U.S. patent application number 13/997107 was filed with the patent office on 2013-11-14 for coryneform bacterium transformant and process for producing aniline using the same.
This patent application is currently assigned to Sumitomo Rubber Industries, Ltd.. The applicant listed for this patent is Masayuki Inui, Hideaki Yukawa. Invention is credited to Masayuki Inui, Hideaki Yukawa.
Application Number | 20130302860 13/997107 |
Document ID | / |
Family ID | 46383073 |
Filed Date | 2013-11-14 |
United States Patent
Application |
20130302860 |
Kind Code |
A1 |
Yukawa; Hideaki ; et
al. |
November 14, 2013 |
Coryneform Bacterium Transformant and Process for Producing Aniline
Using The Same
Abstract
Provided is an aniline-producing transformant constructed by
introducing a gene which encodes an enzyme having aminobenzoate
decarboxylase activity into a coryneform bacterium as a host. Also
provided is a process for producing aniline, which comprises a step
of allowing the transformant to react in a reaction mixture
containing aminobenzoic acid, an ester thereof, and/or a salt
thereof under reducing conditions, and a step of recovering aniline
from the reaction mixture.
Inventors: |
Yukawa; Hideaki; (Kyoto,
JP) ; Inui; Masayuki; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yukawa; Hideaki
Inui; Masayuki |
Kyoto
Kyoto |
|
JP
JP |
|
|
Assignee: |
Sumitomo Rubber Industries,
Ltd.
Hyogo
JP
Research Institute of Innovative Technology for the
Earth
Kyoto
JP
|
Family ID: |
46383073 |
Appl. No.: |
13/997107 |
Filed: |
December 24, 2011 |
PCT Filed: |
December 24, 2011 |
PCT NO: |
PCT/JP2011/080151 |
371 Date: |
July 30, 2013 |
Current U.S.
Class: |
435/128 ;
435/252.32 |
Current CPC
Class: |
C12N 9/88 20130101; C12Y
401/01024 20130101; C12N 15/77 20130101; Y02P 20/52 20151101; C12P
13/001 20130101 |
Class at
Publication: |
435/128 ;
435/252.32 |
International
Class: |
C12P 13/00 20060101
C12P013/00; C12N 15/77 20060101 C12N015/77 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2010 |
JP |
2010-293972 |
Claims
1. An aniline-producing transformant constructed by introducing a
gene which encodes an enzyme having aminobenzoate decarboxylase
activity into a coryneform bacterium as a host.
2. The transformant of claim 1, wherein the gene which encodes an
enzyme having aminobenzoate decarboxylase activity is a gene
derived from Bacillus subtilis, a gene derived from Lactobacillus
rhamnosus, a gene derived from Lactobacillus brevis, a gene derived
from Pseudomonas putida, a gene derived from Escherichia coli, a
gene derived from Saccharomyces cerevisiae, or a gene derived from
Enterobacter cloacae.
3. The transformant of claim 1, wherein the gene which encodes an
enzyme having aminobenzoate decarboxylase activity is the DNA of
the following (a) or (b): (a) a DNA consisting of the base sequence
of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ
ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 34 (b) a DNA which
hybridizes to a DNA consisting of a complementary base sequence of
any of the DNAs of (a) under stringent conditions and which encodes
a polypeptide having aminobenzoate decarboxylase activity.
4. The transformant of claim 1, wherein the coryneform bacterium as
the host is Corynebacterium glutamicum.
5. The transformant of claim 4, wherein the coryneform bacterium as
the host is Corynebacterium glutamicum R (FERM BP-18976),
ATCC13032, or ATCC13869.
6. Corynebacterium glutamicum ANI-1 (Accession Number: NITE
BP-1001), which is a transformant of Corynebacterium
glutamicum.
7. A process for producing aniline, which comprises a step of
allowing the transformant of claim 1 to react in a reaction mixture
containing aminobenzoic acid, an ester thereof, and/or a salt
thereof under reducing conditions, and a step of recovering aniline
from the reaction mixture.
8. The process of claim 7, wherein the transformant does not
substantially grow in the reaction step.
9. The process of claim 7, wherein the oxidation-reduction
potential of the reaction mixture under reducing conditions is -200
mV to -500 mV.
Description
TECHNICAL FIELD
[0001] The present invention relates to a technique for producing
aniline. In more detail, the present invention relates to a
coryneform bacterium transformant constructed by specific gene
recombination and thereby provided with an aniline-producing
function, and relates to an efficient aniline-producing process
using the transformant.
BACKGROUND ART
[0002] Against the backdrop of global warming and exhaustion of
fossil resources, production of chemical products using renewable
resources, along with production of biofuels, is recognized as an
emerging industry, biorefinery, which is an important means for
realizing a low-carbon society, and has attracted keen
attention.
[0003] Aniline is widely used as raw materials for various products
including chemical products, such as dyes and rubber product
materials (a vulcanization accelerator and an antioxidant for
tires, etc.); functional materials, such as and textiles and
conductive polymers; agricultural chemicals; medicinal drugs; or
the like.
[0004] Currently, aniline is chemically produced from crude oil as
a raw material. Chemical processes for producing aniline include a
process in which nitrobenzen is reduced with the use of tin or iron
and hydrochloric acid; a process in which nitrobenzen is reduced by
hydrogen addition with the use of a metal catalyst, such as copper
or nickel; and a process called ammonolysis, in which chlorobenzene
and ammonia are made to react at high temperature and pressure.
They are all typical energy-consumptive processes in the chemical
industry requiring great amounts of solvent and thermal energy.
Therefore, in the light of global environment conservation and
greenhouse gas reduction, there is an urgent need to develop an
environment-conscious, energy saving process that allows production
of aniline from renewable resources and can reduce carbon dioxide
emissions and waste products, that is, to establish bioaniline
production technologies.
[0005] However, production of bioaniline from renewable resources
is less productive as compared to production of lactic acid or
ethanol because the metabolic reaction from a raw material sugar
consists of a great many steps. In addition, there are problems,
such as inhibition of bacterial growth by produced aniline and
cytotoxicity of aniline. Therefore, industrial production of
aniline has been considered to be impossible.
[0006] Specifically known examples of technologies for producing
aniline are as follows.
[0007] For example, Non Patent Literature 1 discloses that a slight
amount of aniline is produced by culturing Mycobacterium smegmatis,
washing the cells, and then adding 4-aminobenzoic acid. However,
the process of Non Patent Literature 1 does not show practically
sufficient aniline productivity. Non Patent Literature 1 does not
mention any enzyme involved in aniline production from
4-aminobenzoic acid, let alone its activity or related gene.
[0008] Non Patent Literature 2 discloses that a slight amount of
aniline is produced by adding anthranilic acid (2-aminobenzoic
acid) or 4-aminobenzoic acid to washed cells of virulent
Escherichia coli O111 or an extract from the cells. However, the
process of Non Patent Literature 2 does not have practically
sufficient aniline productivity. Non Patent Literature 2 does not
mention any enzyme involved in aniline production from
4-aminobenzoic acid, let alone its activity or related gene.
[0009] Patent Literature 1 discloses a technology in which
Streptomyces griseus is cultured in TSB culture medium (Trypticase
Soy Broth) supplemented with glucose (raw material for aniline)
under aerobic conditions for 4 to 5 days for aniline production.
However, Patent Literature 1 does not specifically show the amount
of produced aniline or the productivity. Therefore, the
practicality of the method of Patent Literature 1 is unknown.
CITATION LIST
Patent Literature
[0010] [PTL 1] JP 2008-274225 A
Non Patent Literature
[0010] [0011] [NPL 1] The Journal of Biological Chemistry, Vol.
193, 1951, 453-458. [0012] [NPL 2] Journal of the American Chemical
Society, Vol. 79, 1957, 628-630.
SUMMARY OF INVENTION
Technical Problem
[0013] An object of the present invention is to provide a
microorganism capable of efficiently producing aniline from
aminobenzoic acid, and a process for efficiently producing aniline
from aminobenzoic acid.
Solution to Problem
[0014] The present inventors have wholeheartedly carried out
investigations in order to achieve the object described above and
obtained the findings that a transformant constructed by
introducing an aminobenzoate decarboxylase gene into a coryneform
bacterium can efficiently produce aniline from aminobenzoic acid
and that the transformant has a particularly higher aniline
productivity when growth is substantially inhibited in a reaction
mixture under reducing conditions.
[0015] The present invention, which has been completed based on the
above-mentioned findings, provides the following transformant and
process for producing aniline.
[1] An aniline-producing transformant constructed by introducing a
gene which encodes an enzyme having aminobenzoate decarboxylase
activity into a coryneform bacterium as a host. [2] The
transformant of the above [1], wherein the gene which encodes an
enzyme having aminobenzoate decarboxylase activity is a gene
derived from Bacillus subtilis, a gene derived from Lactobacillus
rhamnosus, a gene derived from Lactobacillus brevis, a gene derived
from Pseudomonas putida, a gene derived from Escherichia coli, a
gene derived from Saccharomyces cerevisiae, or a gene derived from
Enterobacter cloacae. [3] The transformant of the above [1],
wherein the gene which encodes an enzyme having aminobenzoate
decarboxylase activity is the DNA of the following (a) or (b). (a)
a DNA consisting of the base sequence of SEQ ID NO: 16, SEQ ID NO:
19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, or
SEQ ID NO: 34 (b) a DNA which hybridizes to a DNA consisting of a
complementary base sequence of any of the DNAs of (a) under
stringent conditions and which encodes a polypeptide having
aminobenzoate decarboxylase activity [4] The transformant of any
one of the above [1] to [3], wherein the coryneform bacterium as
the host is Corynebacterium glutamicum. [5] The transformant of the
above [4], wherein the coryneform bacterium as the host is
Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, or
ATCC13869. [6] Corynebacterium glutamicum ANI-1 (Accession Number:
NITE BP-1001), which is a transformant of Corynebacterium
glutamicum. [7] A process for producing aniline, which comprises a
step of allowing the transformant of any one of the above [1] to
[6] to react in a reaction mixture containing aminobenzoic acid, an
ester thereof, and/or a salt thereof under reducing conditions, and
a step of recovering aniline from the reaction mixture. [8] The
process of the above [7], wherein the transformant does not
substantially grow in the reaction step. [9] The process of the
above [7] or [8], wherein the oxidation-reduction potential of the
reaction mixture under reducing conditions is -200 mV to -500
mV.
Advantageous Effects of Invention
[0016] With the use of the transformant of the present invention,
aniline can be efficiently produced from aminobenzoic acid, a salt
thereof, and/or an ester thereof.
[0017] Generally, growth of microorganisms is inhibited by a
solvent, such as aniline, because of its cytotoxicity, and
therefore aniline production with the use of microorganisms has
been difficult. According to the process of the present invention,
however, aniline production with the use of microorganisms can be
achieved with a practically sufficient efficiency.
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 shows the constructs of plasmids used in
Examples.
DESCRIPTION OF EMBODIMENTS
[0019] Hereinafter, the present invention will be described in
detail.
(I) Aniline-Producing Transformant
[0020] The transformant of the present invention capable of
producing aniline is a transformant constructed by introducing a
gene which encodes an enzyme having aminobenzoate decarboxylase
activity into a coryneform bacterium as a host.
Host
[0021] The coryneform bacterium is a group of microorganisms
defined in Bergey's Manual of Determinative Bacteriology, Vol. 8,
599 (1974), and is not particularly limited as long as it grows
under normal aerobic conditions.
[0022] The specific examples include Corynebacterium,
Brevibacterium, Arthrobacter, Mycobacterium and Micrococcus. Among
the coryneform bacteria, Corynebacterium is preferred.
[0023] Examples of the Corynebacterium include Corynebacterium
glutamicum, Corynebacterium efficiens, Corynebacterium
ammoniagenes, Corynebacterium halotolerance, and Corynebacterium
alkanolyticum.
[0024] Inter alia, Corynebacterium glutamicum is preferred for
safety and high aniline production. Examples of preferred strains
include Corynebacterium glutamicum R (FERM P-18976), ATCC13032,
ATCC13869, ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286,
ATCC13287, ATCC13655, ATCC13745, ATCC13746, ATCC13761, ATCC14020,
ATCC31831, MJ-233 (FERM BP-1497), and MJ-233AB-41 (FERM BP-1498).
Inter alia, strains R (FERM P-18976), ATCC13032, and ATCC13869 are
preferred.
[0025] According to molecular biological classification, names of
some species of coryneform bacteria, such as Brevibacterium flavum,
Brevibacterium lactofermentum, Brevibacterium divaricatum, and
Corynebacterium lilium are standardized to Corynebacterium
glutamicum (Liebl, W. et al., Transfer of Brevibacterium
divaricatum DSM 20297T, "Brevibacterium flavum" DSM 20411,
"Brevibacterium lactofermentum" DSM 20412 and DSM 1412, and
Corynebacterium glutamicum and their distinction by rRNA gene
restriction patterns. Int. J. Syst. Bacteriol. 41: 255-260. (1991);
and Kazuo Komagata et al., "Classification of the coryneform group
of bacteria", Fermentation and industry, 45: 944-963 (1987)).
[0026] Brevibacterium lactofermentum ATCC13869, Brevibacterium
flavum MJ-233 (FERM BP-1497) and MJ-233AB-41 (FERM BP-1498), etc.
of the old classification are also suitable as Corynebacterium
glutamicum.
[0027] Examples of the Brevibacterium include Brevibacterium
ammoniagenes (for example, ATCC6872).
[0028] Examples of the Arthrobacter include Arthrobacter
globiformis (for example, ATCC8010, ATCC4336, ATCC21056, ATCC31250,
ATCC31738 and ATCC35698).
[0029] Examples of the Mycobacterium include Mycobacterium bovis
(for example, ATCC19210 and ATCC27289).
[0030] Examples of the Micrococcus include Micrococcus
freudenreichii (for example, NO. 239 (FERM P-13221)), Micrococcus
leuteus (for example, NO. 240 (FERM P-13222)), Micrococcus ureae
(for example, IAM1010), and Micrococcus roseus (for example,
IFO3764).
[0031] The coryneform bacteria may be, let alone a wild strain, a
mutant thereof or an artificial recombinant thereof. Examples
thereof include disruptants in which a gene of lactate
dehydrogenase, phosphoenolpyruvate carboxylase, or malate
dehydrogenase is disrupted. Using such a disruptant as a host can
improve aniline productivity and reduce production of
by-products.
[0032] Inter alia, preferred is a disruptant in which a lactate
dehydrogenase gene is disrupted. In the disruptant, the lactate
dehydrogenase gene is disrupted and the metabolic pathway from
pyruvic acid to lactic acid is blocked. Inter alia, especially
preferred is a disruptant of Corynebacterium glutamicum R (FERM
P-18976) strain in which the lactate dehydrogenase gene is
disrupted.
[0033] Such a disruptant can be prepared based on a conventional
gene engineering process. Such a lactate dehydrogenase disruptant
and the preparation process thereof are described in WO 2005/010182
A1.
[0034] Compared with other bacteria, coryneform bacteria are more
resistant to solvents, such as aniline. Further, compared with
other aerobic bacteria, coryneform bacteria more efficiently
produce substances under reducing conditions where growth is
substantially inhibited. In these respects, coryneform bacteria are
suitable for the aniline production by the method of the present
invention.
Aminobenzoate Decarboxylase Gene
[0035] Aminobenzoate decarboxylase is an enzyme that catalyzes a
reaction in which aniline is produced by elimination of carbonic
acid from aminobenzoic acid and the reverse reaction.
[0036] The gene which encodes an enzyme having aminobenzoate
decarboxylase activity may be of any origin without particular
limitation, and preferred examples thereof include a gene derived
from Bacillus subtilis, a gene derived from Lactobacillus
rhamnosus, a gene derived from Lactobacillus brevis, a gene derived
from Pseudomonas putida, a gene derived from Escherichia coli, a
gene derived from Saccharomyces cerevisiae, and a gene derived from
Enterobacter cloacae. Inter alia, more preferred are a gene derived
from Bacillus subtilis and a gene derived from Enterobacter
cloacae. In particular, when the substrate is anthranilic acid
(2-aminobenzoic acid), preferred is a gene derived from Bacillus
subtilis, and when the substrate is 4-aminobenzoic acid, preferred
is a gene derived from Enterobacter cloacae.
[0037] Examples of the gene derived from Bacillus subtilis include
the DNA consisting of the base sequence of SEQ ID NO: 16, examples
of the gene derived from Lactobacillus rhamnosus include the DNA
consisting of the base sequence of SEQ ID NO: 19, examples of the
gene derived from Lactobacillus brevis include the DNA consisting
of the base sequence of SEQ ID NO: 22, examples of the gene derived
from Pseudomonas putida include the DNA consisting of the base
sequence of SEQ ID NO: 25, examples of the gene derived from
Escherichia coli include the DNA consisting of the base sequence of
SEQ ID NO: 28, examples of the gene derived from Saccharomyces
cerevisiae include the DNA consisting of the base sequence of SEQ
ID NO: 31, and examples of the gene derived from Enterobacter
cloacae include the DNA consisting of the base sequence of SEQ ID
NO: 34.
[0038] In the present invention, a DNA which hybridizes to a DNA
consisting of a complementary base sequence of the base sequence of
SEQ ID NO: 16, 19, 22, 25, 28, 31, or 34 under stringent conditions
and which encodes a polypeptide having aminobenzoate decarboxylase
activity can also be used.
[0039] The "stringent conditions" as used herein means general
conditions, for example, the conditions described in Molecular
Cloning, ALaboratory Manual, Second edition, 1989, Vol. 2, p. 11.
45. It means, in particular, conditions where hybridization occurs
at a temperature 5 to 10.degree. C. below the melting temperature
(Tm) of a perfect hybrid.
[0040] The aminobenzoate decarboxylase activity can be measured by
a modified method of the method described in J. Am. Chem. Soc., 79,
628-630 (1957). Briefly, a coryneform bacterium is cultured in a
nutrient medium at 33.degree. C. for 18 hours, washed with minimal
medium twice, and resuspended in minimal medium to prepare intact
cells. Subsequently, for the reaction, HEPES (pH 7.0) as a buffer
solution is added to the intact cells so that the concentration is
25 mM, and anthranilic acid or 4-amino benzoate as a substrate is
added so that the final concentration is 5 mM. After shaking at 200
rpm at 33.degree. C. for 6 hours, the reaction mixture was
centrifuged to separate bacterial cells and supernatant. The
supernatant is filtered through a 0.22-.mu.m filter, and the
filtrate is used as a sample. The produced aniline can be
quantified by GC/MS or HPLC.
[0041] In the present invention, a DNA consisting of a base
sequence which has 90% or more, preferably 95% or more, more
preferably 98% or more homology with the base sequence of SEQ ID
NO: 16, 19, 22, 25, 28, 31, or 34 and which encodes a polypeptide
having aminobenzoate decarboxylase activity can also be used.
[0042] The base sequence homology was calculated using GENETYX Ver.
8 (made by Genetyx).
[0043] The homologue of the DNA consisting of the base sequence of
SEQ ID NO: 16, 19, 22, 25, 28, 31, or 34 can be selected from a DNA
library of a different species by, for example, PCR or
hybridization using a primer or a probe designed based on these
base sequences, according to a conventional method, and as a
result, a DNA which encodes a polypeptide having aminobenzoate
decarboxylase activity can be obtained with a high probability.
Construction of Vector for Transformation
[0044] The DNA which encodes aminobenzoate decarboxylase is
amplified by PCR and then cloned into a suitable vector which is
replicable in a host.
[0045] The plasmid vector may be any plasmid vector as long as it
comprises a gene responsible for autonomously replicating function
in a coryneform bacterium. Specific examples of the plasmid vector
include pAM330 derived from Brevibacterium lactofermentum 2256 (JP
58-67699 A; Miwa, K. et al., Cryptic plasmids in glutamic
acid-producing bacteria. Agric. Biol. Chem. 48:2901-2903 (1984);
and Yamaguchi, R. et al., Determination of the complete nucleotide
sequence of the Brevibacterium lactofermentum plasmid pAM330 and
the analysis of its genetic information. Nucleic Acids Symp. Ser.
16:265-267 (1985)); pHM1519 derived from Corynebacterium glutamicum
ATCC13058 (Miwa, K. et al., Cryptic plasmids in glutamic
acid-producing bacteria. Agric. Biol. Chem. 48:2901-2903 (1984))
and pCRY30 derived from the same (Kurusu, Y. et al., Identification
of plasmid partition function in coryneform bacteria. Appl.
Environ. Microbiol. 57:759-764 (1991)); pCG4 derived from
Corynebacterium glutamicum T250 (JP 57-183799 A; and Katsumata, R.
et al., Protoplast transformation of glutamate-producing bacteria
with plasmid DNA. J. Bacteriol., 159:306-311 (1984)), pAG1, pAG3,
pAG14 and pAG50 derived from the same (JP 62-166890 A), and pEK0,
pEC5 and pEKEx1 derived from the same (Eikmanns, B. J. et al., A
family of Corynebacterium glutamicum/Escherichia coli shuttle
vectors for cloning, controlled gene expression, and promoter
probing. Gene, 102:93-98 (1991)); etc.
[0046] Examples of a preferred promoter include promoter PgapA as a
promoter of the glyceraldehyde-3-phosphate dehydrogenase A gene
(gapA), promoter Pmdh as a promoter of the malate dehydrogenase
gene (mdh), and promoter PldhA as a promoter of lactate
dehydrogenase A gene (ldhA), all of which are derived from
Corynebacterium glutamicum R, and inter alia, PgapA is
preferred.
[0047] Examples of a preferred terminator include terminator rrnB
T1T2 of Escherichia coli rRNA operon, terminator trpA of
Escherichia coli, and terminator trp of Brevibacterium
lactofermentum, and inter alia, terminator rrnB T1T2 is
preferred.
Transformation
[0048] As a method of transformation, any publicly known method can
be used without limitation. Examples of such a known method include
the calcium chloride/rubidium chloride method, the calcium
phosphate method, DEAE-dextran transfection, and electroporation.
Inter alia, preferred for a coryneform bacterium is
electroporation, which can be performed by a known method (Kurusu,
Y. et al., Electroporation-transformation system for Coryneform
bacteria by auxotrophic complementation., Agric. Biol. Chem.
54:443-447 (1990); and Vertes A. A. et al., Presence of mrr- and
mcr-like restriction systems in Coryneform bacteria. Res.
Microbial. 144:181-185 (1993)).
[0049] The transformant is cultured using a culture medium usually
used for culture of microorganisms. The culture medium may be a
natural or synthetic medium containing a carbon source, a nitrogen
source, inorganic salts, other nutritional substances, etc.
[0050] Examples of the carbon source include carbohydrates and
sugar alcohols such as glucose, fructose, sucrose, mannose,
maltose, mannitol, xylose, arabinose, galactose, starch, molasses,
sorbitol and glycerol; organic acids such as acetic acid, citric
acid, lactic acid, fumaric acid, maleic acid and gluconic acid; and
alcohols such as ethanol and propanol. Hydrocarbons, such as normal
paraffin, etc. may also be used as desired. These carbon sources
may be used alone or as a mixture of two or more thereof. The
concentration of these carbon sources in the culture medium is
usually about 0.1 to 10 w/v %.
[0051] Examples of the nitrogen source include inorganic or organic
ammonium compounds, such as ammonium chloride, ammonium sulfate,
ammonium nitrate, and ammonium acetate; urea; aqueous ammonia;
sodium nitrate; and potassium nitrate. Nitrogen-containing organic
compounds, such as corn steep liquor, meat extract, peptone,
N--Z-amine, protein hydrolysate, amino acid, etc. may also be used.
These nitrogen sources may be used alone or as a mixture of two or
more thereof. The concentration of these nitrogen sources in the
culture medium varies depending on the kind of the nitrogen
compound, but is usually about 0.1 to 10 w/v %.
[0052] Examples of the inorganic salts include potassium dihydrogen
phosphate, dipotassium hydrogenphosphate, magnesium sulfate, sodium
chloride, iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt
sulfate, and calcium carbonate. These inorganic salts may be used
alone or as a mixture of two or more thereof. The concentration of
the inorganic salts in the culture medium varies depending on the
kind of the inorganic salts, but is usually about 0.01 to 1 w/v
%.
[0053] Examples of the nutritional substances include meat extract,
peptone, polypeptone, yeast extract, dry yeast, corn steep liquor,
skim milk powder, defatted soybean hydrochloric acid hydrolysate,
and extract from animals, plants or microorganisms, and degradation
products thereof. The concentration of the nutritional substances
in the culture medium varies depending on the kind of the
nutritional substances, but is usually about 0.1 to 10 w/v %.
Further, vitamins may be added as needed. Examples of the vitamins
include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6),
pantothenic acid, inositol, nicotinic acid, etc.
[0054] The pH of the culture medium is preferably about 5 to 8.
[0055] Examples of the preferable microbial culture medium include
A medium (Inui, M. et al., Metabolic analysis of Corynebacterium
glutamicum during lactate and succinate productions under oxygen
deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182-196
(2004)), BT medium (Omumasaba, C. A. et al., Corynebacterium
glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with
opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol.
8:91-103 (2004)), etc.
[0056] The culture temperature is about 15 to 45.degree. C., and
the culture period is about 1 to 7 days.
(II) Process for Producing Aniline
[0057] Aniline can be produced by a process comprising a step of
allowing the above-described transformant of the present invention
to react in a reaction mixture containing aminobenzoic acid, a salt
thereof, and/or an ester thereof, and a step of recovering aniline
from the reaction mixture.
Growth of Microorganism
[0058] Before the reaction, the transformant is preferably cultured
and grown under aerobic conditions at about 25 to 35.degree. C. for
about 12 to 48 hours.
Culture Medium
[0059] The culture medium used for aerobic culture of the
transformant before the reaction may be a natural or synthetic
medium containing a carbon source, a nitrogen source, inorganic
salts, other nutritional substances, etc.
[0060] Examples of the carbon source that can be used include
sugars (monosaccharides such as glucose, fructose, mannose, xylose,
arabinose, and galactose; disaccharides such as sucrose, maltose,
lactose, cellobiose, xylobiose, and trehalose; polysaccharides such
as starch; and molasses); sugar alcohols such as mannitol,
sorbitol, xylitol, and glycerol; organic acids such as acetic acid,
citric acid, lactic acid, fumaric acid, maleic acid and gluconic
acid; alcohols such as ethanol and propanol; and hydrocarbons such
as normal paraffin.
[0061] These carbon sources may be used alone or as a mixture of
two or more thereof.
[0062] Examples of the nitrogen source that can be used include
inorganic or organic ammonium compounds, such as ammonium chloride,
ammonium sulfate, ammonium nitrate, and ammonium acetate; urea;
aqueous ammonia; sodium nitrate; and potassium nitrate.
Nitrogen-containing organic compounds, such as corn steep liquor,
meat extract, peptone, N--Z-amine, protein hydrolysate, amino acid,
etc. may also be used. These nitrogen sources may be used alone or
as a mixture of two or more thereof. The concentration of these
nitrogen sources in the culture medium varies depending on the kind
of the nitrogen compound, but is usually about 0.1 to 10 w/v %.
[0063] Examples of the inorganic salts include potassium dihydrogen
phosphate, dipotassium hydrogenphosphate, magnesium sulfate, sodium
chloride, iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt
sulfate, and calcium carbonate. These inorganic salts may be used
alone or as a mixture of two or more thereof. The concentration of
the inorganic salts in the culture medium varies depending on the
kind of the inorganic salts, but is usually about 0.01 to 1 w/v
%.
[0064] Examples of the nutritional substances include meat extract,
peptone, polypeptone, yeast extract, dry yeast, corn steep liquor,
skim milk powder, defatted soybean hydrochloric acid hydrolysate,
and extract from animals, plants or microorganisms, and degradation
products thereof. The concentration of the nutritional substances
in the culture medium varies depending on the kind of the
nutritional substances, but is usually about 0.1 to 10 w/v %.
[0065] Further, vitamins may be added as needed. Examples of the
vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin
B6), pantothenic acid, inositol, nicotinic acid, etc.
[0066] The pH of the culture medium is preferably about 6 to 8.
[0067] Specific examples of the preferable culture medium for
coryneform bacteria include A medium (Inui, M. et al., Metabolic
analysis of Corynebacterium glutamicum during lactate and succinate
productions under oxygen deprivation conditions. J. Mol. Microbiol.
Biotechnol. 7:182-196 (2004)), BT medium (Omumasaba, C. A. et al.,
Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase
isoforms with opposite, ATP-dependent regulation. J. Mol.
Microbiol. Biotechnol. 8:91-103 (2004)), etc. Such a culture medium
can be used after prepared so as to contain a sugar at a
concentration in the above-mentioned range.
Reaction Mixture
[0068] As the reaction mixture, water, a buffer solution, an
inorganic salt medium, or the like, containing an aniline precursor
(raw material for aniline) can be used.
[0069] As the precursor, aminobenzoic acid, a salt thereof, and/or
an ester thereof may be used. As the aminobenzoic acid,
2-aminobenzoic acid (o-aminobenzoic acid; anthranilic acid),
3-aminobenzoic acid (m-aminobenzoic acid), and 4-aminobenzoic acid
(p-aminobenzoic acid) are all usable. Inter alia, preferred are
2-aminobenzoic acid and 4-aminobenzoic acid because they are
soluble in water and thus easy to use for the reaction.
[0070] Examples of the salt include a sodium salt, a potassium
salt, and a hydrochloride. Examples of the ester include esters
with alcohols having 1 to 4 carbon atoms.
[0071] Salts are preferred because they are highly soluble in the
reaction mixture. These precursors may be used alone or a mixture
of two or more kinds.
[0072] The concentration of aminobenzoic acid, a salt thereof,
and/or an ester thereof in the reaction mixture is preferably about
0.1 to 10 w/v %, more preferably about 0.5 to 7 w/v %, and still
more preferably about 0.5 to 5 w/v %. When the concentration is in
the above range, aniline can be efficiently produced.
[0073] Examples of the buffer solution include a phosphate buffer,
a Tris buffer, a carbonate buffer, etc. The concentration of the
buffer solution is preferably about 10 to 150 mM.
[0074] Examples of the inorganic salt medium include a medium
containing one or more kinds of inorganic salts including potassium
dihydrogen phosphate, dipotassium hydrogenphosphate, magnesium
sulfate, sodium chloride, iron(II) nitrate, manganese sulfate, zinc
sulfate, cobalt sulfate, and calcium carbonate. Inter alia,
preferred is a medium containing magnesium sulfate. Specific
example of the inorganic salt medium include BT medium (Omumasaba,
C. A. et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate
dehydrogenase isoforms with opposite, ATP-dependent regulation. J.
Mol. Microbiol. Biotechnol. 8:91-103 (2004)) etc. The concentration
of the inorganic salts in the culture medium varies depending on
the kind of the inorganic salts, but is usually about 0.01 to 1 w/v
%.
[0075] The pH of the reaction mixture is preferably about 6 to 8.
During the reaction, the pH of the reaction mixture is preferably
kept nearly neutral, in particular at around 7 with the use of
aqueous ammonia, aqueous sodium hydroxide, or the like, under the
control of a pH controller (for example, Type: DT-1023 made by
Able).
Reaction Conditions
[0076] The reaction temperature, that is, the temperature for
keeping the transformant alive during the reaction is preferably
about 20 to 40.degree. C., and more preferably about 25 to
35.degree. C. When the temperature is in the above range, aniline
can be efficiently produced.
[0077] The reaction period is preferably about 1 to 7 days, and
more preferably about 1 to 3 days.
[0078] The culture may be a batch process, a fed-batch process, or
a continuous process. Inter alia, a batch process is preferred.
<Reducing Conditions>
[0079] The reaction may be performed under aerobic conditions or
reducing conditions, but preferably is performed under reducing
conditions. Under reducing conditions, coryneform bacteria do not
substantially grow and can further efficiently produce aniline.
[0080] The "reducing conditions" is defined based on the
oxidation-reduction potential of the reaction mixture. The
oxidation-reduction potential of the reaction mixture is preferably
about -200 mV to -500 mV, and more preferably about -250 mV to -500
mV.
[0081] The reducing conditions of the reaction mixture can be
simply estimated with the use of resazurin indicator (in reducing
conditions, decolorization from blue to colorless is observed).
However, for precise measurement, a redox-potential meter (for
example, ORP Electrodes made by BROADLEY JAMES) is used.
[0082] As a method of preparing a reaction mixture under reducing
conditions, any publicly known method can be used without
limitation. For example, as a liquid medium for preparation of the
reaction mixture, an aqueous solution for a reaction mixture may be
used instead of distillated water or the like. As reference for
preparation of the aqueous solution for a reaction mixture, for
example, the method for preparing a culture medium for strictly
anaerobic microorganisms, such as sulfate-reducing microorganisms
(Pfennig, N. et al.: The dissimilatory sulfate-reducing bacteria,
In The Prokaryotes, A Handbook on Habitats, Isolation and
Identification of Bacteria, Ed. by Starr, M. P. et al. Berlin,
Springer Verlag, 926-940, 1981, or Nogeikagaku Jikkensho, Ed. by
Kyoto Daigaku Nogakubu Nogeikagaku Kyoshitsu, Vol. 3, Sangyo Tosho,
1990, Issue 26) may be used, and such a method provides an aqueous
solution under desired reducing conditions.
[0083] Specifically, by treating distillated water or the like with
heat or under reduced pressure for removal of dissolved gases, an
aqueous solution for a reaction mixture under reducing conditions
can be obtained. In this case, for removal of dissolved gases,
especially dissolved oxygen, distillated water or the like may be
treated under reduced pressure of about 10 mmHg or less, preferably
about 5 mmHg or less, more preferably about 3 mmHg or less, for
about 1 to 60 minutes, preferably for about 5 to 40 minutes.
[0084] Alternatively, by adding a suitable reducing agent (for
example, thioglycolic acid, ascorbic acid, cysteine hydrochloride,
mercaptoacetic acid, thiol acetic acid, glutathione, sodium
sulfide, etc.), an aqueous solution for a reaction mixture under
reducing conditions can be prepared.
[0085] These methods may be suitably combined to prepare an
effective aqueous solution for a reaction mixture under reducing
conditions.
[0086] It is preferred to maintain the reducing conditions of the
reaction mixture during the reaction. For maintenance of reducing
conditions, it is preferred that oxygen from the outside of the
reaction system is prevented to the utmost extent from entering the
system. Specific examples of the method employed for this purpose
include a method comprising encapsulating the reaction system with
inert gas, such as nitrogen gas, carbon dioxide gas, etc. In some
cases, for allowing the metabolic functions in the cells of the
aerobic bacterium of the present invention to work effectively
during the reaction, addition of a solution of various nutrients or
a reagent solution for adjusting and maintaining the pH of the
reaction system may be needed. In such a case, for more effective
prevention of oxygen incorporation, it is effective to remove
oxygen in the solutions to be added, in advance.
Recovery of Aniline
[0087] Through the culture performed in the above manner, aniline
is produced in the reaction mixture. Aniline can be recovered by
collecting the reaction mixture, and it is also feasible to isolate
aniline from the reaction mixture by a known method. Examples of
such a known method include distillation, the membrane permeation
method, and the organic solvent extraction method.
EXAMPLES
[0088] Hereinafter, the present invention will be described in more
detail by way of Examples, but the present invention is not limited
thereto.
Example 1
[0089] Cloning and Expression of Aniline-Producing Genes
(1) Extraction of Chromosomal DNA from Microorganisms
[0090] To extract chromosomal DNA from Bacillus subtilis NBRC14144,
the bacterium was inoculated into NBRC Medium No. 802 (10 g of
polypeptone, 2 g of yeast extract, and 1 g of MgSO.sub.4.7H.sub.2O
were dissolved in 1 L of distilled water) with the use of a
platinum loop, and cultured with shaking at 37.degree. C. until the
logarithmic growth phase. After the bacterial cells were collected,
chromosomal DNA was recovered from the collected cells with the use
of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue
DNA Isolation Kit, made by Amersham) according to the instruction
manual.
[0091] To extract chromosomal DNA from Lactobacillus rhamnosus
NBRC3425, the bacterium was inoculated into NBRC Medium No. 804 (5
g of polypeptone, 5 g of yeast extract, 5 g of glucose, and 1 g of
MgSO.sub.4.7H.sub.2O were dissolved in 1 L of distilled water) with
the use of a platinum loop, and cultured with shaking at 30.degree.
C. until the logarithmic growth phase. After bacterial cells were
collected, chromosomal DNA was recovered from the collected cells
with the use of a DNA extraction kit (trade name: GenomicPrep Cells
and Tissue DNA Isolation Kit, made by Amersham) according to the
instruction manual.
[0092] To extract chromosomal DNA from Lactobacillus brevis
ATCC367, the bacterium was inoculated in Lactobacilli MRS broth
(made by Becton, Dickinson and Company, BD 288130) with use of a
platinum loop, and cultured with shaking at 30.degree. C. until
logarithmic growth phase. After bacterial cells were collected,
chromosomal DNA was recovered from the collected cells with the use
of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue
DNA Isolation Kit, made by Amersham) according to the instruction
manual.
[0093] To extract chromosomal DNA from Pseudomonas putida (KT2440)
ATCC47054, the bacterium was inoculated into LB Medium (10 g of
tryptone, 5 g of yeast extract, and 5 g of NaCl were dissolved in 1
L of distilled water) with the use of a platinum loop, and cultured
with shaking at 37.degree. C. until the logarithmic growth phase.
After the bacterial cells were collected, chromosomal DNA was
recovered from the collected cells with the use of a DNA extraction
kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit,
made by Amersham) according to the instruction manual.
[0094] To extract chromosomal DNA from Escherichia coli (K-12
MG1655), the bacterium was inoculated into LB Medium (10 g of
tryptone, 5 g of yeast extract, and 5 g of NaCl were dissolved in 1
L of distilled water) with the use of a platinum loop, and cultured
with shaking at 37.degree. C. until the logarithmic growth phase.
After the bacterial cells were collected, chromosomal DNA was
recovered from the collected cells with the use of a DNA extraction
kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit,
made by Amersham) according to the instruction manual.
[0095] To extract chromosomal DNA from Saccharomyces cerevisiae
NBRC10217, the bacterium was inoculated into NBRC Medium No. 108
(10 g of glucose, 5 g of polypeptone, 3 g of yeast extract, and 3 g
of malt extract were dissolved in 1 L of distilled water) with the
use of a platinum loop, and cultured with shaking at 24.degree. C.
until the logarithmic growth phase. After bacterial cells were
collected, chromosomal DNA was recovered from the collected cells
with the use of a DNA extraction kit (trade name: GenomicPrep Cells
and Tissue DNA Isolation Kit, made by Amersham) according to the
instruction manual.
[0096] To extract chromosomal DNA from Enterobacter cloacae
NBRC13535, the bacterium was inoculated into NBRC Medium No. 802
(10 g of polypeptone, 2 g of yeast extract, and 1 g of
MgSO.sub.4.7H.sub.2O were dissolved in 1 L of distilled water) with
the use of a platinum loop, and cultured with shaking at 37.degree.
C. until the logarithmic growth phase. After the bacterial cells
were collected, chromosomal DNA was recovered from the collected
cells with the use of a DNA extraction kit (trade name: GenomicPrep
Cells and Tissue DNA Isolation Kit, made by Amersham) according to
the instruction manual.
(2) Construction of Cloning Vectors
[0097] Construction of Cloning Vector pCRB22
[0098] A DNA fragment comprising a DNA replication origin sequence
of pCASE1, a plasmid derived from Corynebacterium casei JCM12072
(hereinafter abbreviated as pCASE1-ori) and a DNA fragment
comprising a cloning vector pHSG298 (made by Takara Bio, Inc.) were
amplified by the following PCR method.
[0099] In the PCR, the following sets of primers were synthesized
based on SEQ ID NO: 1 (pCASE1-ori sequence) and SEQ ID NO: 2
(cloning vector pHSG298) for cloning of the pCASE1-ori sequence and
the cloning vector pHSG298, and were used.
Primers for pCASE1-Ori Sequence Amplification
TABLE-US-00001 (SEQ ID NO: 3) (a-1); 5'-AT AGATCT
AGAACGTCCGTAGGAGC-3' (SEQ ID NO: 4) (b-1); 5'-AT AGATCT
GACTTGGTTACGATGGAC-3'
[0100] Primers (a-1) and (b-1) each have a BglII restriction enzyme
site added thereto.
Primers for Cloning Vector pHSG298 Amplification
TABLE-US-00002 (SEQ ID NO: 5) (a-2): 5'-AT AGATCT
AGGTTTCCCGACTGGAAAG-3' (SEQ ID NO: 6) (b-2): 5'-AT AGATCT
CGTGCCAGCTGCATTAATGA-3'
[0101] Primers (a-2) and (b-2) each have a BglII restriction enzyme
site added thereto.
[0102] As the template DNA, total DNA extracted from
Corynebacterium casei JCM12072 obtained from Japan Collection of
Microorganisms (JCM) and cloning vector pHSG298 (made by Takara
Bio, Inc.) were used.
[0103] Actual PCR was performed with the use of a thermal cycler,
GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA
Taq (made by Takara Bio, Inc.) as a reaction reagent under the
conditions described below.
Reaction Mixture:
TABLE-US-00003 [0104] TaKaRa LA Taq .TM. (5 units/.mu.L) 0.5 .mu.L
10X LA PCR .TM. Buffer II 5 .mu.L (Mg.sup.2+ free) 25 mM MgCl.sub.2
5 .mu.L dNTP Mixture (2.5 mM each) 8 .mu.L Template DNA 5 .mu.L
(DNA content: 1 .mu.g or less) The above 2 primers*.sup.) 0.5 .mu.L
each (final conc.: 1 .mu.M) Sterile distilled water 25.5 .mu.L The
above ingredients were mixed, and 50 .mu.L of the reaction mixture
was subjected to PCR. *.sup.)For amplification of the pCASE1-ori
sequence, a combination of primers (a-1) and (b-1), and for
amplification of the cloning vector pHSG298, a combination of
primers (a-2) and (b-2) were used.
PCR Cycle:
[0105] Denaturation step: 94.degree. C., 60 seconds
[0106] Annealing step: 52.degree. C., 60 seconds
[0107] Extension step: 72.degree. C. [0108] pCASE1-ori sequence:
150 seconds [0109] Cloning vector pHSG298: 180 seconds
[0110] A cycle consisting of the above 3 steps was repeated 30
times.
[0111] Using 10 .mu.L of the above-produced reaction mixture, 0.8%
agarose gel electrophoresis was performed. In the case of the
pCASE1-ori sequence, an about 1.4-kb DNA fragment was detected. In
the case of the cloning vector pHSG298, an about 2.7-kb DNA
fragment was detected.
[0112] 10 .mu.l, of the about 1.4-kb DNA fragment comprising the
pCASE1-ori sequence derived from Corynebacterium casei, and 10
.mu.l, of the about 2.7-kb DNA fragment comprising the cloning
vector pHSG298, both amplified by the above PCR, were each cut with
the use of restriction enzyme BglII and processed at 70.degree. C.
for 10 minutes for deactivation of the restriction enzyme. Both
were mixed, and 1 .mu.L of T4 DNA ligase 10.times. buffer solution
and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added
thereto. Sterile distilled water was added thereto so that the
total volume was 10 .mu.L, and the mixture was allowed to react at
15.degree. C. for 3 hours for ligation. This was named Ligation
Liquid A.
[0113] With the use of the Ligation Liquid A, Escherichia coli
JM109 was transformed by the calcium chloride method (Journal of
Molecular Biology, 53, 159 (1970)) and was applied to LB agar
medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride,
and 1.5% agar) containing 50 .mu.g/mL of kanamycin.
[0114] A growing strain on the culture medium was subjected to
liquid culture in the usual manner. Plasmid DNA was extracted from
the culture and cut with the use of restriction enzyme BglII to
confirm the inserted fragment. As a result, in addition to an about
2.7-kb DNA fragment of the cloning vector pHSG298, an about 1.4-kb
DNA fragment of the pCASE-ori sequence was confirmed.
[0115] The cloning vector comprising the pCASE1-ori sequence was
named pCRB22.
Construction of Cloning Vector pCRB207
[0116] A DNA fragment comprising a promoter sequence of the gapA
gene encoding the glyceraldehyde-3-phosphate dehydrogenase
(hereinafter abbreviated as PgapA) derived from Corynebacterium
glutamicum R, and a DNA fragment comprising an rrnBT1T2
bidirectional terminator sequence (hereinafter abbreviated as
terminator sequence) derived from a cloning vector pKK223-3 (made
by Pharmacia) were amplified by the following method.
[0117] In the PCR, the following sets of primers were synthesized
based on SEQ ID NO: 7 (PgapA sequence) and SEQ ID NO: 8 (terminator
sequence) for cloning of the PgapA sequence and the terminator
sequence, and were used.
Primers for PgapA Sequence Amplification
TABLE-US-00004 [0118] (SEQ ID NO: 9) (a-3); 5'-CTCT GTCGAC
CCGAAGATCTGAAGATTCCTG-3' (SEQ ID NO: 10) (b-3); 5'-CTCT GTCGAC
GGATCC CCATGG TGTGTCTCCTCTAAAGATTGTAGG-3'
[0119] Primer (a-3) has a SalI restriction enzyme site added
thereto, and primer (b-3) has SalI, BamHI, and NcoI restriction
enzyme sites added thereto.
Primers for Terminator Sequence Amplification
TABLE-US-00005 [0120] (SEQ ID NO: 11) (a-4); 5'-CTCT GCATGC CCATGG
CTGTTTTGGCGGATGAGAGA-3' (SEQ ID NO: 12) (b-4); 5'-CTCT GCATGC
TCATGA AAGAGTTTGTAGAAACGCAAAAAGG-3'
[0121] Primer (a-4) has SphI and NcoI restriction enzyme sites
added thereto, and primer (b-4) has SphI and BspHI restriction
enzyme sites added thereto.
[0122] As the template DNA, the chromosomal DNA extracted from
Corynebacterium glutamicum R (FERM P-18976) and the plasmid
pKK223-3 (made by Pharmacia) were used.
[0123] Actual PCR was performed with the use of a thermal cycler,
GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA
Taq (made by Takara Bio, Inc.) as a reaction reagent under the
conditions described below.
Reaction Mixture:
TABLE-US-00006 [0124] TaKaRa LA Taq .TM. (5 units/.mu.L) 0.5 .mu.L
10X LA PCR .TM. Buffer II 5 .mu.L (Mg.sup.2+ free) 25 mM MgCl.sub.2
5 .mu.L dNTP Mixture (2.5 mM each) 8 .mu.L Template DNA 5 .mu.L
(DNA content: 1 .mu.g or less) The above 2 primers*.sup.) 0.5 .mu.L
each (final conc.: 1 .mu.M) Sterile distilled water 25.5 .mu.L The
above ingredients were mixed, and 50 .mu.L of the reaction mixture
was subjected to PCR. *.sup.)For amplification of the PgapA
sequence, a combination of primers (a-3) and (b-3), and for
amplification of the terminator sequence, a combination of primers
(a-4) and (b-4) were used.
PCR Cycle:
[0125] Denaturation step: 94.degree. C., 60 seconds
[0126] Annealing step: 52.degree. C., 60 seconds
[0127] Extension step: 72.degree. C. [0128] PgapA sequence: 45
seconds [0129] Terminator sequence: 30 seconds
[0130] A cycle consisting of the above 3 steps was repeated 30
times.
[0131] Using 10 .mu.l, of the above-produced reaction mixture, 0.8%
agarose gel electrophoresis was performed. In the case of the PgapA
sequence, an about 0.6-kb DNA fragment was detected. In the case of
the terminator sequence, an about 0.4-kb DNA fragment was
detected.
[0132] 10 .mu.l, of the about 0.6-kb DNA fragment comprising the
PgapA sequence derived from Corynebacterium glutamicum R, which was
amplified by the above PCR, and the about 4.1-kb cloning vector
pCRB22 were each cut with the use of restriction enzyme SalI and
processed at 70.degree. C. for 10 minutes for deactivation of the
restriction enzyme. Both were mixed, and 1 .mu.L of T4 DNA ligase
10.times. buffer solution and 1 unit of T4 DNA ligase (made by
Takara Bio, Inc.) were added thereto. Sterile distilled water was
added thereto so that the total volume was 10 .mu.L, and the
mixture was allowed to react at 15.degree. C. for 3 hours for
ligation. This was named Ligation Liquid B.
[0133] With the use of the Ligation Liquid B, Escherichia coli
JM109 was transformed by the calcium chloride method (Journal of
Molecular Biology, 53, 159 (1970)) and was applied to LB agar
medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride,
and 1.5% agar) containing 50 .mu.g/mL of kanamycin.
[0134] A growing strain on the culture medium was subjected to
liquid culture in the usual manner. Plasmid DNA was extracted from
the culture and cut with the use of restriction enzyme SalI to
confirm the inserted fragment. As a result, in addition to an about
4.1-kb DNA fragment of the cloning vector pCRB22, an about 0.6-kb
DNA fragment of the PgapA sequence was confirmed.
[0135] The cloning vector comprising the PgapA sequence was named
pCRB206.
[0136] 10 .mu.L of the about 0.4-kb DNA fragment comprising the
terminator sequence derived from the plasmid pKK223-3, which was
amplified by the above PCR, was cut with the use of restriction
enzymes NcoI and BspHI, 2 .mu.L of the above cloning vector pCRB206
was cut with the use of restriction enzyme NcoI, and both were
processed at 70.degree. C. for 10 minutes for deactivation of the
restriction enzymes. Both were mixed, and 1 .mu.L of T4 DNA ligase
10.times. buffer solution and 1 unit of T4 DNA ligase (made by
Takara Bio, Inc.) were added thereto. Sterile distilled water was
added thereto so that the total volume was 10 .mu.L, and the
mixture was allowed to react at 15.degree. C. for 3 hours for
ligation. This was named Ligation Liquid C.
[0137] With the use of the Ligation Liquid C, Escherichia coli
JM109 was transformed by the calcium chloride method (Journal of
Molecular Biology, 53, 159 (1970)) and was applied to LB agar
medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride,
and 1.5% agar) containing 50 .mu.g/mL of kanamycin.
[0138] A growing strain on the culture medium was subjected to
liquid culture in the usual manner. Plasmid DNA was extracted from
the culture and cut with the use of the restriction enzyme to
confirm the inserted fragment. As a result, in addition to an about
4.7-kb DNA fragment of the cloning vector pCRB206, an about 0.4-kb
DNA fragment of the terminator sequence was confirmed.
[0139] The cloning vector comprising the rrnBT1T2 terminator
sequence was named pCRB207.
Construction of Cloning Vector pCRB209
[0140] A DNA fragment comprising a promoter sequence of the gapA
(glyceraldehyde 3-phosphate dehydrogenase A) gene (hereinafter
abbreviated as PgapA) derived from Corynebacterium glutamicum R was
amplified by the following method.
[0141] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 13 (pCRB207) for cloning of the pCRB207
sequence, and was used.
Primers for pCRB207 Sequence Amplification
TABLE-US-00007 (SEQ ID NO: 14) (a-5); 5'-CTCT CATATG
CTGTTTTGGCGGATGAGAG-3' (SEQ ID NO: 15) (b-5); 5'-CTCT CATATG
GTGTCTCCTCTAAAGATTGTAGG-3'
[0142] Primers (a-5) and (b-5) each have an NdeI restriction enzyme
site added thereto.
[0143] As the template DNA, the cloning vector pCRB207 comprising a
gapA promoter and a rrnBT1T2 terminator sequence was used.
[0144] Actual PCR was performed with the use of a thermal cycler,
GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA
Taq (made by Takara SHUZO) as a reaction reagent under the
conditions described below.
Reaction Mixture:
TABLE-US-00008 [0145] TaKaRa LA Taq .TM. (5 units/.mu.L) 0.5 .mu.L
10X LA PCR .TM. Buffer II 5 .mu.L (Mg.sup.2+ free) 25 mM MgCl.sub.2
5 .mu.L dNTP Mixture (2.5 mM each) 8 .mu.L Template DNA 5 .mu.L
(DNA content: 1 .mu.g or less) The above 2 primers*.sup.) 0.5 .mu.L
each (final conc.: 1 .mu.M) Sterile distilled water 25.5 .mu.L The
above ingredients were mixed, and 50 .mu.L of the reaction mixture
was subjected to PCR. *.sup.)For amplification of the pCRB207
sequence, a combination of primers (a-5) and (b-5) was used.
PCR Cycle:
[0146] Denaturation step: 94.degree. C., 60 seconds
[0147] Annealing step: 52.degree. C., 60 seconds
[0148] Extension step: 72.degree. C., 307 seconds
[0149] A cycle consisting of the above 3 steps was repeated 30
times.
[0150] Using 10 .mu.L of the above-produced reaction mixture, 0.8%
agarose gel electrophoresis was performed, and an about 5.1-kb DNA
fragment comprising the cloning vector pCRB207 was detected.
[0151] 10 .mu.L of the about 5.1-kb DNA fragment comprising the
gene derived from pCRB207, which was amplified by the above PCR,
was cut with the use of restriction enzyme NdeI and processed at
70.degree. C. for 10 minutes for deactivation of the restriction
enzyme. To this, 1 .mu.L of T4 DNA ligase 10.times. buffer solution
and 1 unit of T4 DNA ligase (made by Takara SHUZO) were added.
Sterile distilled water was added thereto so that the total volume
was 10 .mu.L, and the mixture was allowed to react at 15.degree. C.
for 3 hours for ligation. This was named Ligation Liquid D.
[0152] With the use of the Ligation Liquid D, Escherichia coli
JM109 was transformed by the calcium chloride method (Journal of
Molecular Biology, 53, 159 (1970)) and was applied to LB agar
medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride,
and 1.5% agar) containing 50 .mu.g/mL of kanamycin.
[0153] A growing strain on the culture medium was subjected to
liquid culture in the usual manner. Plasmid DNA was extracted from
the culture and cut with the use of restriction enzyme NdeI to
confirm the inserted restriction enzyme site.
[0154] The cloning vector comprising the PgapA sequence and the
rrnBT1T2 terminator sequence was named pCRB209.
(3) Cloning of Aniline-Producing Genes
[0155] Cloning of Aniline-Producing Gene Derived from Bacillus
subtilis
[0156] A DNA fragment comprising the bsdBCD (hereinafter indicated
as dec/BS) gene which encodes aminobenzoate decarboxylase derived
from Bacillus subtilis was amplified by the PCR method as described
below.
[0157] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 16 (the dec/BS gene of Bacillus subtilis) with
the use of "394 DNA/RNA Synthesizer" made by Applied Biosystems for
cloning of the dec/BS gene, and was used.
Primers for dec/BS Gene Amplification
TABLE-US-00009 (SEQ ID NO: 17) (a-6); 5'-CTCT CATATG
AAAGCAGAATTCAAGCGTAAAG-3' (SEQ ID NO: 18) (b-6); 5'-CTCT CATATG
GATCAAGCCTTTCGTTCCG-3'
[0158] Primers (a-6) and (b-6) each have an NdeI restriction enzyme
site added thereto.
Cloning of Aniline-Producing Gene Derived from Lactobacillus
rhamnosus
[0159] A DNA fragment comprising the ubiDX (hereinafter indicated
as dec/LR) gene which encodes aminobenzoate decarboxylase derived
from Lactobacillus rhamnosus was amplified by the PCR method as
described below.
[0160] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 19 (the dec/LR gene of Lactobacillus rhamnosus)
with the use of "394 DNA/RNA Synthesizer" made by Applied
Biosystems for cloning of the dec/LR gene, and was used.
Primers for dec/LR Gene Amplification
TABLE-US-00010 (SEQ ID NO: 20) (a-7); 5'-CTCT CATATG
ACAGCATCACCTTGGG-3' (SEQ ID NO: 21) (b-7); 5'-CTCT CATATG
TCATCTTAACGACGCTCCATTC-3'
[0161] Primers (a-7) and (b-7) each have an NdeI restriction enzyme
site added thereto.
Cloning of Aniline-Producing Gene Derived from Lactobacillus
brevis
[0162] A DNA fragment comprising the
LVIS.sub.--1987-LVIS.sub.--1986 (hereinafter indicated as dec/LB)
gene which encodes aminobenzoate decarboxylase derived from
Lactobacillus brevis was amplified by the PCR method as described
below.
[0163] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 22 (the dec/LB gene of Lactobacillus brevis)
with the use of "394 DNA/RNA Synthesizer" made by Applied
Biosystems for cloning of the dec/LB gene, and was used.
Primers for dec/LB Gene Amplification
TABLE-US-00011 (SEQ ID NO: 23) (a-8); 5'-CTCT CATATG
GTAAATGATCCTTATGATTTACGAAAAG-3' (SEQ ID NO: 24) (b-8); 5'-CTCT
CATATG CTAATCTCCCTCCCAACG-3'
[0164] Primers (a-8) and (b-8) each have an NdeI restriction enzyme
site added thereto.
Cloning of Aniline-Producing Gene Derived from Pseudomonas
putida
[0165] A DNA fragment comprising the ubiD (hereinafter indicated as
dec/PP) gene which encodes aminobenzoate decarboxylase derived from
Pseudomonas putida was amplified by the PCR method as described
below.
[0166] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 25 (the dec/PP gene of Pseudomonas putida) with
the use of "394 DNA/RNA Synthesizer" made by Applied Biosystems for
cloning of the dec/PP gene, and was used.
Primers for dec/PP Gene Amplification
TABLE-US-00012 (SEQ ID NO: 26) (a-9); 5'-CTCT CATATG
AACGGGCCGGAAC-3' (SEQ ID NO: 27) (b-9); 5'-CTCT CATATG
TCAATCATCCACCCCGAAG-3'
[0167] Primers (a-9) and (b-9) each have an NdeI restriction enzyme
site added thereto.
Cloning of Aniline-Producing Gene Derived from Escherichia coli
[0168] A DNA fragment comprising the purEK (hereinafter indicated
as dec/EC) gene which encodes aminobenzoate decarboxylase derived
from Escherichia coli was amplified by the PCR method as described
below.
[0169] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 28 (the dec/EC gene of Escherichia coli) with
the use of "394 DNA/RNA Synthesizer" made by Applied Biosystems for
cloning of the dec/EC gene, and was used.
Primers for dec/EC Gene Amplification
TABLE-US-00013 (SEQ ID NO: 29) (a-10); 5'-CTCT CATATG
TCTTCCCGCAATAATCCG-3' (SEQ ID NO: 30) (b-10); 5'-CTCT CATATG
TTAACCGAACTTACTCTGCGC-3'
[0170] Primers (a-10) and (b-10) each have an NdeI restriction
enzyme site added thereto.
Cloning of Aniline-Producing Gene Derived from Saccharomyces
cerevisiae
[0171] A DNA fragment comprising the ADE2 (hereinafter indicated as
dec/SC) gene which encodes aminobenzoate decarboxylase derived from
Saccharomyces cerevisiae was amplified by the PCR method as
described below.
[0172] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 31 (the dec/SC gene of Saccharomyces
cerevisiae) with use of "394 DNA/RNA Synthesizer" made by Applied
Biosystems for cloning of the dec/SC gene, and was used.
Primers for dec/SC Gene Amplification
TABLE-US-00014 (SEQ ID NO: 32) (a-11); 5'-CTCT CCATGG
ATTCTAGAACAGTTGGTATATTAG-3' (SEQ ID NO: 33) (b-11); 5'-CTCT CCATGG
TTACTTGTTTTCTAGATAAGCTTCGTAAC-3'
[0173] Primers (a-11) and (b-11) each have an NcoI restriction
enzyme site added thereto.
Cloning of Aniline-Producing Gene Derived from Enterobacter
cloacae
[0174] A DNA fragment comprising the
ECL.sub.--04083-ECL.sub.--04082-ECL.sub.--04081 (hereinafter
indicated as dec/ECL) gene which encodes aminobenzoate
decarboxylase derived from Enterobacter cloacae was amplified by
the PCR method as described below.
[0175] In the PCR, the following set of primers was synthesized
based on SEQ ID NO: 34 (the dec/ECL gene of Enterobacter cloacae)
with the use of "394 DNA/RNA Synthesizer" made by Applied
Biosystems for cloning of the dec/ECL gene, and was used.
Primers for dec/ECL Gene Amplification
TABLE-US-00015 (SEQ ID NO: 35) (a-12); 5'-CTCT CATATG
AGATTGATCGTGGGAATGAC-3' (SEQ ID NO: 36) (b-12); 5'-CTCT CATATG
TTACAGCAATGGCGGAATGG-3'
[0176] Primers (a-12) and (b-12) each have an NdeI restriction
enzyme site added thereto.
[0177] As the template DNA for Bacillus subtilis, the chromosomal
DNA extracted from Bacillus subtilis NBRC14144 obtained from NITE
Biological Resource Center (NBRC) was used.
[0178] For Lactobacillus rhamnosus, the chromosomal DNA extracted
from Lactobacillus rhamnosus NBRC3425 obtained from NITE Biological
Resource Center (NBRC) was used.
[0179] For Lactobacillus brevis, the chromosomal DNA extracted from
Lactobacillus brevis ATCC367 obtained from American Type Culture
Collection (ATCC) was used.
[0180] For Pseudomonas putida, the chromosomal DNA extracted from
Pseudomonas putida ATCC47054 obtained from American Type Culture
Collection (ATCC) was used.
[0181] For Escherichia coli, the chromosomal DNA extracted from
Escherichia coli K-12 MG1655 was used.
[0182] For Saccharomyces cerevisiae, the chromosomal DNA extracted
from Saccharomyces cerevisiae NBRC10217 obtained from NITE
Biological Resource Center (NBRC) was used.
[0183] For Enterobacter cloacae, the chromosomal DNA extracted from
Enterobacter cloacae NBRC13535 obtained from NITE Biological
Resource Center (NBRC) was used.
[0184] Actual PCR was performed with the use of a thermal cycler,
GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA
Tag (made by Takara Bio, Inc.) as a reaction reagent under the
conditions described below.
Reaction Mixture:
TABLE-US-00016 [0185] TaKaRa LA Taq .TM. (5 units/.mu.L) 0.5 .mu.L
10X LA PCR .TM. Buffer II 5 .mu.L (Mg.sup.2+ free) 25 mM MgCl.sub.2
5 .mu.L dNTP Mixture (2.5 mM each) 8 .mu.L Template DNA 5 .mu.L
(DNA content: 1 .mu.g or less) The above 2 primers*.sup.) 0.5 .mu.L
each (final conc.: 1 .mu.M) Sterile distilled water 25.5 .mu.L The
above ingredients were mixed, and 50 .mu.L of the reaction mixture
was subjected to PCR. *.sup.)For amplification of the dec/BS gene
of Bacillus subtilis, a combination of primers (a-6) and (b-6); for
amplification of the dec/LR gene of Lactobacillus rhamnosus, a
combination of primers (a-7) and (b-7); for amplification of the
dec/LB gene of Lactobacillus brevis, a combination of primers (a-8)
and (b-8); for amplification of the dec/PP gene of Pseudomonas
putida, a combination of primers (a-9) and (b-9); for amplification
of the dec/EC gene of Escherichia coli, a combination of primers
(a-10) and (b-10); for amplification of the dec/SC gene of
Saccharomyces cervisiae, a combination of primers (a-11) and
(b-11); and for amplification of the dec/ECL gene of Enterobacter
cloacae, a combination of primers (a-12) and (b-12) were used.
PCR Cycle:
[0186] Denaturation step: 94.degree. C., 60 seconds Annealing step:
52.degree. C., 60 seconds Extension step: 72.degree. C.
TABLE-US-00017 Bacillus subtilis dec/BS gene 137 seconds
Lactobacillus rhamnosus dec/LR gene 123 seconds Lactobacillus
brevis dec/LB gene 123 seconds Pseudomonas putida dec/PP gene 45
seconds Escherichia coli dec/EC gene 94 seconds Saccharomyces
cervisiae dec/SC gene 103 seconds Enterobacter cloacae dec/ECL gene
135 seconds
[0187] A cycle consisting of the above 3 steps was repeated 30
times.
[0188] With the use of 10 .mu.l of the reaction mixture produced
above, 0.8% agarose gel electrophoresis was performed. As a result,
detected were an about 2.3-kb DNA fragment in the case of the
Bacillus subtilis dec/BS gene, an about 2.1-kb DNA fragment in the
case of the Lactobacillus rhamnosus dec/LR gene, an about 2.0-kb
DNA fragment in the case of the Lactobacillus brevis dec/LB gene,
an about 0.6-kb DNA fragment in the case of the Pseudomonas putida
dec/PP gene, an about 1.6-kb DNA fragment in the case of the
Escherichia coli dec/EC gene, an about 1.7-kb DNA fragment in the
case of the Saccharomyces cerevisiae dec/SC gene, and an about
2.3-kb DNA fragment in the case of the Enterobacter cloacae dec/ECL
gene.
(4) Construction of Aniline-Producing Gene Expression Plasmids
[0189] Cloning of Aniline-Producing Gene to pCRB207 10 .mu.L of the
about 1.7-kb DNA fragment comprising the dec/SC gene derived from
Saccharomyces cerevisiae amplified by the PCR in the above (3) and
2 .mu.L of the cloning vector pCRB207 comprising a promoter PgapA
were each cut with the use of restriction enzyme NcoI and processed
at 70.degree. C. for 10 minutes for deactivation of the restriction
enzyme. Both were mixed, and 1 .mu.L of T4 DNA ligase 10.times.
buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio,
Inc.) were added thereto. Sterile distilled water was added thereto
so that the total volume was 10 .mu.L, and the mixture was allowed
to react at 15.degree. C. for 3 hours for ligation. This was named
Ligation Liquid E.
[0190] With the use of the Ligation Liquid E, Escherichia coli
JM109 was transformed by the calcium chloride method (Journal of
Molecular Biology, 53, 159 (1970)) and was applied to LB agar
medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride,
and 1.5% agar) containing 50 .mu.g/mL of kanamycin.
[0191] A growing strain on the culture medium was subjected to
liquid culture in the usual manner. Plasmid DNA was extracted from
the culture medium and cut with the use of the restriction enzyme
to confirm the inserted fragment. As a result, in addition to an
about 5.1-kb DNA fragment of the plasmid pCRB207, an about 1.7-kb
inserted fragment of the dec/SC gene derived from Saccharomyces
cerevisiae (Ligation Liquid E) was confirmed.
[0192] The plasmid comprising the dec/SC gene derived from
Saccharomyces cerevisiae was named pCRB207-dec/SC (FIG. 1).
Cloning of Aniline-Producing Genes to pCRB209
[0193] 10 .mu.L of the about 2.3-kb DNA fragment comprising the
dec/BS gene derived from Bacillus subtilis, the about 2.1-kb DNA
fragment comprising the dec/LR gene derived from Lactobacillus
rhamnosus, the about 2.0-kb DNA fragment comprising the dec/LB gene
derived from Lactobacillus brevis, the about 0.6-kb DNA fragment
comprising the dec/PP gene derived from Pseudomonas putida, the
about 1.6-kb DNA fragment comprising the dec/EC gene derived from
Escherichia coli, or the about 2.3-kb DNA fragment comprising the
dec/ECL gene derived from Enterobacter cloacae amplified by the PCR
in the above (3) and 2 .mu.L of the cloning vector pCRB209
comprising a promoter PgapA were each cut with the use of
restriction enzyme NdeI and processed at 70.degree. C. for 10
minutes for deactivation of the restriction enzyme. Both were
mixed, and 1 .mu.L of T4 DNA ligase 10.times. buffer solution and 1
unit of T4 DNA ligase (made by Takara Bio, Inc.) were added
thereto. Sterile distilled water was added thereto so that the
total volume was 10 .mu.L, and the mixture was allowed to react at
15.degree. C. for 3 hours for ligation. The resulting liquid was
named Ligation Liquid F, G, H, I, J, or K.
[0194] With the use of each of the obtained 6 kinds of Ligation
Liquids F, G, H, I, J, and K, Escherichia coli JM109 was
transformed by the calcium chloride method (Journal of Molecular
Biology, 53, 159 (1970)) and was applied to LB agar medium (1%
polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5%
agar) containing 50 .mu.g/mL of kanamycin.
[0195] A growing strain on the culture medium was subjected to
liquid culture in the usual manner. Plasmid DNA was extracted from
the culture and cut with the use of restriction enzyme to confirm
the inserted fragment. As a result, in addition to an about 5.1-kb
DNA fragment of the plasmid pCRB209, confirmed were an about 2.3-kb
inserted fragment in the case of the dec/BS gene derived from
Bacillus subtilis (Ligation Liquid F), an about 2.1-kb inserted
fragment in the case of the dec/LR derived from Lactobacillus
rhamnosus (Ligation Liquid G), an about 2.0-kb inserted fragment in
the case of the dec/LB gene derived from Lactobacillus brevis
(Ligation Liquid H), an about 0.6-kb inserted fragment in the case
of the dec/PP gene derived from Pseudomonas putida (Ligation Liquid
I), an about 1.6-kb inserted fragment in the case of the dec/EC
gene derived from Escherichia coli (Ligation Liquid J), and an
about 2.3-kb inserted fragment in the case of the dec/ECL gene
derived from Enterobacter cloacae (Ligation Liquid K).
[0196] The plasmid comprising the dec/BS gene derived from Bacillus
subtilis was named pCRB209-dec/BS, the plasmid comprising the
dec/LR gene derived from Lactobacillus rhamnosus was named
pCRB209-dec/LR, the plasmid comprising the dec/LB gene derived from
Lactobacillus brevis was named pCRB209-dec/LB, the plasmid
comprising the dec/PP gene derived from Pseudomonas putida was
named pCRB209-dec/PP, the plasmid comprising the dec/EC gene
derived from Escherichia coli was named pCRB209-dec/EC, and the
plasmid comprising the dec/ECL gene derived from Enterobacter
cloacae was named pCRB209-dec/ECL (FIG. 1).
(5) Construction of Transgenic Strains for Aniline-Producing
Gene
[0197] With the use of the above-described 7 kinds of plasmids
pCRB209-dec/BS, pCRB209-dec/LR, pCRB209-dec/LB, pCRB209-dec/PP,
pCRB209-dec/EC, pCRB207-dec/SC, and pCRB209-dec/ECL, transformation
of Corynebacterium glutamicum R was performed by electroporation
(Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol.,
Vol. 144, 181-185 (1993)), and each strain was applied to A agar
medium containing 50 .mu.g/mL of kanamycin.
[0198] A growing strain on the culture medium was subjected to
liquid culture in the usual manner. Plasmid DNA was extracted from
the culture and cut with the use of restriction enzyme to confirm
the inserted plasmid. As a result, introduction of the
above-constructed plasmids pCRB209-dec/BS, pCRB209-dec/LR,
pCRB209-dec/LB, pCRB209-dec/PP, pCRB209-dec/EC, pCRB207-dec/SC, and
pCRB209-dec/ECL was confirmed.
[0199] The strain to which the plasmid pCRB209-dec/BS had been
introduced was named Corynebacterium glutamicum ANI-1, the strain
to which the plasmid pCRB209-dec/LR had been introduced was named
Corynebacterium glutamicum ANI-2, the strain to which the plasmid
pCRB209-dec/LB had been introduced was named Corynebacterium
glutamicum ANI-3, the strain to which the plasmid pCRB209-dec/PP
had been introduced was named Corynebacterium glutamicum ANI-4, the
strain to which the plasmid pCRB209-dec/EC had been introduced was
named Corynebacterium glutamicum ANI-5, the strain to which the
plasmid pCRB207-dec/SC had been introduced was named
Corynebacterium glutamicum ANI-6, and the strain to which the
plasmid pCRB209-dec/ECL had been introduced was named
Corynebacterium glutamicum ANI-7.
[0200] Corynebacterium glutamicum ANI-1 was deposited in
Incorporated Administrative Agency National Institute of Technology
and Evaluation, NITE Patent Microorganisms Depositary (2-5-8
Kazusakamatari, Kisarazu-shi, Chiba 292-0818 Japan) under Accession
Number NITE BP-1001 on Nov. 16, 2010.
Example 2
Experiment of Aniline Production from Anthranilic Acid Using
Corynebacterium glutamicum Aniline-Producing Gene Transgenic
Strains
[0201] Each of the Corynebacterium glutamicum ANI-1 to ANI-7
strains constructed in Example 1 was applied to A agar medium (2 g
of (NH.sub.2).sub.2CO, 7 g of (NH.sub.4).sub.2SO.sub.4, 0.5 g of
KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4, 0.5 g of
MgSO.sub.4.7H.sub.2O, 1 mL of 0.06% (w/v)
Fe.sub.2SO.sub.4.7H.sub.2O+0.042% (w/v) MnSO.sub.4.2H.sub.2O, 1 mL
of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin
solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid,
40 g of glucose, and 15 g of agar were suspended in 1 L of
distilled water) containing 50 .mu.g/mL of kanamycin, and left
stand in the dark at 33.degree. C. for 20 hours.
[0202] An inoculation loop of each of the Corynebacterium
glutamicum ANI-1 to ANI-7 strains grown on a plate as above was
inoculated into a test tube containing 10 mL of A liquid medium
containing 50 .mu.g/mL of kanamycin, and aerobically cultured with
shaking at 33.degree. C. for 20 hours. The bacterial cells of each
strain cultured and grown as above were collected by centrifugation
(15,000.times.g at 4.degree. C. for 10 minutes). The obtained
bacterial cells were washed twice with 10 mL of BT liquid medium (2
g of (NH.sub.2).sub.2CO, 7 g of (NH.sub.4).sub.2SO.sub.4, 0.5 g of
KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4, 0.5 g of
MgSO.sub.4.7H.sub.2O, 1 mL of 0.06% (w/v)
Fe.sub.2SO.sub.4.7H.sub.2O+0.042% (w/v) MnSO.sub.4.2H.sub.2O, 1 mL
of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin
solution) and then suspended in the BT liquid medium in such a way
that the bacterial cell concentration would be OD.sub.610=10. To a
15-mL centrifuge tube, the cell suspension was transferred,
anthranilic acid as a substrate was added so as to be 25 mM in
concentration, and the reaction was allowed to proceed under
reducing conditions (the ORP of the reaction mixture: -450 mV) in a
water bath kept at 33.degree. C. with stirring for 6 hours. A
sample of the reaction mixture was centrifuged (15,000.times.g at
4.degree. C. for 10 minutes), and the obtained supernatant was used
for quantitative determination of aniline by GC/MS.
[0203] As a result, in the reaction under reducing conditions, the
Corynebacterium glutamicum ANI-1 to ANI-7 strains had produced
aniline as shown in Table 1 below.
TABLE-US-00018 TABLE 1 Experiment of aniline production of
Corynebacterium glutamicum ANI-1 to ANI-7 strains with use of
anthranilic acid as a substrate Amount of aniline Origin of
aminobenzoate production Strain Host strain decarboxylase gene (mM)
ANI-1 Corynebacterium Bacillus subtilis 0.75 ANI-2 glutamicum
Lactobacillus rhamnosus 0.7 ANI-3 (Wild strain) Lactobacillus
brevis 0.6 ANI-4 Pseudomonas putida 0.6 ANI-5 Escherichia coli 0.5
ANI-6 Saccharomyces cerevisiae 0.5 ANI-7 Enterobacter cloacae
0.5
[0204] Without the addition of kanamycin to the culture medium, the
same experiment as above was conducted using Corynebacterium
glutamicum wild strain. In this case, aniline production was not
observed.
Example 3
Experiment of Aniline Production from 4-Aminobenzoate Using
Corynebacterium glutamicum Aniline-Producing Gene Transgenic
Strains
[0205] Each of the Corynebacterium glutamicum ANI-1 to ANI-7
strains constructed in Example 1 was applied to A agar medium (2 g
of (NH.sub.2).sub.2CO, 7 g of (NH.sub.4).sub.2SO.sub.4, 0.5 g of
KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4, 0.5 g of
MgSO.sub.4.7H.sub.2O, 1 mL of 0.06% (w/v)
Fe.sub.2SO.sub.4.7H.sub.2O+0.042% (w/v) MnSO.sub.4.2H.sub.2O, 1 mL
of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin
solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid,
40 g of glucose, and 15 g of agar were suspended in 1 L of
distilled water) containing 50 .mu.g/mL of kanamycin, and left
stand in the dark at 33.degree. C. for 20 hours.
[0206] An inoculation loop of each of the Corynebacterium
glutamicum ANI-1 to ANI-7 strains grown on a plate as above was
inoculated into a test tube containing 10 mL of A liquid medium
containing 50 .mu.g/mL of kanamycin, and aerobically cultured with
shaking at 33.degree. C. for 20 hours. The bacterial cells of each
strain cultured and grown as above were collected by centrifugation
(15,000.times.g at 4.degree. C. for 10 minutes). The obtained
bacterial cells were washed twice with 10 mL of BT liquid medium (2
g of (NH.sub.2).sub.2CO, 7 g of (NH.sub.4).sub.2SO.sub.4, 0.5 g of
KH.sub.2PO.sub.4, 0.5 g of K.sub.2HPO.sub.4, 0.5 g of
MgSO.sub.4.7H.sub.2O, 1 mL of 0.06% (w/v)
Fe.sub.2SO.sub.4.7H.sub.2O+0.042% (w/v) MnSO.sub.4.2H.sub.2O, 1 mL
of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin
solution) and then suspended in BT liquid medium in such a way that
the bacterial cell concentration would be OD.sub.610=10. To a 15-mL
centrifuge tube, the cell suspension was transferred,
4-aminobenzoate as a substrate was added so as to be 5 mM in
concentration, and the reaction was allowed to proceed under
reducing conditions (the ORP of the reaction mixture: -450 mV) in a
water bath kept at 33.degree. C. with stirring for 6 hours. A
sample of the reaction mixture was centrifuged (15,000.times.g at
4.degree. C. for 10 minutes), and the obtained supernatant was used
for quantitative determination of aniline by GC/MS.
[0207] As a result, in the reaction under reducing conditions, the
Corynebacterium glutamicum ANI-1 to ANI-7 strains had produced
aniline as shown in Table 2 below.
TABLE-US-00019 TABLE 2 Experiment of aniline production of
Corynebacterium glutamicum ANI-1 to ANI-7 strains with use of
4-aminobenzoate as a substrate Amount of aniline Origin of
aminobenzoate production Strain Host strain decarboxylase gene (mM)
ANI-1 Corynebacterium Bacillus subtilis 0.7 ANI-2 glutamicum
Lactobacillus rhamnosus 0.65 ANI-3 (Wild strain) Lactobacillus
brevis 0.6 ANI-4 Pseudomonas putida 0.6 ANI-5 Escherichia coli 0.5
ANI-6 Saccharomyces cerevisiae 0.5 ANI-7 Enterobacter cloacae
1.25
[0208] Without the addition of kanamycin to the culture medium, the
same experiment as above was conducted using Corynebacterium
glutamicum wild strain. In this case, aniline production was not
observed.
INDUSTRIAL APPLICABILITY
[0209] According to the process of the present invention, aniline
can be produced from aminobenzoic acid with a practical efficiency
using microorganisms.
Sequence CWU 1
1
3611195DNAArtificial sequencepCASE1-ori 1atgaaaaccg accgtgcacg
ctcgtgtgag aaagtcagct acatgagacc aactacccgc 60cctgagggac gctttgagca
gctgtggctg ccgctgtggc cattggcaag cgatgacctc 120cgtgagggca
tttaccgcac ctcacggaag aacgcgctgg ataagcgcta cgtcgaagcc
180aatcccgacg cgctctctaa cctcctggtc gttgacatcg accaggagga
cgcgcttttg 240cgctctttgt gggacaggga ggactggaga cctaacgcgg
tggttgaaaa ccccttaaac 300gggcacgcac acgctgtctg ggcgctcgcg
gagccattta cccgcaccga atacgccaaa 360cgcaagcctt tggcctatgc
cgcggctgtc accgaaggcc tacggcgctc tgtcgatggc 420gatagcggat
actccgggct gatcaccaaa aaccccgagc acactgcatg ggatagtcac
480tggatcaccg ataagctgta tacgctcgat gagctgcgct tttggctcga
agaaaccggc 540tttatgccgc ctgcgtcctg gaggaaaacg cggcggttct
cgccagttgg tctaggtcgt 600aattgcgcac tctttgaaag cgcacgtacg
tgggcatatc gggaggtcag aaagcatttt 660ggagacgctg acggcctagg
ccgcgcaatc caaaccaccg cgcaagcact taaccaagag 720ctgtttgatg
aaccactacc tgtggccgaa gttgactgta ttgccaggtc aatccataaa
780tggatcatca ccaagtcacg catgtggaca gacggcgccg ccgtctacga
cgccacattc 840accgcaatgc aatccgcacg cgggaagaaa ggctggcaac
gaagcgctga ggtgcgtcgt 900gaggctggac atactctttg gaggaacatt
ggctaaggtt tatgcacgtt atccacgcaa 960cggaaaaaca gcccgcgagc
tggcagaacg tgccggtatg tcggtgagaa cagctcaacg 1020atggacttcc
gaaccgcgtg aagtgttcat taaacgtgcc aacgagaagc gtgctcgcgt
1080ccaggagctg cgcgccaaag gtctgtccat gcgcgctatc gcggcagaga
ttggttgctc 1140ggtgggcacg gttcaccgct acgtcaaaga agttgaagag
aagaaaaccg cgtaa 119522675DNAArtificial sequencepHSG298 2gaggtctgcc
tcgtgaagaa ggtgttgctg actcatacca ggcctgaatc gccccatcat 60ccagccagaa
agtgagggag ccacggttga tgagagcttt gttgtaggtg gaccagttgg
120tgattttgaa cttttgcttt gccacggaac ggtctgcgtt gtcgggaaga
tgcgtgatct 180gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag
ccacgttgtg tctcaaaatc 240tctgatgtta cattgcacaa gataaaaata
tatcatcatg aacaataaaa ctgtctgctt 300acataaacag taatacaagg
ggtgttatga gccatattca acgggaaacg tcttgctcga 360agccgcgatt
aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
420atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
gcgccagagt 480tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
tacagatgag atggtcagac 540taaactggct gacggaattt atgcctcttc
cgaccatcaa gcattttatc cgtactcctg 600atgatgcatg gttactcacc
actgcgatcc ccgggaaaac agcattccag gtattagaag 660aatatcctga
ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
720attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
ctcgctcagg 780cgcaatcacg aatgaataac ggtttggttg atgcgagtga
ttttgatgac gagcgtaatg 840gctggcctgt tgaacaagtc tggaaagaaa
tgcataagct tttgccattc tcaccggatt 900cagtcgtcac tcatggtgat
ttctcacttg ataaccttat ttttgacgag gggaaattaa 960taggttgtat
tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
1020tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
caaaaatatg 1080gtattgataa tcctgatatg aataaattgc agtttcattt
gatgctcgat gagtttttct 1140aatcagaatt ggttaattgg ttgtaacact
ggcagagcat tacgctgact tgacgggacg 1200gcggctttgt tgaataaatc
gcattcgcca ttcaggctgc gcaactgttg ggaagggcga 1260tcggtgcggg
cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
1320ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
ggccagtgcc 1380aagcttgcat gcctgcaggt cgactctaga ggatccccgg
gtaccgagct cgaattcgta 1440atcatgtcat agctgtttcc tgtgtgaaat
tgttatccgc tcacaattcc acacaacata 1500cgagccggaa gcataaagtg
taaagcctgg ggtgcctaat gagtgagcta actcacatta 1560attgcgttgc
gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
1620tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg gcgaactttt
gctgagttga 1680aggatcagat cacgcatctt cccgacaacg cagaccgttc
cgtggcaaag caaaagttca 1740aaatcagtaa ccgtcagtgc cgataagttc
aaagttaaac ctggtgttga taccaacatt 1800gaaacgctga tcgaaaacgc
gctgaaaaac gctgctgaat gtgcgagctt cttccgcttc 1860ctcgctcact
gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
1920aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
acatgtgagc 1980aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
ttgctggcgt ttttccatag 2040gctccgcccc cctgacgagc atcacaaaaa
tcgacgctca agtcagaggt ggcgaaaccc 2100gacaggacta taaagatacc
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2160tccgaccctg
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
2220ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
ccaagctggg 2280ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
ttatccggta actatcgtct 2340tgagtccaac ccggtaagac acgacttatc
gccactggca gcagccactg gtaacaggat 2400tagcagagcg aggtatgtag
gcggtgctac agagttcttg aagtggtggc ctaactacgg 2460ctacactaga
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
2520aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
gtttttttgt 2580ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
gaagatcctt tgatcttttc 2640tacggggtct gacgctcagt ggaacgatcc gtcga
2675325DNAArtificial sequencePCR primer 3atagatctag aacgtccgta
ggagc 25426DNAArtificial sequencePCR primer 4atagatctga cttggttacg
atggac 26527DNAArtificial sequencePCR primer 5atagatctag gtttcccgac
tggaaag 27628DNAArtificial sequencePCR primer 6atagatctcg
tgccagctgc attaatga 287551DNACorynebacterium glutamicum 7ccgaagatct
gaagattcct gatacaaatt ctgttgtgac ggaagatttg ttggaagaaa 60tctagtcgct
cgtctcataa aaacgaccga gcctattggg attaccattg aagccagtgt
120gagttgcatc acactggctt caaatctgag actttacttt gtggattcac
gggggtgtag 180tgcaattcat aattagcccc attcggggga gcagatcgcg
gcgcgaacga tttcaggttc 240gttccctgca aaaactattt agcgcaagtg
ttggaaatgc ccccgtctgg ggtcaatgtc 300tatttttgaa tgtgtttgta
tgattttgaa tccgctgcaa aatctttgtt tccccgctaa 360agttggggac
aggttgacac ggagttgact cgacgaatta tccaatgtga gtaggtttgg
420tgcgtgagtt ggaaaatttc gccatactcg cccttgggtt ctgtcagctc
aagaattctt 480gagtgaccga tgctctgatt gacctaactg cttgacacat
tgcatttcct acaatcttta 540gaggagacac a 5518425DNAArtificial
sequencerrnBT1T2 terminator 8ctgttttggc ggatgagaga agattttcag
cctgatacag attaaatcag aacgcagaag 60cggtctgata aaacagaatt tgcctggcgg
cagtagcgcg gtggtcccac ctgaccccat 120gccgaactca gaagtgaaac
gccgtagcgc cgatggtagt gtggggtctc cccatgcgag 180agtagggaac
tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
240gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
ccgggagcgg 300atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc
aggacgcccg ccataaactg 360ccaggcatca aattaagcag aaggccatcc
tgacggatgg cctttttgcg tttctacaaa 420ctctt 425931DNAArtificial
sequencePCR primer 9ctctgtcgac ccgaagatct gaagattcct g
311046DNAArtificial sequencePCR primer 10ctctgtcgac ggatccccat
ggtgtgtctc ctctaaagat tgtagg 461136DNAArtificial sequencePCR primer
11ctctgcatgc ccatggctgt tttggcggat gagaga 361241DNAArtificial
sequencePCR primer 12ctctgcatgc tcatgaaaga gtttgtagaa acgcaaaaag g
41135118DNAArtificial sequencepCRB207 13agatctaggt ttcccgactg
gaaagcgggc agtgagcgca acgcaattaa tgtgagttag 60ctcactcatt aggcacccca
ggctttacac tttatgcttc cggctcgtat gttgtgtgga 120attgtgagcg
gataacaatt tcacacagga aacagctatg accatgatta cgaattcgag
180ctcggtaccc ggggatcctc tagagtcgac ccgaagatct gaagattcct
gatacaaatt 240ctgttgtgac ggaagatttg ttggaagaaa tctagtcgct
cgtctcataa aaacgaccga 300gcctattggg attaccattg aagccagtgt
gagttgcatc acactggctt caaatctgag 360actttacttt gtggattcac
gggggtgtag tgcaattcat aattagcccc attcggggga 420gcagatcgcg
gcgcgaacga tttcaggttc gttccctgca aaaactattt agcgcaagtg
480ttggaaatgc ccccgtctgg ggtcaatgtc tatttttgaa tgtgtttgta
tgattttgaa 540tccgctgcaa aatctttgtt tccccgctaa agttggggac
aggttgacac ggagttgact 600cgacgaatta tccaatgtga gtaggtttgg
tgcgtgagtt ggaaaatttc gccatactcg 660cccttgggtt ctgtcagctc
aagaattctt gagtgaccga tgctctgatt gacctaactg 720cttgacacat
tgcatttcct acaatcttta gaggagacac accatggctg ttttggcgga
780tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
tctgataaaa 840cagaatttgc ctggcggcag tagcgcggtg gtcccacctg
accccatgcc gaactcagaa 900gtgaaacgcc gtagcgccga tggtagtgtg
gggtctcccc atgcgagagt agggaactgc 960caggcatcaa ataaaacgaa
aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 1020tttgtcggtg
aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc
1080gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
ggcatcaaat 1140taagcagaag gccatcctga cggatggcct ttttgcgttt
ctacaaactc tttcatgggg 1200atccgtcgac ctgcaggcat gcaagcttgg
cactggccgt cgttttacaa cgtcgtgact 1260gggaaaaccc tggcgttacc
caacttaatc gccttgcagc acatccccct ttcgccagct 1320ggcgtaatag
cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
1380gcgaatgcga tttattcaac aaagccgccg tcccgtcaag tcagcgtaat
gctctgccag 1440tgttacaacc aattaaccaa ttctgattag aaaaactcat
cgagcatcaa atgaaactgc 1500aatttattca tatcaggatt atcaatacca
tatttttgaa aaagccgttt ctgtaatgaa 1560ggagaaaact caccgaggca
gttccatagg atggcaagat cctggtatcg gtctgcgatt 1620ccgactcgtc
caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
1680agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
cttatgcatt 1740tctttccaga cttgttcaac aggccagcca ttacgctcgt
catcaaaatc actcgcatca 1800accaaaccgt tattcattcg tgattgcgcc
tgagcgagac gaaatacgcg atcgctgtta 1860aaaggacaat tacaaacagg
aatcgaatgc aaccggcgca ggaacactgc cagcgcatca 1920acaatatttt
cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
1980atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
gatggtcgga 2040agaggcataa attccgtcag ccagtttagt ctgaccatct
catctgtaac atcattggca 2100acgctacctt tgccatgttt cagaaacaac
tctggcgcat cgggcttccc atacaatcga 2160tagattgtcg cacctgattg
cccgacatta tcgcgagccc atttataccc atataaatca 2220gcatccatgt
tggaatttaa tcgcggcttc gagcaagacg tttcccgttg aatatggctc
2280ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
tgatgatata 2340tttttatctt gtgcaatgta acatcagaga ttttgagaca
caacgtggct ttgttgaata 2400aatcgaactt ttgctgagtt gaaggatcag
atcacgcatc ttcccgacaa cgcagaccgt 2460tccgtggcaa agcaaaagtt
caaaatcacc aactggtcca cctacaacaa agctctcatc 2520aaccgtggct
ccctcacttt ctggctggat gatggggcga ttcaggcctg gtatgagtca
2580gcaacacctt cttcacgagg cagacctctc gacggagttc cactgagcgt
cagaccccgt 2640agaaaagatc aaaggatctt cttgagatcc tttttttctg
cgcgtaatct gctgcttgca 2700aacaaaaaaa ccaccgctac cagcggtggt
ttgtttgccg gatcaagagc taccaactct 2760ttttccgaag gtaactggct
tcagcagagc gcagatacca aatactgttc ttctagtgta 2820gccgtagtta
ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
2880aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
ggttggactc 2940aagacgatag ttaccggata aggcgcagcg gtcgggctga
acggggggtt cgtgcacaca 3000gcccagcttg gagcgaacga cctacaccga
actgagatac ctacagcgtg agctatgaga 3060aagcgccacg cttcccgaag
ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3120aacaggagag
cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
3180cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
gggggcggag 3240cctatggaaa aacgccagca acgcggcctt tttacggttc
ctggcctttt gctggccttt 3300tgctcacatg ttctttcctg cgttatcccc
tgattctgtg gataaccgta ttaccgcctt 3360tgagtgagct gataccgctc
gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 3420ggaagcggaa
gaagctcgca cattcagcag cgtttttcag cgcgttttcg atcaacgttt
3480caatgttggt atcaacacca ggtttaactt tgaacttatc ggcactgacg
gttactgatt 3540ttgaactttt gctttgccac ggaacggtct gcgttgtcgg
gaagatgcgt gatctgatcc 3600ttcaactcag caaaagttcg ccaatacgca
aaccgcctct ccccgcgcgt tggccgattc 3660attaatgcag ctggcacgag
atctgacttg gttacgatgg actttgaaca cgccgagggt 3720gactaaaccg
ctggatttac gcggttttct tctcttcaac ttctttgacg tagcggtgaa
3780ccgtgcccac cgagcaacca atctctgccg cgatagcgcg catggacaga
cctttggcgc 3840gcagctcctg gacgcgagca cgcttctcgt tggcacgttt
aatgaacact tcacgcggtt 3900cggaagtcca tcgttgagct gttctcaccg
acataccggc acgttctgcc agctcgcggg 3960ctgtttttcc gttgcgtgga
taacgtgcat aaaccttagc caatgttcct ccaaagagta 4020tgtccagcct
cacgacgcac ctcagcgctt cgttgccagc ctttcttccc gcgtgcggat
4080tgcattgcgg tgaatgtggc gtcgtagacg gcggcgccgt ctgtccacat
gcgtgacttg 4140gtgatgatcc atttatggat tgacctggca atacagtcaa
cttcggccac aggtagtggt 4200tcatcaaaca gctcttggtt aagtgcttgc
gcggtggttt ggattgcgcg gcctaggccg 4260tcagcgtctc caaaatgctt
tctgacctcc cgatatgccc acgtacgtgc gctttcaaag 4320agtgcgcaat
tacgacctag accaactggc gagaaccgcc gcgttttcct ccaggacgca
4380ggcggcataa agccggtttc ttcgagccaa aagcgcagct catcgagcgt
atacagctta 4440tcggtgatcc agtgactatc ccatgcagtg tgctcggggt
ttttggtgat cagcccggag 4500tatccgctat cgccatcgac agagcgccgt
aggccttcgg tgacagccgc ggcataggcc 4560aaaggcttgc gtttggcgta
ttcggtgcgg gtaaatggct ccgcgagcgc ccagacagcg 4620tgtgcgtgcc
cgtttaaggg gttttcaacc accgcgttag gtctccagtc ctccctgtcc
4680cacaaagagc gcaaaagcgc gtcctcctgg tcgatgtcaa cgaccaggag
gttagagagc 4740gcgtcgggat tggcttcgac gtagcgctta tccagcgcgt
tcttccgtga ggtgcggtaa 4800atgccctcac ggaggtcatc gcttgccaat
ggccacagcg gcagccacag ctgctcaaag 4860cgtccctcag ggcgggtagt
tggtctcatg tagctgactt tctcacacga gcgtgcacgg 4920tcggttttca
ttcataatac gacatttaac caagtcagat gtttccccgg tttccggggg
4980ttcccctgaa gaacccttcc agtgcgagcg aagcgagctc ctttggccgg
cgcccctcag 5040gtagccctct aaggctccca gggctccgcc cctccctgag
gttggctcaa gcctcctggt 5100ggctcctacg gacgttct 51181429DNAArtificial
sequencePCR primer 14ctctcatatg ctgttttggc ggatgagag
291533DNAArtificial sequencePCR primer 15ctctcatatg gtgtctcctc
taaagattgt agg 33162285DNABacillus subtilis 16atgaaagcag aattcaagcg
taaaggaggg ggcaaagtga aactcgttgt cggaatgaca 60ggggcaacag gggccatttt
cggggtcagg ctgctgcagt ggctgaaggc cgccggagtg 120gaaacccatc
tcgttgtgtc tccttgggca aacgtcacga tcaaacacga aacaggctat
180acgttacaag aagtagaaca actggccaca tacacttact cacataagga
tcaggcggca 240gccatttcaa gcgggtcgtt tgataccgat ggaatgattg
ttgcgccgtg cagcatgaaa 300tctctcgcaa gcattcgcac aggaatggcg
gataatctgc tgacacgtgc ggcggatgtc 360atgctcaagg agagaaaaaa
actcgtcctc ttaacgagag agacgccttt gaaccaaatt 420catctcgaaa
atatgctagc gcttacgaaa atgggcacca tcattcttcc tccgatgccg
480gcattttata atcggccgag aagcttagag gaaatggttg accatattgt
ttttagaacg 540ttggaccaat tcggcattcg gcttcctgaa gcgaagcgct
ggaatgggat tgaaaaacaa 600aaaggaggag cttgatcatg gcttatcaag
atttcagaga atttctcgct gcccttgaaa 660aagaaggaca gctgcttaca
gtgaatgaag aggtaaagcc ggaaccggat ttaggggcct 720ccgcacgggc
agccagcaat cttggcgata aaagccctgc gctcttattt aacaacattt
780acggctatca taacgcgcga attgcgatga atgtcatcgg ctcttggcca
aaccatgcca 840tgatgctggg catgccgaaa gacacaccgg taaaagaaca
gttttttgaa ttcgcaaagc 900gttatgacca gtttccgatg ccggtcaaac
gtgaggaaac agcgccattt catgaaaatg 960aaatcacaga agatatcaat
ttgttcgata tactgcctct tttcagaatt aaccagggtg 1020atggaggcta
ctatttagac aaagcatgtg tcatttcccg tgatcttgag gaccctgaca
1080acttcggcaa acaaaatgtc ggcatttaca gaatgcaagt caaaggaaaa
gaccgccttg 1140gcattcagcc tgtcccgcag cacgatattg caatccatct
gcgccaagct gaagaacgcg 1200gcatcaacct tccggtcact attgcgctcg
gctgtgagcc ggtcattaca acggcggcat 1260cgactccgct tctctatgat
caatcagaat acgaaatggc aggtgcgatt caaggcgaac 1320catatcgcat
cgtcaaatca aagctgtctg atcttgatgt tccgtggggc gctgaagtgg
1380tgcttgaagg tgagattatt gccggagagc gcgaatatga agggccgttc
ggtgaattca 1440caggccatta ttccggcgga cgcagcatgc cgattatcaa
aattaaacgc gtctatcaca 1500gaaacaatcc gatctttgaa catttatact
taggcatgcc ttggacagaa tgcgattaca 1560tgatcggcat taacacatgc
gtgccgcttt atcagcagtt aaaagaagcg tatccgaacg 1620aaattgtggc
agtgaacgcc atgtacacac acggtttaat cgcgattgtt tccacaaaaa
1680cccgctatgg cggatttgcg aaagcggtcg gcatgcgcgc actcacaacg
ccgcacggac 1740tcggctactg caaaatggtc atagtcgttg atgaggatgt
cgatccattc aaccttccgc 1800aggtcatgtg ggcgctttcg accaaaatgc
atccgaaaca tgatgcggtc atcattccgg 1860acttatctgt cctgccgctt
gatccgggat ccaatccatc aggaatcact cacaaaatga 1920ttctcgacgc
cactacaccg gttgcgccgg aaacaagagg ccattattca cagccgcttg
1980attctccgct aacaacgaaa gaatgggaac aaaaactaat ggacttaatg
aataaataag 2040gaaaggatgt tcgaaatgca tacatgtcct cgatgcgact
caaaaaaggg agaagtcatg 2100agcaaatcgc ctgtagaagg cgcatgggaa
gtttatcagt gccaaacatg cttttttaca 2160tggagatcct gtgaaccgga
aagcattaca aatcccgaaa aatacaatcc agcgtttaaa 2220attgatccaa
aggaaacaga aacagcaatt gaagttccgg cggtgccgga acgaaaggct 2280tgatc
22851732DNAArtificial sequencePCR primer 17ctctcatatg aaagcagaat
tcaagcgtaa ag 321829DNAArtificial sequencePCR primer 18ctctcatatg
gatcaagcct ttcgttccg 29192058DNALactobacillus rhamnosus
19atgacagcat caccttggga cttaagaaaa gtattggatg aactaaaaca ggatccgcag
60caatatcatg aaacagaggt gcaagtcgat cccgatgcag agcttgctgg cgtttatcgt
120tacatcggtg ccggtgggac ggtcgaacgt ccgacacagg aaggtccggc
aatgatgttt 180aacaacgttg tcggcttccc aacgacaagg gttttgatcg
gtttaatggc cagtcgcaag 240cgggttggca agatgtttca ccaagactat
cacacacttg gtcgattctt gaacaaagcg 300gttttaaatc ctattcaacc
cgttacagtc gaagaatcag cagcgcctgc gcatgaagtc 360gttgccaagg
ctagtgaccc ggactttgac attagaaaac tcgttgcagc accaaccaat
420acgccacaag atgccggccc atacatcaca tgcggcgtag ttttgggttc
caatatggcc 480aaaacaatga ctgatgtgac gattcatcgc atggttttgg
aagataagga tacgcttggt 540atttatatca tgcccggtgg tcgccacatt
ggtcattttg ctgaagaata tgaaaaagcc 600aataagccga tgccggtgac
catcaacatt ggcttggatc cggccattac cattggtgcc 660acttttgaac
cgcctaccac gccgcttggc tacgatgaac taggagttgc cggagccatt
720cgccaagaac ccgtgcaact ggttcaggct gtgaccgtca atgaaaaagc
cattgcgcgt 780tcagaattta cactggaagg ctatatcatg cctaacacgc
gtatccaaga agatatcaat 840acccataccg gcaaagccat gccagagttt
cccggctatg acggtgatgc caatccggct 900ttgcaagtga ttaaagtgac
ggctgtaacc catcggcgcg atcatcccat tatgcaaagt 960gtcatcggac
ctagtgaaga acatgtctcc atggccggca ttccaaccga agccagcatt
1020ttacaacttg ttgatcgtgc catccccggc aaggtcaaga atgtgtacaa
tcccccagct 1080ggtggcggca aactcatgac catcatgcaa attcacaaag
ataatccagc tgatgaaggg 1140attcaacgtc aagctgcatt actcgctttt
tcggcattca aagaactaaa aactgtttgg 1200ctggtcgatg
atgatgtcga tatttttgac atgaatgatg tcgtctggac aatgaacacg
1260cgttttcaag gtgatcagga catcatggta ttacctggca tgcgcaacca
tccgcttgat 1320ccgtcagaac gaccgcaata tgatcccaag tctattcggg
tacgcggaat gagttcgaag 1380acggtcattg atggtaccgt accatttgat
atgcgcgatc aattcaaacg agcagccttt 1440aaaaaagttt ccgactggca
aaaatatttg aaataggtga ttgaattgaa acgtattatc 1500gtagggatta
ccggggcatc cggaactatt tacgctgtta acctgctcca gcatttacat
1560cgcctgcctg atgtcgaagt tcatttggtg atgagtgctt gggcaaagca
aaacctgtca 1620cttgagaccg acatgaaaca aagcgaactc gaagctttgg
cggattatgt ttatcctgtt 1680caaaaccaag gggcaaccat tgcaagcggc
agttttttaa ccgatgcaat ggtcattgtt 1740ccggcaagca tgaaaaccat
tgcgggcatt gcgatgggct ttgatgataa tctcattgga 1800cgagcagccg
atgtcacgat taaagaacag cggcaattga ttattgtgcc gcgggaaaca
1860ccgcttagtc caattcatct ggataacctc gctaaactag cccacattgg
cgttcaaatc 1920attccgccta ttccagcttt ctatcagcat ccccaaacca
tccaggattt aattgagcat 1980cacaccatga aactattaga cgccttgcat
attaaaaccg aaaccgctag tcgctggaat 2040ggagcgtcgt taagatga
20582026DNAArtificial sequencePCR primer 20ctctcatatg acagcatcac
cttggg 262132DNAArtificial sequencePCR primer 21ctctcatatg
tcatcttaac gacgctccat tc 32222045DNALactobacillus brevis
22atggtaaatg atccttatga tttacgaaaa gtattggccg agctaaaaac ccatgccaac
60cagtaccatg agacgaacgt agctgtgaat ccaaatgctg aattggctgg tgtttaccgc
120tatattggtg ctggagggac ggtaaaacgg ccgacgcaag aaggtccagc
aatgatgttt 180aataacgttg aaggcttctc cgatacgaaa gtcctaatgg
gattaatggc aaatcggcga 240cgagtggggc tcatgttcca tcatgattac
caaaccctgg gaaaatttct aaatacagcg 300gttgaaaaac caatcccccc
agtgatggtt actgacgcac ctacccacga ggtggttcac 360aaggccactg
atccggactt tgatattcgt aaactcgttg cagcacctac gaatacaccg
420gaggacgcgg gaccttacat tacagtgggt gtcgtgttgg gatctaatat
ggccaagact 480atgtcagatg tgacgattca ccgaatggtt ctagaagata
aagacaagtt agggatttac 540attatgcctg gcggtcggca tattggtgcc
tttgctaaag aatacgaggc cgctaataaa 600ccgatgccca tcacgattaa
tattgggtta gatccggcca ttacgattgg gtgcaccttt 660gagccaccaa
ctacaccatt ggggtataac gagctggggg tggctggtgc gatccgacaa
720gaagctgtgg gtctgaccaa agcgctaacc gttgatgaga atgctattgc
ccgttctgaa 780ttcacgttgg aagggtatat catgcccaac gaacggatgc
aagaggatat caatacccag 840acaggtaagg caatgcctga atttccgggt
tacgacggtg atgctaatcc agccgtacag 900gtcattaaag taacggctgt
cactcaccgg aagcatccaa ttatgcaaag tgtgattggg 960ccatccgaag
agcatgtcag catggcagga attcccaccg aagcgagcat cttagaatta
1020acggatcgcg ctatcccggg taaagtttta aatgtttata atccacctgc
aggcggagga 1080aaactgatga caattatgca gatccataag gatgatgcgg
ccgatgaagg tattcagcgg 1140caagcagcac tgctggcatt ttcggcgttc
aaagagttga agacagttat tttggtggat 1200gaagacgttg atatttttga
tatgaacgat gtaatgtgga ccgtgaatac gcgtttccag 1260gccgatcagg
atttaatgat attaccgggg atgcggaatc acccactgga cccgtcggaa
1320cgaccagagt atgatctgaa atctattcga acgcgaggca tgtcatcgaa
gttggtgatt 1380gatggcacgg taccctttga tatgagggaa caatttgaac
gcgcgaagtt taagccagtt 1440gctgactggg aaaaatattt gaaataaaag
ggtgatggct gatgaaacgg attgtgattg 1500gggtgactgg tgcgtccggt
acgatttacg cgattgattt gttaaaaaag ttacgggata 1560agccaggcgt
tgaaacacat ttggtaatga gtccgtgggc caccaaaaac ttggcactag
1620aaacaagtta tacattagcc caagttaaag cgatggccga ctacacgtac
agtgatcggg 1680accaaggggc taagattgct agcggttcat tcctacacga
tgggatggtt attgttcccg 1740ctagcatgaa aacggtggcg ggtgttgcct
atgggtttgg cgataatcta attgcgcggg 1800ctgccgatgt aactattaag
gaacatcgac aattgatcat tgtcccacgg gaaacgccac 1860tgagtgtgat
tcatctagag aatttaacga aactagccaa actaggcgcg caaattattc
1920cacctattcc cgccttttat aatcagccac aaaccattca agacttagtg
gatcatcaga 1980ctatgaaggt actgggtgca tttggcattc agcaagtgac
cgctaagcgt tgggagggag 2040attag 20452338DNAArtificial sequencePCR
primer 23ctctcatatg gtaaatgatc cttatgattt acgaaaag
382428DNAArtificial sequencePCR primer 24ctctcatatg ctaatctccc
tcccaacg 2825630DNAPseudomonas putida 25atgaacgggc cggaacgcat
caccctggcc atgacgggcg cctcgggtgc ccagtatggc 60cttcgcctgc tcgattgcct
ggtacgcgaa gaccgcgagg tgcacttcct gatttccaag 120gccgcacagt
tggtgatggc caccgagacg gatgttgtgt tgccggccaa gccccaggcg
180atgcaggcct tcctgaccga atacaccggc gcggccgacg ggcagatccg
tgtgtatggc 240aaggaagact ggatgtcgcc ggtagcctcg ggttctggcg
ccccggcggc aatggtggtg 300gtcccctgtt ccactggcac cttgtcggcc
attgccactg gcgcctgcaa caacctgatc 360gagcgtgctg ccgacgttac
cctcaaggag cgtcgccagc tgatcctggt gccacgcgaa 420gcgccattct
ccaccatcca cctggaaaac atgctcaagc tgtcgcaaat gggcgcggtg
480atcctgccgg cggcaccggg gttctatcac cagccgcaga ccatcgacga
cctggtcgac 540tttgtcgtgg cgcgtatcct caacctgctg aacatccccc
aggatatgtt gccgcgttgg 600ggcgagcacc acttcggggt ggatgattga
6302623DNAArtificial sequencePCR primer 26ctctcatatg aacgggccgg aac
232729DNAArtificial sequencePCR primer 27ctctcatatg tcaatcatcc
accccgaag 29281574DNAEscerichia coli 28atgtcttccc gcaataatcc
ggcgcgtgtc gccatcgtga tggggtccaa aagcgactgg 60gctaccatgc agttcgccgc
cgaaatcttc gaaatcctga atgtcccgca ccacgttgaa 120gtggtttctg
ctcaccgcac ccccgataaa ctgttcagct tcgccgaaag cgccgaagag
180aacggttatc aggtgattat tgcgggcgca ggcggcgcag cgcatctgcc
aggcatgatt 240gccgccaaaa cgctggtgcc ggtgctgggc gtgccagtac
agagcgccgc actgagcggt 300gtcgatagcc tctactccat cgtacaaatg
ccgcgcggca ttccggtggg tacgctggcg 360attggtaaag ctggcgcggc
aaacgcggcg ttactggcag cacaaattct tgcgactcat 420gataaagaac
tgcaccagcg tctgaatgac tggcgcaaag cccagaccga cgaagtgctg
480gaaaacccgg acccgcgagg tgcggcatga aacaggtttg cgtcctcggt
aacgggcagt 540taggccgtat gctgcgtcag gcaggcgaac cgttaggcat
tgctgtctgg ccagtcgggc 600tggacgctga accggcggcg gtgccttttc
aacaaagcgt gattaccgct gagatagaac 660gctggccgga aaccgcatta
acccgcgagc tggcgcgcca tccggccttt gtgaaccgcg 720atgtgttccc
gattattgct gaccgtctga ctcagaagca gcttttcgat aagctccacc
780tgccgactgc accgtggcag ttacttgccg aacgcagcga gtggcctgcg
gtgtttgatc 840gtttaggtga gctggcgatt gttaagcgtc gcactggtgg
ttatgacggt cgcggtcaat 900ggcgtttacg cgcaaatgaa accgaacagt
taccggcaga gtgttacggc gaatgtattg 960tcgagcaggg cattaacttc
tctggtgaag tgtcgctggt tggcgcgcgc ggctttgatg 1020gcagcaccgt
gttttatccg ctgacgcata acctgcatca ggacggtatt ttgcgcacca
1080gcgtcgcttt tccgcaggcc aacgcacagc agcaggcgca agccgaagag
atgctgtcgg 1140cgattatgca ggagctgggc tatgtgggcg tgatggcgat
ggagtgtttt gtcaccccgc 1200aaggtctgtt gatcaacgaa ctggcaccgc
gtgtgcataa cagcggtcac tggacacaaa 1260acggtgccag catcagccag
tttgagctgc atctgcgggc gattaccgat ctgccgttac 1320cgcaaccagt
ggtgaataat ccgtcggtga tgatcaatct gattggtagc gatgtgaatt
1380atgactggct gaaactgccg ctggtgcatc tgcactggta cgacaaagaa
gtccgtccgg 1440ggcgtaaagt ggggcatctg aatttgaccg acagcgacac
atcgcgtctg actgcgacgc 1500tggaagcctt aatcccgctg ctgccgccgg
aatatgccag cggcgtgatt tgggcgcaga 1560gtaagttcgg ttaa
15742928DNAArtificial sequencePCR primer 29ctctcatatg tcttcccgca
ataatccg 283031DNAArtificial sequencePCR primer 30ctctcatatg
ttaaccgaac ttactctgcg c 31311716DNASaccharomyces cerevisiae
31atggattcta gaacagttgg tatattagga gggggacaat tgggacgtat gattgttgag
60gcagcaaaca ggctcaacat taagacggta atactagatg ctgaaaattc tcctgccaaa
120caaataagca actccaatga ccacgttaat ggctcctttt ccaatcctct
tgatatcgaa 180aaactagctg aaaaatgtga tgtgctaacg attgagattg
agcatgttga tgttcctaca 240ctaaagaatc ttcaagtaaa acatcccaaa
ttaaaaattt acccttctcc agaaacaatc 300ggattgatac aagacaaata
tattcaaaaa gagcatttaa tcaaaaatgg tatagcagtt 360acccaaagtg
ttcctgtgga acaagccagt gagacgtccc tattgaatgt tggaagagat
420ttgggttttc cattcgtctt gaaatcgagg actttggcat acgatggaag
aggtaacttc 480gttgtaaaga ataaggaaat gattccggaa gctttggaag
tactgaagga tcgtcctttg 540tacgccgaaa aatgggcacc atttactaaa
gaattagcag tcatgattgt gagatctgtt 600aacggtttag tgttttctta
cccaattgta gagactatcc acaaggacaa tatttgtgac 660ttatgttatg
cgcctgctag agttccggac tccgttcaac ttaaggcgaa gttgttggca
720gaaaatgcaa tcaaatcttt tcccggttgt ggtatatttg gtgtggaaat
gttctattta 780gaaacagggg aattgcttat taacgaaatt gccccaaggc
ctcacaactc tggacattat 840accattgatg cttgcgtcac ttctcaattt
gaagctcatt tgagatcaat attggatttg 900ccaatgccaa agaatttcac
atctttctcc accattacaa cgaacgccat tatgctaaat 960gttcttggag
acaaacatac aaaagataaa gagctagaaa cttgcgaaag agcattggcg
1020actccaggtt cctcagtgta cttatatgga aaagagtcta gacctaacag
aaaagtaggt 1080cacataaata ttattgcctc cagtatggcg gaatgtgaac
aaaggcttaa ctacattaca 1140ggtagaactg atattccaat caaaatctct
gtcgctcaaa agttggactt ggaagcaatg 1200gtcaaaccat tggttggaat
catcatggga tcagactctg acttgccggt aatgtctgcc 1260gcatgtgcgg
ttttaaaaga ttttggcgtt ccatttgaag tgacaatagt ctctgctcat
1320agaactccac ataggatgtc agcatatgct atttccgcaa gcaagcgtgg
aattaaaaca 1380attatcgctg gagctggtgg ggctgctcac ttgccaggta
tggtggctgc aatgacacca 1440cttcctgtca tcggtgtgcc cgtaaaaggt
tcttgtctag atggagtaga ttctttacat 1500tcaattgtgc aaatgcctag
aggtgttcca gtagctaccg tcgctattaa taatagtacg 1560aacgctgcgc
tgttggctgt cagactgctt ggcgcttatg attcaagtta tacaacaaaa
1620atggaacagt ttttattgaa gcaagaagaa gaagttcttg tcaaagcaca
aaagttagaa 1680actgtcggtt acgaagctta tctagaaaac aagtaa
17163234DNAArtificial sequencePCR primer 32ctctccatgg attctagaac
agttggtata ttag 343339DNAArtificial sequencePCR primer 33ctctccatgg
ttacttgttt tctagataag cttcgtaac 39342252DNAEnterobacter cloacae
34atgagattga tcgtgggaat gacgggagca acaggtgctc cgctgggtgt ggctttactg
60caggcgttac gtgacatgcc agaggttgaa acccatctgg tgatgtcgaa atgggcgaaa
120accaccattg agctggaaac gccttatacc gcgcaggatg tcgccgccct
ggcagatgtc 180gttcacagtc ctgccgatca ggctgccacc atctcctccg
gctcgtttcg taccgacggc 240atgatcgtca ttccctgcag catgaaaacg
ctggcgggta tccgcgcggg ctatgccgaa 300gggctggtgg gccgtgcggc
agacgtggtg ctgaaagagg ggcgcaagct ggtgctggtc 360ccgcgtgaaa
cgccgctcag caccattcat ctggagaaca tgctcgcgct ttcccgcatg
420ggggtggcga tggtgccgcc catgcctgcg tattacaacc acccgcaaac
cgccgatgat 480atcacccagc atatcgtgac ccgcgtactc gaccagtttg
gtctggagca caaaaaggcg 540cgtcgctgga acggcctgca ggcggcgaaa
catttttcac aggagaataa cgatggcatt 600tgatgatttg agaagcttcc
tgcaggcgct agatgagcaa gggcaactgc tgaaaattga 660agaagaggtc
aatgcggagc cggatctggc ggcggccgct aacgcgacgg gacgtatcgg
720tgatggtgcg cctgcgctgt ggttcgataa cattcgcggg tttaccgatg
ccagggtggt 780gatgaacacc atcggctcct ggcagaacca cgccatttcg
atggggctgc cggcgaatac 840cccggtcaaa aagcagatcg atgagtttat
tcgccgctgg gataaattcc cggtcgcacc 900ggagcgccgg gccaaccccg
catgggcgca gaatacggtg gacggtgagg agattaacct 960gttcgacatc
ctgccgctgt ttcgcctgaa cgacggggac ggcggttttt atctcgacaa
1020agcgtgcgtt gtctcgcgcg atccgctcga cccggaccat ttcggcaagc
agaacgtcgg 1080tatttaccgc atggaagtga agggcaaacg taagctcggc
ctgcagccgg tgccgatgca 1140tgatatcgcc ctgcatctgc ataaagccga
agagcgtggt gaagacctgc cgattgcgat 1200tacgttgggc aacgatccga
tcatcaccct gatgggcgca acgccgctga aatacgatca 1260gtccgagtat
gaaatggccg gggcgctgcg tgaaagcccg tacccgattg cgaccgcgcc
1320gttgaccggc ttcgatgtgc cgtgggggtc tgaagtgatc ctggaagggg
tgattgaagg 1380ccgtaaacgt gaaattgaag ggccgttcgg tgagtttacc
gggcactatt cgggcggacg 1440caatatgacg gtggtccgta ttgataaagt
ctcgtaccgc accaaaccga ttttcgaatc 1500cctctatctc gggatgccct
ggaccgagat cgactacctg atggggccag ccacctgtgt 1560gccgctttac
cagcaactga aagcggagtt ccctgaagtg caggcggtga acgcgatgta
1620tacccacggt ctgctggcga tcatctccac caaaaaacgc tacggtggtt
ttgcccgcgc 1680ggtcggttta cgcgccatga ccacgccgca tggcctgggc
tatgtgaaga tggtgattat 1740ggtggatgaa gatgtcgatc cgttcaacct
gccgcaggtg atgtgggcgc tgtcatcaaa 1800agtgaacccg gcaggggatc
tggtgcagct gccgaacatg tcggttcttg agcttgatcc 1860tgggtccagc
ccggcaggca tcaccgacaa gctgattatt gatgccacca cgcctgttgc
1920gccggataac cgcggtcact acagccagcc ggtgcaggat ttacctgaaa
ccaaagcctg 1980ggctgaaaag ctgactgcga tgctggcagc acgccaataa
ggaggaaaag atgatttgtc 2040cacgttgtgc cgatgagcaa attgaggtga
tggccacatc accggtgaaa gggatctgga 2100cggtttatca gtgccagcat
tgcctgtata cctggcgcga tactgagccg ctgcgtcgta 2160ccagccgcga
acattaccct gaagcgttcc gcatgacgca gaaggatatt gatgaggcgc
2220cgcaggtacc gaccattccg ccattgctgt aa 22523530DNAArtificial
sequencePCR primer 35ctctcatatg agattgatcg tgggaatgac
303630DNAArtificial sequencePCR primer 36ctctcatatg ttacagcaat
ggcggaatgg 30
* * * * *