Gly Asn Glu Pro Lys Cys Gly Arg1 5 10 15Glu Gly Arg Ser
Ala Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly 20 25 30Ala Val Gly
Ala Ala Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu 35 40 45Met Ala
Lys Leu Gly Lys Ser Ala Gln Pro Val Leu Thr His Ala Glu 50 55 60Gly
Ile Tyr Val His Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro65 70 75
80Ala Gly Met Trp Cys Ala Gln Val Gly Tyr Gly Arg Arg Glu Ile Val
85 90 95Asp Ala Met Ala His Gln Ala Met Val Leu Pro Tyr Ala Ser Pro
Trp 100 105 110Tyr Met Ala Thr Ser Pro Ala Ala Arg Leu Ala Glu Lys
Ile Ala Thr 115 120 125Leu Thr Pro Gly Asp Leu Asn Arg Ile Phe Phe
Thr Thr Gly Gly Ser 130 135 140Thr Ala Val Asp Ser Ala Leu Arg Phe
Ser Glu Phe Tyr Asn Asn Val145 150 155 160Leu Gly Arg Pro Gln Lys
Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr 165 170 175His Gly Ser Thr
Ala Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn 180 185 190Trp Pro
Asn Phe Asp Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser 195 200
205Pro Asn Pro Arg His Ala Gly Asn Arg Ser Gln Glu Ala Phe Leu Asp
210 215 220Asp Leu Val Gln Glu Phe Glu Asp Arg Ile Glu Ser Leu Gly
Pro Asp225 230 235 240Thr Ile Ala Ala Phe Leu Ala Glu Pro Ile Leu
Ala Ser Gly Gly Val 245 250 255Ile Ile Pro Pro Ala Gly Tyr His Ala
Arg Phe Lys Ala Ile Cys Glu 260 265 270Lys His Asp Ile Leu Tyr Ile
Ser Asp Glu Val Val Thr Gly Phe Gly 275 280 285Arg Cys Gly Glu Trp
Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro 290 295 300Asp Ile Ile
Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu305 310 315
320Gly Gly Leu Ala Ile Ser Glu Ala Val Leu Ala Arg Ile Ser Gly Glu
325 330 335Asn Ala Lys Gly Ser Trp Phe Thr Asn Gly Tyr Thr Tyr Ser
Asn Gln 340 345 350Pro Val Ala Cys Ala Ala Ala Leu Ala Asn Ile Glu
Leu Met Glu Arg 355 360 365Glu Gly Ile Val Asp Gln Ala Arg Glu Met
Ala Asp Tyr Phe Ala Ala 370 375 380Ala Leu Ala Ser Leu Arg Asp Leu
Pro Gly Val Ala Glu Thr Arg Ser385 390 395 400Val Gly Leu Val Gly
Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala 405 410 415Asp Gly Thr
Ala Glu Asp Lys Ala Phe Thr Leu Lys Ile Asp Glu Arg 420 425 430Cys
Phe Glu Leu Gly Leu Ile Val Arg Pro Leu Gly Asp Leu Cys Val 435 440
445Ile Ser Pro Pro Leu Ile Ile Ser Arg Ala Gln Ile Asp Glu Met Val
450 455 460Ala Ile Met Arg Gln Ala Ile Thr Glu Val Ser Ala Ala His
Gly Leu465 470 475 480Thr Ala Lys Glu Pro Ala Ala Val
48520837DNALegionella 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 Ala1 5 10 15aaa 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 30gaa 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 45ctt
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
60aat 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
Asn65 70 75 80ggc 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 95gtg 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 110ttc 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 125atg 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 140aaa 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 Val145 150 155 160tct 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 175gag
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
190aag 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 205gtt 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 220gaa 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 Thr225 230 235 240agt 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 255att 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 270tac caa caa cta gta
aac taa 837Tyr Gln Gln Leu Val Asn 27521278PRTLegionella
pneumophila 21Met Ser Ile Ala Phe Val Asn Gly Lys Tyr Cys Cys Gln
Ser Glu Ala1 5 10 15Lys Ile Ser Ile Phe Asp Arg Gly Phe Leu Phe Gly
Asp Ser Val Tyr 20 25 30Glu Val Leu Pro Val Tyr His Gly Gln Pro Tyr
Phe Val Asp Gln His 35 40 45Leu Asp Arg Leu Phe Ser Asn Met Lys Lys
Ile Lys Met Ile Ile Pro 50 55 60Asn Tyr Asp Trp His Gly Leu Ile His
Arg Leu Ile Ser Glu Asn Asn65 70 75 80Gly Gly Asn Leu Gln Val Tyr
Ile Gln Val Thr Arg Gly Asn Gln Gly 85 90 95Val Arg Lys His Asp Ile
Pro Thr Ser Ile Thr Pro Ser Val Ile Ala 100 105 110Phe Thr Met His
Asn Pro Phe Pro Thr Leu Glu Asp Lys Glu Gln Gly 115 120 125Met Ser
Ala Lys Leu Val Glu Asp Phe Arg Trp Met Arg Cys Asp Ile 130 135
140Lys Thr Thr Ser Leu Ile Ala Asn Ile Leu Leu Asn Asp Glu Ala
Val145 150 155 160Ser Ala Gly Phe His Thr Ala Ile Leu Ala Arg Asn
Gly Leu Ile Thr 165 170 175Glu Gly Ser Ser Thr Asn Val Phe Ile Val
Ala Gln Asp Gly Val Ile 180 185 190Lys Thr Pro Pro Met Asn Asn Phe
Cys Leu Pro Gly Ile Thr Arg Gln 195 200 205Val Val Ile Glu Ile Ile
Lys Lys Leu Asp Leu Lys Phe Arg Glu Ile 210 215 220Glu Ile Ser Ile
Ser Glu Leu Phe Ser Ala Gln Glu Val Trp Ile Thr225 230 235 240Ser
Thr Thr Lys Glu Val Phe Pro Ile Thr Lys Ile Asn Asp Ser Leu 245 250
255Ile Asn Gly Gly Lys Val Gly Glu Tyr Trp Arg Ile Ile Asn Asp Ser
260 265 270Tyr Gln Gln Leu Val Asn 27522861DNANitrosomonas
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 Val1 5 10 15cca 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 30ata 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 45cgg 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 60gaa 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 Glu65 70 75 80ggt 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 95cgt 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 110agc 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
125tca 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 140gcc 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 Asp145 150 155 160gca 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 175ggg 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 190ccc 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 205ctg 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 220tca 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 Ser225 230 235
240acc 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 255aat 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 270gca 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 28523286PRTNitrosomonas
europaea 23Met Ile Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gln Ala
Thr Val1 5 10 15Pro Val Leu Asp Arg Gly Phe Ile Phe Gly Asp Gly Val
Tyr Glu Val 20 25 30Ile Pro Val Tyr Ser Arg Lys Pro Phe Arg Leu Gly
Glu His Leu Ser 35 40 45Arg Leu Gln His Ser Leu Asp Gly Ile Arg Leu
Gln Asn Pro His Thr 50 55 60Glu Glu Gln Trp Ala Gly Leu Ile Glu Arg
Ile Ile Glu Leu Asn Glu65 70 75 80Gly Asp Asp Gln Tyr Leu Tyr Leu
His Ile Thr Arg Gly Val Ala Lys 85 90 95Arg Asp His Ala Phe Pro Arg
Glu Val Thr Pro Thr Val Phe Ile Met 100 105 110Ser Asn Pro Leu Pro
Ala Pro Pro Ala Lys Leu Leu Val Ser Gly Val 115 120 125Ser Ala Ile
Thr Ala Arg Asp Asn Arg Trp Gly Arg Cys Asp Ile Lys 130 135 140Ala
Ile Ser Leu Leu Pro Asn Ile Leu Leu Arg Gln Leu Ala Val Asp145 150
155 160Ala Gln Ala Met Glu Thr Ile Leu Leu Arg Asp Gly Leu Leu Thr
Glu 165 170 175Gly Ala Ala Ser Asn Ile Phe Ile Val Lys Asp Asp Leu
Leu Leu Thr 180 185 190Pro Pro Lys Asp His Arg Ile Leu Pro Gly Ile
Thr Tyr Asp Val Val 195 200 205Leu Glu Leu Ala Glu Thr His Gly Val
Pro His Ala Thr Arg Glu Ile 210 215 220Ser Glu Leu Glu Leu Arg Thr
Ala Arg Glu Ile Met Leu Thr Ser Ser225 230 235 240Thr Lys Glu Ile
Leu Pro Ile Thr Gln Leu Asp Gly Gln Pro Ile Gly 245 250 255Asn Gly
Thr Pro Gly Pro Val Phe Gln Gln Leu Asp Arg Leu Tyr Gln 260 265
270Ala Tyr Lys Leu Glu Val Met Arg Gly His Ala Pro Arg Gln 275 280
285241293DNANeisseria 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 Lys1 5 10 15gcc 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 30gtc 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 45tgg
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
60cct 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
Arg65 70 75 80gaa 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 95atc 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 110ctg 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 125ctg 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 140tgc 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 Gly145 150 155 160ctg 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 175acc
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
190gaa 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 205ccc 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 220aaa 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 Tyr225 230 235 240gac 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 255ctg 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 270ggc 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 285tgt 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 300aac
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 Gln305 310
315 320cgc 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 335aac 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 350agc 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 365aac 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 380ttc 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 Tyr385 390 395 400gaa 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 415acg 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
43025430PRTNeisseria gonorrhoeae 25Met Arg Ile Asn Met Asn Arg Asn
Glu Ile Leu Phe Asp Arg Ala Lys1 5 10 15Ala Ile Ile Pro Gly Gly Val
Asn Ser Pro Val Arg Ala Phe Gly Ser 20 25 30Val Gly Gly Val Pro Arg
Phe Ile Lys Lys Ala Glu Gly Ala Tyr Val 35 40 45Trp Asp Glu Asn Gly
Thr Arg Tyr Thr Asp Tyr Val Gly Ser Trp Gly 50 55 60Pro Ala Ile Val
Gly His Ala His Pro Glu Val Val Glu Ala Val Arg65 70 75 80Glu Ala
Ala Leu Gly Gly Leu Ser Phe Gly Ala Pro Thr Glu Gly Glu 85 90 95Ile
Ala Ile Ala Glu Gln Ile Ala Glu Ile Met Pro Ser Val Glu Arg 100 105
110Leu Arg Leu Val Ser Ser Gly Thr Glu Ala Thr Met Thr Ala Ile Arg
115 120 125Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys Ile Ile Lys Phe
Glu Gly 130 135 140Cys Tyr His Gly His Ser Asp Ser Leu Leu Val Lys
Ala Gly Ser Gly145 150 155 160Leu Leu Thr Phe Gly Asn Pro Ser Ser
Ala Gly Val Pro Ala Asp Phe 165 170 175Thr Lys His Thr Leu Val Leu
Glu Tyr Asn Asn Ile Ala Gln Leu Glu 180 185 190Glu Ala Phe Ala Gln
Ser Gly Asp Glu Ile Ala Cys Val Ile Val Glu 195 200 205Pro Phe Val
Gly Asn Met Asn Leu Val Arg Pro Thr Glu Ala Phe Val 210 215 220Lys
Ala Leu Arg Gly Leu Thr Glu Lys His Gly Ala Val Leu Ile Tyr225 230
235 240Asp Glu Val Met Thr Gly Phe Arg Val Ala Leu Gly Gly Ala Gln
Ser 245 250 255Leu His Gly Ile Thr Pro Asp Leu Thr Thr Met Gly Lys
Val Ile Gly 260 265 270Gly Gly Met Pro Leu Ala Ala Phe Gly Gly Arg
Lys Asp Ile Met Glu 275 280 285Cys Ile Ser Pro Leu Gly Gly Val Tyr
Gln Ala Gly Thr Leu Ser Gly 290 295 300Asn Pro Ile Ala Val Ala Ala
Gly Leu Lys Thr Leu Glu Ile Ile Gln305 310 315 320Arg Glu Gly Phe
Tyr Glu Asn Leu Thr Ala Leu Thr Gln Arg Leu Ala 325 330 335Asn Gly
Ile Ala Ala Ala Lys Ala His Gly Ile Glu Phe Ala Ala Asp 340 345
350Ser Val Gly Gly Met Phe Gly Leu Tyr Phe Ala Ala His Val Pro Arg
355 360 365Asn Tyr Ala Asp Met Ala Arg Ser Asn Ile Asp Ala Phe Lys
Arg Phe 370 375 380Phe His Gly Met Leu Asp Arg Gly Ile Ala Phe Gly
Pro Ser Ala Tyr385 390 395 400Glu Ala Gly Phe Val Ser Ala Ala His
Thr Pro Glu Leu Ile Asp Glu 405 410 415Thr Val Ala Val Ala Val Glu
Val Phe Lys Ala Met Ala Ala 420 425 43026924DNAPseudomonas
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 Leu1 5 10 15gtg 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 30tat 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 45ggc 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 60tcc 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 Ile65 70 75 80aac 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 95tat 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 110gcc
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
125gcc 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 140agt 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 Lys145 150 155 160tcc 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 175tcc 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 190gcc 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 205tac 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 220atc 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 Arg225 230 235
240atc 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 255act 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 270ggc 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 285ttc 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 300ctg gtc aag taa 924Leu Val
Lys30527307PRTPseudomonas aeruginosa 27Met Ser Met Ala Asp Arg Asp
Gly Val Ile Trp Tyr Asp Gly Glu Leu1 5 10 15Val Gln Trp Arg Asp Ala
Thr Thr His Val Leu Thr His Thr Leu His 20 25 30Tyr Gly Met Gly Val
Phe Glu Gly Val Arg Ala Tyr Asp Thr Pro Gln 35 40 45Gly Thr Ala Ile
Phe Arg Leu Gln Ala His Thr Asp Arg Leu Phe Asp 50 55 60Ser Ala His
Ile Met Asn Met Gln Ile Pro Tyr Ser Arg Asp Glu Ile65 70 75 80Asn
Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala 85 90
95Tyr Ile Arg Pro Met Val Phe Tyr Gly Ser Glu Gly Met Gly Leu Arg
100 105 110Ala Ser Gly Leu Lys Val His Val Ile Ile Ala Ala Trp Ser
Trp Gly 115 120 125Ala Tyr Met Gly Glu Glu Ala Leu Gln Gln Gly Ile
Lys Val Arg Thr 130 135 140Ser Ser Phe Thr Arg His His Val Asn Ile
Ser Met Thr Arg Ala Lys145 150 155 160Ser Asn Gly Ala Tyr Ile Asn
Ser Met Leu Ala Leu Gln Glu Ala Ile 165 170 175Ser Gly Gly Ala Asp
Glu Ala Met Met Leu Asp Pro Glu Gly Tyr Val 180 185 190Ala Glu Gly
Ser Gly Glu Asn Ile Phe Ile Ile Lys Asp Gly Val Ile 195 200 205Tyr
Thr Pro Glu Val Thr Ala Cys Leu Asn Gly Ile Thr Arg Asn Thr 210 215
220Ile Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu Lys
Arg225 230 235 240Ile Thr Arg Asp Glu Val Tyr Ile Ala Asp Glu Ala
Phe Phe Thr Gly 245 250 255Thr Ala Ala Glu Val Thr Pro Ile Arg Glu
Val Asp Gly Arg Lys Ile 260 265 270Gly Ala Gly Arg Arg Gly Pro Val
Thr Glu Lys Leu Gln Lys Ala Tyr 275 280 285Phe Asp Leu Val Ser Gly
Lys Thr Glu Ala His Ala Glu Trp Arg Thr 290 295 300Leu Val
Lys305281407DNARhodopseudomonas 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 Cys1 5 10 15atg
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
30gcg 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 45gcc 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 60tac 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 Met65 70 75 80tgg 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 95aag 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 110cag 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 125gaa 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
140gac 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
Gln145 150 155 160ggc 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 175ggc 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 190ttc 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 205cgc 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 220ttc 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 Thr225 230 235 240atc
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
255ccg 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 270cac 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 285ctc 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 300atc 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 Gly305 310 315 320gtg 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 335cac 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 350gcc 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 365ctg
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
380atg 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
Gly385 390 395 400ctg 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 415cgt 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 430ctg 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 445agc 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 460cgc gcg gtc gcc
tga 1407Arg Ala Val Ala46529468PRTRhodopseudomonas palustris 29Met
Lys Leu Ile Pro Cys Arg Ala Phe His Pro Pro Ala Ala Gln Cys1 5 10
15Met Arg Ser Ala Met Leu Asp Lys Ile Lys Pro Thr Ser Ala Val Asn
20 25 30Ala Pro Asn Asp Leu Asn Ala Phe Trp Met Pro Phe Thr Ala Asn
Arg 35 40 45Ala Phe Lys Arg Ala Pro Lys Met Val Val Gly Ala Glu Gly
Met His 50 55 60Tyr Ile Thr Ala Asp Gly Arg Lys Ile Ile Asp Ala Ala
Ser Gly Met65 70 75 80Trp Cys Thr Asn Ala Gly His Gly Arg Lys Glu
Ile Ala Glu Ala Ile 85 90 95Lys Ala Gln Ala Asp Glu Leu Asp Phe Ser
Pro Pro Phe Gln Phe Gly 100 105 110Gln Pro Lys Ala Phe Glu Leu Ala
Ser Arg Ile Ala Asp Leu Ala Pro 115 120 125Glu Gly Leu Asp His Val
Phe Phe Cys Asn Ser Gly Ser Glu Ala Gly 130 135 140Asp Thr Ala Leu
Lys Ile Ala Val Ala Tyr Gln Gln Ile Lys Gly Gln145 150 155 160Gly
Ser Arg Thr Arg Leu Ile Gly Arg Glu Arg Gly Tyr His Gly Val 165 170
175Gly Phe Gly Gly Thr Ala Val Gly Gly Ile Gly Asn Asn Arg Lys Met
180 185 190Phe Gly Pro Leu Leu Asn Gly Val Asp His Leu Pro Ala Thr
Tyr Asp 195 200 205Arg Asp Lys Gln Ala Phe Thr Ile Gly Glu Pro Glu
Tyr Gly Ala His 210 215 220Phe Ala Glu Ala Leu Glu Gly Leu Val Asn
Leu His Gly Ala Asn Thr225 230 235 240Ile Ala Ala Val Ile Val Glu
Pro Met Ala Gly Ser Thr Gly Val Leu 245 250 255Pro Ala Pro Lys Gly
Tyr Leu Lys Lys Leu Arg Glu Ile Thr Lys Lys 260 265 270His Gly Ile
Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Tyr Gly Arg 275 280 285Leu
Gly Tyr Ala Phe Ala Ser Glu Arg Tyr Gly Val Thr Pro Asp Met 290 295
300Ile Thr Phe Ala Lys Gly Val Thr Asn Gly Ala Val Pro Met Gly
Gly305 310 315 320Val Ile Thr Ser Ala Glu Ile His Asp Ala Phe Met
Thr Gly Pro Glu 325 330 335His Ala Val Glu Leu Ala His Gly Tyr Thr
Tyr Ser Ala His Pro Leu 340 345 350Ala Cys Ala Ala Gly Ile Ala Thr
Leu Asp Ile Tyr Arg Asp Glu Lys 355 360 365Leu Phe Glu Arg Ala Lys
Ala Leu Glu Pro Lys Phe Ala Glu Ala Val 370 375 380Met Ser Leu Lys
Ser Ala Pro Asn Val Val Asp Ile Arg Thr Val Gly385 390 395 400Leu
Thr Ala Gly Ile Asp Leu Ala Ser Ile Ala Asp Ala Val Gly Lys 405 410
415Arg Gly Phe Glu Ala Met Asn Ala Gly Phe His Asp His Glu Leu Met
420 425 430Leu Arg Ile Ala Gly Asp Thr Leu Ala Leu Thr Pro Pro Leu
Ile Leu 435 440 445Ser Glu Asp His Ile Gly Glu Ile Val Asp Lys Val
Gly Lys Val Ile 450 455 460Arg Ala Val Ala465301263DNAEscherichia
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 Asn1 5 10 15ctg 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 30gcg 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 45gtg 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 60atg 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 Ile65 70 75 80gag 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 95att 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 110agt 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
125caa 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 140ccg 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 Glu145 150 155 160aac 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 175gtg 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 190ggt 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 205gtg 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 220ggc 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 Asp225 230 235
240acc 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 255cgc 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 270ctg 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 285caa 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 300ctg 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 Leu305 310 315 320gca 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 335gtc 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 350ggg
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
365tat 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 380aac 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 Gly385 390 395 400cag 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 415ctg gaa ttg ctt taa 1263Leu Glu Leu
Leu 42031420PRTEscherichia coli 31Met Pro His Ser Leu Phe Ser Thr
Asp Thr Asp Leu Thr Ala Glu Asn1 5 10 15Leu Leu Arg Leu Pro Ala Glu
Phe Gly Cys Pro Val Trp Val Tyr Asp 20 25 30Ala Gln Ile Ile Arg Arg
Gln Ile Ala Ala Leu Lys Gln Phe Asp Val 35 40 45Val Arg Phe Ala Gln
Lys Ala Cys Ser Asn Ile His Ile Leu Arg Leu 50 55 60Met Arg Glu Gln
Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu Ile65 70 75 80Glu Arg
Ala Leu Ala Ala Gly Tyr Asn Pro Gln Thr His Pro Asp Asp 85 90 95Ile
Val Phe Thr Ala Asp Val Ile Asp Gln Ala Thr Leu Glu Arg Val 100 105
110Ser Glu Leu Gln Ile Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp
115 120 125Gln Leu Gly Gln Val Ser Pro Gly His Arg Val Trp Leu Arg
Val Asn 130 135 140Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn
Thr Gly Gly Glu145 150 155 160Asn Ser Lys His Gly Ile Trp Tyr Thr
Asp Leu Pro Ala Ala Leu Asp 165 170 175Val Ile Gln Arg His His Leu
Gln Leu Val Gly Ile His Met His Ile 180 185 190Gly Ser Gly Val Asp
Tyr Ala His Leu Glu Gln Val Cys Gly Ala Met 195 200 205Val Arg Gln
Val Ile Glu Phe Gly Gln Asp Leu Gln Ala Ile Ser Ala 210 215 220Gly
Gly Gly Leu Ser Val Pro Tyr Gln Gln Gly Glu Glu Ala Val Asp225 230
235 240Thr Glu His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg Glu Gln Ile
Ala 245 250 255Arg His Leu Gly His Pro Val Lys Leu Glu Ile Glu Pro
Gly Arg Phe 260 265 270Leu Val Ala Gln Ser Gly Val Leu Ile Thr Gln
Val Arg Ser Val Lys 275 280 285Gln Met Gly Ser Arg His Phe Val Leu
Val Asp Ala Gly Phe Asn Asp 290 295 300Leu Met Arg Pro Ala Met Tyr
Gly Ser Tyr His His Ile Ser Ala Leu305 310 315 320Ala Ala Asp Gly
Arg Ser Leu Glu His Ala Pro Thr Val Glu Thr Val 325 330 335Val Ala
Gly Pro Leu Cys Glu Ser Gly Asp Val Phe Thr Gln Gln Glu 340 345
350Gly Gly Asn Val Glu Thr Arg Ala Leu Pro Glu Val Lys Ala Gly Asp
355 360 365Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met
Ser Ser 370 375 380Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu
Phe Asp Asn Gly385 390 395 400Gln Ala Arg Leu Ile Arg Arg Arg Gln
Thr Ile Glu Glu Leu Leu Ala 405 410 415Leu Glu Leu Leu
420321265DNAArtificialEscherichia.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 Gln1 5 10 15gtc 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 30ttg
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 45gcc 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 60aag 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 Ser65 70 75 80gct 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 95cac 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
110ttg 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 125tct 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 140gcc 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 Gln145 150 155 160aga 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 175cca 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 190gat 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 205aag 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 220cac
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 Phe225 230
235 240cca 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 255cca 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 270aag 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 285ttg 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 300aac 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 Thr305 310 315 320ttc 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 335att 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
350act 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 365tgg 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 380att 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 Phe385 390 395 400cca 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 415ttc 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 430gat 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 445ttg 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 460tac
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 Ile465 470
475 480cac 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 495tcc 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 510gct 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 525gac 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 540gct 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 Asn545 550 555 560gct aag caa taa
1692Ala Lys Gln34563PRTSaccharomyces cerevisiae 34Met Ser Glu Ile
Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln1 5 10 15Val Asn Val
Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30Leu Leu
Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45Ala
Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55
60Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser65
70 75 80Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val
Leu 85 90 95His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln
Leu Leu 100 105 110Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val
Phe His Arg Met 115 120 125Ser Ala Asn Ile Ser Glu Thr Thr Ala Met
Ile Thr Asp Ile Ala Thr 130 135 140Ala Pro Ala Glu Ile Asp Arg Cys
Ile Arg Thr Thr Tyr Val Thr Gln145 150 155 160Arg Pro Val Tyr Leu
Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175Pro Ala Lys
Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190Asp
Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val 195 200
205Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg
210 215 220His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr
Gln Phe225 230 235 240Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser
Ile Asp Glu Gln His 245 250 255Pro Arg Tyr Gly Gly Val Tyr Val Gly
Thr Leu Ser Lys Pro Glu Val 260 265 270Lys Glu Ala Val Glu Ser Ala
Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285Leu Ser Asp Phe Asn
Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300Asn Ile Val
Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr305 310 315
320Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr
325 330 335Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro
Ala Arg 340 345 350Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro
Leu Lys Gln Glu 355 360 365Trp Met Trp Asn Gln Leu Gly Asn Phe Leu
Gln Glu Gly Asp Val Val 370 375 380Ile Ala Glu Thr Gly Thr Ser Ala
Phe Gly Ile Asn Gln Thr Thr Phe385 390 395 400Pro Asn Asn Thr Tyr
Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415Phe Thr Thr
Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430Asp
Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440
445Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro
450 455 460Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys
Leu Ile465 470 475 480His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln
Gly Trp Asp His Leu 485 490 495Ser Leu Leu Pro Thr Phe Gly Ala Lys
Asp Tyr Glu Thr His Arg Val 500 505 510Ala Thr Thr Gly Glu Trp Asp
Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525Asp Asn Ser Lys Ile
Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp 530 535 540Ala Pro Gln
Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn545 550 555
560Ala Lys Gln351692DNAArtificialSaccharomyces 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 Ile1
5 10 15ggt 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 30ctt 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 45aac 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 60ggc 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 Ala65 70 75 80ttt 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 95atc 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 110cat 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 125aag 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 140ccg 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 Lys145 150 155
160ccg 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 175gct 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 190gct 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 205cgc 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 220gct 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 Val225 230 235 240gct 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 255tac 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 270acg
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
285gac 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 300gtt 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 Pro305 310 315 320agc 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 335aag 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 350ctg 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 365gaa 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 380att 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 Leu385 390 395
400ccg 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 415tgg 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 430cgc 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 445gaa 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 460atc 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 Pro465 470 475 480tac 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
495aac 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 510aaa 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 525acc 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 540act 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 Ser545 550 555 560cgt aag cct gtt aac aag ctc
ctc tag 1707Arg Lys Pro Val Asn Lys Leu Leu 56537568PRTZymomonas
mobilis 37Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val
Gln Ile1 5 10 15Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn
Leu Val Leu 20 25 30Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln
Val Tyr Cys Cys 35 40 45Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly
Tyr Ala Arg Ala Lys 50 55 60Gly Ala Ala Ala Ala Val Val Thr Tyr Ser
Val Gly Ala Leu Ser Ala65 70 75 80Phe Asp Ala Ile Gly Gly Ala Tyr
Ala Glu Asn Leu Pro Val Ile Leu 85 90 95Ile Ser Gly Ala Pro Asn Asn
Asn Asp His Ala Ala Gly His Val Leu 100 105 110His His Ala Leu Gly
Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala 115 120 125Lys Asn Ile
Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala 130 135 140Pro
Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys145 150
155 160Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys
Ala 165 170 175Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala
Ser Asp Glu 180 185 190Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu
Lys Phe Ile Ala Asn 195 200 205Arg Asp Lys Val Ala Val Leu Val Gly
Ser Lys Leu Arg Ala Ala Gly 210 215 220Ala Glu Glu Ala Ala Val Lys
Phe Ala Asp Ala Leu Gly Gly Ala Val225 230 235 240Ala Thr Met Ala
Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro His 245 250 255Tyr Ile
Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys 260 265
270Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn
275 280 285Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys
Lys Leu 290 295 300Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly
Ile Arg Phe Pro305 310 315 320Ser Val His Leu Lys Asp Tyr Leu Thr
Arg Leu Ala Gln Lys Val Ser 325 330 335Lys Lys Thr Gly Ala Leu Asp
Phe Phe Lys Ser Leu Asn Ala Gly Glu 340 345 350Leu Lys Lys Ala Ala
Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala 355 360 365Glu Ile Ala
Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val 370 375 380Ile
Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu385 390
395 400Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile
Gly 405 410 415Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala
Pro Glu Arg 420 425 430Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe
Gln Leu Thr Ala Gln 435 440 445Glu Val Ala Gln Met Val Arg Leu Lys
Leu Pro Val Ile Ile Phe Leu 450 455 460Ile Asn Asn Tyr Gly Tyr Thr
Ala Glu Val Met Ile His Asp Gly Pro465 470 475 480Tyr Asn Asn Ile
Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe 485 490 495Asn Gly
Asn Gly Gly Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys Ala 500 505
510Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn
515 520 525Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu
Asp Cys 530 535 540Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala
Ala Ala Asn Ser545 550 555 560Arg Lys Pro Val Asn Lys Leu Leu
565381707DNAArtificialZymomonas 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 Gly1
5 10 15att 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 30gat 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 45gaa 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 60gct 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 Ile65 70 75 80aat 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 95gtt 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 110cat 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 125cct 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 140gaa 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 Val145 150 155
160tat 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 175gca 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 190gtg 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 205gta 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 220gta 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 Asn225 230 235 240ttt 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 255tat 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 270gca
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
285ggt 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 300ata 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 Phe305 310 315 320aga 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 335gga 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 350ccc 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 365agc 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 380tca 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 Leu385 390 395
400tgg 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 415gcg 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 430caa 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 445cca 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 460atc 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 Tyr465 470 475 480tcg 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 495aaa 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 510caa
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
525gaa 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 540caa aat aaa tag 1644Gln Asn Lys54540547PRTLactococcus
lactis 40Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu
Leu Gly1 5 10 15Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu
Gln Phe Leu 20 25 30Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile
Gly Asn Ala Asn 35 40 45Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr
Ala Arg Thr Lys Lys 50 55 60Ala Ala Ala Phe Leu Thr Thr Phe Gly Val
Gly Glu Leu Ser Ala Ile65 70 75 80Asn Gly Leu Ala Gly Ser Tyr Ala
Glu Asn Leu Pro Val Val Glu Ile 85 90 95Val Gly Ser Pro Thr Ser Lys
Val Gln Asn Asp Gly Lys Phe Val His 100 105 110His Thr Leu Ala Asp
Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125Pro Val Thr
Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr 130 135 140Glu
Ile Asp Arg Val Leu Ser Gln Leu Leu Lys Glu Arg Lys Pro Val145 150
155 160Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys
Pro 165 170 175Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr
Thr Glu Gln 180 185 190Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys
Asn Ala Gln Lys Pro 195 200 205Val Val Ile Ala Gly His Glu Val Ile
Ser Phe Gly Leu Glu Lys Thr 210 215 220Val Thr Gln Phe Val Ser Glu
Thr Lys Leu Pro Ile Thr Thr Leu Asn225 230 235 240Phe Gly Lys Ser
Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile 245 250 255Tyr Asn
Gly Lys Leu Ser Glu Ile Ser Leu Lys Asn Phe Val Glu Ser 260 265
270Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr
275 280 285Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met Ile Ser
Leu Asn 290 295 300Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu
Asp Phe Asp Phe305 310 315 320Arg Ala Val Val Ser Ser Leu Ser Glu
Leu Lys Gly Ile Glu Tyr Glu 325 330 335Gly Gln Tyr Ile Asp Lys Gln
Tyr Glu Glu Phe Ile Pro Ser Ser Ala 340 345 350Pro Leu Ser Gln Asp
Arg Leu Trp Gln Ala Val Glu Ser Leu Thr Gln 355 360 365Ser Asn Glu
Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380Ser
Thr Ile Phe Leu Lys Ser Asn Ser Arg Phe Ile Gly Gln Pro Leu385 390
395 400Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln
Ile 405 410 415Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp
Gly Ser Leu 420 425 430Gln Leu Thr Val Gln Glu Leu Gly Leu Ser Ile
Arg Glu Lys Leu Asn 435 440 445Pro Ile Cys Phe Ile Ile Asn Asn Asp
Gly Tyr Thr Val Glu Arg Glu 450 455 460Ile His Gly Pro Thr Gln Ser
Tyr Asn Asp Ile Pro Met Trp Asn Tyr465 470 475 480Ser Lys Leu Pro
Glu Thr Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495Lys Ile
Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505
510Gln Ala Asp Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys
515 520 525Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe
Ala Glu 530
535 540Gln Asn Lys545411644DNAArtificialLactococcus 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 Gly1 5 10 15att 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 30gat 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 45gaa 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 60gct 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 Val65 70 75 80aat 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 95gtg 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 110cat 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
125cct 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 140gaa 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 Val145 150 155 160tat 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 175tca 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 190gaa 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 205atc 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 220gtc 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 Asn225 230 235
240ttt 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 255tat 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 270gcc 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 285gga 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 300ata 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 Phe305 310 315 320gaa 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 335gga 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 350ctt
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
365agc 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 380tca 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 Leu385 390 395 400tgg 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 415gca 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 430caa 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 445cca 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 460att 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 Tyr465 470 475
480tca 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 495aaa 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 510caa 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 525gaa 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 540caa aat aaa tca taa 1647Gln Asn
Lys Ser54543548PRTLactococcus lactis 43Met Tyr Thr Val Gly Asp Tyr
Leu Leu Asp Arg Leu His Glu Leu Gly1 5 10 15Ile Glu Glu Ile Phe Gly
Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30Asp Gln Ile Ile Ser
His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45Glu Leu Asn Ala
Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60Ala Ala Ala
Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val65 70 75 80Asn
Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90
95Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His
100 105 110His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met
His Glu 115 120 125Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu
Asn Ala Thr Val 130 135 140Glu Ile Asp Arg Val Leu Ser Ala Leu Leu
Lys Glu Arg Lys Pro Val145 150 155 160Tyr Ile Asn Leu Pro Val Asp
Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175Ser Leu Pro Leu Lys
Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln 180 185 190Glu Ile Leu
Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205Ile
Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr 210 215
220Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu
Asn225 230 235 240Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser
Phe Leu Gly Ile 245 250 255Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu
Lys Glu Phe Val Glu Ser 260 265 270Ala Asp Phe Ile Leu Met Leu Gly
Val Lys Leu Thr Asp Ser Ser Thr 275 280 285Gly Ala Phe Thr His His
Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295 300Ile Asp Glu Gly
Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe305 310 315 320Glu
Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys 325 330
335Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala
340 345 350Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu
Thr Gln 355 360 365Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser
Phe Phe Gly Ala 370 375 380Ser Ser Ile Phe Leu Lys Ser Lys Ser His
Phe Ile Gly Gln Pro Leu385 390 395 400Trp Gly Ser Ile Gly Tyr Thr
Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415Ala Asp Lys Glu Ser
Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430Gln Leu Thr
Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445Pro
Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455
460Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn
Tyr465 470 475 480Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp
Arg Val Val Ser 485 490 495Lys Ile Val Arg Thr Glu Asn Glu Phe Val
Ser Val Met Lys Glu Ala 500 505 510Gln Ala Asp Pro Asn Arg Met Tyr
Trp Ile Glu Leu Ile Leu Ala Lys 515 520 525Glu Gly Ala Pro Lys Val
Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540Gln Asn Lys
Ser545441647DNAArtificialLactococcus 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 Glu1 5 10 15gag 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 30tgg 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 45gct 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 60gct 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 Ala65 70 75 80gcg 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 95ccg 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
110gcc 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 125agc 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 140atc 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 Thr145 150 155 160cat 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 175atg 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 190ccg 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 205ggg 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 220cga
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 Ala225 230
235 240cgc 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 255ctg 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 270atg 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 285gcc 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 300ggc 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 Gln305 310 315 320ggc 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 335tcg 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 350ctg 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 365aac
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
380ctg 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
Arg385 390 395 400agt 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 415ctc 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 430aaa 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 445atc 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 460ctc 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 Gln465 470 475 480cag
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
495ttc 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 510gcc 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 525gag 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 540ctc 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 Phe545 550 555 560acc 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 575gac 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 590ggc 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 605gag
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
620ctg 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
Ser625 630 635 640gtg 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 655gtg 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 670ggc 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 685gcg 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 700atg 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 Ala705 710 715 720tgt
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
735aag 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 750gag 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 765gac 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 780cgt 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 Tyr785 790 795 800cag 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 815cac 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 830gcg 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 845gat
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
860caa 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
Ile865 870 875 880gac 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 895ggc 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 910tcc 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 925aca 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 940gga 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 Val945 950 955 960ggc
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
975tgg 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 990gac 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 1005gtc 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 1020cac 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 1035ggt 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 1050cac 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 1065atc 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 1080agc 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 1095gag 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 1110agc 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 1125gcc 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 1140ctt 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 1155ctg
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
1170gaa 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 1185ccc 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 1200cgc 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 1215gcc 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 1230461231PRTMycobacterium tuberculosis 46Val Ala Asn Ile
Ser Ser Pro Phe Gly Gln Asn Glu Trp Leu Val Glu1 5 10 15Glu Met Tyr
Arg Lys Phe Arg Asp Asp Pro Ser Ser Val Asp Pro Ser 20 25 30Trp His
Glu Phe Leu Val Asp Tyr Ser Pro Glu Pro Thr Ser Gln Pro 35 40 45Ala
Ala Glu Pro Thr Arg Val Thr Ser Pro Leu Val Ala Glu Arg Ala 50 55
60Ala Ala Ala Ala Pro Gln Ala Pro Pro Lys Pro Ala Asp Thr Ala Ala65
70 75 80Ala Gly Asn Gly Val Val Ala Ala Leu Ala Ala Lys Thr Ala Val
Pro 85 90 95Pro Pro Ala Glu Gly Asp Glu Val Ala Val Leu Arg Gly Ala
Ala Ala 100 105 110Ala Val Val Lys Asn Met Ser Ala Ser Leu Glu Val
Pro Thr Ala Thr 115 120 125Ser Val Arg Ala Val Pro Ala Lys Leu Leu
Ile Asp Asn Arg Ile Val 130 135 140Ile Asn Asn Gln Leu Lys Arg Thr
Arg Gly Gly Lys Ile Ser Phe Thr145 150 155 160His Leu Leu Gly Tyr
Ala Leu Val Gln Ala Val Lys Lys Phe Pro Asn 165 170 175Met Asn Arg
His Tyr Thr Glu Val Asp Gly Lys Pro Thr Ala Val Thr 180 185 190Pro
Ala His Thr Asn Leu Gly Leu Ala Ile Asp Leu Gln Gly Lys Asp 195 200
205Gly Lys Arg Ser Leu Val Val Ala Gly Ile Lys Arg Cys Glu Thr Met
210 215 220Arg Phe Ala Gln Phe Val Thr Ala Tyr Glu Asp Ile Val Arg
Arg Ala225 230 235 240Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala
Gly Val Thr Ile Ser 245 250 255Leu Thr Asn Pro Gly Thr Ile Gly Thr
Val His Ser Val Pro Arg Leu 260 265 270Met Pro Gly Gln Gly Ala Ile
Ile Gly Val Gly Ala Met Glu Tyr Pro 275 280 285Ala Glu Phe Gln Gly
Ala Ser Glu Glu Arg Ile Ala Glu Leu Gly Ile 290 295 300Gly Lys Leu
Ile Thr Leu Thr Ser Thr Tyr Asp His Arg Ile Ile Gln305 310 315
320Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr Ile His Glu Leu Leu Leu
325 330 335Ser Asp Gly Phe Trp Asp Glu Val Phe Arg Glu Leu Ser Ile
Pro Tyr 340 345 350Leu Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser
Ile Val Asp Lys 355 360 365Asn Ala Arg Val Met Asn Leu Ile Ala Ala
Tyr Arg Asn Arg Gly His 370 375 380Leu Met Ala Asp Thr Asp Pro Leu
Arg Leu Asp Lys Ala Arg Phe Arg385 390 395 400Ser His Pro Asp Leu
Glu Val Leu Thr His Gly Leu Thr Leu Trp Asp 405 410 415Leu Asp Arg
Val Phe Lys Val Asp Gly Phe Ala Gly Ala Gln Tyr Lys 420 425 430Lys
Leu Arg Asp Val Leu Gly Leu Leu Arg Asp Ala Tyr Cys Arg His 435 440
445Ile Gly Val Glu Tyr Ala His Ile Leu Asp Pro Glu Gln Lys Glu Trp
450 455 460Leu Glu Gln Arg Val Glu Thr Lys His Val Lys Pro Thr Val
Ala Gln465 470 475 480Gln Lys Tyr Ile Leu Ser Lys Leu Asn Ala Ala
Glu Ala Phe Glu Thr 485 490 495Phe Leu Gln Thr Lys Tyr Val Gly Gln
Lys Arg Phe Ser Leu Glu Gly 500 505 510Ala Glu Ser Val Ile Pro Met
Met Asp Ala Ala Ile Asp Gln Cys Ala 515 520 525Glu His Gly Leu Asp
Glu Val Val Ile Gly Met Pro His Arg Gly Arg 530 535 540Leu Asn Val
Leu Ala Asn Ile Val Gly Lys Pro Tyr Ser Gln Ile Phe545 550 555
560Thr Glu Phe Glu Gly Asn Leu Asn Pro Ser Gln Ala His Gly Ser Gly
565 570 575Asp Val Lys Tyr His Leu Gly Ala Thr Gly Leu Tyr Leu Gln
Met Phe 580 585 590Gly Asp Asn Asp Ile Gln Val Ser Leu Thr Ala Asn
Pro Ser His Leu 595 600 605Glu Ala Val Asp Pro Val Leu Glu Gly Leu
Val Arg Ala Lys Gln Asp 610 615 620Leu Leu Asp His Gly Ser Ile Asp
Ser Asp Gly Gln Arg Ala Phe Ser625 630 635 640Val Val Pro Leu Met
Leu His Gly Asp Ala Ala Phe Ala Gly Gln Gly 645 650 655Val Val Ala
Glu Thr Leu Asn Leu Ala Asn Leu Pro Gly Tyr Arg Val 660 665 670Gly
Gly Thr Ile His Ile Ile Val Asn Asn Gln Ile Gly Phe Thr Thr 675 680
685Ala Pro Glu Tyr Ser Arg Ser Ser Glu Tyr Cys Thr Asp Val Ala Lys
690 695 700Met Ile Gly Ala Pro Ile Phe His Val Asn Gly Asp Asp Pro
Glu Ala705 710 715 720Cys Val Trp Val Ala Arg Leu Ala Val Asp Phe
Arg Gln Arg Phe Lys 725 730 735Lys Asp Val Val Ile Asp Met Leu Cys
Tyr Arg Arg Arg Gly His Asn 740 745 750Glu Gly Asp Asp Pro Ser Met
Thr Asn Pro Tyr Val Tyr Asp Val Val 755 760 765Asp Thr Lys Arg Gly
Ala Arg Lys Ser Tyr Thr Glu Ala Leu Ile Gly 770 775 780Arg Gly Asp
Ile Ser Met Lys Glu Ala Glu Asp Ala Leu Arg Asp Tyr785 790 795
800Gln Gly Gln Leu Glu Arg Val Phe Asn Glu Val Arg Glu Leu Glu Lys
805 810 815His Gly Val Gln Pro Ser Glu Ser Val Glu Ser Asp Gln Met
Ile Pro 820 825 830Ala Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu
Ala Arg Ile Gly 835 840 845Asp Ala Phe Leu Ala Leu Pro Asn Gly Phe
Thr Ala His Pro Arg Val 850 855 860Gln Pro Val Leu Glu Lys Arg Arg
Glu Met Ala Tyr Glu Gly Lys Ile865 870 875 880Asp Trp Ala Phe Gly
Glu Leu Leu Ala Leu Gly Ser Leu Val Ala Glu 885 890 895Gly Lys Leu
Val Arg Leu Ser Gly Gln Asp Ser Arg Arg Gly Thr Phe 900
905 910Ser Gln Arg His Ser Val Leu Ile Asp Arg His Thr Gly Glu Glu
Phe 915 920 925Thr Pro Leu Gln Leu Leu Ala Thr Asn Ser Asp Gly Ser
Pro Thr Gly 930 935 940Gly Lys Phe Leu Val Tyr Asp Ser Pro Leu Ser
Glu Tyr Ala Ala Val945 950 955 960Gly Phe Glu Tyr Gly Tyr Thr Val
Gly Asn Pro Asp Ala Val Val Leu 965 970 975Trp Glu Ala Gln Phe Gly
Asp Phe Val Asn Gly Ala Gln Ser Ile Ile 980 985 990Asp Glu Phe Ile
Ser Ser Gly Glu Ala Lys Trp Gly Gln Leu Ser Asn 995 1000 1005Val
Val Leu Leu Leu Pro His Gly His Glu Gly Gln Gly Pro Asp 1010 1015
1020His Thr Ser Ala Arg Ile Glu Arg Phe Leu Gln Leu Trp Ala Glu
1025 1030 1035Gly Ser Met Thr Ile Ala Met Pro Ser Thr Pro Ser Asn
Tyr Phe 1040 1045 1050His Leu Leu Arg Arg His Ala Leu Asp Gly Ile
Gln Arg Pro Leu 1055 1060 1065Ile Val Phe Thr Pro Lys Ser Met Leu
Arg His Lys Ala Ala Val 1070 1075 1080Ser Glu Ile Lys Asp Phe Thr
Glu Ile Lys Phe Arg Ser Val Leu 1085 1090 1095Glu Glu Pro Thr Tyr
Glu Asp Gly Ile Gly Asp Arg Asn Lys Val 1100 1105 1110Ser Arg Ile
Leu Leu Thr Ser Gly Lys Leu Tyr Tyr Glu Leu Ala 1115 1120 1125Ala
Arg Lys Ala Lys Asp Asn Arg Asn Asp Leu Ala Ile Val Arg 1130 1135
1140Leu Glu Gln Leu Ala Pro Leu Pro Arg Arg Arg Leu Arg Glu Thr
1145 1150 1155Leu Asp Arg Tyr Glu Asn Val Lys Glu Phe Phe Trp Val
Gln Glu 1160 1165 1170Glu Pro Ala Asn Gln Gly Ala Trp Pro Arg Phe
Gly Leu Glu Leu 1175 1180 1185Pro Glu Leu Leu Pro Asp Lys Leu Ala
Gly Ile Lys Arg Ile Ser 1190 1195 1200Arg Arg Ala Met Ser Ala Pro
Ser Ser Gly Ser Ser Lys Val His 1205 1210 1215Ala Val Glu Gln Gln
Glu Ile Leu Asp Glu Ala Phe Gly 1220 1225
1230473696DNAArtificialMycobacterium 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 Lys1 5 10 15Gln Lys Asp Glu Gln Phe Val Trp His Ala Met Lys Gly
Ala His Gln 20 25 30Ala Asp Ser Leu Ile Ala Gln Lys Ala Glu Gly Ala
Trp Val Thr Asp 35 40 45Thr Asp Gly Arg Arg Tyr Leu Asp Ala Met Ser
Gly Leu Trp Cys Val 50 55 60Asn Ile Gly Tyr Gly Arg Lys Glu Leu Ala
Glu Ala Ala Tyr Glu Gln65 70 75 80Leu Lys Glu Leu Pro Tyr Tyr Pro
Leu Thr Gln Ser His Ala Pro Ala 85 90 95Ile Gln Leu Ala Glu Lys Leu
Asn Glu Trp Leu Gly Gly Asp Tyr Val 100 105 110Ile Phe Phe Ser Asn
Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys 115 120 125Ile Ala Arg
Gln Tyr His Leu Gln Asn Gly Asp His Ser Arg Tyr Lys 130 135 140Phe
Ile Ser Arg Tyr Arg Ala Tyr His Gly Asn Thr Leu Gly Ala Leu145 150
155 160Ser Ala Thr Gly Gln Ala Gln Arg Lys Tyr Lys Tyr Glu Pro Leu
Ser 165 170 175Gln Gly Phe Leu His Ala Ala Pro Pro Asp Ile Tyr Arg
Asn Pro Asp 180 185 190Asp Ala Asp Thr Leu Glu Ser Ala Asn Glu Ile
Asp Arg Ile Met Thr 195 200 205Trp Glu Leu Ser Glu Thr Ile Ala Gly
Val Ile Met Glu Pro Ile Ile 210 215 220Thr Gly Gly Gly Ile Leu Met
Pro Pro Asp Gly Tyr Met Lys Lys Val225 230 235 240Glu Asp Ile Cys
Arg Arg His Gly Ala Leu Leu Ile Cys Asp Glu Val 245 250 255Ile Cys
Gly Phe Gly Arg Thr Gly Glu Pro Phe Gly Phe Met His Tyr 260 265
270Gly Val Lys Pro Asp Ile Ile Thr Met Ala Lys Gly Ile Thr Ser Ala
275 280 285Tyr Leu Pro Leu Ser Ala Thr Ala Val Lys Arg Asp Ile Phe
Glu Ala 290 295 300Tyr Gln Gly Glu Ala Pro Tyr Asp Arg Phe Arg His
Val Asn Thr Phe305 310 315 320Gly Gly Ser Pro Ala Ala Cys Ala Leu
Ala Leu Lys Asn Leu Gln Ile 325 330 335Met Glu Asp Glu Gln Leu Ile
Gln Arg Ser Arg Asp Leu Gly Ala Lys 340 345 350Leu Leu Gly Glu Leu
Gln Ala Leu Arg Glu His Pro Ala Val Gly Asp 355 360 365Val Arg Gly
Lys Gly Leu Leu Ile Gly Ile Glu Leu Val Lys Asp Lys 370 375 380Leu
Thr Lys Glu Pro Ala Asp Ala Ala Lys Val Asn Gln Val Val Ala385 390
395 400Ala Cys Lys Glu Lys Gly Leu Ile Ile Gly Lys Asn Gly Asp Thr
Val 405 410 415Ala Gly Tyr Asn Asn Val Ile His Val Ala Pro Pro Phe
Cys Leu Thr 420 425 430Glu Glu Asp Leu Ser Phe Ile Val Lys Thr Val
Lys Glu Ser Phe Gln 435 440 445Thr Ile 450661407DNAPseudomonas
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 Leu1 5 10 15Val Arg Ala Asp Gln Ala
His Tyr Met His Gly Tyr His Val Phe Asp 20 25 30Asp His Arg Val Asn
Gly Ser Leu Asn Ile Ala Ala Gly Asp Gly Ala 35 40 45Tyr Ile Tyr Asp
Thr Ala Gly Asn Arg Tyr Leu Asp Ala Val Gly Gly 50 55 60Met Trp Cys
Thr Asn Ile Gly Leu Gly Arg Glu Glu Met Ala Arg Thr65 70 75 80Val
Ala Glu Gln Thr Arg Leu Leu
Ala Tyr Ser Asn Pro Phe Cys Asp 85 90 95Met Ala Asn Pro Arg Ala Ile
Glu Leu Cys Arg Lys Leu Ala Glu Leu 100 105 110Ala Pro Gly Asp Leu
Asp His Val Phe Leu Thr Thr Gly Gly Ser Thr 115 120 125Ala Val Asp
Thr Ala Ile Arg Leu Met His Tyr Tyr Gln Asn Cys Arg 130 135 140Gly
Lys Arg Ala Lys Lys His Val Ile Thr Arg Ile Asn Ala Tyr His145 150
155 160Gly Ser Thr Phe Leu Gly Met Ser Leu Gly Gly Lys Ser Ala Asp
Arg 165 170 175Pro Ala Glu Phe Asp Phe Leu Asp Glu Arg Ile His His
Leu Ala Cys 180 185 190Pro Tyr Tyr Tyr Arg Ala Pro Glu Gly Leu Gly
Glu Ala Glu Phe Leu 195 200 205Asp Gly Leu Val Asp Glu Phe Glu Arg
Lys Ile Leu Glu Leu Gly Ala 210 215 220Asp Arg Val Gly Ala Phe Ile
Ser Glu Pro Val Phe Gly Ser Gly Gly225 230 235 240Val Ile Val Pro
Pro Ala Gly Tyr His Arg Arg Met Trp Glu Leu Cys 245 250 255Gln Arg
Tyr Asp Val Leu Tyr Ile Ser Asp Glu Val Val Thr Ser Phe 260 265
270Gly Arg Leu Gly His Phe Phe Ala Ser Gln Ala Val Phe Gly Val Gln
275 280 285Pro Asp Ile Ile Leu Thr Ala Lys Gly Leu Thr Ser Gly Tyr
Gln Pro 290 295 300Leu Gly Ala Cys Ile Phe Ser Arg Arg Ile Trp Glu
Val Ile Ala Glu305 310 315 320Pro Asp Lys Gly Arg Cys Phe Ser His
Gly Phe Thr Tyr Ser Gly His 325 330 335Pro Val Ala Cys Ala Ala Ala
Leu Lys Asn Ile Glu Ile Ile Glu Arg 340 345 350Glu Gly Leu Leu Ala
His Ala Asp Glu Val Gly Arg Tyr Phe Glu Glu 355 360 365Arg Leu Gln
Ser Leu Arg Asp Leu Pro Ile Val Gly Asp Val Arg Gly 370 375 380Met
Arg Phe Met Ala Cys Val Glu Phe Val Ala Asp Lys Ala Ser Lys385 390
395 400Ala Leu Phe Pro Glu Ser Leu Asn Ile Gly Glu Trp Val His Leu
Arg 405 410 415Ala Gln Lys Arg Gly Leu Leu Val Arg Pro Ile Val His
Leu Asn Val 420 425 430Met Ser Pro Pro Leu Ile Leu Thr Arg Glu Gln
Val Asp Thr Val Val 435 440 445Arg Val Leu Arg Glu Ser Ile Glu Glu
Thr Val Glu Asp Leu Val Arg 450 455 460Ala Gly His
Arg465681335DNAPseudomonas 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 Trp1 5 10 15Met Pro Phe Thr Ala Asn
Arg Gln Phe Lys Ala Arg Pro Arg Leu Leu 20 25 30Glu Ser Ala Glu Gly
Ile His Tyr Ile Ala Gln Gly Gly Arg Arg Ile 35 40 45Leu Asp Gly Thr
Ala Gly Leu Trp Cys Cys Asn Ala Gly His Gly Arg 50 55 60Arg Glu Ile
Ser Glu Ala Val Ala Arg Gln Ile Ala Thr Leu Asp Tyr65 70 75 80Ala
Pro Pro Phe Gln Met Gly His Pro Leu Pro Phe Glu Leu Ala Ala 85 90
95Arg Leu Thr Glu Ile Ala Pro Pro Ser Leu Asn Lys Val Phe Phe Thr
100 105 110Asn Ser Gly Ser Glu Ser Ala Asp Thr Ala Leu Lys Ile Ala
Leu Ala 115 120 125Tyr Gln Arg Ala Ile Gly Gln Gly Thr Arg Thr Arg
Leu Ile Gly Arg 130 135 140Glu Leu Gly Tyr His Gly Val Gly Phe Gly
Gly Leu Ser Val Gly Gly145 150 155 160Met Val Asn Asn Arg Lys Ala
Phe Ser Ala Asn Leu Leu Pro Gly Val 165 170 175Asp His Leu Pro His
Thr Leu Asp Val Ala Arg Asn Ala Phe Thr Val 180 185 190Gly Leu Pro
Glu His Gly Val Glu Lys Ala Glu Glu Leu Glu Arg Leu 195 200 205Val
Thr Leu His Gly Ala Glu Asn Ile Ala Ala Val Ile Val Glu Pro 210 215
220Met Ser Gly Ser Ala Gly Val Val Leu Pro Pro Lys Gly Tyr Leu
Gln225 230 235 240Arg Leu Arg Glu Ile Thr Arg Lys His Gly Ile Leu
Leu Ile Phe Asp 245 250 255Glu Val Ile Thr Gly Phe Gly Arg Val Gly
Glu Ala Phe Ala Ala Gln 260 265 270Arg Trp Gly Val Val Pro Asp Leu
Leu Thr Cys Ala Lys Gly Leu Thr 275 280 285Asn Gly Ser Ile Pro Met
Gly Ala Val Phe Val Asp Glu Lys Ile His 290 295 300Ala Ala Phe Met
Gln Gly Pro Gln Gly Ala Ile Glu Phe Phe His Gly305 310 315 320Tyr
Thr Tyr Ser Gly His Pro Val Ala Cys Ala Ala Ala Leu Ala Thr 325 330
335Leu Asp Ile Tyr Arg Arg Asp Asp Leu Phe Gln Arg Ala Val Glu Leu
340 345 350Glu Gly Tyr Trp Gln Asp Ala Leu Phe Ser Leu Arg Asp Leu
Pro Asn 355 360 365Val Val Asp Ile Arg Ala Val Gly Leu Val Gly Gly
Val Gln Leu Ala 370 375 380Pro His Ala Asp Gly Pro Gly Lys Arg Gly
Tyr Asp Val Phe Glu Arg385 390 395 400Cys Phe Trp Glu His Asp Leu
Met Val Arg Val Thr Gly Asp Ile Ile 405 410 415Ala Met Ser Pro Pro
Leu Ile Ile Asp Lys Pro His Ile Asp Gln Ile 420 425 430Val Glu Arg
Leu Ala Gln Ala Ile Arg Ala Ser Val 435 4407071DNAArtificial
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