U.S. patent application number 15/958548 was filed with the patent office on 2018-08-23 for method of screening gene for 1,4-bdo production.
The applicant listed for this patent is Samsung Electronics Co., Ltd.. Invention is credited to Hwayoung Cho, Yukyung Jung, Jaechan Park, Jinhwan Park.
Application Number | 20180237807 15/958548 |
Document ID | / |
Family ID | 52483909 |
Filed Date | 2018-08-23 |
United States Patent
Application |
20180237807 |
Kind Code |
A1 |
Cho; Hwayoung ; et
al. |
August 23, 2018 |
METHOD OF SCREENING GENE FOR 1,4-BDO PRODUCTION
Abstract
Provided is a screening method of discovering genes capable of
increasing 1,4-BDO production on the basis of proteomics data.
Over-expression of proteins screened by the method, NCgl0630
(citrate synthase) and NCgl2145 (hyperthetical protein), increase
1,4-BDO productivity. The method may lead to screening of a protein
associated with 1,4-BDO productivity, thereby increasing 1,4-BDO
productivity, and thus, the method may be recognized as being
industrially applicable.
Inventors: |
Cho; Hwayoung; (Hwaseong-si,
KR) ; Park; Jinhwan; (Suwon-si, KR) ; Jung;
Yukyung; (Hwaseong-si, KR) ; Park; Jaechan;
(Yongin-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Samsung Electronics Co., Ltd. |
Suwon-si |
|
KR |
|
|
Family ID: |
52483909 |
Appl. No.: |
15/958548 |
Filed: |
April 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14913767 |
Feb 23, 2016 |
9963721 |
|
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PCT/KR2014/007821 |
Aug 22, 2014 |
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15958548 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/34 20130101;
C12Y 203/03001 20130101; C12N 9/1025 20130101; C12N 9/0006
20130101; C12Y 101/01027 20130101; C12P 7/18 20130101; G01N
2333/91045 20130101; C12Q 1/48 20130101 |
International
Class: |
C12P 7/18 20060101
C12P007/18; C12N 9/10 20060101 C12N009/10; C07K 14/34 20060101
C07K014/34; C12N 9/04 20060101 C12N009/04; C12Q 1/48 20060101
C12Q001/48 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 2013 |
KR |
10-2013-0100567 |
Claims
1. A method of screening for a protein positively involved in
1,4-BDO production, comprising: culturing a microorganism that
produces 1,4-BDO in a culture medium comprising 1,4-BDO and in a
culture medium not comprising 1,4-BDO; analyzing protein expression
in the microorganisms; and selecting a protein showing increased
expression in the microorganism cultured with 1,4-BDO as compared
to the microorganism cultured without 1,4-BDO as a protein
positively involved in 1,4-BDO production.
2. The method of claim 1, wherein the microorganism that produces
1,4-BDO is a mutant microorganism, and the method further comprises
culturing a wild type microorganism incapable of producing 1,4-BDO
in a culture medium comprising 1,4-BDO and in a culture medium not
comprising 1,4-BDO a protein showing (a) increased expression in
the microorganism cultured with 1,4-BDO as compared to the
microorganism cultured without 1,4-BDO, and (b) increased
expression in the mutant microorganism capable of producing 1,4-BDO
than in the wild type microorganism.
3. The method of claim 1, wherein the microorganism is
Corynebacterium glutamicum.
4. The method of claim 1, wherein the microorganism does not have
lactate dehydrogenase activity.
5. The method of claim 1, wherein the microorganism comprises genes
encoding coenzyme A-dependent succinate semialdehyde dehydrogenase,
4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl CoA:acetyl-CoA
transferase, and alcohol dehydrogenase.
6. The method of claim 1, wherein the 1,4-BDO is added to the
culture medium during an exponential growth phase of the
microorganism.
7-18. (canceled)
19. The method of claim 2, wherein the mutant microorganism is
Corynebacterium glutamicum modified to produce 1,4-BDO, and the
wild-type microorganism is Corynebacterium glutamicum.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of Korean Patent
Application No. 10-2013-0100567, filed on Aug. 23, 2013, in the
Korean Intellectual Property Office, the disclosure of which is
incorporated herein in its entirety by reference.
[0002] BACKGROUND
1. Field
[0003] One aspect relates to a method of screening a protein
involved in efficiently producing 1,4-BDO. Another aspect relates
to a microorganism the nucleic acid encoding the protein screened
by the method. Another aspect also relates to a method of producing
1,4-BDO at a high efficiency using the microorganism.
2. Description of the Related Art
[0004] 1,4-butandiol (1,4-BDO) is used not only as a solvent for
manufacturing plastics and fiber but also as a raw material for
producing fiber such as spandex. About 1.3 million tons of 1,4-BDO
is produced in a year worldwide from petroleum-based materials such
as acetylene, butane, propylene, and butadiene. In addition, about
6% of consumption increase is anticipated each year. 1,4-butandiol
is important as it is used throughout the entire chemical industry
for the production of various chemicals such as polymers, solvents,
and fine chemistry intermediates. Most of the chemicals having a
carbon number of four are currently synthesized by being derived
from 1,4-butandiol or maleic anhydride, but the chemical production
process needs to be improved or replaced by a newly developed
process as production costs are increasing due to rising oil
prices. Thus, biological processes using microorganisms are
suggested as the alternative processes.
[0005] Different from the method of producing 1,4-BDO chemically,
Genomatica Inc. established in 2011 a biosynthetic pathway of
producing 1,4-BDO using succinyl-CoA synthetase (Cat1), succinate
semialdehyde dehydrogenase (SucCD), NAD-dependent 4-hydroxybutyrate
dehydrogenase (4Hbd), 4-hydroxybutyryl CoA:acetyl-CoA transferase
(Cat2), and alcohol dehydrogenase (AdhE2) genes in an Escherichia
coli. However, there has been an attempt to establish a new
biosynthetic pathway by altering the biological pathways which have
already been shown in an Escherichia coli in order to produce
1,4-BDO more efficiently. The attempt is much focused on
discovering enzymes of high efficiency by inducing various genetic
mutations in enzyme genes.
[0006] However, such an approach alone is limited in effectively
discovering a protein or a gene related to producing of 1,4-BDO. A
novel approach was tried to solve the problem, and the genes
screened by the approach were verified to enable a significant
increase of 1,4-BDO production.
SUMMARY
[0007] An aspect provides a screening method of a protein involved
in efficient production of 1,4-BDO. Another aspect provides a
microorganism including a nucleic acid encoding a protein screened
in the method. Another aspect provides a method of producing
1,4-BDO at a high efficiency using the microorganism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] These and/or other aspects will become apparent and more
readily appreciated from the following description of the
embodiments, taken in conjunction with the accompanying drawings of
which:
[0009] FIG. 1 compares growth curves of a wild type CGL strain and
a CGL strain capable of producing 1,4-BDO in the cases where wild
type CGL strain and the CGL strain capable of producing 1,4-BDO
were treated with 1,4-BDO of different concentrations. WT+BDO 25,
50, and 100 denote that 1,4-BDO was added to the wild type CGL at
the concentrations of 25 g/L, 50 g/L, and 100 g/L, respectively.
adhE2+BDO 25, 50, and 100 denote that 1,4-BDO was added to the CGL
transformed to produce 1,4-BDO at the concentrations of 25 g/L, 50
g/L, and 100 g/L, respectively;
[0010] FIG. 2 shows the result of 2D-gel electrophoresis was
obtained from proteins produced in the CGL strain cultured in a LB
culture medium at 30.degree. C.;
[0011] FIGS. 3a and 3b respectively show the result of 2D-gel
electrophoresis of proteins produced in the wild type CGL and the
mutant strain CGL (.DELTA.ldh 4G adhE2) after each of the CGL
strain was cultured in a LB culture medium at 30.degree. C.;
[0012] FIG. 4 shows 2D-gel electrophoresis spots of CGL strain
wherein intensity was increased. The left shows proteins which were
expressed when the wild type CGL was treated with 1,4-BDO of a
concentration of 100 g/L. The right shows proteins which were
expressed when the mutant strain CGL (.DELTA.ldh 4G adhE2)
producing 1,4-BDO was treated with 1,4-BDO of a concentration of
100 g/L; and
[0013] FIG. 5 compares the 1,4-BDO productivity of CGL wherein an
identified gene is over-expressed. pEKEx1 denotes a null vector as
a control group. pEk0827 denotes a vector expressing NCgl0827.
pEk2145 denotes a vector expressing NCgl2145. pEk0630 denotes a
vector expressing NCgl0630. pEk2826 denotes a vector expressing
NCgl2826.
DETAILED DESCRIPTION
[0014] An aspect of the present invention provides a method of
screening a protein involved in efficient production of
1,4-BDO.
[0015] An embodiment of the present invention provides a method of
screening a protein positively involved in 1,4-BDO production,
including culturing a microorganism producing 1,4-BDO in a culture
medium either including 1,4-BDO or not including 1,4-BDO; screening
a protein showing an increased expression according to increase of
1,4-BDO concentration from culture solution; and selecting the
screened protein as a protein positively involved in 1,4-BDO
production.
[0016] The protein screening method is described in detail
below.
[0017] First, the protein screening method includes culturing a
microorganism producing 1,4-BDO in a culture medium either
including 1,4-BDO or not including 1,4-BDO. The microorganism,
which is a microorganism producing 1,4-BDO, may be a wild type
microorganism or a transformed mutant microorganism. The
microorganism may be a microorganism capable of producing 1,4-BDO.
The microorganism may be a wild type microorganism capable of
producing 1,4-BDO. Also, the microorganism may be a microorganism
where introduction of genes associated with 1,4-BDO biosynthesis
makes the microorganism be capable of producing 1,4-BDO. The
microorganism may be a microorganism of a Corynebacterium genus.
The microorganism of Corynebacterium genus may be an
Corynebacterium glutamicum.
[0018] The microorganism capable of producing 1,4-BDO may include
an enzyme converting succinyl CoA to succinyl semialdehyde, an
enzyme converting succinyl semialdehyde to 4-hydroxybutyrate, an
enzyme converting 4-hydroxybutyrate to 4-hydroxybutyrate-CoA, an
enzyme converting 4-hydroxybutyrate-CoA to 1,4-BDO, or the
combination thereof.
[0019] The enzyme converting succinyl CoA to succinyl semialdehyde
may be CoA-dependent succinate semialdehyde dehydrogenase. The
enzyme may be an enzyme classified as EC.1.2.1.76. An example of
the enzyme may be SucD. The enzyme converting succinyl semialdehyde
to 4-hydroxybutyrate may be 4-hydroxybutyrate dehydrogenase. The
enzyme may be an enzyme classified as EC.1.1.1.61. The enzyme may
be 4Hbd. In addition, the enzyme converting 4-hydroxybutyrate to
4-hydroxybutyrate-CoA may be 4-hydroxybutyryl CoA:acetyl-CoA
transferase. The enzyme may be an enzyme classified as EC.2.8.3.-.
An example of the enzyme may be Cat2. The enzyme converting
4-hydroxybutyrate-CoA to 1,4-BDO may be alcohol dehydrogenase. The
alcohol dehydrogenase may be an enzyme classified as EC.1.1.1.-.
The enzyme may be AdhE or AdhE2. As an example, the microorganism
producing 1,4-BDO may be an microorganism expressing the SucD
protein, the 4Hbd protein, the Cat2 protein, and the AdhE
protein.
[0020] The term "protein expression" herein means that a protein or
an enzyme exists and has activity in a microorganism. The protein
or enzyme may exist through a transcription and a translation where
a polynucleotide encoding the protein, existing in the
microorganism, is transcribed to an mRNA which is in turn
translated into the protein. The polynucleotide encoding the
protein may exist either by being inserted in a chromosome of a
microorganism or by being inserted in a plasmid vector.
[0021] The CoA-dependent succinate semialdehyde dehydrogenase may
be a protein derived from an Escherichia genus, a Corynebacterium
genus or a Porphyromonas genus. The SucD protein may have an amino
acid sequence of SEQ ID NO:10. The polynucleotide encoding the SucD
may have a nucleotide sequence of SEQ ID NO:15.
[0022] The 4-hydroxybutyrate dehydrogenase may be a protein derived
from an Escherichia genus, a Corynebacterium genus or a
Porphyromonas genus. The 4Hbd protein may have an amino acid
sequence of SEQ ID NO:7. The polynucleotide encoding the 4HbD may
have a nucleotide sequence of SEQ ID NO:12.
[0023] The 4-hydroxybutyryl CoA:acetyl-CoA transferase may be a
protein derived from an Escherichia genus, a Corynebacterium genus
or a Porphyromonas genus. The Cat2 protein may have an amino acid
sequence of SEQ ID NO:8. The polynucleotide encoding the Cat2 may
have a nucleotide sequence of SEQ ID NO:13.
[0024] The alcohol dehydrogenase may be a protein derived from
Clostridium acetobutylicum. The AdhE protein may have an amino acid
sequence of SEQ ID NO:9. The polynucleotide encoding the AdhE may
have a nucleotide sequence of SEQ ID NO:14.
[0025] The microorganism may additionally include succinyl
CoA:coenzyme A transferase. The succinyl CoA:coenzyme A transferase
may have an activity to catalyze a reaction converting succinate to
succinyl CoA. The enzyme may be an enzyme classified as EC.2.8.3.-.
As an example, the enzyme may be Cat1. The Cat1 may have an amino
acid sequence of SEQ ID NO:11. The polynucleotide encoding the Cat1
may have a nucleotide sequence of SEQ ID NO:16.
[0026] The microorganism may be a microorganism wherein a pathway
synthesizing lactate from pyruvate is inactivated or decreased. The
microorganism may have the eliminated or decreased activity of
lactate dehydrogenase (Ldh). The Ldh may have an activity
catalyzing a reaction converting pyruvate to lactate. The Ldh may
be an enzyme classified as EC.1.1.1.27.The microorganism may have
the inactivated or attenuated gene encoding lactate
dehydrogenase.
[0027] The term "inactivation" herein may mean that a gene which is
not expressed or which is expressed but produces the enzyme or the
protein without activity, is produced. The term "attenuation" may
mean that a gene of which expression is decreased to a level lower
than an expression level of wild type strain, a strain which is not
genetically engineered or a parent strain or a gene which is
expressed but produces the enzyme or the protein with a decreased
activity, is produced. A decreased Ldh activity in the
microorganism may be lower than 30%, 20% or 10% of the Ldh activity
of wild type microorganism. The Ldh activity in the microorganism
may be completely eliminated. The inactivation or the attenuation
may be caused by homologous recombination. The inactivation or
attenuation may be performed by introducing a vector including a
part of the sequence of the genes into a cell, culturing the cell
so that homologous recombination between the sequence and an
endogenous gene of the cell may occur, and then selecting a cell
wherein homologous recombination has occurred using a selection
marker. The microorganism may be a microorganism wherein activity
of an enzyme encoded by the gene may be eliminated or decreased by
inactivation or attenuation of the gene. The term "decrease" may
relatively represent the activity of the genetically engineered
microorganism in comparison to the activity of a microorganism
which is not genetically engineered.
[0028] Activity of the lactate dehydrogenase may be inactivated or
attenuated in the microorganism by a mutation of gene encoding the
lactate dehydrogenase. The mutation may be performed by
substitution, partial or total deletion, or addition of a
nucleotide. Activity of the lactate dehydrogenase in the
microorganism may be decreased by eliminating endogenous lactate
dehydrogenase gene. The elimination includes not only physical
elimination of the gene but also prevention of functional
expression of the gene. The elimination may be performed by
homologous recombination.
[0029] The term "transformation" herein refers to introducing a
gene to a microorganism so that the gene may be expressed in the
microorganism. The introduced gene, if the gene is expressed in the
microorganism, may be inserted into a chromosome of the
microorganism or exists outside a chromosome. The gene may be a
polynucleotide capable of encoding a polypeptide, which may be DNA
or RNA. The introduction of the gene may be any type of
introduction, only if the gene may be introduced into and expressed
in the microorganism. For example, the gene may be introduced into
a microorganism in the form of an expression cassette, a
polynucleotide construct including all elements necessary to be
expressed by itself. The expression cassette usually includes a
promoter, a transcription termination signal, a ribosome binding
site, and a translation termination signal operably linked with the
gene. The expression cassette may be an expression vector capable
of self-replication. The gene may be introduced as itself or in the
form of a polynucleotide construct to a host cell and be operably
linked with a sequence required for an expression in the
microorganism.
[0030] The term "sequence identity" of a nucleic acid or a
polypeptide herein means the degree of identity with reference to
base-to-base or amino acid-to-amino acid comparison or with
reference to function or structure in a whole window of comparison.
Therefore, "percentage of sequence identity" may be calculated, for
example, by comparing two optimally aligned sequences in a whole
window of comparison, determining the number of positions wherein
the same base or the same amino acid is located in both sequences
and obtaining the number of matched positions, and by dividing the
number of the matched positions with the total number of positions
(i.e., window size) and then multiplying 100 with the resulting
value. The percent sequence identity may be determined by using
known sequence comparing software such as BLASTn (NCBI) and
MEGALIGN.TM. (DNASTAR Inc). Various levels of sequence identity may
be used to identify many polypeptides or genes having an identical
or similar function or activity. For example, a percent sequence
identity of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% may
be used.
[0031] 1,4-BDO may be added to culture medium in any phase in the
growth curve of a microorganism. 1,4-BDO may be added to culture
medium in exponential phase wherein a microorganism grows most
actively. In addition, concentration of the added 1,4-BDO may be
from about 0 to about 500 g/L. For example, the concentration of
the added 1,4-BDO may be selected from the range from about 0 to
about 400 g/L, from about 0 to about 300 g/L, from about 0 to about
200 g/L, or from about 0 to about 100 g/L. Two or n different
concentrations of 1,4-BDO may be treated (n is a integer equal to
or greater than 2.). When 1,4-BDO is added, at least two different
concentrations, for example, three or more, four or more, or five
or more different concentrations of 1,4-BDO may be added to culture
medium.
[0032] Microorganism culture conditions may be dependent on the
microorganism. The term "culture conditions" refers to conditions
to culture a microorganism. The culture condition may be, for
example, carbon source, nitrogen source or oxygen conditions.
Carbon sources which may be used by a microorganism include
monosaccharide, disaccharide or polysaccharide. Specifically,
glucose, fructose, mannose, or galactose etc. may be used. Nitrogen
sources which may be used by a microorganism include organic
nitrogen compounds and inorganic nitrogen compounds. Specifically,
amino acids, amides, amines, nitrates or ammonium salts etc. may be
used.
[0033] The protein screening method also includes screening a
protein showing an increased expression according to increase of
1,4-BDO concentration from culture solution. A protein produced by
a microorganism refers to all proteins produced by a microorganism,
and may be proteins existing in or secreted by a microorganism.
[0034] The protein screening method may additionally include a step
of collecting cultured microorganism and a step of extracting a
protein from the microorganism in order to compare the protein
quantity. Methods including SDS-PAGE or Western blot may be used to
compare the protein quantity. In addition, the protein quantity may
be verified through two-dimensional gel electrophoresis or
matrix-assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF/MS).
[0035] A protein expressed to a higher level at a higher 1,4-BDO
concentration may be screened in the steps. After treating with 1,
4-BDO of two different concentrations, a protein expressed to a
higher level under a higher 1,4-BDO concentration may be screened.
In addition, when 1,4-BDO of two or n different concentrations is
treated, a protein commonly expressed to a higher level in
comparison to an expression level at the lowest 1,4-BDO
concentration may be screened (n is a integer equal to or greater
than 2.). In addition, a protein of which expression level is
increased as the 1,4-BDO concentration is increased may be
screened.
[0036] The protein screening method also includes selecting the
screened protein as a protein positively involved in 1,4-BDO
production. The selected protein in the above steps may be a
protein involved in 1,4-BDO production. The protein may be directly
or indirectly involved in 1,4-BDO production by a microorganism.
The protein with an increased expression may be expressed in a
cell.
[0037] The culturing may include culturing a wild type
microorganism incapable of producing 1,4-BDO and a mutant
microorganism thereof capable of producing 1,4-BDO. The screening
may additionally include screening of a protein of which expression
is higher in the mutant microorganism capable of producing 1,4-BDO
than that in the wild type microorganism incapable of producing
1,4-BDO.
[0038] The method may include culturing a wild type microorganism
incapable of producing 1,4-BDO and a mutant thereof capable of
producing 1,4-BDO, analyzing a protein produced by the
microorganisms, and screening a protein of which expression is
higher in a mutant capable of producing 1,4-BDO than that in a wild
type microorganism.
[0039] A wild type microorganism incapable of producing 1,4-BDO and
a mutant microorganism capable of producing 1,4-BDO may be the same
type as the microorganism producing 1,4-BDO used in the method. In
addition, the wild type microorganism refers to a microorganism
which is not yet mutated to produce 1,4-BDO. The culture conditions
may be the same as the culture conditions used in the screening
method. A wild type microorganism and a mutant thereof capable of
producing 1,4-BDO may be cultured at the same 1,4-BDO concentration
or in the absence of 1,4-BDO. The protein of which expression is
increased may be expressed in a cell.
[0040] The protein screening method also includes comparing
proteins expressed at a high level in the culturing and selecting a
protein commonly expressed in the screening.
[0041] When 1,4-BDO of different concentrations is added, a protein
expressed at a higher level in a microorganism to which 1,4-BDO of
a higher concentration is added may be screened. In addition, a
protein expressed at a higher level in a microorganism to which
1,4-BDO is added than that in a microorganism to which 1,4-BDO is
not added may be screened. When 1,4-BDO of a higher concentration
is added, a protein expressed at a higher level may be screened. In
addition, a protein commonly expressed in all microorganisms to
which 1,4-BDO of different concentrations is added may be screened.
In addition, a protein of which expression level is increased as
1,4-BDO is increased may be screened.
[0042] An over-expressed protein may be verified by the screening.
For example, the protein may be citrate synthase NCgl0630 or
NCgl2145. Production of 1,4-BDO may be increased by introducing a
nucleic acid sequence encoding an over-expressed protein such as
NCgl0630 or NCgl2145 into a microorganism capable of producing
1,4-BDO. In an Example of the present invention, 1,4-BDO
productivity of a strain wherein NCgl0630 was introduced was 49%
higher than that of the control group. In addition, 1,4-BDO
productivity of a strain wherein NCgl2145 was introduced was 18%
higher than that of the control group. These results verified that
the a protein involved in 1,4-BDO production may be effectively
screened by the screening method.
[0043] An aspect relates to a microorganism including a nucleic
acid encoding a protein screened by the method. An Example of the
present invention provides a microorganism, which is capable of
producing 1,4-BDO, wherein activity of citrate synthase is
increased. The activity of citrate synthase may be increased in
comparison with that of a wild type of the microorganism. In
addition, the microorganism is capable of producing 1,4-BDO at a
high level.
[0044] The microorganism, which is a microorganism producing
1,4-BDO, may be a wild type microorganism or a transformed
microorganism. The transformed microorganism may be prepared by an
addition, deletion, or substitution of a gene to produce 1,4-BDO in
a wild type microorganism. In addition, the transformation may be
performed by mutating one or more genes. The microorganism may be
Corynebacterium glutamicum (CGL). Activity of Ldh in a wild type
CGL may be eliminated for producing 1,4-BDO. In addition, cat1,
sucD, 4hbD, cat2, and adhE genes may be introduced into a wild type
CGL for producing 1,4-BDO.
[0045] The citrate synthase may be a gene derived from a CGL. In
amino acids constituting the citrate synthase, part of the amino
acid may be substituted, altered or deleted, as long as the
sequence retains original activity of citrate synthase. In
addition, the citrate synthase may include an amino acid sequence
having at least 95% of sequence identity with an amino acid
sequence of SEQ ID NO:1. For example, the citrate synthase may
include an amino acid sequence of SEQ ID NO:1.
[0046] A nucleic acid sequence encoding the citrate synthase may
include a nucleic acid sequence encoding an amino acid sequence
having at least 95% of sequence identity with an amino acid
sequence of SEQ ID NO:1. For example, the nucleic acid may have a
nucleic acid sequence of SEQ ID NO:2. Part of the nucleic acid
sequence may be substituted, altered, or deleted, as long as the
protein encoded by the nucleic acid sequence retains original
activity of citrate synthase. The nucleic acid may be a nucleic
acid sequence having 80% or higher, 90% or higher, or 95% or higher
sequence identity with the nucleic acid sequence, as long as the
protein encoded by the nucleic acid sequence retains original
activity of citrate synthase.
[0047] Activity of citrate synthase may be increased by
over-expressing a nucleic acid encoding citrate synthase in a cell.
The nucleic acid may be introduced to a microorganism as itself or
as it is inserted to a vector. The nucleic acid may be expressed
within a vector or over-expressed as it is inserted into a
chromosome of a microorganism.
[0048] The term "vector" herein refers to a DNA product including a
DNA sequence operably linked to an appropriate regulatory sequence
capable of expressing DNA in an appropriate host. The vector may be
a plasmid vector, a bacteriophage vector, or a cosmid vector.
[0049] To operate as an expression vector, a vector may include a
replication origin, a promoter, a multi-cloning site (MCS), and a
selection marker. A replication origin gives a function to a
plasmid to replicate itself independently of host cell chromosome.
A promoter operates in transcription process of an inserted foreign
gene. An MCS enables a foreign gene to be inserted through various
restriction enzyme sites. A selection marker verifies whether a
vector has been properly introduced to a host cell or not. A
selection includes an antibiotic-resistant gene generally used in
the art. For example, a selection marker may include a gene
resistant to ampicillin, gentamycin, carbenicillin,
chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, or
tetracycline. Considering the cost, ampicillin or
gentamycin-resistant gene may be used.
[0050] When a vector of an aspect of the present invention uses a
prokaryotic cell as host cell, a strong promoter, for example,
lamda-PL promoter, trp promoter, lac promoter, T7 promoter, or tac
promoter is included in the vector. If a vector uses a eukaryotic
cell as host cell, the vector may include a promoter derived from
genome of a mammal (metallothionin promoter, e.g.) or a promoter
derived from a mammal virus (adenovirus late promoter, vaccinia
virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter or tk
promoter of HSV promoter, e.g.). The promoter may be a lamda-PL
promoter, trp promoter, lac promoter, T7 promoter, or tac promoter.
In this manner, a promoter is operably linked with a sequence
encoding a gene.
[0051] The term "operably linked" herein may mean a functional
linkage between a nucleic acid expression regulatory sequence
(promoter, signal sequence, or a sequence at transcription
regulation factor binding site) and another nucleic acid sequence.
Through the functional linkage, the regulatory sequence may control
transcription and/or translation of a nucleic acid encoding the
gene.
[0052] A microorganism in another Example of the present invention
may include NCgl2145 protein which is not included in a wild type
microorganism. The microorganism provides a microorganism, which is
capable of producing 1,4-BDO, including NCgl2145 protein. NCgl2145
protein may include an amino acid sequence having at least 95% of
sequence identity with an amino acid sequence of SEQ ID NO:3. For
example, NCgl2145 protein may include an amino acid sequence of SEQ
ID NO:3.
[0053] A nucleic acid sequence encoding the NCgl2145 protein may
include a nucleic acid sequence encoding an amino acid sequence
having at least 95% of sequence identity with an amino acid
sequence of SEQ ID NO:3. For example, the nucleic acid encoding the
NCgl2145 protein may have a nucleic acid sequence of SEQ ID NO:4.
The nucleic acid encoding amino acid sequence of SEQ ID NO:3 may be
a nucleic acid sequence having 80% or higher, 85% or higher, 90% or
higher, 95% or higher, or 99% or higher sequence identity with a
nucleic acid sequence of SEQ ID NO:4 or a fragment thereof. For
example, the amino acid sequence of SEQ ID NO:3 may be encoded by
the sequence of SEQ ID NO:4. In addition, the microorganism
provides a microorganism, which is capable of producing 1,4-BDO,
wherein a nucleic acid sequence encoding an amino acid sequence
having at least 95% of sequence identity with an amino acid
sequence of SEQ ID NO:3 is introduced. The microorganism may
additionally include a nucleic acid encoding an amino acid sequence
having at least 95% of sequence identity with an amino acid
sequence of SEQ ID NO:3, in comparison with a wild type
microorganism. The microorganism is capable of producing 1,4-BDO at
a high level.
[0054] The microorganism may be CGL. Ldh activity may be eliminated
in a wild type CGL for producing 1,4-BDO. For example, cat1, sucD,
4hbD, cat2, and adhE genes may be introduced into a wild type CGL
for producing 1,4-BDO.
[0055] Another example of the present invention provides a
microorganism wherein nucleic acids encoding citrate synthase and
NCgl2145 (hypothetical protein) are introduced. The microorganism
overexpressing the two enzymes at the same time is capable of
producing 1,4-BDO at a high level.
[0056] The microorganism may be CGL. The citrate synthase may have
an amino acid sequence of SEQ ID NO:1. The amino acid sequence of
SEQ ID NO:1 may be encoded by a nucleic acid sequence of SEQ ID
NO:2. In addition, the citrate synthase may be derived from CGL.
The NCgl2145 may have an amino acid sequence of SEQ ID NO:3. In
addition, the amino acid sequence of SEQ ID NO:3 may be encoded by
a nucleic acid sequence of SEQ ID NO:4. The microorganism may be
CGL. Ldh activity may be eliminated in a wild type CGL for
producing 1,4-BDO. Nucleic acids encoding Cat1, SucD, 4HbD, Cat2,
and AdhE may be introduced into a wild type CGL for producing
1,4-BDO.
[0057] Another aspect provides a method of producing a
microorganism having an increased capability of producing 1,4-BDO,
the method including introduction of a nucleic acid encoding a
screened protein positively involved in 1,4-BDO production to a
microorganism capable of producing 1,4-BDO.
[0058] The protein positively involved in 1,4-BDO production may be
citrate synthase or a protein having an amino acid sequence of SEQ
ID NO:3. The citrate synthase may have an amino acid sequence of
SEQ ID NO:1. The citrate synthase may be encoded by a nucleic acid
sequence of SEQ ID NO:2. In addition, the amino acid sequence of
SEQ ID NO:3 may be encoded by a nucleic acid sequence of SEQ ID
NO:4. The microorganism may be CGL. Ldh activity may be eliminated
in a wild type CGL for producing 1,4-BDO. A nucleic acid encoding
one protein selected from the group consisting of Cat1, SucD, 4HbD,
Cat2 , and AdhE may be introduced into a wild type CGL for
producing 1,4-BDO. In addition, the microorganism may include Cat1,
SucD, 4HbD, Cat2 , and AdhE all together.
[0059] Another aspect provides a method of producing 1,4-BDO
including culturing a microorganism wherein a nucleic acid encoding
a protein positively involved in 1,4-BDO production is introduced;
and obtaining 1,4-BDO from culture medium.
[0060] The microorganism may be CGL. Ldh activity may be eliminated
in a wild type CGL for producing 1,4-BDO. sucD, 4hbD, cat2, and
adhE genes may be introduced into a wild type CGL for producing
1,4-BDO. In addition, the microorganism may additionally include a
nucleic acid encoding Cat1 or nucleic acid encoding SucCD.
[0061] To over-expressing in a cell a nucleic acid encoding a
protein screened by the screening method, a nucleic acid may be
introduced to a microorganism as itself or as it is inserted to a
vector. The nucleic acid may be expressed within a vector or
over-expressed as it is inserted into a chromosome of a
microorganism. A vector for expression may include a replication
origin, a promoter, an MCS, and a selection marker.
[0062] Another example of the present invention provides a method
of producing 1,4-BDO at a high yield including culturing a
microorganism wherein citrate synthase is over-expressed, Ncgl2145
is introduced, or a nucleic acid encoding a screened protein is
introduced; and obtaining 1,4-BDO from culture medium.
[0063] The culturing may be performed under an appropriate culture
medium and culture conditions known in this art. The culture medium
and culture conditions may be conveniently adjusted according to
the selected microorganism. The culturing method may include batch
culturing, continuous culturing, fed-batch culturing or a
combination thereof.
[0064] The culture medium may include various carbon sources,
nitrogen sources, and trace elements.
[0065] The carbon source may include a carbohydrate such as
glucose, sucrose, lactose, fructose, maltose, starch, and
cellulose, a lipid such as soybean oil, sunflower oil, castor oil,
and coconut oil, a fatty acid such as palmitic acid, stearic acid,
and linoleic acid, an organic acid such as acetic acid or a
combination thereof. The culturing may be performed by using
glucose as a carbon source. The nitrogen source may include an
organic nitrogen source such as peptone, yeast extract, meat
extract, malt extract, corn steep liquid, and soybean, an inorganic
nitrogen source such as urea, ammonium sulfate, ammonium chloride,
ammonium phosphate, ammonium carbonate, and ammonium nitrate or a
combination thereof. The culture medium may include as a
phosphorous source, for example, potassium dihydrogen phosphate,
dipotassium phosphate, a sodium-containing salt corresponding to
potassium dihydrogen phosphate, and dipotassium phosphate, and a
metal salt such as magnesium sulfate and iron sulfate. The culture
medium or an individual component may be added to the culture in a
batch mode or a continuous mode. The culture medium or an
individual component may be added to the culture solution in a
batch mode or a continuous mode.
[0066] In addition, pH of the culture may be adjusted during the
culturing by adding a compound such as ammonium hydroxide,
potassium hydroxide, ammonia, phosphoric acid or sulfuric acid to
the culture in an appropriate mode. In addition, bubble formation
may be repressed by using an endoplasmic reticulum such as fatty
acid polyglycol ester.
[0067] The culturing may be performed under anaerobic conditions.
The term "anaerobic conditions" herein refers to a state wherein
oxygen content is lower than that of normal atmospheric state.
Anaerobic conditions may be formed, for example, by supplying
carbon dioxide or nitrogen at a flow rate range from about 0.1 vvm
(Volume per Volume per Minute) to about 0.4 vvm, from about 0.2 vvm
to about 0.3 vvm or at a flow rate of 0.25 vvm. In addition,
anaerobic conditions may be formed by setting an aeration rate in
the range from about 0 vvm and to 0.4 vvm, from about 0.1 vvm to
about 0.3 vvm or from 0.15 vvm to about 0.25 vvm.
[0068] The method of producing 1,4-BDO includes recovering of the
produced 1,4-BDO from the culture medium. For example, the recovery
of 1,4-BDO may be performed by using known separation and
purification methods. The recovery may be performed by
centrifugation, ion exchange chromatography, filtration,
precipitation or a combination thereof.
[0069] As described above, according to a screening method of one
Example of the present invention, a gene for producing 1,4-BDO at a
high efficiency may be effectively screened. In addition, a
microorganism over-expressing NCgl0630 gene encoding citrate
synthase and NCgl2145 gene screened by the screening method is
capable of producing 1,4-BDO effectively. 1,4-BDO may be
effectively produced by the method and with the genes.
[0070] It should be understood that the exemplary embodiments
described therein should be considered in a descriptive sense only
and not for purposes of limitation. Descriptions of features or
aspects within each embodiment should typically be considered as
available for other similar features or aspects in other
embodiments.
Example 1. Preparation of Corynebacterium Microorganism Wherein
Endogenous Lactate Dehydrogenase Gene is Deleted
[0071] A decrease in intracellular acetyl-CoA concentration was
found when culturing Corynebacterium glutamicum ATCC13032 under
anaerobic conditions. Therefore, it was assumed that decrease in
TCA cycle activity may be caused by the decrease in the acetyl-CoA
concentration. In addition, an experiment was designed in search of
a method to resolve the problem. For this, a .DELTA.ldh
Corynebacterium microorganism ATCC13032 wherein endogenous lactate
dehydrogenase gene is deleted ("basic strain" hereinafter) was
prepared by deleting the endogenous lactate dehydrogenase gene so
that the Pdh enzyme activity might be conveniently measured in the
natural Corynebacterium glutamicum.
1.1 Preparation of replacement vector
[0072] The L-lactate dehydrogenase gene of Corynebacterium
glutamicum (CGL) ATCC13032 was inactivated by homologous
recombination using a pK19 mobsacB (ATCC87098) vector. The two
homologous regions for the elimination of the ldhA gene were
obtained by PCR amplification using the genome DNA of CGL
ATCC13032. Two homologous regions for the elimination of the ldh
gene were located upstream and downstream from the gene and
obtained by PCR amplification using a primer set including
ldhA_5'_HindIII (SEQ ID NO:17) and ldhA_up_3'_Xhol (SEQ ID NO:18)
and a primer set including ldhA_dn_5'_Xhol (SEQ ID NO:19) and
ldhA_3'_EcoRI (SEQ ID NO:20). The PCR amplification was performed
by repeating, 30 times, a cycle including a denaturation step at
95.degree. C. for 30 seconds, an annealing step at 55.degree. C.
for 30 seconds, and an extension step at 72.degree. C. for 30
seconds. All the PCR amplifications hereinafter were performed
under the same conditions. A pK19_.DELTA.ldhA vector was prepared
by cloning the obtained amplification product to the HindIII and
EcoRI restriction enzyme positions of pK19 mobsacB vector.
1.2 Preparation of CGL (.DELTA.ldhA) strain
[0073] The pK19_.DELTA.ldhA vector was introduced to CGL ATCC13032
by electroporation. The strain wherein the pK19_.DELTA.ldhA vector
was introduced was cultured at 30.degree. C. by streaking the
strain on a lactobacillus selection (LBHIS) culture medium
including kanamycin 25 .mu.g/ml. The LBHIS culture medium included
brain-heart infusion broth 18.5 g/L, 0.5 M sorbitol, 5 g/L
bacto-tryptone, 2.5 g/L bacto-yeast extract, 5 g/L NaCl, and 18 g/L
bacto-agar. Hereinafter, the composition of the LBHIS culture
medium is the same. Colonies on the culture medium were streaked on
an LB-sucrose culture medium and cultured at 30.degree. C., and
then only the colonies wherein double crossing-over occurred were
selected. After separating genomic DNA from the selected colonies,
deletion of the ldhA gene was verified by PCR using a primer set
including ldhA up (SEQ ID NO:21) and ldhA down (SEQ ID NO:22). CGL
(.DELTA.ldhA) strain (B005) was obtained as a result.
Example 2. Introduction of Genes for 1,4-BDO Production 2.1
Preparation of pK19 gapA::4G vector
[0074] A CGL strain capable of producing 1,4-BDO was prepared on
the basis of the strain prepared above. To insert four genes of
cat1, sucD, 4hbD, and cat2 into a chromosome of the strain, pK19
gapA::4G vector for the insertion of cat1, sucD 4hbD, and cat2
genes was prepared on the basis of pK19 mobsacB. The pK19 gapA::4G
vector was prepared by synthesizing whole 4G gene having a
nucleotide sequence of SEQ ID NO:23 and cloning the 4G gene into
the Nhel and Xbal restriction enzyme sites of the pK19 mobsacB
vector.
2.2 Preparation of CGL (.DELTA.ldhA) strain
[0075] The pK19 gapA::4G vector was introduced to CGL (.DELTA.ldh)
by electroporation. The strain wherein the pK19 gapA::4G vector was
introduced was cultured at 30.degree. C. by streaking the strain on
LBHIS culture medium including kanamycin 25 .mu.g/ml. The colony
was streaked on LB-sucrose culture medium and cultured at
30.degree. C. Then, only the colonies wherein double crossing-over
occurred were selected. The genome DNA was separated from the
selected colonies, and introduction of the 4G genes was verified
through PCR by using primer sets 0049-1 for (SEQ ID NO:24) and
0049-2 rev (SEQ ID NO:25). CGL (.DELTA.ldh 4G) strain was obtained
as a result.
Example 3. Preparation of Strain Wherein adhE2 is Introduced
[0076] 3.1 Preparation of pK19 gapA::adhE2 vector
[0077] To insert the adhE2 gene to the chromosome, the pK19
gapA::adhE2 vector for insertion of adhE2 gene was prepared on the
basis of pK19 mobsacB. The pK19 gapA::adhE2 was prepared by
synthesizing whole adhE2 gene having a nucleotide sequence of SEQ
ID NO:26 and the cloning the adhE2 gene into the Smal restriction
enzyme site of the pK19 mobsacB vector.
3.2 Preparation of CGL (.DELTA.ldhA 4G adhE2) strain
[0078] The pK19 gapA::adhE2 vector was introduced to CGL
(.DELTA.ldh 4G) by electroporation. The strain wherein the pK19
gapA::adhE2 vector was introduced was cultured at 30 .degree. C. by
streaking the strain on LBHIS culture medium including kanamycin 25
.mu./ml. The colony was streaked on LB-sucrose culture medium and
cultured at 30.degree. C. Then, only the colonies wherein double
crossing over occurred were selected. The genome DNA was separated
from the selected colonies, and introduction of the adhE2 gene was
verified through PCR by using primer sets AdhE2_1_F for (SEQ ID
NO:27) and AdhE2_2260_R (SEQ ID NO:28). CGL (.DELTA.ldhA 4G adhE2)
strain capable of producing 1,4-BDO was obtained as a result.
Example 4. Screening of a Protein Related to Producing of
1,4-BDO
[0079] A wild type CGL and the mutant capable of producing 1,4-BDO,
which was prepared above, were cultured in LB culture medium at
30.degree. C. In an exponential phase of the wild type CGL and the
mutant capable of producing 1,4-BDO (.DELTA.ldhA, cat1, sucD 4hbD,
cat2, and adhE), 1,4-BDO of a concentration of 0, 25, 50, and 100
g/L was added to the culture medium at the time when the value of
OD.sub.600 was in the range from about 1.5 to about 2.5. Samples
were taken at the time lapse of 0, 1, 3, and 5 hours, and
expression of the total proteins was compared by 2D-gel
electrophoresis (FIG. 3). Seven spots wherein electrophoresis
expression intensity was increased in redundancy were selected
(FIG. 4). Proteins of which electrophoresis expression level was
increased in redundancy were identified as NCgl0827, NCgl2145,
NCgl0630, and NCgl2826 by MALDI/MS.
Example 5. Preparation of a Strain, which is Capable of Producing
1,4-BDO, Over-Expressing a Screened Protein
[0080] To verify whether or not a screened protein actually affects
1,4-BDO production, a microorganism wherein a screened protein is
introduced was prepared. For this, a sequence encoding a screened
protein was introduced into a vector, and the vector was in turn
introduced into a CGL. The CGL was a strain which was transformed
to be capable of producing 1,4-BDO. NCgl0630 gene (SEQ ID NO:2),
NCgl2145 gene (SEQ ID NO:4), NCgl0827 gene (SEQ ID NO:5), and
NCgl2826 gene (SEQ ID NO:6) were inserted into a MCS of pEKEx1
vector (a family of Corynebacterium glutamicum/Escherichia coli
shuttle vectors for cloning, controlled gene expression and
promoter probing. Gene, 102 (1991) 93-98) and then expressed by tac
promoter. NCgl0630 gene was inserted by using the restriction
enzymes EcoRI and SalI recognition sequences. NCgl2145, NCgl0827,
and NCgl2826 genes were inserted by using the restriction enzymes
BamHI and PstI recognition sites; restriction enzyme EcoRI (single)
recognition site; and restriction enzymes EcoRI and BamHI
recognition sites, respectively. A total of five strains were
prepared by introducing expression vectors wherein each of the
genes was introduced (pEK0630, pEK2145, pEK0827, and pEK2826) and a
pEKEx1null vector (Ref) as a control group to a mutant strain CO24
(.DELTA.ldhA, cat1 sucD 4hbD cat2 adhE2) capable of producing
1,4-BDO.
Example 6. Comparison Of 1,4-BDO Productivity of CGL
Over-Expressing an Identified Gene
[0081] 1,4-BDO productivity was compared after fermenting a total
of five CGL mutants prepared above, including the control group.
Firstly, to provide equal growth conditions, the strains were
fermented under aerobic conditions. Fermentation was performed by
changing the conditions into anaerobic conditions by reducing
oxygen in culture medium after a predetermined time passed.
[0082] Colonies of the five mutants were injected respectively
inoculated to LB culture medium 3 mL including kanamycin 25
.mu.g/ml and cultured at 30.degree. C. at a stirring rate of 220
rpm for 12 hours. The culture solutions were respectively
inoculated to LB culture medium 50 mL including kanamycin 25
.mu.g/ml and isopropyl .beta.-D-1-thiogalactopyranoside (IPTG),
which is a substance to induce expression of tac promoter, and
cultured in 250 mL flasks at 30.degree. C. at a stirring rate of
220 rpm for 24 hours. To change the culture conditions into
anaerobic conditions, the culture solutions were respectively
transported to 125 mL flasks and the flasks were sealed with film
so that air might not be supplied. Then, the culture solutions were
cultured at 30.degree. C. at a stirring rate of 90 rpm for 72
hours. Cells of the strains were separated from the final culture
solutions by centrifugation, and 1,4-BDO was quantified by
analyzing supernatants by HPLC. 1,4-BDO productivity of the strains
wherein NCgl0630 was expressed was 49% higher than that of the
control group. 1,4-BDO productivity of the strains wherein NCgl2145
was expressed was 18% higher than that of the control group.
Sequence CWU 1
1
281383PRTCorynebacterium glutamicum 1Met Ser Ser Ala Thr Thr Thr
Asp Val Arg Lys Gly Leu Tyr Gly Val 1 5 10 15 Ile Ala Asp Tyr Thr
Ala Val Ser Lys Val Met Pro Glu Thr Asn Ser 20 25 30 Leu Thr Tyr
Arg Gly Tyr Ala Val Glu Asp Leu Val Glu Asn Cys Ser 35 40 45 Phe
Glu Glu Val Phe Tyr Leu Leu Trp His Gly Glu Leu Pro Thr Ala 50 55
60 Gln Gln Leu Ala Glu Phe Asn Glu Arg Gly Arg Ser Tyr Arg Ser
Leu65 70 75 80 Asp Ala Gly Leu Ile Ser Leu Ile His Ser Leu Pro Lys
Glu Ala His 85 90 95 Pro Met Asp Val Met Arg Thr Ala Val Ser Tyr
Met Gly Thr Lys Asp 100 105 110 Ser Glu Tyr Phe Thr Thr Asp Ser Glu
His Ile Arg Lys Val Gly His 115 120 125 Thr Leu Leu Ala Gln Leu Pro
Met Val Leu Ala Met Asp Ile Arg Arg 130 135 140 Arg Lys Gly Leu Asp
Ile Ile Ala Pro Asp Ser Ser Lys Ser Val Ala145 150 155 160 Glu Asn
Leu Leu Ser Met Val Phe Gly Thr Gly Pro Glu Ser Pro Ala 165 170 175
Ser Asn Pro Ala Asp Val Arg Asp Phe Glu Lys Ser Leu Ile Leu Tyr 180
185 190 Ala Glu His Ser Phe Asn Ala Ser Thr Phe Thr Ala Arg Val Ile
Thr 195 200 205 Ser Thr Lys Ser Asp Val Tyr Ser Ala Ile Thr Gly Ala
Ile Gly Ala 210 215 220 Leu Lys Gly Pro Leu His Gly Gly Ala Asn Glu
Phe Val Met His Thr225 230 235 240 Met Leu Ala Ile Asp Asp Pro Asn
Lys Ala Ala Ala Trp Ile Asn Asn 245 250 255 Ala Leu Asp Asn Lys Asn
Val Val Met Gly Phe Gly His Arg Val Tyr 260 265 270 Lys Arg Gly Asp
Ser Arg Val Pro Ser Met Glu Lys Ser Phe Arg Glu 275 280 285 Leu Ala
Ala Arg His Asp Gly Glu Lys Trp Val Ala Met Tyr Glu Asn 290 295 300
Met Arg Asp Ala Met Asp Ala Arg Thr Gly Ile Lys Pro Asn Leu Asp305
310 315 320 Phe Pro Ala Gly Pro Ala Tyr His Leu Leu Gly Phe Pro Val
Asp Phe 325 330 335 Phe Thr Pro Leu Phe Val Ile Ala Arg Val Ala Gly
Trp Thr Ala His 340 345 350 Ile Val Glu Gln Tyr Glu Asn Asn Ser Leu
Ile Arg Pro Leu Ser Glu 355 360 365 Tyr Asn Gly Glu Glu Gln Arg Glu
Val Ala Pro Ile Glu Lys Arg 370 375 380 21152DNACorynebacterium
glutamicum 2atgtccagcg ccacaaccac tgatgttcgc aaagggctct acggagtcat
cgccgattac 60acggccgttt ccaaagtcat gccagagacc aattcactga cctaccgtgg
ctacgcggtg 120gaagatttgg tggaaaactg cagcttcgag gaggtgtttt
acctcctgtg gcacggcgag 180ctgcccactg cgcaacaact tgcggagttc
aatgagcgtg gccgttccta ccgctccctg 240gatgccggtt tgatctccct
gatccactct ttgcccaaag aagcccaccc gatggatgtt 300atgcgcaccg
cggtgtccta catgggcacc aaggattccg agtatttcac caccgattct
360gagcacatcc gcaaagttgg ccacaccttg ttggcgcagc ttccgatggt
gctagccatg 420gatattcgtc gccgcaaggg cctcgatatc atcgcccctg
actccagcaa gtcagtcgcc 480gaaaacctgc tgtctatggt gtttggtact
ggcccggaat cacctgcatc caacccagct 540gacgtccgcg attttgagaa
atcactgatc ctctacgccg agcactcctt caacgcctcc 600accttcaccg
cccgcgtgat cacctccacc aaatcggatg tgtactccgc aatcaccggc
660gcgatcggtg ctctcaaggg cccattgcac ggtggcgcca acgagtttgt
catgcacacc 720atgttggcga tcgacgatcc caacaaggcc gccgcctgga
tcaacaacgc tttggacaac 780aagaatgtgg tcatgggctt tggccaccgc
gtgtacaagc gcggcgattc ccgcgtgcca 840tcaatggaga agtccttccg
ggaattagct gcccgccacg acggcgaaaa gtgggttgcc 900atgtatgaaa
acatgcgcga cgccatggac gcccgcaccg gcatcaagcc gaatctcgat
960ttccctgctg gccctgccta ccacctgctc ggtttcccgg tcgatttctt
caccccgctg 1020ttcgtcatcg cccgcgtcgc cggctggacg gcccacatcg
tggagcagta cgaaaacaac 1080tcgctcatcc gcccactgtc cgagtacaac
ggcgaggagc agcgcgaggt cgcgcccatt 1140gaaaagcgct aa
11523160PRTCorynebacterium glutamicum 3Met Ala Ile Lys Leu Ser Ile
Asp Leu Ser Asp Ala Thr Phe Ala Glu 1 5 10 15 Leu Ser Ala Val Ile
Gly Tyr Ala His Gln Leu Gly Val Asp Ala Asp 20 25 30 Glu Lys Leu
Thr Phe Glu Gly Thr Val Leu Asn Ile Glu Phe Asp Gly 35 40 45 Asp
Leu Gln Phe Asp Asp Val Phe Asp Ala Phe Asp Glu Ala Glu Ile 50 55
60 Glu Leu Asp Asn Pro Arg Glu Asp Gly Pro Ile Tyr Ala Asp Asp
Leu65 70 75 80 Ile Asp Glu Asp Glu Asp Tyr Arg Ala Gln Thr Lys Ser
Gln Ile Asn 85 90 95 Asp Glu Val Ile Asn Glu Ile Arg Asp Gly Ile
Ser Ser Phe Val Asp 100 105 110 Gly Ile Val Asn Gly Leu Gly Gln Gly
Arg Arg Gly Gly Arg Tyr Gly 115 120 125 Asp Phe Gly Gly Pro Arg Gly
Pro Arg Gly Pro Arg Asn Asp Gly Pro 130 135 140 Phe Gly Pro Phe Gly
Pro Phe Gly Pro Gly Tyr Arg Gly Pro Arg Phe145 150 155
1604483DNACorynebacterium glutamicum 4atggcaatca agctgtccat
tgacctatca gatgcaacat tcgcagaact ttcggcagtc 60atcggttacg cacatcagtt
gggtgttgat gcggatgaga agctcacctt tgaaggtaca 120gtccttaaca
ttgaattcga cggcgacctt cagtttgatg atgtttttga tgcctttgat
180gaggcggaaa ttgagctcga caaccctcgc gaagacggcc ccatctacgc
agatgatctg 240atcgatgagg atgaggacta ccgcgcacag accaagagcc
agatcaacga cgaggttatc 300aacgagatcc gcgatggtat ttcaagcttc
gttgatggca tcgtaaatgg ccttggccag 360ggtcgccgcg gtggacgtta
cggtgatttc ggtgggccac gcggccctcg cggtccacgc 420aatgacggtc
cattcggccc atttggacca ttcggtccgg gataccgcgg tccgcgtttc 480tag
48351563DNACorynebacterium glutamicum 5atgagcgatg atcgtaaggc
aattaaacgc gcactaatta gcgtgtatga caagactggc 60ctggaggatc tagcccaggc
acttcaccgc gagaacgtgg aaattgtttc caccggatcc 120actgcggcga
agattgctga gcttggtatt cctgttaccc cggttgagga gctcaccggt
180ttccctgagt gccttgaggg ccgtgtgaag acactgcacc ctaaggttca
cgctggcatc 240ttggcggaca cccgcaagga agaccacctg cgtcagctca
aggaacttga ggtcgcccca 300ttccagcttg tcgtggtgaa cctgtaccca
tttgctgaga ccgttgcgtc cggcgccgat 360ttcgatgctt gcgttgagca
gatcgacatc ggaggcccat ccatggttcg tgctgcggca 420aagaaccacc
catctgtcgc tgtggttgtt tcaccgaacc gctacgagga tgtccaggaa
480gctttgaaga ccggtggatt ctcccgcgcg gagcgcacca agttggctgc
tgaggctttc 540cgccacaccg caacctacga tgtcaccgtt gcaacctgga
tgagcgagca gctggctgcc 600gaagattctg agactgagtt cccaggttgg
atcggcacca ccaacacctt gtcccgcagc 660ttgcgttacg gtgagaaccc
tcaccagtct gcagctttgt acgtgggcaa cacccgcgga 720cttgcacagg
ctaagcagtt ccacggcaag gaaatgagct acaacaacta caccgattct
780gatgctgcat ggcgtgcagc gtgggatcac gagcgtcctt gtgtagctat
catcaagcat 840gcaaaccctt gtggcattgc tgtttctgat gagtccatcg
cagcggcaca ccgcgaggca 900cacgcatgtg actctgtgtc cgcattcggt
ggcgtcatcg cgtccaaccg tgaagtcagc 960gttgagatgg ctaaccaggt
tgcagagatc ttcactgagg tcatcatcgc tccttcctat 1020gaagagggcg
ctgtggagat cctgagccag aagaagaaca tccgtattct tcaggctgaa
1080gcacctgtgc gtaagggctt tgagtcccgt gagatctccg gcggtctgct
tgttcaggaa 1140cgcgacttga tccacgctga gggcgacaac tccgcaaact
ggactcttgc tgccggctct 1200gctgtttctc ctgaggttct gaaggacctg
gagttcgcgt ggactgcagt tcgttccgtg 1260aagtccaacg caattctgtt
ggctaagaac ggcgctaccg ttggcgttgg catgggacag 1320gtcaaccgcg
ttgactctgc tcgcttggct gtcgaccgtg caggtgcaga gcgcgctacc
1380ggttccgttg ctgcttccga tgcgttcttc ccattcgctg acggctttga
ggttctcgct 1440gaggctggca tcactgctgt tgtgcagcct ggtggatcca
ttcgcgacaa cgaggtcatt 1500gaggcagcca acaaggctgg cgtgaccatg
tacctgactg gtgcgcgaca cttcgctcac 1560taa 15636603DNACorynebacterium
glutamicum 6atggctgtat acgaactccc agaactcgac tacgcatacg acgctctcga
gccacacatc 60gccgctgaaa tcatggagct tcaccactcc aagcaccacg caacctacgt
tgcaggcgca 120aatgcagcac tcgaggcact agagaaggca cgcgaagagg
gcaccaaccc tgaccagatc 180cgcgcactgt ccaagaacct tgcattcaac
ctcggtggac acaccaacca ctccgttttc 240tggaagaacc tctcccctaa
cggtggtggc gagcctaccg gcgaactggc tgaggctatc 300aaccgcgact
tcggttcttt cgctaagttc caggatcact tcaactccgc agcactcggc
360ctgcagggct ccggctgggc agttctcggc tacgaccaca tctccggtcg
cctggttatc 420gagcagctca ccgaccagca gggcaacatc tccgtcgaca
tcaccccagt tctgatgctc 480gatatgtggg agcacgcttt ctacctgcag
tacaagaacg ttaaggcaga ttacgtcaag 540gctgtttgga acgtcttcaa
ctgggacgac gcagcagcac gcttcgcagc agcttccaag 600taa
6037371PRTClostridium kluyveri 7Met Gln Leu Phe Lys Leu Lys Ser Val
Thr His His Phe Asp Thr Phe 1 5 10 15 Ala Glu Phe Ala Lys Glu Phe
Cys Leu Gly Glu Arg Asp Leu Val Ile 20 25 30 Thr Asn Glu Phe Ile
Tyr Glu Pro Tyr Met Lys Ala Cys Gln Leu Pro 35 40 45 Cys His Phe
Val Met Gln Glu Lys Tyr Gly Gln Gly Glu Pro Ser Asp 50 55 60 Glu
Met Met Asn Asn Ile Leu Ala Asp Ile Arg Asn Ile Gln Phe Asp65 70 75
80 Arg Val Ile Gly Ile Gly Gly Gly Thr Val Ile Asp Ile Ser Lys Leu
85 90 95 Phe Val Leu Lys Gly Leu Asn Asp Val Leu Asp Ala Phe Asp
Arg Lys 100 105 110 Ile Pro Leu Ile Lys Glu Lys Glu Leu Ile Ile Val
Pro Thr Thr Cys 115 120 125 Gly Thr Gly Ser Glu Val Thr Asn Ile Ser
Ile Ala Glu Ile Lys Ser 130 135 140 Arg His Thr Lys Met Gly Leu Ala
Asp Asp Ala Ile Val Ala Asp His145 150 155 160 Ala Ile Ile Ile Pro
Glu Leu Leu Lys Ser Leu Pro Phe His Phe Tyr 165 170 175 Ala Cys Ser
Ala Ile Asp Ala Leu Ile His Ala Ile Glu Ser Tyr Val 180 185 190 Ser
Pro Lys Ala Ser Pro Tyr Ser Arg Leu Phe Ser Glu Ala Ala Trp 195 200
205 Asp Ile Ile Leu Glu Val Phe Lys Lys Ile Ala Glu His Gly Pro Glu
210 215 220 Tyr Arg Phe Glu Lys Leu Gly Glu Met Ile Met Ala Ser Asn
Tyr Ala225 230 235 240 Gly Ile Ala Phe Gly Asn Ala Gly Val Gly Ala
Val His Ala Leu Ser 245 250 255 Tyr Pro Leu Gly Gly Asn Tyr His Val
Pro His Gly Glu Ala Asn Tyr 260 265 270 Gln Phe Phe Thr Glu Val Phe
Lys Val Tyr Gln Lys Lys Asn Pro Phe 275 280 285 Gly Tyr Ile Val Glu
Leu Asn Trp Lys Leu Ser Lys Ile Leu Asn Cys 290 295 300 Gln Pro Glu
Tyr Val Tyr Pro Lys Leu Asp Glu Leu Leu Gly Cys Leu305 310 315 320
Leu Thr Lys Lys Pro Leu His Glu Tyr Gly Met Lys Asp Glu Glu Val 325
330 335 Arg Gly Phe Ala Glu Ser Val Leu Lys Thr Gln Gln Arg Leu Leu
Ala 340 345 350 Asn Asn Tyr Val Glu Leu Thr Val Asp Glu Ile Glu Gly
Ile Tyr Arg 355 360 365 Arg Leu Tyr 370 8431PRTPorphyromonas
gingivalis 8Met Lys Asp Val Leu Ala Glu Tyr Ala Ser Arg Ile Val Ser
Ala Glu 1 5 10 15 Glu Ala Val Lys His Ile Lys Asn Gly Glu Arg Val
Ala Leu Ser His 20 25 30 Ala Ala Gly Val Pro Gln Ser Cys Val Asp
Ala Leu Val Gln Gln Ala 35 40 45 Asp Leu Phe Gln Asn Val Glu Ile
Tyr His Met Leu Cys Leu Gly Glu 50 55 60 Gly Lys Tyr Met Ala Pro
Glu Met Ala Pro His Phe Arg His Ile Thr65 70 75 80 Asn Phe Val Gly
Gly Asn Ser Arg Lys Ala Val Glu Glu Asn Arg Ala 85 90 95 Asp Phe
Ile Pro Val Phe Phe Tyr Glu Val Pro Ser Met Ile Arg Lys 100 105 110
Asp Ile Leu His Ile Asp Val Ala Ile Val Gln Leu Ser Met Pro Asp 115
120 125 Glu Asn Gly Tyr Cys Ser Phe Gly Val Ser Cys Asp Tyr Ser Lys
Pro 130 135 140 Ala Ala Glu Ser Ala His Leu Val Ile Gly Glu Ile Asn
Arg Gln Met145 150 155 160 Pro Tyr Val His Gly Asp Asn Leu Ile His
Ile Ser Lys Leu Asp Tyr 165 170 175 Ile Val Met Ala Asp Tyr Pro Ile
Tyr Ser Leu Ala Lys Pro Lys Ile 180 185 190 Gly Glu Val Glu Glu Ala
Ile Gly Arg Asn Cys Ala Glu Leu Ile Glu 195 200 205 Asp Gly Ala Thr
Leu Gln Leu Gly Ile Gly Ala Ile Pro Asp Ala Ala 210 215 220 Leu Leu
Phe Leu Lys Asp Lys Lys Asp Leu Gly Ile His Thr Glu Met225 230 235
240 Phe Ser Asp Gly Val Val Glu Leu Val Arg Ser Gly Val Ile Thr Gly
245 250 255 Lys Lys Lys Thr Leu His Pro Gly Lys Met Val Ala Thr Phe
Leu Met 260 265 270 Gly Ser Glu Asp Val Tyr His Phe Ile Asp Lys Asn
Pro Asp Val Glu 275 280 285 Leu Tyr Pro Val Asp Tyr Val Asn Asp Pro
Arg Val Ile Ala Gln Asn 290 295 300 Asp Asn Met Val Ser Ile Asn Ser
Cys Ile Glu Ile Asp Leu Met Gly305 310 315 320 Gln Val Val Ser Glu
Cys Ile Gly Ser Lys Gln Phe Ser Gly Thr Gly 325 330 335 Gly Gln Val
Asp Tyr Val Arg Gly Ala Ala Trp Ser Lys Asn Gly Lys 340 345 350 Ser
Ile Met Ala Ile Pro Ser Thr Ala Lys Asn Gly Thr Ala Ser Arg 355 360
365 Ile Val Pro Ile Ile Ala Glu Gly Ala Ala Val Thr Thr Leu Arg Asn
370 375 380 Glu Val Asp Tyr Val Val Thr Glu Tyr Gly Ile Ala Gln Leu
Lys Gly385 390 395 400 Lys Ser Leu Arg Gln Arg Ala Glu Ala Leu Ile
Ala Ile Ala His Pro 405 410 415 Asp Phe Arg Glu Glu Leu Thr Lys His
Leu Arg Lys Arg Phe Gly 420 425 430 9858PRTClostridium
acetobutyricum 9Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu
Asn Glu Leu 1 5 10 15 Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr
Gln Glu Gln Val Asp 20 25 30 Lys Ile Phe Lys Gln Cys Ala Ile Ala
Ala Ala Lys Glu Arg Ile Asn 35 40 45 Leu Ala Lys Leu Ala Val Glu
Glu Thr Gly Ile Gly Leu Val Glu Asp 50 55 60 Lys Ile Ile Lys Asn
His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr65 70 75 80 Lys Asn Glu
Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly 85 90 95 Ile
Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro 100 105
110 Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125 Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala
Lys Lys 130 135 140 Ser Thr Ile Ala Ala Ala Lys Leu Ile Leu Asp Ala
Ala Val Lys Ala145 150 155 160 Gly Ala Pro Lys Asn Ile Ile Gly Trp
Ile Asp Glu Pro Ser Ile Glu 165 170 175 Leu Ser Gln Asp Leu Met Ser
Glu Ala Asp Ile Ile Leu Ala Thr Gly 180 185 190 Gly Pro Ser Met Val
Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200 205 Gly Val Gly
Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp 210 215 220 Ile
Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn225 230
235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser
Ile 245 250 255 Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser
Tyr Ile Leu 260 265 270 Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr
Met Phe Lys Asn Gly 275 280 285 Ala Ile Asn Ala Asp Ile Val Gly Lys
Ser Ala Tyr Ile Ile Ala Lys 290 295 300 Met Ala Gly Ile Glu Val Pro
Gln Thr Thr Lys Ile Leu Ile Gly Glu305 310 315 320 Val Gln Ser Val
Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser 325 330 335 Pro Val
Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys 340 345 350
Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser 355
360 365 Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe
Gly 370 375 380 Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro
Ser Ser Gln385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala
Ile Ala Pro Ser Phe Thr 405 410 415 Leu Gly
Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu 420 425 430
Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn 435
440 445 Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly
Cys 450 455 460 Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys
Lys Arg Ala465 470 475 480 Phe Ile Val Thr Asp Lys Asp Leu Phe Lys
Leu Gly Tyr Val Asn Lys 485 490 495 Ile Thr Lys Val Leu Asp Glu Ile
Asp Ile Lys Tyr Ser Ile Phe Thr 500 505 510 Asp Ile Lys Ser Asp Pro
Thr Ile Asp Ser Val Lys Lys Gly Ala Lys 515 520 525 Glu Met Leu Asn
Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly 530 535 540 Ser Pro
Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro545 550 555
560 Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys
565 570 575 Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser
Val Ala 580 585 590 Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr
Pro Phe Ala Val 595 600 605 Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr
Pro Leu Thr Ser Tyr Glu 610 615 620 Leu Thr Pro Asn Met Ala Ile Ile
Asp Thr Glu Leu Met Leu Asn Met625 630 635 640 Pro Arg Lys Leu Thr
Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala 645 650 655 Ile Glu Ala
Tyr Val Ser Val Met Ala Thr Asp Tyr Thr Asp Glu Leu 660 665 670 Ala
Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr 675 680
685 Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala
690 695 700 Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly
Val Cys705 710 715 720 His Ser Met Ala His Lys Leu Gly Ala Met His
His Val Pro His Gly 725 730 735 Ile Ala Cys Ala Val Leu Ile Glu Glu
Val Ile Lys Tyr Asn Ala Thr 740 745 750 Asp Cys Pro Thr Lys Gln Thr
Ala Phe Pro Gln Tyr Lys Ser Pro Asn 755 760 765 Ala Lys Arg Lys Tyr
Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly 770 775 780 Thr Ser Asp
Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys785 790 795 800
Leu Lys Ile Asp Leu Ser Ile Pro Gln Asn Ile Ser Ala Ala Gly Ile 805
810 815 Asn Lys Lys Asp Phe Tyr Asn Thr Leu Asp Lys Met Ser Glu Leu
Ala 820 825 830 Phe Asp Asp Gln Cys Thr Thr Ala Asn Pro Arg Tyr Pro
Leu Ile Ser 835 840 845 Glu Leu Lys Asp Ile Tyr Ile Lys Ser Phe 850
855 10451PRTPorphyromonas gingivalis 10Met Glu Ile Lys Glu Met Val
Ser Leu Ala Arg Lys Ala Gln Lys Glu 1 5 10 15 Tyr Gln Ala Thr His
Asn Gln Glu Ala Val Asp Asn Ile Cys Arg Ala 20 25 30 Ala Ala Lys
Val Ile Tyr Glu Asn Ala Ala Ile Leu Ala Arg Glu Ala 35 40 45 Val
Asp Glu Thr Gly Met Gly Val Tyr Glu His Lys Val Ala Lys Asn 50 55
60 Gln Gly Lys Ser Lys Gly Val Trp Tyr Asn Leu His Asn Lys Lys
Ser65 70 75 80 Ile Gly Ile Leu Asn Ile Asp Glu Arg Thr Gly Met Ile
Glu Ile Ala 85 90 95 Lys Pro Ile Gly Val Val Gly Ala Val Thr Pro
Thr Thr Asn Pro Ile 100 105 110 Val Thr Pro Met Ser Asn Ile Ile Phe
Ala Leu Lys Thr Cys Asn Ala 115 120 125 Ile Ile Ile Ala Pro His Pro
Arg Ser Lys Lys Cys Ser Ala His Ala 130 135 140 Val Arg Leu Ile Lys
Glu Ala Ile Ala Pro Phe Asn Val Pro Glu Gly145 150 155 160 Met Val
Gln Ile Ile Glu Glu Pro Ser Ile Glu Lys Thr Gln Glu Leu 165 170 175
Met Gly Ala Val Asp Val Val Val Ala Thr Gly Gly Met Gly Met Val 180
185 190 Lys Ser Ala Tyr Ser Ser Gly Lys Pro Ser Phe Gly Val Gly Ala
Gly 195 200 205 Asn Val Gln Val Ile Val Asp Ser Asn Ile Asp Phe Glu
Ala Ala Ala 210 215 220 Glu Lys Ile Ile Thr Gly Arg Ala Phe Asp Asn
Gly Ile Ile Cys Ser225 230 235 240 Gly Glu Gln Ser Ile Ile Tyr Asn
Glu Ala Asp Lys Glu Ala Val Phe 245 250 255 Thr Ala Phe Arg Asn His
Gly Ala Tyr Phe Cys Asp Glu Ala Glu Gly 260 265 270 Asp Arg Ala Arg
Ala Ala Ile Phe Glu Asn Gly Ala Ile Ala Lys Asp 275 280 285 Val Val
Gly Gln Ser Val Ala Phe Ile Ala Lys Lys Ala Asn Ile Asn 290 295 300
Ile Pro Glu Gly Thr Arg Ile Leu Val Val Glu Ala Arg Gly Val Gly305
310 315 320 Ala Glu Asp Val Ile Cys Lys Glu Lys Met Cys Pro Val Met
Cys Ala 325 330 335 Leu Ser Tyr Lys His Phe Glu Glu Gly Val Glu Ile
Ala Arg Thr Asn 340 345 350 Leu Ala Asn Glu Gly Asn Gly His Thr Cys
Ala Ile His Ser Asn Asn 355 360 365 Gln Ala His Ile Ile Leu Ala Gly
Ser Glu Leu Thr Val Ser Arg Ile 370 375 380 Val Val Asn Ala Pro Ser
Ala Thr Thr Ala Gly Gly His Ile Gln Asn385 390 395 400 Gly Leu Ala
Val Thr Asn Thr Leu Gly Cys Gly Ser Trp Gly Asn Asn 405 410 415 Ser
Ile Ser Glu Asn Phe Thr Tyr Lys His Leu Leu Asn Ile Ser Arg 420 425
430 Ile Ala Pro Leu Asn Ser Ser Ile His Ile Pro Asp Asp Lys Glu Ile
435 440 445 Trp Glu Leu 450 11538PRTClostridium kluyveri 11Met Ser
Lys Gly Ile Lys Asn Ser Gln Leu Lys Lys Lys Asn Val Lys 1 5 10 15
Ala Ser Asn Val Ala Glu Lys Ile Glu Glu Lys Val Glu Lys Thr Asp 20
25 30 Lys Val Val Glu Lys Ala Ala Glu Val Thr Glu Lys Arg Ile Arg
Asn 35 40 45 Leu Lys Leu Gln Glu Lys Val Val Thr Ala Asp Val Ala
Ala Asp Met 50 55 60 Ile Glu Asn Gly Met Ile Val Ala Ile Ser Gly
Phe Thr Pro Ser Gly65 70 75 80 Tyr Pro Lys Glu Val Pro Lys Ala Leu
Thr Lys Lys Val Asn Ala Leu 85 90 95 Glu Glu Glu Phe Lys Val Thr
Leu Tyr Thr Gly Ser Ser Thr Gly Ala 100 105 110 Asp Ile Asp Gly Glu
Trp Ala Lys Ala Gly Ile Ile Glu Arg Arg Ile 115 120 125 Pro Tyr Gln
Thr Asn Ser Asp Met Arg Lys Lys Ile Asn Asp Gly Ser 130 135 140 Ile
Lys Tyr Ala Asp Met His Leu Ser His Met Ala Gln Tyr Ile Asn145 150
155 160 Tyr Ser Val Ile Pro Lys Val Asp Ile Ala Ile Ile Glu Ala Val
Ala 165 170 175 Ile Thr Glu Glu Gly Asp Ile Ile Pro Ser Thr Gly Ile
Gly Asn Thr 180 185 190 Ala Thr Phe Val Glu Asn Ala Asp Lys Val Ile
Val Glu Ile Asn Glu 195 200 205 Ala Gln Pro Leu Glu Leu Glu Gly Met
Ala Asp Ile Tyr Thr Leu Lys 210 215 220 Asn Pro Pro Arg Arg Glu Pro
Ile Pro Ile Val Asn Ala Gly Asn Arg225 230 235 240 Ile Gly Thr Thr
Tyr Val Thr Cys Gly Ser Glu Lys Ile Cys Ala Ile 245 250 255 Val Met
Thr Asn Thr Gln Asp Lys Thr Arg Pro Leu Thr Glu Val Ser 260 265 270
Pro Val Ser Gln Ala Ile Ser Asp Asn Leu Ile Gly Phe Leu Asn Lys 275
280 285 Glu Val Glu Glu Gly Lys Leu Pro Lys Asn Leu Leu Pro Ile Gln
Ser 290 295 300 Gly Val Gly Ser Val Ala Asn Ala Val Leu Ala Gly Leu
Cys Glu Ser305 310 315 320 Asn Phe Lys Asn Leu Ser Cys Tyr Thr Glu
Val Ile Gln Asp Ser Met 325 330 335 Leu Lys Leu Ile Lys Cys Gly Lys
Ala Asp Val Val Ser Gly Thr Ser 340 345 350 Ile Ser Pro Ser Pro Glu
Met Leu Pro Glu Phe Ile Lys Asp Ile Asn 355 360 365 Phe Phe Arg Glu
Lys Ile Val Leu Arg Pro Gln Glu Ile Ser Asn Asn 370 375 380 Pro Glu
Ile Ala Arg Arg Ile Gly Val Ile Ser Ile Asn Thr Ala Leu385 390 395
400 Glu Val Asp Ile Tyr Gly Asn Val Asn Ser Thr His Val Met Gly Ser
405 410 415 Lys Met Met Asn Gly Ile Gly Gly Ser Gly Asp Phe Ala Arg
Asn Ala 420 425 430 Tyr Leu Thr Ile Phe Thr Thr Glu Ser Ile Ala Lys
Lys Gly Asp Ile 435 440 445 Ser Ser Ile Val Pro Met Val Ser His Val
Asp His Thr Glu His Asp 450 455 460 Val Met Val Ile Val Thr Glu Gln
Gly Val Ala Asp Leu Arg Gly Leu465 470 475 480 Ser Pro Arg Glu Lys
Ala Val Ala Ile Ile Glu Asn Cys Val His Pro 485 490 495 Asp Tyr Lys
Asp Met Leu Met Glu Tyr Phe Glu Glu Ala Cys Lys Ser 500 505 510 Ser
Gly Gly Asn Thr Pro His Asn Leu Glu Lys Ala Leu Ser Trp His 515 520
525 Thr Lys Phe Ile Lys Thr Gly Ser Met Lys 530 535
121116DNAClostridium kluyveri 12atgcagcttt tcaagctcaa gagcgtcaca
catcactttg atacttttgc agagtttgcc 60aaggagttct gtctcggtga acgcgacttg
gtaattacca acgagttcat ctacgaaccg 120tatatgaagg catgccagct
gccttgtcat tttgtgatgc aggagaaata cggccaaggc 180gagccttctg
acgagatgat gaacaacatc ctagcagata tccgtaatat ccagttcgac
240cgcgtgatcg ggatcggagg tggtacggtt attgacatct caaaactctt
tgttctgaag 300ggattaaatg atgttctcga cgcgttcgat cgcaagattc
cccttatcaa agagaaagaa 360ctgatcattg tgcccaccac ctgcggaacc
ggctcggagg tgacgaacat ttccatcgcc 420gagatcaagt cccggcacac
caagatgggt ttggctgacg atgcaattgt tgctgaccac 480gccataatca
tccctgaact tctgaagagc ttgcccttcc acttctatgc atgctccgca
540atcgacgctc ttattcatgc catcgagtca tacgtttctc caaaagcgtc
tccatactcc 600cgtctgttca gtgaggcggc gtgggacatt atcctggaag
ttttcaagaa aatcgccgaa 660cacggcccag agtaccgctt cgagaagctg
ggggaaatga tcatggccag caactatgcc 720ggtatcgctt tcggcaacgc
aggcgttggc gccgtccacg ctctatccta cccgttgggc 780ggcaactatc
acgtgccgca tggagaagca aactatcagt tcttcaccga ggtctttaaa
840gtataccaaa agaagaatcc gttcggctat attgtcgaac tcaactggaa
gctctccaag 900attctgaact gccagccaga gtacgtgtac ccgaagctgg
atgaactgct cggttgcctt 960cttaccaaga aacctttgca cgaatacggc
atgaaggacg aagaggttcg tggcttcgcg 1020gaatcggtcc tgaagaccca
gcaacgcttg ctcgccaaca actacgtcga acttactgtc 1080gatgagatcg
aaggtatcta ccgacgtctc tactag 1116131296DNAPorphyromonas gingivalis
13atgaaggatg tactggcgga atacgcctcc cgcattgttt cggcggagga ggccgttaag
60cacatcaaaa acggtgaacg ggtagctttg tcacacgctg ccggcgtgcc tcagagttgc
120gttgacgcac tggtgcagca ggccgacctt ttccagaatg tggaaatcta
tcacatgctg 180tgcctcggtg agggtaagta tatggcgcct gagatggccc
ctcacttccg ccacatcacc 240aactttgtcg gtggtaactc ccgtaaggcg
gtcgaagaaa accgggccga tttcattccg 300gtattctttt acgaggtgcc
aagcatgatt cgcaaagaca tcctccacat tgatgtcgcc 360atcgttcagc
tttcaatgcc tgacgaaaat ggttactgtt cctttggagt atcttgcgat
420tactccaagc cggcagcaga gagcgctcac ctggttatcg gagaaatcaa
ccgtcaaatg 480ccatacgtac acggcgacaa cttgattcat atctccaagt
tggattacat cgtgatggca 540gactacccca tctactctct tgcaaagccc
aagatcgggg aagtcgagga agctatcggg 600aggaattgtg ccgagcttat
tgaagatggt gccactctcc agctgggaat cggcgcgatt 660cctgatgcgg
ccctgttatt tctcaaggac aaaaaggatc tgggcatcca taccgaaatg
720ttctccgatg gtgttgtcga attggttcgc tccggcgtta tcacaggcaa
gaaaaagact 780cttcaccccg gaaagatggt cgcaaccttc ctgatgggaa
gcgaggacgt gtatcatttc 840atcgataaaa accccgatgt agaactgtat
ccagtagatt acgtgaatga cccgcgtgtg 900atcgcccaaa acgacaatat
ggtctcgatt aacagctgca tcgaaatcga ccttatggga 960caggtcgtgt
ccgagtgcat cggctcaaag caattcagcg gcaccggcgg ccaagttgac
1020tacgtgcgtg gcgcagcatg gtctaaaaac ggcaaatcga tcatggcaat
cccgtccact 1080gcaaaaaacg gtacggcatc tcgaattgta cctatcatcg
cggagggcgc tgctgtcacc 1140accctgcgca acgaggtcga ttacgttgta
accgagtacg gtatcgctca gctcaagggc 1200aagagcctgc gccagcgcgc
agaggctttg atcgcgatag cccaccccga cttccgtgag 1260gaactaacga
aacatctccg caagcgattc ggataa 1296142577DNAClostridium
acetobutyricum 14atgaaagtaa ccaatcagaa agagttgaag cagaagttga
acgagctgcg agaggctcag 60aagaagttcg caacctacac ccaggaacag gtggacaaga
tctttaagca gtgtgccatt 120gcagccgcga aagaacgtat taatctcgcg
aaacttgcgg tcgaggaaac cggtattggg 180ctggtagaag acaagatcat
caagaaccac ttcgccgctg aatacatcta caacaagtac 240aaaaacgaaa
agacatgtgg tatcatcgac cacgacgaca gcttgggcat caccaaggta
300gcggagccaa tcggtatcgt cgcagctatc gtgcccacta ctaaccctac
ctccactgct 360attttcaagt cactcatctc cctgaaaacc cgcaatgcta
tcttcttctc acctcaccca 420cgcgctaaga aatcaactat cgctgcagct
aaacttatcc tggatgcagc cgtgaaagcc 480ggggctccga aaaacatcat
cggttggatc gacgaacctt ccattgaact ctctcaagac 540ctcatgtccg
aggcagacat tatcctggca accggaggcc catccatggt taaagcagct
600tacagctcag gcaagccggc tatcggcgtt ggagctggta acactccagc
aatcatcgac 660gagtcggccg atatcgacat ggcagtgtcc tctattatcc
tgtccaaaac ttatgacaac 720ggcgttattt gcgcgtccga gcagtctatt
ctcgtcatga actctattta cgagaaggta 780aaggaggagt ttgtgaagcg
ggggtcgtac attctgaacc agaacgagat cgctaagatc 840aaagagacta
tgtttaaaaa cggagccatc aacgcagata tcgtagggaa gtccgcgtac
900atcattgcta agatggctgg aatcgaagtc cctcaaacca cgaaaattct
gatcggcgag 960gtgcaatcgg tcgaaaagtc cgagctgttc tcgcatgaaa
agttgtcccc ggtcctcgcg 1020atgtataaag ttaaggattt tgatgaagca
ctcaagaaag ctcagcgcct gatcgaattg 1080ggtggctcgg gtcacacctc
ttccctctac attgactccc agaacaataa agataaggtg 1140aaagagttcg
gcctggctat gaagacgtct cgtaccttca tcaatatgcc ctcttcacag
1200ggcgccagcg gtgaccttta caatttcgct atcgctccta gctttaccct
cggctgcggc 1260acctggggcg gtaattctgt gtcccaaaac gtcgaaccaa
agcatctgct caacattaaa 1320agcgtcgccg aacgtcgcga gaacatgttg
tggttcaagg tcccgcaaaa aatctacttc 1380aagtatggtt gcttgcgctt
tgcacttaaa gagcttaagg acatgaataa aaagcgggcg 1440ttcatcgtca
ctgataagga tctgttcaaa ctgggctatg ttaacaagat taccaaggtc
1500ctggatgaga tcgatatcaa gtattccatc ttcaccgata ttaagtccga
tccgaccatt 1560gattccgtga agaagggcgc gaaggagatg ctcaactttg
aacccgacac gattatttct 1620attggcggag gcagcccaat ggacgcagct
aaggttatgc acctgctgta tgagtaccca 1680gaagcagaga tcgagaacct
tgcaatcaat ttcatggata ttcgcaaacg catttgcaac 1740tttcctaagc
ttggtacaaa agctatctct gttgcgatcc ctaccaccgc aggaaccggc
1800agcgaagcga caccattcgc cgttattacc aacgatgaaa caggtatgaa
gtacccactt 1860acctcttatg aacttacccc gaacatggct atcattgata
cggaattgat gctgaacatg 1920ccgcggaagt tgaccgcagc tacgggaatc
gatgcattgg ttcatgcaat cgaggcatac 1980gtttccgtca tggcaaccga
ttacaccgac gagctcgcgt tgcgtgcgat taaaatgatc 2040ttcaagtacc
ttccacgcgc atacaagaat ggcacaaacg atattgaagc ccgagaaaag
2100atggcacacg cttcgaacat cgctggtatg gccttcgcga atgcgtttct
cggagtgtgt 2160cactccatgg cgcacaaact gggagccatg catcacgtgc
cccacggtat cgcatgcgcc 2220gttcttattg aagaggtgat caagtataat
gccaccgatt gccccactaa gcagacggcc 2280ttccctcagt acaaatcgcc
caatgccaag cgtaaatacg cggaaattgc cgagtacttg 2340aaccttaagg
ggaccagcga cacggaaaag gtgaccgcac tgattgaagc catctccaag
2400cttaagatcg acctgagcat cccacaaaac atctcagcag ccggcattaa
caagaaggac 2460ttctacaaca ctctcgacaa gatgtcagag ctcgccttcg
atgatcagtg cactaccgca 2520aacccacgtt atccgctcat ctctgaactg
aaggatatct acatcaagtc gttttaa 2577151356DNAPorphyromonas gingivalis
15atggagatta aagagatggt cagtcttgcg cgcaaagctc agaaggagta tcaggccacc
60cataaccaag aagctgtgga caacatctgc cgagcagcag cgaaggttat ttacgaaaat
120gcagcaattc tggcacgcga ggcagtggac gaaaccggca tgggtgttta
cgagcacaag 180gtggccaaga atcaaggcaa gtccaaaggt gtttggtaca
acctgcataa caagaagtcg 240attggcatcc tcaatatcga cgagcgtacc
ggcatgatcg agatcgcaaa acctatcggg 300gttgtaggcg ccgttacgcc
aaccaccaac cctatcgtta ctccgatgag caacatcatc 360tttgctctta
agacctgcaa cgccatcatt atcgccccac acccgcgctc caaaaagtgc
420tctgcccacg cagttcggct gatcaaagag gctatcgctc cgttcaacgt
gcccgaaggt 480atggttcaga tcatcgagga gcctagcatc gagaagacgc
aggaattgat gggcgccgta 540gacgtggtcg ttgctaccgg gggcatgggc
atggtcaagt ctgcctactc ctcagggaag 600ccttctttcg gtgtcggagc
cggcaatgtt caggtgatag tggacagcaa catcgacttc 660gaagcggcag
cagaaaagat catcaccgga cgtgccttcg acaacggtat catctgctca
720ggcgaacagt ccatcatcta caacgaggct gacaaggaag cagttttcac
agcattccgc 780aaccacggtg cgtacttttg cgacgaggcc gagggagatc
gggctcgtgc agcgatcttc 840gaaaatggag ccatcgcgaa agatgttgtg
ggccagtccg ttgcctttat tgcaaagaag 900gcgaacatta atatccccga
gggtactcgt attctcgtgg tcgaagctcg cggagtaggc 960gccgaagatg
tcatctgtaa agaaaagatg tgtccagtca tgtgcgccct ctcctacaag
1020cacttcgaag agggggtaga gatcgcaagg acgaacctcg caaacgaagg
caatggccat 1080acctgtgcta
tccactccaa caaccaagca cacatcatct tggcaggctc ggagctgacc
1140gtgtctcgca tcgtggtcaa cgcgccaagt gctaccacag caggcggtca
catccagaac 1200ggtcttgccg tcaccaatac tctaggctgc ggctcttggg
gtaacaactc gatctccgaa 1260aacttcactt ataaacacct gctcaacatt
tcacgcatcg ccccgttgaa ctccagcatt 1320catatcccag atgataagga
aatctgggaa ctctaa 1356161617DNAClostridium kluyveri 16atgtctaaag
gaatcaagaa tagccaattg aaaaaaaaga acgtcaaggc cagtaacgtt 60gctgagaaga
tcgaagagaa ggtggaaaag accgacaagg tcgttgagaa ggctgctgag
120gtgaccgaaa agcgcattcg aaacttaaag ctccaggaaa aagttgtgac
cgcagatgtc 180gcagctgaca tgatcgagaa tggcatgatc gtcgcaatta
gcggcttcac gccatccggg 240tatccaaagg aggttccaaa agcccttact
aagaaggtta atgcgctgga ggaggagttc 300aaggtgacgc tgtataccgg
ttctagcaca ggcgctgata ttgacggaga atgggcgaag 360gcaggaataa
tcgaacggcg tatcccatac cagaccaact ctgacatgag gaaaaaaata
420aacgatggtt caatcaagta cgcagatatg cacctgagcc acatggctca
atacattaac 480tattctgtga ttcctaaggt tgacattgcc atcatcgagg
cggtggccat taccgaggaa 540ggggatatta ttcctagtac tggaatcggc
aacacagcta cgtttgtcga gaatgcggat 600aaggtaattg tggaaataaa
cgaggctcag ccgcttgagt tggaaggcat ggcagatatc 660tataccctga
agaaccctcc acgtcgcgag cccatcccga tagtcaacgc aggcaaccgc
720atagggacca cttacgtcac ctgtggctct gaaaaaatct gcgcgatcgt
catgaccaac 780acccaagaca aaacccgccc actcaccgaa gtttctcctg
tcagtcaggc aatctccgat 840aacctgattg gcttcctgaa caaagaagta
gaggagggta aactcccaaa aaacctgctc 900cccatacagt caggtgtcgg
ttcggttgct aacgccgttc tagccggact ctgcgaatca 960aacttcaaaa
atttgagctg ctacacagaa gtgatccagg attcgatgtt gaagctcatc
1020aaatgtggaa aggcagatgt ggtgtccggc acctcgatct cgccatcacc
ggaaatgctg 1080cccgagttca taaaggacat aaattttttt cgcgagaaga
tagtactgcg cccccaggaa 1140atatctaata atccggaaat agctcgtcgt
ataggagtga tctccataaa cactgctttg 1200gaagtagaca tctacggtaa
tgtgaactcc acgcatgtca tgggctccaa gatgatgaac 1260ggcatcggcg
gcagcggcga ctttgcccgc aacgcatacc tcaccatatt cactacggag
1320tccatcgcga agaagggcga catttcctct atcgttccta tggtttccca
cgtggaccac 1380accgagcatg acgtaatggt catcgttacc gaacaggggg
ttgcggatct gcgcggtctt 1440tcccctcggg aaaaggccgt ggcgataatt
gagaattgcg tccacccgga ttacaaggat 1500atgctcatgg agtacttcga
ggaggcttgt aagtcctcag gtggcaacac cccacacaac 1560cttgaaaaag
ccctatcctg gcacactaag ttcataaaaa ctggctcgat gaagtaa
16171743DNAArtificial SequenceSynthetic ldhA_5'_HindIII
17catgattacg ccaagcttga gagcccacca cattgcgatt tcc
431842DNAArtificial SequenceSynthetic ldhA_up_3'_XhoI 18tcgaaactcg
agtttcgatc ccacttcctg atttccctaa cc 421939DNAArtificial
SequenceSynthetic ldhA_dn_5'_XhoI 19tcgaaactcg agtaaatctt
tggcgcctag ttggcgacg 392046DNAArtificial SequenceSynthetic
ldhA_3'_EcoRI 20acgacggcca gtgaattcga cgacatctga gggtggataa agtggg
462120DNAArtificial SequenceSynthetic ldhA up 21atcgggcata
attaaaggtg 202222DNAArtificial SequenceSynthetic ldhA down
22gtcacctcat caagttctag aa 22236702DNAArtificial SequenceSynthetic
4gene_cat1_sucD_4hbd_cat2 23tctagaatga ctattaatgt ctccgaacta
cttgccaaag tccccacggg tctactgatt 60ggtgattcct gggtggaagc atccgacggc
ggtactttcg atgtggaaaa cccagcgacg 120ggtgaaacaa tcgcaacgct
cgcgtctgct acttccgagg atgcactggc tgctcttgat 180gctgcatgcg
ctgttcaggc cgagtgggct aggatgccag cgcgcgagcg ttctaatatt
240ttacgccgcg gttttgagct cgtagcagaa cgtgcagaag agttcgccac
cctcatgacc 300ttggaaatgg gcaagccttt ggctgaagct cgcggcgaag
tcacctacgg caacgaattc 360ctgcgctggt tctctgagga agcagttcgt
ctgtatggcc gttacggaac cacaccagaa 420ggcaacttgc ggatgctgac
cgccctcaag ccagttggcc cgtgcctcct gatcacccca 480tggaacttcc
cactagcaat ggctactaga tgattttgca tctgctgcga aatctttgtt
540tccccgctaa agttgaggac aggttgacac ggagttgact cgacgaatta
tccaatgtga 600gtaggtttgg tgcgtgagtt ggaaaaattc gccatactcg
cccttgggtt ctgtcagctc 660aagaattctt gagtgaccga tgctctgatt
gacctaactg cttgacacat tgcatttcct 720acaatcttta gaggagacac
aacatgtcta aaggaatcaa gaatagccaa ttgaaaaaaa 780agaacgtcaa
ggccagtaac gttgctgaga agatcgaaga gaaggtggaa aagaccgaca
840aggtcgttga gaaggctgct gaggtgaccg aaaagcgaat tcgaaactta
aagctccagg 900aaaaagttgt gaccgcagat gtcgcagctg acatgatcga
gaatggcatg atcgtcgcaa 960ttagcggctt cacgccatcc gggtatccaa
aggaggttcc aaaagccctt actaagaagg 1020ttaatgcgct ggaggaggag
ttcaaggtga cgctgtatac cggttctagc acaggcgctg 1080atattgacgg
agaatgggcg aaggcaggaa taatcgaacg gcgtatccca taccagacca
1140actctgacat gaggaaaaaa ataaacgatg gttcaatcaa gtacgcagat
atgcacctga 1200gccacatggc tcaatacatt aactattctg tgattcctaa
ggttgacatt gccatcatcg 1260aggcggtggc cattaccgag gaaggggata
ttattcctag tactggaatc ggcaacacag 1320ctacgtttgt cgagaatgcg
gataaggtaa ttgtggaaat aaacgaggct cagccgcttg 1380agttggaagg
catggcagat atctataccc tgaagaaccc tccacgtcgc gagcccatcc
1440cgatagtcaa cgcaggcaac cgcataggga ccacttacgt cacctgtggc
tctgaaaaaa 1500tctgcgcgat cgtcatgacc aacacccaag acaaaacccg
cccactcacc gaagtttctc 1560ctgtcagtca ggcaatctcc gataacctga
ttggcttcct gaacaaagaa gtagaggagg 1620gtaaactccc aaaaaacctg
ctccccatac agtcaggtgt cggttcggtt gctaacgccg 1680tgcatcccgg
actctgcgaa tcaaacttca aaaatttgag ctgctacaca gaagtgatcc
1740aggattcgat gttgaagctg atcaaatgtg gaaaggcaga tgtggtgtcc
ggcacctcga 1800tctcgccatc accggaaatg ctgcccgagt tcataaagga
cataaatttt tttcgcgaga 1860agatagtact gcgcccccag gaaatatcta
ataatccgga aatagctcgt cgtataggag 1920tgatctccat aaacactgct
ttggaagtag acatctacgg taatgtgaac tccacgcatg 1980tcatgggctc
caagatgatg aacggcatcg gcggcagcgg cgactttgcc cgcaacgcat
2040acctcaccat attcactacg gagtccatcg cgaagaaggg cgacatttcc
tctatcgttc 2100ctatggtttc ccacgtggac cacaccgagc atgacgtaat
ggtcatcgtt accgaacagg 2160gggttgcgga tctccgcggt ctttcccctc
gggaaaaggc cgtggcgata attgagaatt 2220gcgtccaccc ggattacaag
gatatgctca tggagtactt cgaggaggct tgtaagtcct 2280caggtggcaa
caccccacac aaccttgaaa aagccctatc ctggcacact aagttcataa
2340aaactggctc gatgaagtaa ttagaggaga cacaacatgg agattaaaga
gatggtcagt 2400cttgcgcgca aagctcagaa ggagtatcag gccacccata
accaagaagc tgtggacaac 2460atctgccgag ctgcagcgaa ggttatttac
gaaaatgcag caattctggc ccgcgaggca 2520gtggacgaaa ccggcatggg
tgtttacgag cacaaggtgg ccaagaatca aggcaagtcc 2580aaaggtgttt
ggtacaacct gcataacaag aagtcgattg gcatcctcaa tatcgatgag
2640cgtaccggca tgatcgagat cgcaaaacct atcggggttg taggcgccgt
tacgccaacc 2700accaacccta tcgttactcc gatgagcaac atcatctttg
ctcttaagac ctgcaacgcc 2760atcattatcg ccccacaccc gcgctccaaa
aagtgctctg cccacgcagt tcggctgatc 2820aaagaggcta tcgctccgtt
caacgtgccc gaaggtatgg ttcagatcat cgaggagcct 2880agcatcgaga
agacgcagga attgatgggc gccgtagacg tggtcgttgc taccgggggc
2940atgggcatgg tcaagtctgc ctactcctca gggaagcctt ctttcggtgt
cggagccggc 3000aatgttcagg tgatagtgga cagcaacatc gatttcgaag
cggctgcaga aaagatcatc 3060accggacgtg ccttcgacaa cggtatcatc
tgctcaggcg aacagtccat catctacaac 3120gaggctgaca aggaagcagt
tttcacagca ttccgcaacc acggtgcgta cttttgcgac 3180gaggccgagg
gagatcgggc tcgtgcagcg atcttcgaaa atggagccat cgcgaaagat
3240gttgtgggcc agtccgttgc ctttattgcc aagaaggcga acattaatat
ccccgagggt 3300actcgtattc tcgtggtcga agctcgcgga gtaggcgccg
aagatgtcat ctgtaaagaa 3360aagatgtgtc cagtcatgtg cgccctctcc
tacaagcact tcgaagaggg ggtagagatc 3420gcaaggacga acctcgcaaa
cgaaggcaat ggccatacct gtgctatcca ctccaacaac 3480caagcacaca
tcatcttggc aggctcggag ctgaccgtgt ctcgcatcgt ggtcaacgcg
3540ccaagtgcta ccacagcagg cggtcacatc cagaacggtc ttgccgtcac
caatactcta 3600ggctgcggct cttggggtaa caactcgatc tccgaaaact
tcacttataa acacctgctc 3660aacatttcac gcatcgcccc gttgaactcc
agcattcata tcccagatga taaggaaatc 3720tgggaactct aattagagga
gacacaacat gcagcttttc aagctcaaga gcgtcacaca 3780tcactttgat
acttttgcag agtttgccaa ggaattctgt ctcggtgaac gcgacttggt
3840aattaccaac gagttcatct acgaaccgta tatgaaggca tgccagctgc
cttgtcattt 3900tgtgatgcag gagaaatacg gccaaggcga gccttctgac
gagatgatga acaacatcct 3960agcagatatc cgtaatatcc agttcgaccg
cgtgatcggg atcggaggtg gtacggttat 4020tgacatctca aaactctttg
ttctgaaggg attaaatgat gttctcgacg cgttcgatcg 4080caagattccc
cttatcaaag agaaagaact gatcattgtg cccaccacct gcggaaccgg
4140ctcggaggtg acgaacattt ccatcgccga gatcaagtcc cggcacacca
agatgggttt 4200ggctgacgat gcaattgttg ctgaccacgc cataatcatc
cctgaacttc tgaagagctt 4260gcccttccac ttctatgcat gctccgcaat
cgatgctctt attcatgcca tcgagtcata 4320cgtttctcca aaagcgtctc
catactcccg tctgttcagt gaggcggcgt gggacattat 4380cctggaagtt
ttcaagaaaa tcgccgaaca cggcccagag taccgcttcg agaagctggg
4440ggaaatgatc atggccagca actatgccgg tatcgctttc ggcaacgcag
gcgttggcgc 4500cgtccacgct ctatcctacc cgttgggcgg caactatcac
gtgccgcatg gagaagcaaa 4560ctatcagttc ttcaccgagg tctttaaagt
ataccaaaag aagaatccgt tcggctatat 4620tgtcgaactc aactggaagc
tctccaagat tctgaactgc cagccagagt acgtgtaccc 4680gaagctggat
gaactgctcg gttgccttct taccaagaaa cctttgcacg aatacggcat
4740gaaggacgaa gaggttcgtg gcttcgcgga atcggtcctg aagacccagc
aacgcttgct 4800cgccaacaac tacgtcgaac ttactgtcga tgagatcgaa
ggtatctacc gacgtctcta 4860ctaattagag gagacacaac atgaaggatg
tactggcgga atacgcctcc cgcattgttt 4920cggcggagga ggccgttaag
cacatcaaaa acggtgaacg ggtagctttg tcacacgctg 4980ccggcgtgcc
tcagagttgc gttgacgcac tggtgcagca ggccgacctt ttccagaatg
5040tggaaatcta tcacatgctg tgcctcggtg agggtaagta tatggcgcct
gagatggccc 5100ctcacttccg ccacatcacc aactttgtcg gtggtaactc
ccgtaaggcg gtcgaagaaa 5160accgggccga tttcattccg gtattctttt
acgaggtgcc aagcatgatt cgcaaagaca 5220tcctccacat tgatgtcgcc
atcgttcagc tttcaatgcc tgacgaaaat ggttactgtt 5280cctttggagt
atcttgcgat tactccaagc cggcagcaga gagcgctcac ctggttatcg
5340gagaaatcaa ccgtcaaatg ccatacgtac acggcgacaa cttgattcat
atctccaagt 5400tggattacat cgtgatggca gactacccca tctactctct
tgcaaagccc aagatcgggg 5460aagtcgagga agctatcggg aggaattgtg
ccgagcttat tgaagatggt gccactctcc 5520agctgggaat cggcgcgatt
cctgatgcgg ccctgttatt tctcaaggac aaaaaggatc 5580tgggcatcca
taccgaaatg ttctccgatg gtgttgtcga attggttcgc tccggcgtta
5640tcacaggcaa gaaaaagact cttcaccccg gaaagatggt cgcaaccttc
ctgatgggaa 5700gcgaggacgt gtatcatttc atcgataaaa accccgatgt
agaactgtat ccagtagatt 5760acgtgaatga cccgcgtgtg atcgcccaaa
acgacaatat ggtctcgatt aacagctgca 5820tcgaaatcga ccttatggga
caggtcgtgt ccgagtgcat cggctcaaag caattcagcg 5880gcaccggcgg
ccaagttgac tacgtgcgtg gcgcagcatg gtctaaaaac ggcaaatcga
5940tcatggcaat cccgtccact gcaaaaaacg gtacggcatc tcgaattgta
cctatcatcg 6000cggagggcgc tgctgtcacc accctgcgca acgaggtcga
ttacgttgta accgagtacg 6060gtatcgctca gctcaagggc aagagcctgc
gccagcgcgc agaggctttg atcgcgatag 6120cccaccccga cttccgtgag
gaactaacga aacatctccg caagcgattc ggataacata 6180tggcggccgc
aagcttgcct cgacgaaggc gtcaccgtgg gccccctggt tgaggaaaaa
6240gcacgagaca gcgttgcatc gcttgtcgac gccgccgtcg ccgaaggtgc
caccgtcctc 6300accggcggca aggccggcac aggtgcaggc tacttctacg
aaccaacggt gctcacggga 6360gtttcaacag atgcggctat cctgaacgaa
gagatcttcg gtcccgtcgc accgatcgtc 6420accttccaaa ccgaggaaga
agccctgcgt ctagccaact ccaccgaata cggactggcc 6480tcctatgtgt
tcacccagga cacctcacgt attttccgcg tctccgatgg tctcgagttc
6540ggcctagtgg gcgtcaattc cggtgtcatc tctaacgctg ctgcaccttt
tggtggcgta 6600aaacaatccg gaatgggccg cgaaggtggt ctcgaaggaa
tcgaggagta cacctccgtg 6660cagtacatcg gtatccggga tccttacgcc
ggctaggcta gc 67022436DNAArtificial SequenceSynthetic 0049-1 for
24gcaggcatgc aagcttaaag tccccacggg tctact 362536DNAArtificial
SequenceSynthetic 0049-2 rev 25ggccagtgcc aagctttacc gatgtactgc
acggag 36263608DNAArtificial SequenceSynthetic adhE2_nt
26aagcttgcat gcctgcaggt cgactctaga ggatccccgg gaggcacctc acaggtgcaa
60ttattacaca accccacagc gatgtccgca tcctttgatg accccaacct catctcgctt
120gctggactgg ttccaaccat gcacttagcc gatgctgcca gcctgtccac
cttggcccag 180gaccggttga gcatcaccgg tgataaaggt gccaatgctg
gtgcgaagat cgcctcccta 240gtcgcgggca tggtcgccgg tgctgattcc
atcgatgaca tggatgtact ccgccacgga 300ggtatgcgcc gacttttcga
ccggatctac gccccatcca cattggggtc ttttctgcgg 360gccttcactt
tcggccacgt acgccaactc gatgattttg catctgctgc gaaatctttg
420tttccccgct aaagttgagg acaggttgac acggagttga ctcgacgaat
tatccaatgt 480gagtaggttt ggtgcgtgag ttggaaaaat tcgccatact
cgcccttggg ttctgtcagc 540tcaagaattc ttgagtgacc gatgctctga
ttgacctaac tgcttgacac attgcatttc 600ctacaatctt tagaggagac
acaacatgaa agtaaccaat cagaaagagt tgaagcagaa 660gttgaacgag
ctgcgagagg ctcagaagaa gttcgcaacc tacacccagg aacaggtgga
720caagatcttt aagcagtgtg ccattgcagc cgcgaaagaa cgtattaatc
tcgcgaaact 780tgcggtcgag gaaaccggta ttgggctggt agaagacaag
atcatcaaga accacttcgc 840cgctgaatac atctacaaca agtacaaaaa
cgaaaagaca tgtggtatca tcgaccacga 900cgacagcttg ggcatcacca
aggtagcgga gccaatcggt atcgtcgcag ctatcgtgcc 960cactactaac
cctacctcca ctgctatttt caagtcactc atctccctga aaacccgcaa
1020tgctatcttc ttctcacctc acccacgcgc taagaaatca actatcgctg
cagctaaact 1080tatcctggat gcagccgtga aagccggggc tccgaaaaac
atcatcggtt ggatcgacga 1140accttccatt gaactctctc aagacctcat
gtccgaggca gacattatcc tggcaaccgg 1200aggcccatcc atggttaaag
cagcttacag ctcaggcaag ccggctatcg gcgttggagc 1260tggtaacact
ccagcaatca tcgacgagtc ggccgatatc gacatggcag tgtcctctat
1320tatcctgtcc aaaacttatg acaacggcgt tatttgcgcg tccgagcagt
ctattctcgt 1380catgaactct atttacgaga aggtaaagga ggagtttgtg
aagcgggggt cgtacattct 1440gaaccagaac gagatcgcta agatcaaaga
gactatgttt aaaaacggag ccatcaacgc 1500agatatcgta gggaagtccg
cgtacatcat tgctaagatg gctggaatcg aagtccctca 1560aaccacgaaa
attctgatcg gcgaggtgca atcggtcgaa aagtccgagc tgttctcgca
1620tgaaaagttg tccccggtcc tcgcgatgta taaagttaag gattttgatg
aagcactcaa 1680gaaagctcag cgcctgatcg aattgggtgg ctcgggtcac
acctcttccc tctacattga 1740ctcccagaac aataaagata aggtgaaaga
gttcggcctg gctatgaaga cgtctcgtac 1800cttcatcaat atgccctctt
cacagggcgc cagcggtgac ctttacaatt tcgctatcgc 1860tcctagcttt
accctcggct gcggcacctg gggcggtaat tctgtgtccc aaaacgtcga
1920accaaagcat ctgctcaaca ttaaaagcgt cgccgaacgt cgcgagaaca
tgttgtggtt 1980caaggtcccg caaaaaatct acttcaagta tggttgcttg
cgctttgcac ttaaagagct 2040taaggacatg aataaaaagc gggcgttcat
cgtcactgat aaggatctgt tcaaactggg 2100ctatgttaac aagattacca
aggtcctgga tgagatcgat atcaagtatt ccatcttcac 2160cgatattaag
tccgatccga ccattgattc cgtgaagaag ggcgcgaagg agatgctcaa
2220ctttgaaccc gacacgatta tttctattgg cggaggcagc ccaatggacg
cagctaaggt 2280tatgcacctg ctgtatgagt acccagaagc agagatcgag
aaccttgcaa tcaatttcat 2340ggatattcgc aaacgcattt gcaactttcc
taagcttggt acaaaagcta tctctgttgc 2400gatccctacc accgcaggaa
ccggcagcga agcgacacca ttcgccgtta ttaccaacga 2460tgaaacaggt
atgaagtacc cacttacctc ttatgaactt accccgaaca tggctatcat
2520tgatacggaa ttgatgctga acatgccgcg gaagttgacc gcagctacgg
gaatcgatgc 2580attggttcat gcaatcgagg catacgtttc cgtcatggca
accgattaca ccgacgagct 2640cgcgttgcgt gcgattaaaa tgatcttcaa
gtaccttcca cgcgcataca agaatggcac 2700aaacgatatt gaagcccgag
aaaagatggc acacgcttcg aacatcgctg gtatggcctt 2760cgcgaatgcg
tttctcggag tgtgtcactc catggcgcac aaactgggag ccatgcatca
2820cgtgccccac ggtatcgcat gcgccgttct tattgaagag gtgatcaagt
ataatgccac 2880cgattgcccc actaagcaga cggccttccc tcagtacaaa
tcgcccaatg ccaagcgtaa 2940atacgcggaa attgccgagt acttgaacct
taaggggacc agcgacacgg aaaaggtgac 3000cgcactgatt gaagccatct
ccaagcttaa gatcgacctg agcatcccac aaaacatctc 3060agcagccggc
attaacaaga aggacttcta caacactctc gacaagatgt cagagctcgc
3120cttcgatgat cagtgcacta ccgcaaaccc acgttatccg ctcatctctg
aactgaagga 3180tatctacatc aagtcgtttt aatttgatca cggccattca
ccaccgtaac cggtagctcc 3240ctgaccaccc agccgagctt tcggcgtgag
atgacaacaa ttcgtggaac aaccagaaca 3300agacgtgatc tggcgatcac
ccctacccga aaattccgga cccgcccgga accgggatca 3360ggacatcacc
gagggcacat cggtggatcg aggcttaatg gaacgcccca ctcatccaat
3420ccggcaattt tgatgctgta cccatcgacg catggtgctc caaatacgtg
gaagccatca 3480cggtcacgga tgaagcatgg caggttttcc ggttggaagt
ccactggatt gttgggcagg 3540aaccaggtga gcgcctgaat ggcgaatggc
gataagctag aggatccccg ggtaccgagc 3600tcgaattc 36082720DNAArtificial
SequenceSynthetic AdhE2_1_F 27atgaaagtaa ccaatcagaa
202820DNAArtificial SequenceSynthetic AdhE2_2260_R 28aatcggtggc
attatacttg 20
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