U.S. patent application number 10/320647 was filed with the patent office on 2003-09-18 for method of producing l-serine by fermentation.
This patent application is currently assigned to AJINOMOTO CO. INC. Invention is credited to Hibino, Wataru, Ito, Mika, Nakamatsu, Tsuyoshi, Osumi, Tsuyoshi, Suga, Mikiko, Sugimoto, Masakazu.
Application Number | 20030175912 10/320647 |
Document ID | / |
Family ID | 26337388 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175912 |
Kind Code |
A1 |
Suga, Mikiko ; et
al. |
September 18, 2003 |
Method of producing L-serine by fermentation
Abstract
L-serine is produced by cultivating in a medium a coryneform
bacterium having L-serine productivity in which an activity of at
least one of phosphoserine phosphatase and phosphoserine
transaminase is enhanced, preferably, further having introduced
therein a gene coding for D-3-phosophoglycerate dehydrogenase in
which feedback inhibition by L-serine is desensitizied, allowing
L-serine to accumulate in the medium, and collecting the L-serine
from the medium.
Inventors: |
Suga, Mikiko; (Kawasaki-shi,
JP) ; Sugimoto, Masakazu; (Kawasaki-shi, JP) ;
Osumi, Tsuyoshi; (Tokyo, JP) ; Nakamatsu,
Tsuyoshi; (Kawasaki-shi, JP) ; Hibino, Wataru;
(Kawasaki-shi, JP) ; Ito, Mika; (Yokkaichi-shi,
JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
AJINOMOTO CO. INC
15-1, Kyobashi 1-chome Chuo-ku
Tokyo
JP
104-8315
|
Family ID: |
26337388 |
Appl. No.: |
10/320647 |
Filed: |
December 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10320647 |
Dec 17, 2002 |
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09517331 |
Mar 2, 2000 |
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09517331 |
Mar 2, 2000 |
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09222817 |
Dec 30, 1998 |
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6037154 |
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Current U.S.
Class: |
435/116 ;
435/193; 435/196; 435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/0006 20130101;
C12P 13/06 20130101; C12R 2001/19 20210501; C12N 1/205 20210501;
C12R 2001/15 20210501; C12R 2001/13 20210501 |
Class at
Publication: |
435/116 ;
435/69.1; 435/193; 435/196; 435/252.3; 435/320.1; 536/23.2 |
International
Class: |
C12P 021/02; C12P
013/06; C12N 009/10; C12N 009/16; C07H 021/04; C12N 001/21; C12N
015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 12, 1998 |
JP |
10-3751 |
Dec 11, 1998 |
JP |
10-353521 |
Claims
What is claimed is:
1. A coryneform bacterium having L-serine productivity in which an
activity of at least one of phosphoserine phosphatase and
phosphoserine transaminase is enhanced.
2. The coryneform bacterium as claimed in claim 1, wherein said
bacterium is enhanced the activitise of both phosphoserine
phosphatase and phosphoserine transaminase.
3. The coryneform bacterium as claimed in claim 1, wherein said
bacterium has L-serine productivity due to deficiency in L-serine
decomposing activity.
4. The coryneform bacterium as claimed in claim 1 or 3, wherein
said bacterium has L-serine productivity due to its resistance to
L-serine analogue(s).
5. The coryneform bacterium as claimed in claim 1, wherein an
activity of phosphoserine phosphatase or phosphoserine transaminase
is enhanced by increasing a copy number of a gene coding for
phosphoserine phosphatase or a gene coding for phosphoserine
transaminase in said coryneform bacterium in its cell.
6. The coryneform bacterium as claimed in any one of claims 1 to 5,
wherein said bacterium has introduced therein a gene coding for
D-3-phosophoglycerate dehydrogenase in which feedback inhibition by
L-serine is desensitized.
7. A method of producing L-serine, comprising the steps of
cultivating the coryneform bacterium as claimed in any one of
claims 1 to 6 ndescribed above in a medium to accumulate L-serine
in the medium and collecting the L-serine from the medium.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method of producing
L-serine for use in the production of amino acid mixtures utilized
in the field of pharmaceuticals, chemicals, and cosmetics and to
coryneform bacteria constituting the method.
BACKGROUND OF THE INVENTION
[0002] As a conventional method of producing L-serine by
fermentation, there has been reported the method in which a
bacterial strain capable of converting glycine and sugar into
L-serine is used in a medium containing 30 g/L of glycine to
produce at most 14 g/L of L-serine. The conversion yield of glycine
into L-serine by this method amounted to 46% (Kubota K.
Agricultural Biological Chemistry, 49, 7-12 (1985)). Using a
bacterial strain capable of converting glycine and methanol into
L-serine, 53 g/L of L-serine can be produced from 100 g/L of
glycine (T. Yoshida et al., Journal of Fermentation and
Bioengineering, Vol. 79, No. 2, 181-183, 1995). In the method using
a bacterium belonging to the genus Nocardia, it has been known that
the L-serine productivity of the bacterium can be improved by
breeding those strains resistant to serine hydroxamate, azaserine
or the like (Japanese Patent Publication No. 57-1235). However,
these methods involve use of glycine that is a precursor of
L-serine and include complicated operation and is disadvantageous
from the viewpoint of costs.
[0003] As strains that can ferment L-serine directly from a sugar
and do not need addition of the precursor of L-serine to the
medium, there has been known Corynebacterium glutamicum that is
resistant to D-serine, .alpha.-methylserine, o-methylserine,
isoserine, serine hydroxamate, and 3-chloroalanine but the
accumulation of L-serine is as low as 0.8 g/L (Nogei Kagakukaishi,
Vol. 48, No. 3, p201-208, 1974). Accordingly, a further strain
improvements of are needed for direct fermentation of L-serine on
an industrial scale.
[0004] On the other hand, regarding coryneform bacteria, there have
been disclosed a vector plasmid that is capable of autonomous
replication in the cell and having a drug resistance marker gene
(cf. U.S. Pat. No. 4,514,502) and a method of introducing a gene
into the cell (Japanese Patent Application Laid-open No. 2-207791),
and the possibility of growing L-threonine or L-isoleucine
producing bacteria (U.S. Pat. Nos. 4,452,890 and 4,442,208). Also,
regarding the growth of L-lysine producing bacteria, there has been
known a technology involving the incorporation of a gene
participating in the biosynthesis of L-lysine into a vector plasmid
and the amplification of the plasmid in the cell (Japanese Patent
Application Laid-open No. 56-160997).
[0005] In the case of Escherichia coli, the enzymes participating
in the biosynthesis of L-serine include an enzyme that is
susceptible to feedback inhibition relative to L-serine production
in the wild type and an example has been known in which the
introduction of a mutant gene that has been mutated so that the
feedback inhibition could be desensitized resulted in an
enhancement in the L-serine (Japanese Patent No. 2584409). As such
genes, there has been known specifically 3-PGDH gene (hereafter,
the gene coding for 3-PGDH protein will also be referred to
"serA").
[0006] Further, in the case of coryneform bacteria, an example has
been known in which the amplification of 3-PGDH gene influences the
productivity of L-tryptophane (Japanese Patent Application
Laid-open No. 3-7591).
SUMMARY OF THE INVENTION
[0007] An object of the present invention is to provide a
microorganism that converts a sugar into L-serine and to provide a
method of accumulating L-serine in a culture medium utilizing the
ability of the microorganism to convert the sugar into L-serine,
i.e., a method of producing L-serine that is advantageous in
practicing on an industrial scale.
[0008] As a result of intensive investigation with view to
achieving the above object, it has now been discovered by the
present inventors that screening a strain in which an activity of
at least one of phosphoserine phosphatase and phosphoserine
transaminase is enhanced from coryneform bacteria having L-serine
productivity, preferably the bacteria deficient in L-serine
decomposing activity or a mutant thereof having resistance to an
L-serine analogue, and L-serine fermentation using the screened
strain will enhance the accumulation of L-serine drastically. The
present invention has been completed based on this discovery.
[0009] That is, the present invention relates to a coryneform
bacterium having L-serine productivity in which an activity of at
least one of phosphoserine phosphatase and phosphoserine
transaminase is enhanced.
[0010] Further, the present invention relates to the coryneform
bacterium as described above, which is enhanced the activitise of
both phosphoserine phosphatase and phosphoserine transaminase; the
coryneform bacterium as described above, having L-serine
productivity due to deficiency in L-serine decomposing activity;
the coryneform bacterium as described above, having L-serine
productivity due to its resistance to L-serine analogue(s); the
coryneform bacterium as described above, in which an activity of
phosphoserine phosphatase or phosphoserine transaminase is enhanced
by increasing a copy number of a gene coding for phosphoserine
phosphatase or a gene coding for phosphoserine transaminase in the
coryneform bacterium described above in its cell; and the
coryneform bacterium as described above, having introduced therein
a gene coding for D-3-phosophoglycerate dehydrogenase in which
feedback inhibition by L-serine is desensitized.
[0011] Further, the present invention relates to a method of
producing L-serine, comprising the steps of cultivating the
coryneform bacterium as described above in a medium to accumulate
L-serine in the medium and collecting the L-serine from the
medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 illustrates a manner of feedback inhibition of 3-PGDH
derived from various strains by L-serine. The horizontal axis
indicates the concentration of L-serine in the enzyme solution. The
vertical axis indicates percentage of the 3-PGDH activity in the
presence of L-serine to that in the absence of L-serine. Symbol
.diamond-solid. illustrates a manner of feedback inhibition of
3-PGDH derived from ATCC14067 strain by L-serine. Symbol
.box-solid. illustrates a manner of feedback inhibition of 3-PGDH
derived from AJ13377 strain by L-serine. Symbol .tangle-solidup.
illustrates a manner of feedback inhibition of 3-PGDH derived from
AJ13324 strain by L-serine. Symbol X illustrates a manner of
feedback inhibition of 3-PGDH derived from AJ13325 strain by
L-serine. Symbol * illustrates a manner of feedback inhibition of
3-PGDH derived from AJ13327 strain by L-serine.
[0013] FIG. 2 illustrates the construction of plasmids pVK7 and
pVK6.
[0014] FIG. 3 illustrates the construction of plasmid pSB on which
serB is carried.
[0015] FIG. 4 illustrates the construction of plasmid pSC on which
serC is carried.
[0016] FIG. 5 illustrates the construction of plasmid pBC8 and
pBC14 on which serB and serC are carried.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The coryneform bacteria referred to in the present invention
are a group of microorganisms as defined in Bergey's Manual of
Determinative Bacteriology, 8th ed., p. 599 (1974), which are
aerobic Gram-positive rods having no acid resistance and no
spore-forming ability. The coryneform bacteria include bacteria
belonging to the genus Corynebacterium, bacteria belonging to the
genus Brevibacterium having been hitherto classified into the genus
Brevibacterium but united as bacteria belonging to the genus
Corynebacterium at present, and bacteria belonging to the genus
Brevibacterium closely relative to bacteria belonging to the genus
Corynebacterium and bacteria belonging to the genus
Microbacterium.
[0018] The coryneform bacteria of the present invention are
coryneform bacteria that have L-serine productivity in which an
activity of phosphoserine phosphatase or phosphoserine transaminase
is enhanced. Such bacteria can be obtained, for example, by
increasing the copy number of a gene coding for phosphoserine
phosphatase (hereafter, referred to as "serB") or a gene coding for
phosphoserine transaminase (hereafter, referred to as "serC") in a
coryneform bacterial cell having L-serine productivity.
[0019] Also, the coryneform bacteria of the present invention can
be obtained by imparting L-serine productivity to a coryneform
bacterium having an enhanced activity of phosphoserine phosphatase
or phosphoserine transaminase.
[0020] As the coryneform bacteria having L-serine productivity,
there can be cited, for example, coryneform bacterial deficient in
L-serine decomposing activity, coryneform bacteria resistant to
L-serine analogues, and coryneform bacteria deficient in L-serine
decomposing activity and being resistant to L-serine analogues.
[0021] In the present invention, the L-serine analogue includes
azaserine or .beta.-(2-thienyl)-DL-alanine.
[0022] The coryneform bacteria resistant to L-serine analogues and
having L-serine productivity, more preferably the coryneform
bacteria deficient in L-serine decomposing activity from among them
can be artificially mutated or induced using wild type or
coryneform bacteria having L-serine productivity as a parent
strain.
[0023] The coryneform bacteria having resistance to an L-serine
analogue, deficient in L-serine decomposing activity, and having
L-serine productivity can be collected, for example, as follows.
Brevibacterium flavum ATCC14067 is subjected to mutation treatment
by a conventional method (contact with
N-methyl-N'-nitro-N-nitrosoguanidine, etc.) to obtain a mutant that
is deficient in L-serine decomposing activity, and then a bacterium
resistant to an L-serine analogue such as azaserine or
.beta.-(2-thienyl)-DL-alanine is collected from the mutant as a
parent strain. Also, after L-serine analogue-resistant bacterium is
obtained, a mutant deficient in L-serine decomposing activity may
be obtained. Among the mutants obtained by the methods described
above, there are strains that accumulate L-serine in high
concentrations.
[0024] The L-serine analogue-resistant bacteria can be obtained by
introducing the mutant serA described later on into a parent strain
or L-serine decomposing activity deficient mutant.
[0025] By the term "L-serine analogue resistance" is meant the
property that a bacterium grows faster than the wild type in a
medium containing an L-serine analogue.
[0026] More specifically, for example, the term "azaserine
resistance" refers to the property that a bacterium grows faster
than the wild type in a medium containing azaserine. For example,
those strains that form colonies on a solid medium containing 0.25
g/L of azaserine at 30.degree. C. within 4 to 5 days are said to
have azaserine resistance.
[0027] Similarly, the term ".beta.-(2-thienyl)-DL-alanine
resistance" refers to the property that a bacterium grows faster
than the wild type in a medium containing
.beta.-(2-thienyl)-DL-alanine. For example, those strains that form
colonies on a solid medium containing 0.25 g/L of
.beta.-(2-thienyl)-DL-alanine at 30.degree. C. within 4 to 5 days
are said to have .beta.-(2-thienyl)-DL-alanine resistance.
[0028] Next, the enhancement of phosphoserine phosphatase activity
or phosphoserine transaminase activity will be described.
[0029] The enhancement of phosphoserine phosphatase activity or
phosphoserine transaminase activity can be performed by introducing
serB or serC each in an expressible form into a coryneform
bacterium. This is possible either by forced expression of genes
coding for respective enzymes by means of separate promoters or by
forced expression of the both genes under the control of a single
promoter. Regardless of whether these genes are on a plasmid or
chromosome, the expression may be enhanced by enhancement of an
expression control sequence such as promoter of a gene, or
improvement in translation efficiency. Alternatively, the enzyme
activity can be enhanced by amplification of the number of genes on
a chromosome. Further, the enhancement of these enzyme activities
can be achieved by use of a modified gene coding for phosphoserine
phosphatase or phosphoserine transaminase modified in such a manner
that a modified enzyme having an increased specific activity is
coded for.
[0030] In order to introduce serB or serC into a coryneform
bacterium, a DNA fragment containing serB or serC may be ligated
with a vector that functions in coryneform bacteria to generate a
recombinant DNA, followed by introduction of it into a coryneform
bacterium host having L-serine productivity to transform it. As a
result of an increase in copy number of serB or serC in the cell of
transformed strain, the phosphoserine phosphatase activity or
phosphoserine transaminase activity thereof is amplified.
Introduction of a recombinant DNA containing both serB and serC or
both a recombinant DNA containing serB and a recombinant DNA
containing serC into a coryneform bacterium will amplify the both
phosphoserine phosphatase activity and phosphoserine transaminase
activity.
[0031] The base sequences of serB and serC are known (serB:
GenBank; X03046 M30784, serC: GenBank; D90728). It is possible to
synthesize primers based on their base sequences and collect the
serB gene or serC gene of these microorganisms by the PCR method
using the chromosomal DNA of Escherichia coli, Brevibacterium
flavum or other microorganisms as a template. As such a primer,
there can be cited the primer having the base sequence shown in
Sequence ID No. 15 to 18.
[0032] It is preferred that serB gene or serC gene is ligated with
vector DNA autonomously replicable in cells of Escherichia coli
and/or coryneform bacteria to prepare recombinant DNA, and the
recombinant DNA is introduced into cells of Escherichia coli
beforehand. Such provision makes following operations easy. The
vector autonomously replicable in cells of Escherichia coli is
preferably a plasmid vector which is preferably autonomously
replicable in cells of a host, including, for example, pUC19,
pUC18, pBR322, pHSG299, pHSG399, pHSG398, and RSF1010.
[0033] In the case where serB gene and serC gene are loaded on
separate vectors for introduction into a coryneform bacterium, it
is preferred to use two vectors having respective marker genes
differing one from another.
[0034] Recombinant DNA may be prepared by utilizing transposon (WO
02/02627 International Publication Pamphlet, WO 93/18151
International Publication Pamphlet, European Patent Application
Laid-open No. 0445385, Japanese Patent Application Laid-open No.
6-46867, Vertes, A. A. et al., Mol. Microbiol., 11, 739-746 (1994),
Bonamy, C., et al., Mol. Microbiol., 14, 571-581 (1994), Vertes, A.
A. et al., Mol. Gen. Genet., 245, 397-405 (1994), Jagar, W. et al.,
FEMS Microbiology Letters, 126, 1-6 (1995), Japanese Patent
Application Laid-open No. 7-107976, Japanese Patent Application
Laid-open No. 7-327680, etc.), phage vectors, recombination of
chromosomes (Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory Press (1972); Matsuyama, S. and Mizushima, S., J.
Bacteriol., 162, 1196 (1985)) and the like.
[0035] When a DNA fragment having an ability to allow a plasmid to
be autonomously replicable in coryneform bacteria is inserted into
these vectors, they can be used as a so-called shuttle vector
autonomously replicable in both Escherichia coli and coryneform
bacteria.
[0036] Such a shuttle vector includes the followings.
Microorganisms harboring each of vectors and deposition numbers in
international deposition facilities are shown in parentheses.
[0037] pHC4: Escherichia coli AJ12617 (FERM BP-3532)
[0038] pAJ655: Escherichia coli AJ11882 (FERM BP-136)
[0039] Corynebacterium glutamicum SR8201 (ATCC 39135)
[0040] pAJ1844: Escherichia coli AJ11883 (FERM BP-137)
[0041] Corynebacterium glutamicum SR8202 (ATCC 39136)
[0042] pAJ611: Escherichia coli AJ11884 (FERM BP-138)
[0043] pAJ3148: Corynebacterium glutamicum SR8203 (ATCC 39137)
[0044] pAJ440: Bacillus subtilis AJ11901 (FERM BP-140)
[0045] These vectors are obtainable from the deposited
microorganisms as follows. Cells collected at a logarithmic growth
phase were lysed by using lysozyme and SDS, followed by separation
from a lysate by centrifugation at 30,000.times.g to obtain a
supernatant to which polyethylene glycol is added, followed by
fractionation and purification by means of cesium chloride-ethidium
bromide equilibrium density gradient centrifugation.
[0046] Escherichia coli can be transformed by introducing a plasmid
in accordance with, for example, a method of D. M. Morrison
(Methods in Enzymology, 68, 326 (1979)) or a method in which
recipient cells are treated with calcium chloride to increase
permeability for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53,
159 (1970)).
[0047] Introduction of plasmids to coryneform bacteria to cause
transformation can be performed by the electric pulse method
(Sugimoto et al., Japanese Patent Application Laid-open No.
2-207791).
[0048] Examples of the coryneform bacterium used to introduce the
DNA described above include, for example, the following wild type
strains:
[0049] Corynebacterium acetoacidophilum ATCC 13870;
[0050] Corynebacterium acetoglutamicum ATCC 15806;
[0051] Corynebacterium callunae ATCC 15991;
[0052] Corynebacterium glutamicum ATCC 13032;
[0053] (Brevibacterium divaricatum) ATCC 14020;
[0054] (Brevibacterium lactofermentum) ATCC 13869;
[0055] (Corynebacterium lilium) ATCC 15990;
[0056] (Brevibacterium flavum) ATCC 14067;
[0057] Corynebacterium melassecola ATCC 17965;
[0058] Brevibacterium saccharolyticum ATCC 14066;
[0059] Brevibacterium immariophilum ATCC 14068;
[0060] Brevibacterium roseum ATCC 13825;
[0061] Brevibacterium thiogenitalis ATCC 19240;
[0062] Microbacterium ammoniaphilum ATCC 15354;
[0063] Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539).
[0064] Enhancement of phosphoserine phosphatase activity or
phosphoserine transaminase activity can also be achieved by
introducing multiple copies of the serB gene or serC gene into the
chromosomal DNA of the above-described host strains. In order to
introduce multiple copies of the serB gene or serC gene in the
chromosomal DNA of coryneform bacterium, the homologous
recombination is carried out using a sequence whose multiple copies
exist in the chromosomal DNA as targets. As sequences whose
multiple copies exist in the chromosomal DNA, repetitive DNA,
inverted repeats exist at the end of a transposable element can be
used. Also, as disclosed in Japanese Patent Publication Laid-Open
No. 2-109985, it is possible to incorporate the serB gene or serC
gene into transposon, and allow it to be transferred to introduce
multiple copies of the serB gene or serC gene into the chromosomal
DNA. By either method, the number of copies of the serB gene or
serC gene within cells of the transformant strain increases, and as
a result, phosphoserine phosphatase activity or phosphoserine
transaminase activity is enhanced.
[0065] Other than the above-described gene amplification,
enhancement of phosphoserine phosphatase activity or phosphoserine
transaminase activity can also be achieved by substituting the
expression regulation sequence such as promoter of the serB gene or
serC gene with a more potent one. For example, lac promoter, trp
promoter, trc promoter, tac promoter, and P.sub.R promoter and
P.sub.L promoter of lambda phage are known as potent promoters. By
substituting the promoter inherent in serB gene or serC gene with
these promoters, the expression of serB gene or serC gene is
enhanced, thereby enhancing phosphoserine phosphatase activity or
phosphoserine transaminase activity.
[0066] In a preferred embodiment, the coryneform bacteria of the
present invention is a strain obtained by introducing a gene coding
for D-3-phosphoglycerate dehydrogenase (hereafter, also referred to
as "3-PGDH") in which feedback inhibition by L-serine is
desensitized, into a coryneform bacterium having L-serine
productivity and an enhanced activity of phosphoserine phosphatase
or phosphoserine transaminase.
[0067] 3-PGDH catalyzes reaction in which 3-phosphoglycerate is
oxidized into 3-phosphohydroxylpyruvic acid in the presence of
nicotinamide adenine dinucleotide (NAD) as a coenzyme.
[0068] 3-PGDH derived from a wild type coryneform bacterium is
susceptible to feedback inhibition by L-serine and its activity is
almost completely inhibited in the presence of 10 mM of L-serine.
By the term "3-PGDH in which feedback inhibition by L-serine is
desensitized" is meant 3-PGDH having 20% or more, preferably 40% or
more, more preferably 90% or more of the activity in the absence of
L-serine even in the presence of 10 mM of L-serine. 3-PGDH derived
from Brevibacterium flavum AJ13327 described in the examples
hereinbelow retains substantially 100% of the activity in the
presence of 80 mM of L-serine and therefore one of the most
preferred 3-PGDHs.
[0069] The gene coding for 3-PGDH in which feedback inhibition by
L-serine is desensitized can be prepared from the chromosomal DNA
of L-serine analogue resistant coryneform bacteria, for example,
azaserine resistant strain AJ13327 of Brevibacterium flavum
obtained in the examples described below.
[0070] 3-PGDH derived from a wild type coryneform bacterium
(hereafter, DNA coding for this is also referred to as "wild type
serA") has the amino acid sequence described by SEQ ID NO: 12 in
the Sequence Listing. Specific examples of the 3-PGDH in which
feedback inhibition by L-serine is desensitized (hereafter, DNA
coding for this is also referred to as "mutant serA") include
D-3-phosphoglycerate dehydrogenase characterized in that in
D-3-phosphoglycerate dehydrogenase having the amino acid sequence
described by SEQ ID NO: 12 in the Sequence Listing or the same
amino acid sequence as above but has substitution, addition or
deletion of one or more amino acids, the amino acid residue
corresponding to the 325th glutamic acid residue of the amino acid
sequence in the SEQ ID NO: 12 has been substituted by other amino
acid. Most preferred as the other amino acid residue is a lysine
residue.
[0071] The DNA fragment containing serA gene from a coryneform
bacterium can be isolated, for example, by preparing chromosomal
DNA according to the method of Saito and Miura (H. Saito and K.
Miura, Biochem. Biophys. Acta, 72, 619 (1963)) or the like and then
amplifying serA gene by polymerase chain reaction method (PCR:
polymerase chain reaction; cf. White, T. J. et al.; Trends Genet.
5, 185 (1989)). For example, in order to amplify DNA fragment
containing ORF (172 to 1705) of SEQ ID NO: 11 in the Sequence
Listing, any 20 to 30 bases are selected from the region from the
first base in SEQ ID NO: 11 to the base immediately before ATG to
obtain one primer. Further, any 20 to 30 bases are selected from
the region from the base immediately after the termination codon to
the last base in SEQ ID NO: 11 to obtain another primer.
[0072] When serA is isolated from a wild type strain of 3-PGDH,
wild type serA is obtained and isolation of serA from a mutant
retaining 3-PGDH in which feedback inhibition by L-serine is
desensitized (3-PGDH mutant) gives mutant serA. Specifically, the
wild type serA has the sequence described by SEQ ID NO: 11 in the
Sequence Listing, and mutant serA has the sequence described by SEQ
ID NO: 13 in the Sequence Listing.
[0073] The mutant serA may be introduced into a coryneform
bacterium by transformation of the coryneform bacterium with a
mutant serA-containing recombinant vector in the same manner as in
the introduction of serB or serC. The mutant serA is preferably
introduced in multiple copies. The mutant serA and serB or serC may
be loaded on a single vector or on separate two or three vectors,
respectively.
[0074] For L-serine production using the strain of the present
invention, the following methods may be used. As the medium to be
used, there can be used conventional liquid mediums containing
carbon sources, nitrogen sources, inorganic salts, and optionally
organic trace nutrients such as amino acids, vitamins, etc., if
desired.
[0075] As carbon sources, it is possible to use sugars such as
glucose, sucrose, fructose, galactose; saccharified starch
solutions, sweet potato molasses, sugar beet molasses and hightest
molasses which are including the sugars described above; organic
acids such as acetic acid; alcohols such as ethanol; glycerol and
the like.
[0076] As nitrogen sources, it is possible to use ammonia gas,
aqueous ammonia, ammonium salts, urea, nitrates and the like.
Further, organic nitrogen sources for supplemental use, for
example, oil cakes, soybean hydrolysate liquids, decomposed casein,
other amino acids, corn steep liquor, yeast or yeast extract,
peptides such as peptone, and the like, may be used.
[0077] As inorganic ions, phosphoric ion, magnesium ion, calcium
ion, iron ion, manganese ion and the like may be added
optionally.
[0078] In case of using the microorganism of the present invention
which requires nutrients such as amino acids for its growth, the
required nutrients should be supplemented.
[0079] The microorganisms are incubated usually under aerobic
conditions at pH 5 to 8 and temperature ranges of 25 to 40.degree.
C. The pH of the culture medium is controlled at a predetermined
value within the above-described ranges depending on the presence
or absence of inorganic or organic acids, alkaline substances,
urea, calcium carbonate, ammonia gas, and the like.
[0080] L-Serine can be collected from the fermentation liquid, for
example, by separating and removing the cells, subjecting to ion
exchange resin treatment, concentration cooling crystallization,
membrane separation, and other known methods in any suitable
combination. In order to remove impurities, activated carbon
adsorption and recrystallization may be used for purification.
[0081] The present invention provides a coryneform bacterium that
synthesizes L-serine from a sugar. The coryneform bacterium can be
utilized in a method of producing L-serine that is advantageous
industrially.
DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
Construction of L-serine Producing Bacteria Brevibacterium flavum
AJ13324 and AJ13327
[0082] Brevibacterium flavum AJ13324 and AJ13327 were constructed
from Brevibacterium flavum AJ13377 that is deficient in L-serine
decomposing activity obtained from wild type strain Brevibacterium
flavumATCC 14067.
[0083] To obtain a mutant, cells proliferated for 24 hours in a
bouillon medium (a medium containing 1 g of fish meat extract, 1 g
of polypeptone, 0.5 g of yeast extract, and 0.5 g of sodium
chloride in 1 liter of water, adjusted to pH 7.0) were suspended in
100 mM phosphate buffer (pH 7.0) (containing 10.sup.9 to 10.sup.10
cells/ml). NG (N-methyl-N'-nitro-N-nitr- osoguanidine) was added to
the suspension to a concentration of 200 .mu.g/ml and left to stand
at 30.degree. C. for 30 minutes. The thus NG treated cells were
washed well with the above-described buffer.
[0084] To select strains having no L-serine decomposing activity
from the NG treated cells, NG treated cells of Brevibacterium
flavum ATCC 14067 after washed were spread on a bouillon agar
medium and incubated at 30.degree. C. for 24 hours to allow colony
formation. Then, the colonies on the bouillon agar medium were used
as a negative and replica formation was performed on a minimal
medium and a minimal medium for selection. Then, strains were
screened that grow on the minimal medium but do not grow on the
minimal medium for selection. The minimal medium was a medium that
contained 20 g of glucose, 1 g of ammonium sulfate, 1 g of
potassium dihydrogen phosphate, 2.5 g of urea, 0.4 g of magnesium
sulfate heptahydrate, 0.01 g of iron (II) sulfate heptahydrate,
0.01 g of manganese sulfate tetra- to pentahydrate, 50 .mu.g of
biotin, 200 .mu.g of thiamin hydrochloride, 200 .mu.g of nicotinic
acid amide, and 2.0 g of agar per liter of distilled water. The
minimal medium for selection was a medium that contained 1 g of
ammonium sulfate, 1 g of potassium dihydrogen phosphate, 2.5 g of
urea, 0.4 g of magnesium sulfate heptahydrate, 0.01 g of iron (II)
sulfate heptahydrate, 0.01 g of manganese sulfate tetra- to
pentahydrate, 50 .mu.g of biotin, 200 .mu.g of thiamin
hydrochloride, 200 .mu.g of nicotinic acid amide, 0.5 g of L-serine
and 2.0 g of agar per liter of distilled water. Among the mutants
obtained by this method were found many strains that have no
L-serine decomposing activity and Brevibacterium flavum AJ13377 was
obtained as one of such strains.
[0085] To select azaserine resistant strains from NG treated
strains using Brevibacterium flavum AJ13377 as a parent strain, NG
treated Brevibacterium flavum AJ13377 cells after washed were
inoculated on a minimal medium for selection. The minimal medium
for selection was a medium that contained 20 g of glucose, 1 g of
ammonium sulfate, 1 g of potassium dihydrogen phosphate, 2.5 g of
urea, 0.4 g of magnesium sulfate heptahydrate, 0.01 g of iron (II)
sulfate heptahydrate, 0.01 g of manganese sulfate tetra- to
pentahydrate, 50 .mu.g of biotin, 200 .mu.g of thiamin
hydrochloride, 200 .mu.g of nicotinic acid amide, and 250 mg of
azaserine per liter of distilled water. The NG treated mutant was
incubated in the above-described medium at 30.degree. C. for 5 to
10 days. The cell culture thus obtained was spread on a bouillon
agar medium and incubated at 30.degree. C. for 24 hours for colony
formation. Azaserine resistant strains were obtained from the
strains that formed colonies. The mutants thus obtained included
many strains that accumulated L-serine in considerable amounts at
high yields. From the strains were obtained two strains, i.e.,
Brevibacterium flavum AJ13324 and AJ13327. It was confirmed that
these strains were able to grow in the presence of 0.25 g/L of
azaserine.
EXAMPLE 2
Construction of Novel L-serine Producing Bacterium Brevibacterium
flavum AJ13325
[0086] Brevibacterium flavum AJ13325 was constructed from
Brevibacterium flavum AJ13377 lacking L-serine decomposing
activity, which was obtained from the wild type strain
Brevibacterium flavum ATCC 14067.
[0087] To select .beta.-(2-thienyl)-DL-alanine resistant strains
from NG treated strains using Brevibacterium flavum AJ13377 as a
parent strain, Brevibacterium flavum AJ13377 cells were NG treated
and washed before their inoculation on a minimal medium for
selection. The minimal medium for selection was a medium that
contained 20 g of glucose, 1 g of ammonium sulfate, 1 g of
potassium dihydrogen phosphate, 2.5 g of urea, 0.4 g of magnesium
sulfate heptahydrate, 0.01 g of iron (II) sulfate heptahydrate,
0.01 g of manganese sulfate tetra- to pentahydrate, 50 .mu.g of
biotin, 200 .mu.g of thiamin hydrochloride, 200 .mu.g of nicotinic
acid amide, and 250 mg of .beta.-(2-thienyl)-DL-alanine per liter
of distilled water. The NG treated mutant was incubated in the
above-described medium at 30.degree. C. for 5 to 10 days. The cell
culture thus obtained was spread on a bouillon agar medium and
incubated at 30.degree. C. for 24 hours for colony formation.
.beta.-(2-Thienyl)-DL-alanine resistant strains were obtained from
the strains that formed colonies. The mutants thus obtained
included many strains that accumulated L-serine in considerable
amounts at high yields. Brevibacterium flavum AJ13325 was obtained
as one of such strains. It was confirmed that these strains were
able to grow in the presence of 0.25 g/L of
.beta.-(2-thienyl)-DL-alanine.
EXAMPLE 3
Production of L-serine by L-serine Producing Bacteria
Brevibacterium flavum AJ13324, AJ13325 and AJ13327
[0088] Brevibacterium flavum AJ13324, AJ13325 and AJ13327 were each
incubated on a bouillon agar medium at 30.degree. C. for 24 hours
and a loopful of each microorganism was inoculated in a 500 ml
shaking flask containing 20 ml of a fermentation medium having the
composition shown in Table 1. As a control, the parent strains
Brevibacterium flavum ATCC 14067 and AJ13377 were inoculated as a
same manner as described above. The medium was adjusted to pH 7.0
with potassium hydroxide and autoclaved at 115.degree. C. for 15
minutes. After the sterilization and cooling, calcium carbonate
that had been dry air sterilized at 180.degree. C. for 3 hours was
added in an amount of 5 g/L.
1 TABLE 1 Component Content/liter Glucose 110.0 g Potassium
dihydrogen phosphate 0.4 g Magnesium sulfate heptahydrate 0.4 g
Iron (II) sulfate heptahydrate 0.01 g Manganese sulfate tetra- to
penta- 0.01 g hydrate Ammonium sulfate 25.0 g Thiamin hydrochloride
100 .mu.g Biotin 100 .mu.g Soy bean protein hydrochloric acid 40 ml
hydrolysate ("Mieki" (registered trademark) pH 7.0
[0089] Determination of L-serine using high performance liquid
chromatography (Hitachi L-8500 Amino Acid Autoanalyzer) revealed
that Brevibacterium flavum AJ13324, AJ13325 and AJ13327 accumulated
L-serine in the medium in amounts of 15.2 g/L, 14.3 g/L, and 15.4
g/L, respectively. On the other hand, Brevibacterium flavum strains
ATCC 14067 and AJ13377 incubated as a control accumulated L-serine
in amounts of 0 g/L and 5.0 g/L, respectively.
[0090] The culture broth of Brevibacterium flavum AJ13324 was
centrifuged and the supernatant was subjected to desalting
treatment using cation exchange resin, followed by chromatographic
separation with cation exchange resin and anion exchange resin to
remove byproducts and purification by crystallization to obtain
L-serine crystals of at least 99% purity at a yield from broth of
55%.
EXAMPLE 4
Measurement of 3-PGDH Activity
[0091] Brevibacterium flavum AJ13324, AJ13325 and AJ13327 were each
incubated on a bouillon agar medium at 30.degree. C. for 24 hours
and a loopful of each microorganism was inoculated in a 500 ml
shaking flask containing 50 ml of a fermentation medium having the
composition shown in Table 2. As a control, the parent strains
Brevibacterium flavum ATCC 14067 and AJ13377 were inoculated as a
same manner as described above. The medium for inoculation was
adjusted to pH 5.5 with sodium hydroxide and autoclaved at
115.degree. C. for 15 minutes.
2 TABLE 2 Component Content/liter Glucose 30.0 g Potassium
dihydrogen phosphate 1.0 g Magnesium sulfate heptahydrate 0.4 g
Iron (II) sulfate heptahydrate 0.01 g Manganese sulfate tetra- to
penta- 0.01 g hydrate Ammonium sulfate 3.0 g Soy bean protein
hydrochloric acid 3.0 ml hydrolysate ("Mieki" (registered
trademark) Thiamin hydrochloride 200 .mu.g Biotin 50 .mu.g Urea 3.0
g Yeast extract 2.0 g pH 5.5
[0092] After collecting cells from the culture broth of each
strain, the cells were washed twice with physiological saline and
suspended in 50 mM sodium phosphate buffer (pH 7.0) containing 2 mM
dithiothreitol. After ice cooling, the suspension was subjected to
a sonicator to fragment the cells and the resulting liquid was
ultracentrifuged. The ultracentrifugaton was run at 45,000 rpm for
1 hour to obtain a crude enzyme solution.
[0093] The enzyme activity of 3-PGDH was measured by the method of
Salach H. J. et al. (Method in Enzymology, vol 9, 216-220
(1966)).
[0094] More specifically, 0.4 ml of 0.015 M NAD, 0.12 ml of 0.25 M
EDTA (pH 9, NaOH), 0.1 ml of 0.05 M glutathione (pH 6, KOH), 0.5 ml
of 1 M hydrazine (pH 9, acetate), 0.6 ml of 1 M Tris (pH 9, HCl), a
suitable concentration of L-serine (0 to 40 mM), and water to make
2.3 ml, warmed to 25.degree. C. in advance, were added. Then, 0.2
ml of the crude enzyme solution was added and the temperature was
kept the same for 5 minutes. Thereafter, 0.5 ml of 0.1 M 3-PGA
(3-phosphoglycerate disodium salt, pH 7, NaOH) was added. After
stirring, the absorbance at 340 nm of the reaction mixture was
measured for 30 seconds. The reaction was carried out at 25.degree.
C.
[0095] For the measurement of activity, Hitachi U-2000A
spectrophotometer was used.
[0096] FIG. 1 illustrates the results obtained. AJ13377 strain was
relieved of L-serine sensitivity as compared with the wild type
strain ATCC 14067. The AJ13324 strain was more relieved of L-serine
sensitivity and the AJ13325 strain was of the same level as the
AJ13324 strain in this respect. The AJ13327 strain was relieved of
L-serine sensitivity greatly. And the inhibition was completely
desensitized even in the presence of 80 mM L-serine.
[0097] Although some examples of desensitization of the inhibition
of 3-PGDH by L-serine were reported on Escherichia coli (Tosa and
Pizer, J. Bacteriol. 106: 972-982 (1971) or Japanese Patent
Application Laid-open No. 6-510911), there has been known no
example of complete desensitization of the inhibition in the
presence of such a high concentration of L-serine.
EXAMPLE 5
Cloning of Coryneform Bacteria-Derived Wild Type and Mutant
serA
[0098] As shown in Example 4, the feedback inhibition by L-serine
was completely desensitized in the AJ13327 strain. Accordingly,
cloning of serA gene coding for wild type 3-PGDH derived from the
ATCC 14067 strain and mutant 3-PGDH derived from the AJ13327 strain
was attempted in order to elucidate what the variation was like and
confirm the amplification effect of 3-PGDH.
[0099] To amplify serA from the chromosome of Brevibacterium flavum
using a PCR method, it is necessary to make a corresponding primer.
Since no report has been made on the cloning and nucleotide
sequence of serA of Brevibacterium flavum, the sequence of serA
derived from Corynebacterium was used. Plasmid pDTS9901 was
extracted from the strain Corynebacterium glutamicum K82 (cf. FERM
BP-2444 and Japanese Patent Application Laid-open No. 3-7591) in
which the serA fragment derived from Corynebacterium was cloned
using Wizard Minipreps DNA Purification System (manufactured by
Promega) and a DNA fragment of about 1.4 kb containing serA was
cleaved with restriction enzyme BamHI (manufactured by Takara Shuzo
Co., Ltd.).
[0100] As a vector for cloning the gene fragment, there was used a
newly constructed cloning vector pVK7 for coryneform bacteria.
[0101] pVK7 was constructed by ligating (a cloning vector for
Escherichia coli) pHSG299 (Kmr; Takeshita, S. et al., Gene, 61,
63-74 (1987), Japanese Patent Application Laid-open No. 10-215883),
to pAM330, a cryptic plasmid of Brevibacterium lactofermentum, in
the manner described below. pHSG299 was cleaved with monospecific
restriction enzyme AvaII (manufactured by Takara Shuzo Co., Ltd.)
and blunt ended with T4 DNA polymerase. This was ligated with
pAM330 that had been cleaved with HindIII (manufactured by Takara
Shuzo Co., Ltd.) and blunt ended with T4 DNA polymerase. The two
types of plasmids obtained were designated pVK6 and pVK7 depending
on the direction of pAM330 insertion relative to pHSG299, and pVK7
was used in the following experiments. pVK7 was capable of
autonomous replication in Escherichia coli and Brevibacterium
lactofermentum and retains the multiple cloning site and lacZ'
derived from pHSG299. FIG. 2 illustrates the process of
constructing pVK6 and pVK7.
[0102] To the shuttle vector pVK7 thus constructed was ligated a
DNA fragment of about 1.4 kb containing serA. pDTS9901 was cleaved
with restriction enzyme BamHI (manufactured by Takara Shuzo Co.,
Ltd.) and ligated to pVK7 also cleaved with restriction enzyme
BamHI. The ligation of DNA was performed using DNA Ligation Kit
(manufactured by Takara Shuzo Co., Ltd.) according to the
prescribed method.
[0103] For the sequencing reaction, use was made of PCR thermal
cycler MP type (manufactured by Takara Shuzo Co., Ltd.) and of Dye
Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by
Perkin Elmer). As the DNA primer, there were used M13(-21), RV
primer (manufactured by Takara Shuzo Co., Ltd.). The SEQ ID NO: 1
in the Sequence Listing shows the sequence thus obtained. SEQ ID
NO: 2 shows an amino acid sequence that can be coded for by this
sequence.
[0104] A primer was synthesized based on the base sequence thus
determined and serA was amplified by a PCR method using the
chromosomal DNA of the mutant Brevibacterium flavum AJ13327 as a
template. The SEQ ID NOS: 3 and 4 in the Sequence Listing show the
N-terminal side and C terminal side sequences, respectively, of the
DNA primer that were synthesized for gene amplification.
[0105] In the preparation of the chromosomal DNA of Brevibacterium
flavum, use is made of Genomic DNA Purification Kit (Bacterial)
(manufactured by Advanced Genetic Technologies Corp.) and the
preparation method was according to the annexed protocol.
[0106] For the PCR reaction, use is made of PCR Thermal Cycler MP
type (Takara Shuzo Co., Ltd.) and of TaKaRa Taq (manufactured by
Takara Shuzo Co., Ltd.).
[0107] The PCR product was ligated directly to plasmid pCR2.1
vector using Original TA Cloning Kit (manufactured by Invitrogen)
and transformation was performed using competent cell of
INV.alpha.F'. The transformed cells were spread on L medium (10 g/L
of bactotryptone, 5 g/L of bactoyeast extract, 15 g/L of NaCl, and
15 g/L of agar) further containing 40 .mu.g/ml of X-Gal
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and 25 .mu.g/ml
of Kanamycin, and incubated overnight. The white colonies, which
appeared, were collected and separated to single colonies to obtain
a transformed strain.
[0108] Plasmids were extracted from the transformed strain and
those plasmids of which insertion of the serA fragment was
confirmed by a PCR method were treated with restriction enzyme
EcoRI and ligated to the shuttle vector pVK. Determination of the
base sequence of the product suggested that no full-length sequence
be contained on the C-terminal side. The sequence thus obtained
corresponds to the region from 277 bases upstream of SEQ ID NO: 13
on the 5' side to the 1134th base of SEQ ID NO: 13 in the Sequence
Listing on the 3' side.
[0109] To obtain a fragment containing the full length serA gene,
cloning of a deleted part from the chromosomal DNA of
Brevibacterium flavum AJ13327 strain was performed according to the
annexed protocol using TaKaRa LA PCR in vitro Cloning Kit
(manufactured by Takara Shuzo Co., Ltd.)
[0110] First, the chromosomal DNA thus prepared was completely
digested with various restriction enzymes and ligated with
cassettes having respective restriction enzyme sites corresponding
thereto. Cassette primer (C1) (SEQ ID NO: 5 in the Sequence
Listing) and a primer complementary to a known region of DNA (S1)
(SEQ ID NO: 6 in the Sequence Listing) were used for carrying out
first PCR. Using a portion of the reaction mixture, second PCR was
carried out with inner primer C2 (SEQ ID NO: 7 in the Sequence
Listing) and S2 (SEQ ID NO: 8 in the Sequence Listing) to amplify
only the targeted DNA.
[0111] When EcoRI (manufactured by Takara Shuzo Co., Ltd.) was used
as the restriction enzyme, the amplification of the targeted DNA
was confirmed and the base sequence of the PCR product was
determined directly. Based on the base sequence thus obtained, a
primer coding for the C-terminal side was made and the fragments
containing full length serA were collected from Brevibacterium
flavum ATCC 14067 as a wild type strain and Brevibacterium flavum
AJ13327 as a mutant strain. SEQ ID NOS: 9 and 10 in the Sequence
Listing show the sequences of N-terminal and C-terminal side DNA
primers, respectively.
[0112] The gene fragments containing wild type serA and mutant
serA, respectively, in their full length were ligated to
EcoRI-cleaved shuttle vector pVK7 using Original TA Cloning Kit
(manufactured by Invitrogen). Plasmids harboring respective gene
fragments were made separately and their base sequence was
determined. SEQ ID NOS: 11 and 13 indicate the sequences of the
wild type and of mutant, respectively. SEQ ID NOS: 12 and 14
indicate amino acid sequences that these sequences can code for.
Comparing the base sequences thus determined, it was confirmed that
in the mutant serA, the 1087th base, G, was mutated into A and as a
result, the 325th amino acid, glutamic acid, was changed to
lysine.
EXAMPLE 6
Introduction of Plasmid Containing 3-PGDH Gene into Brevibacterium
flavum
[0113] Plasmids harboring wild type serA or mutant serA were each
introduced into Brevibacterium flavum AJ13377. The plasmids were
introduced by the electric pulse method (Sugimoto et al., Japanese
Patent Application Laid-open No. 2-207791). Transformed cells were
selected in a complete medium containing 25 .mu.g/ml of
kanamycin.
EXAMPLE 7
Production of L-serine by Transformed Cells
[0114] Transformed cells each having introduced therein plasmids
harboring gene fragments containing wild serA or mutant serA in
their full-length were incubated in a 500 ml shaking flask
according to Example 3, and L-serine produced was determined. As a
control, the AJ13377 strain as a host was incubated similarly.
[0115] In the transformed cell having introduced therein the wild
type serA was observed no influence on its L-serine productivity
whereas in the transformed cell having introduced therein the
mutant serA was confirmed an increase in L-serine productivity
(Table 3).
[0116] Brevibacterium flavum AJ13377 has been deposited since Oct.
15, 1997 in National Institute of Bioscience and Human Technology
of Agency of Industrial Science and Technology of Ministry of
International Trade and Industry (zip code: 305-8566, 1-3 Higashi
1-Chome, Tsukuba-shi, Ibaraki-ken, Japan), as accession number of
FERM P-16471, and transferred from the original deposition to
international deposition based on Budapest Treaty on Nov. 20, 1998,
and has been deposited as accession number of FERM BP-6576.
[0117] Further, the plasmid containing the mutant serA was retained
in Brevibacterium flavum ATCC 14067. The plasmid-retaining strain
has been awarded Brevibacterium flavum AJ13378 and deposited since
Oct. 15, 1997 in National Institute of Bioscience and Human
Technology of Agency of Industrial Science and Technology of
Ministry of International Trade and Industry (zip code: 305-8566,
1-3 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan), as accession
number of FERM P-16472, and transferred from the original
deposition to international deposition based on Budapest Treaty on
Nov. 20, 1998, and has been deposited as accession number of FERM
BP-6577.
EXAMPLE 8
Amplification of serB and/or serC in Brevibacterium flavum L-serine
Producing Strains
[0118] (1) Construction of Plasmid Expressing serB or serC
[0119] Plasmids pSB that express serB and plasmids pSC that express
serC were constructed as illustrated in FIGS. 3 and 4.
[0120] For the serB gene was made a primer (SEQ ID NOS: 15 and 16
in the Sequence Listing indicate N-terminal and C-terminal sides,
respectively) based on the known base sequence (GenBank; X03046,
M30784). On the other hand, for the serC gene, a primer (SEQ ID
NOS: 17 and 18 in the Sequence Listing indicate N-terminal and
C-terminal sides, respectively) was prepared based on the known
base sequence (GenBank; D90728) and PCR was carried out using the
chromosomal DNA of Escherichia coli JM109 as a template to obtain a
gene fragment (1197 bp) containing ORF coding for serB and a gene
fragment (1380 bp) containing ORF coding for serC.
[0121] The base sequences of SEQ ID NOS: 15 and 16 correspond to
the regions of base Nos. 1197 to 1175 and of base Nos. 1 to 23 in
the sequence GenBank; X03046, M30784 and the base sequences of SEQ
ID NOS: 17 and 18 correspond to the regions of base Nos. 13205 to
13227 and of base Nos. 14584 to 14562 in the sequence GenBank;
D90728.
[0122] The serB fragment, after blunt ended, was inserted into the
SmaI site of pHSG399, a high copy type vector, to obtain p399B. To
render this plasmid to be capable of autonomic replication in
bacteria belonging to the genus Corynebacterium, a replicator
(hereafter, referred to "Brev.-ori") was cleaved form pBK4
retaining the replicator derived from pHM1519 and inserted to p399B
to obtain pSB (FIG. 3). pBK4 was made as follows. That is, a
plasmid pHC4 containing Brev.-ori was prepared from Escherichia
coli AJ12617 strain containing this plasmid (FERM BP-3532) and
cleaved with KpnI (manufactured by Takara Shuzo Co., Ltd.) and
BamHI (manufactured by Takara Shuzo Co., Ltd.) to extract Brev.-ori
fragment, which was then blunt ended. Blunting of the ends was
carried out using DNA Blunting Kit (manufactured by Takara Shuzo
Co., Ltd.) according to the prescribed method. Thereafter, the
product was ligated to an already phosphorylated BamHI linker and
cleaved again with BamHI. This was ligated to pHSG298 that was also
cleaved with BamHI to obtain pBK4. pBK4 may be used for cleaving
Brev.-ori fragment with BamHI.
[0123] Further, a serC fragment was inserted to the SrfI site of
pPCR-Script SK(+) to obtain pScript-serC. To the SacI site of the
resulting plasmid was inserted a PstI linker. Then, the serC
fragment was cleaved with PstI and inserted to the PstI site of
pHSG399 to obtain p399C. A replicator was cleaved from pBK4
retaining the replicator derived from pHM1519 and inserted into
p399C to obtain pSC (FIG. 4).
[0124] (2) Construction of Plasmid that Expresses serB and serC
[0125] Next, plasmids pBC8 and pBC14 that express serB and serC,
respectively, were made (FIG. 5). To the SacI site existing outside
the serC fragment of the above-described pScript-serC was inserted
a PstI linker to introduce a PstI site. This plasmid was treated
with PstI to cleave a serC fragment, which was then inserted to the
PstI site of the serB-containing plasmid pSB. The base sequence was
confirmed and the plasmid in which the serC fragment was inserted
in the reverse direction to lacZ was named pBC8, and the plasmid in
which it was inserted in the forward direction to lacZ was named
pBC14.
[0126] (3) L-Serine Production by serB and serC Amplified
Strains
[0127] Using the plasmids pSB, pSC and pBC8 made as described
above, the wild type strain of Brevibacterium flavum ATCC 14067 was
transformed and plasmids were extracted from the transformed cells.
The plasmids were used for transforming Brevibacterium flavum
AJ13377 and AJ13327 having L-serine productivity. Also,
Brevibacterium flavum AJ13377 and AJ13327 strains retaining pBC8
were transformed with a plasmid containing the mutant serA that
Brevibacterium flavum AJ13378 (FERM P-16472) retained.
[0128] Each of the transformed strains was incubated on an agar
medium containing 10 mg/L of chloramphenicol and the colonies
formed were each incubated in the same manner as in Example 3,
followed by measurement of L-serine that accumulated in the medium.
The transformed strains that contained mutant serA were incubated
by adding 25 mg/L of kanamycin to the medium. Table 3 shows the
results obtained.
3 TABLE 3 Amount of L-serine Strain Amplified Gene that accumulated
(g/L) AJ13377 -- 5.0 serA 5.0 serA* 12.0 SerB 19.3 serC 8.3
serB,serC 19.5 serA*,serB,serC 24.8 AJ13327 -- 15.4 serB 24.2 serC
19.8 serB,serC 26.4 serA*,serB,serC 35.2 serA*: Mutant serA
gene
[0129] As described above, amplification of serB or serC increased
the amount of L-serine that accumulated. Also, amplification of the
both serB and serC genes further increased the amount of L-serine
that accumulated. In addition, amplification of the genes together
with mutant serA gene increased the amount of L-serine that
accumulated more. Similar results were obtained by using pBC14
instead of pBC8.
[0130] In the present example, although L-serine decomposing
activity deficient strain (AJ13377) or L-serine decomposing
activity deficient, azaserine resistant strain (AJ13327) of
Brevibacterium flavum was used as coryneform bacterium host having
L-serine productivity for amplifying each gene, other azaserine
resistant strain (AJ13324) or L-serine decomposing activity
deficient, .beta.-(2-thienyl)-DL-alanine resistant strain (AJ13325)
may also be used.
Sequence CWU 1
1
18 1 1432 DNA Corynebacterium glutamicum CDS (398)..(1432) 1
ggatccggac acacgtgaca aaattgtaga aaattggatg attttgtcac gcctgtctgg
60 tttagctctg gttcgggacg ggcgtggaat ggaggtagcg caccgagacc
ttgacccgcg 120 gcccgacaag ccaaaagtcc ccaaaacaaa cccacctcgc
cggagacgtg aataaaattc 180 gcagctcatt ccatcagcgt aaacgcagct
ttttgcatgg tgagacacct ttgggggtaa 240 atctcacagc atgaatctct
gggttagatg actttctggg tgggggaggg tttagaatgt 300 ttctagtcgc
acgccaaaac ccggcgtgga cacgtctgca gccgacgcgg tcgtgcctgt 360
tgtaggcgga cattcctagt ttttccagga gtaactt gtg agc cag aat ggc cgt
415 Val Ser Gln Asn Gly Arg 1 5 ccg gta gtc ctc atc gcc gat aag ctt
gcg cag tcc act gtt gac gcg 463 Pro Val Val Leu Ile Ala Asp Lys Leu
Ala Gln Ser Thr Val Asp Ala 10 15 20 ctt gga gat gca gta gaa gtc
cgt tgg gtt gac gga cct aac cgc cca 511 Leu Gly Asp Ala Val Glu Val
Arg Trp Val Asp Gly Pro Asn Arg Pro 25 30 35 gaa ctg ctt gat gca
gtt aag gaa gcg gac gca ctg ctc gtg cgt tct 559 Glu Leu Leu Asp Ala
Val Lys Glu Ala Asp Ala Leu Leu Val Arg Ser 40 45 50 gct acc act
gtc gat gct gaa gtc atc gcc gct gcc cct aac ttg aag 607 Ala Thr Thr
Val Asp Ala Glu Val Ile Ala Ala Ala Pro Asn Leu Lys 55 60 65 70 atc
gtc ggt cgt gcc ggc gtg ggc ttg gac aac gtt gac atc cct gct 655 Ile
Val Gly Arg Ala Gly Val Gly Leu Asp Asn Val Asp Ile Pro Ala 75 80
85 gcc act gaa gct ggc gtc atg gtt gct aac gca ccg acc tct aac att
703 Ala Thr Glu Ala Gly Val Met Val Ala Asn Ala Pro Thr Ser Asn Ile
90 95 100 cac tct gct tgt gag cac gca att tct ttg ctg ctg tct act
gct cgc 751 His Ser Ala Cys Glu His Ala Ile Ser Leu Leu Leu Ser Thr
Ala Arg 105 110 115 cag atc cct gct gct gat gcg acg ctg cgt gag ggc
gag tgg aag cgg 799 Gln Ile Pro Ala Ala Asp Ala Thr Leu Arg Glu Gly
Glu Trp Lys Arg 120 125 130 tct tct ttc aac ggt gtg gaa att ttc gga
aaa act gtc ggt atc gtc 847 Ser Ser Phe Asn Gly Val Glu Ile Phe Gly
Lys Thr Val Gly Ile Val 135 140 145 150 ggt ttt ggc cac att ggt cag
ttg ttt gct cag cgt ctt gct gcg ttt 895 Gly Phe Gly His Ile Gly Gln
Leu Phe Ala Gln Arg Leu Ala Ala Phe 155 160 165 gag acc acc att gtt
gct tac gat cct tac gcc aac cct gct cgt gca 943 Glu Thr Thr Ile Val
Ala Tyr Asp Pro Tyr Ala Asn Pro Ala Arg Ala 170 175 180 gct cag ctg
aac gtt gag ttg gtt gag ttg gat gag ctg atg agc cgt 991 Ala Gln Leu
Asn Val Glu Leu Val Glu Leu Asp Glu Leu Met Ser Arg 185 190 195 tct
gac ttt gtc acc att cac ctt cct aag acc aag gaa act gct ggc 1039
Ser Asp Phe Val Thr Ile His Leu Pro Lys Thr Lys Glu Thr Ala Gly 200
205 210 atg ttt gat gcg cag ctc ctt gct aag tcc aag aag ggc cag atc
atc 1087 Met Phe Asp Ala Gln Leu Leu Ala Lys Ser Lys Lys Gly Gln
Ile Ile 215 220 225 230 atc aac gct gct cgt ggt ggc ctt gtt gat gag
cag gct ttg gct gat 1135 Ile Asn Ala Ala Arg Gly Gly Leu Val Asp
Glu Gln Ala Leu Ala Asp 235 240 245 gcg att gag tcc ggt cac att cgt
ggc gct ggt ttc gat gtg tac tcc 1183 Ala Ile Glu Ser Gly His Ile
Arg Gly Ala Gly Phe Asp Val Tyr Ser 250 255 260 acc gag cct tgc act
gat tct cct ttg ttc aag ttg cct cag gtt gtt 1231 Thr Glu Pro Cys
Thr Asp Ser Pro Leu Phe Lys Leu Pro Gln Val Val 265 270 275 gtg act
cct cac ttg ggt gct tct act gaa gag gct cag gat cgt gcg 1279 Val
Thr Pro His Leu Gly Ala Ser Thr Glu Glu Ala Gln Asp Arg Ala 280 285
290 ggt act gac gtt gct gat tct gtg ctc aag gcg ctg gct ggc gag ttc
1327 Gly Thr Asp Val Ala Asp Ser Val Leu Lys Ala Leu Ala Gly Glu
Phe 295 300 305 310 gtg gcg gat gct gtg aac gtt tcc ggt ggt cgc gtg
ggc gaa gag gtt 1375 Val Ala Asp Ala Val Asn Val Ser Gly Gly Arg
Val Gly Glu Glu Val 315 320 325 gct gtg tgg atg gat ctg gct cgc aag
ctt ggt ctt ctt gct ggc aag 1423 Ala Val Trp Met Asp Leu Ala Arg
Lys Leu Gly Leu Leu Ala Gly Lys 330 335 340 ctt gtc gac 1432 Leu
Val Asp 345 2 345 PRT Corynebacterium glutamicum 2 Val Ser Gln Asn
Gly Arg Pro Val Val Leu Ile Ala Asp Lys Leu Ala 1 5 10 15 Gln Ser
Thr Val Asp Ala Leu Gly Asp Ala Val Glu Val Arg Trp Val 20 25 30
Asp Gly Pro Asn Arg Pro Glu Leu Leu Asp Ala Val Lys Glu Ala Asp 35
40 45 Ala Leu Leu Val Arg Ser Ala Thr Thr Val Asp Ala Glu Val Ile
Ala 50 55 60 Ala Ala Pro Asn Leu Lys Ile Val Gly Arg Ala Gly Val
Gly Leu Asp 65 70 75 80 Asn Val Asp Ile Pro Ala Ala Thr Glu Ala Gly
Val Met Val Ala Asn 85 90 95 Ala Pro Thr Ser Asn Ile His Ser Ala
Cys Glu His Ala Ile Ser Leu 100 105 110 Leu Leu Ser Thr Ala Arg Gln
Ile Pro Ala Ala Asp Ala Thr Leu Arg 115 120 125 Glu Gly Glu Trp Lys
Arg Ser Ser Phe Asn Gly Val Glu Ile Phe Gly 130 135 140 Lys Thr Val
Gly Ile Val Gly Phe Gly His Ile Gly Gln Leu Phe Ala 145 150 155 160
Gln Arg Leu Ala Ala Phe Glu Thr Thr Ile Val Ala Tyr Asp Pro Tyr 165
170 175 Ala Asn Pro Ala Arg Ala Ala Gln Leu Asn Val Glu Leu Val Glu
Leu 180 185 190 Asp Glu Leu Met Ser Arg Ser Asp Phe Val Thr Ile His
Leu Pro Lys 195 200 205 Thr Lys Glu Thr Ala Gly Met Phe Asp Ala Gln
Leu Leu Ala Lys Ser 210 215 220 Lys Lys Gly Gln Ile Ile Ile Asn Ala
Ala Arg Gly Gly Leu Val Asp 225 230 235 240 Glu Gln Ala Leu Ala Asp
Ala Ile Glu Ser Gly His Ile Arg Gly Ala 245 250 255 Gly Phe Asp Val
Tyr Ser Thr Glu Pro Cys Thr Asp Ser Pro Leu Phe 260 265 270 Lys Leu
Pro Gln Val Val Val Thr Pro His Leu Gly Ala Ser Thr Glu 275 280 285
Glu Ala Gln Asp Arg Ala Gly Thr Asp Val Ala Asp Ser Val Leu Lys 290
295 300 Ala Leu Ala Gly Glu Phe Val Ala Asp Ala Val Asn Val Ser Gly
Gly 305 310 315 320 Arg Val Gly Glu Glu Val Ala Val Trp Met Asp Leu
Ala Arg Lys Leu 325 330 335 Gly Leu Leu Ala Gly Lys Leu Val Asp 340
345 3 23 DNA Artificial Sequence Description of Artificial
SequencePrimer 3 ggacacacgt gacaaaattg tag 23 4 23 DNA Artificial
Sequence Description of Artificial SequencePrimer 4 gccagcaaga
agaccaagct tgc 23 5 35 DNA Artificial Sequence Description of
Artificial SequencePrimer 5 gtacatattg tcgttagaac gcgtaatacg actca
35 6 23 DNA Artificial Sequence Description of Artificial
SequencePrimer 6 tcatcaacgc tgctcgtggt ggc 23 7 35 DNA Artificial
Sequence Description of Artificial SequencePrimer 7 cgttagaacg
cgtaatacga ctcactatag ggaga 35 8 23 DNA Artificial Sequence
Description of Artificial SequencePrimer 8 gacgttgctg attctgtgct
caa 23 9 23 DNA Artificial Sequence Description of Artificial
SequencePrimer 9 gggagggttt agaatgtttc tag 23 10 23 DNA Artificial
Sequence Description of Artificial SequencePrimer 10 ggttcaagca
aatggatctc taa 23 11 1730 DNA Brevibacterium flavum CDS
(115)..(1704) 11 gggagggttt agaatgtttc tagtcgcacg ccaaaacccg
gcgtggacac gtctgcagcc 60 gacgcggtcg tgcctgttgt aggcggacat
tcctagtttt tccaggagta actt gtg 117 Val 1 agc cag aat ggc cgt ccg
gta gtc ctc atc gcc gat aag ctt gcg cag 165 Ser Gln Asn Gly Arg Pro
Val Val Leu Ile Ala Asp Lys Leu Ala Gln 5 10 15 tcc act gtt gac gcg
ctt gga gat gca gta gaa gtc cgt tgg gtt gac 213 Ser Thr Val Asp Ala
Leu Gly Asp Ala Val Glu Val Arg Trp Val Asp 20 25 30 gga cct aac
cgc cca gaa ctg ctt gat aca gtt aag gaa gcg gac gca 261 Gly Pro Asn
Arg Pro Glu Leu Leu Asp Thr Val Lys Glu Ala Asp Ala 35 40 45 ctg
ctc gtg cgt tct gct acc act gtc gat gct gaa gtc atc gcc gct 309 Leu
Leu Val Arg Ser Ala Thr Thr Val Asp Ala Glu Val Ile Ala Ala 50 55
60 65 gcc cct aac ttg aag atc gtc ggt cgt gcc ggc gtg ggc ttg gac
aac 357 Ala Pro Asn Leu Lys Ile Val Gly Arg Ala Gly Val Gly Leu Asp
Asn 70 75 80 gtt gac atc cct gct gcc act gaa gct ggc gtc atg gtt
gct aac gca 405 Val Asp Ile Pro Ala Ala Thr Glu Ala Gly Val Met Val
Ala Asn Ala 85 90 95 ccg acc tct aac att cac tct gct tgt gag cac
gca att tct ttg ctg 453 Pro Thr Ser Asn Ile His Ser Ala Cys Glu His
Ala Ile Ser Leu Leu 100 105 110 ctg tct act gct cgc cag atc cct gct
gct gat gcg acg ctg cgt gag 501 Leu Ser Thr Ala Arg Gln Ile Pro Ala
Ala Asp Ala Thr Leu Arg Glu 115 120 125 ggc gag tgg aag cgg tct tct
ttc aac ggt gtg gaa att ttc gga aaa 549 Gly Glu Trp Lys Arg Ser Ser
Phe Asn Gly Val Glu Ile Phe Gly Lys 130 135 140 145 act gtc ggt atc
gtc ggt ttt ggc cac att ggt cag ttg ttt gct cag 597 Thr Val Gly Ile
Val Gly Phe Gly His Ile Gly Gln Leu Phe Ala Gln 150 155 160 cgt ctt
gct gcg ttt gag acc acc att gtt gct tac gat cct tac gct 645 Arg Leu
Ala Ala Phe Glu Thr Thr Ile Val Ala Tyr Asp Pro Tyr Ala 165 170 175
aac cct gct cgt gcg gct cag ctg aac gtt gag ttg gtt gag ttg gat 693
Asn Pro Ala Arg Ala Ala Gln Leu Asn Val Glu Leu Val Glu Leu Asp 180
185 190 gag ctg atg agc cgt tct gac ttt gtc acc att cac ctt cct aag
acc 741 Glu Leu Met Ser Arg Ser Asp Phe Val Thr Ile His Leu Pro Lys
Thr 195 200 205 aag gaa act gct ggc atg ttt gat gcg cag ctc ctt gct
aag tcc aag 789 Lys Glu Thr Ala Gly Met Phe Asp Ala Gln Leu Leu Ala
Lys Ser Lys 210 215 220 225 aag ggc cag atc atc atc aac gct gct cgt
ggt ggc ctt gtt gat gaa 837 Lys Gly Gln Ile Ile Ile Asn Ala Ala Arg
Gly Gly Leu Val Asp Glu 230 235 240 cag gct ttg gct gat gcg att gag
tcc ggt cac att cgt ggc gct ggt 885 Gln Ala Leu Ala Asp Ala Ile Glu
Ser Gly His Ile Arg Gly Ala Gly 245 250 255 ttc gat gtg tac tcc acc
gag cct tgc act gat tct cct ttg ttc aag 933 Phe Asp Val Tyr Ser Thr
Glu Pro Cys Thr Asp Ser Pro Leu Phe Lys 260 265 270 ttg cct cag gtt
gtt gtg act cct cac ttg ggt gct tct act gaa gag 981 Leu Pro Gln Val
Val Val Thr Pro His Leu Gly Ala Ser Thr Glu Glu 275 280 285 gct cag
gat cgt gcg ggt act gac gtt gct gat tct gtg ctc aag gcg 1029 Ala
Gln Asp Arg Ala Gly Thr Asp Val Ala Asp Ser Val Leu Lys Ala 290 295
300 305 ctg gct ggc gag ttc gtg gcg gat gct gtg aac gtt tcc ggt ggt
cgc 1077 Leu Ala Gly Glu Phe Val Ala Asp Ala Val Asn Val Ser Gly
Gly Arg 310 315 320 gtg ggc gaa gag gtt gct gtg tgg atg gat ctg gct
cgc aag ctt ggt 1125 Val Gly Glu Glu Val Ala Val Trp Met Asp Leu
Ala Arg Lys Leu Gly 325 330 335 ctt ctt gct ggc aag ctt gtc gac gcc
gcc cca gtc tcc att gag gtt 1173 Leu Leu Ala Gly Lys Leu Val Asp
Ala Ala Pro Val Ser Ile Glu Val 340 345 350 gag gct cga ggc gag ctt
tct tcc gag cag gtc gat gca ctt ggt ttg 1221 Glu Ala Arg Gly Glu
Leu Ser Ser Glu Gln Val Asp Ala Leu Gly Leu 355 360 365 tcc gct gtt
cgt ggt ttg ttc tcc gga att atc gaa gag tcc gtt act 1269 Ser Ala
Val Arg Gly Leu Phe Ser Gly Ile Ile Glu Glu Ser Val Thr 370 375 380
385 ttc gtc aac gct cct cgc att gct gaa gag cgt ggc ctg gac atc tcc
1317 Phe Val Asn Ala Pro Arg Ile Ala Glu Glu Arg Gly Leu Asp Ile
Ser 390 395 400 gtg aag acc aac tct gag tct gtt act cac cgt tcc gtc
ctg cag gtc 1365 Val Lys Thr Asn Ser Glu Ser Val Thr His Arg Ser
Val Leu Gln Val 405 410 415 aag gtc att act ggc agc ggc gcg agc gca
act gtt gtt ggt gcc ctg 1413 Lys Val Ile Thr Gly Ser Gly Ala Ser
Ala Thr Val Val Gly Ala Leu 420 425 430 act ggt ctt gag cgc gtt gag
aag atc acc cgc atc aat ggc cgt ggc 1461 Thr Gly Leu Glu Arg Val
Glu Lys Ile Thr Arg Ile Asn Gly Arg Gly 435 440 445 ctg gat ctg cgc
gca gag ggt ctg aac ctc ttc ctg cag tac act gac 1509 Leu Asp Leu
Arg Ala Glu Gly Leu Asn Leu Phe Leu Gln Tyr Thr Asp 450 455 460 465
gct cct ggt gca ctg ggt acc gtt ggt acc aag ctg ggt gct gct ggc
1557 Ala Pro Gly Ala Leu Gly Thr Val Gly Thr Lys Leu Gly Ala Ala
Gly 470 475 480 atc aac atc gag gct gct gcg ttg act cag gct gag aag
ggt gac ggc 1605 Ile Asn Ile Glu Ala Ala Ala Leu Thr Gln Ala Glu
Lys Gly Asp Gly 485 490 495 gct gtc ctg atc ctg cgt gtt gag tcc gct
gtc tcc gaa gag ctg gaa 1653 Ala Val Leu Ile Leu Arg Val Glu Ser
Ala Val Ser Glu Glu Leu Glu 500 505 510 gct gaa atc aac gct gag ttg
ggt gct act tcc ttc cag gtt gat ctt 1701 Ala Glu Ile Asn Ala Glu
Leu Gly Ala Thr Ser Phe Gln Val Asp Leu 515 520 525 gac taattagaga
tccattttct agaacc 1730 Asp 530 12 530 PRT Brevibacterium flavum 12
Val Ser Gln Asn Gly Arg Pro Val Val Leu Ile Ala Asp Lys Leu Ala 1 5
10 15 Gln Ser Thr Val Asp Ala Leu Gly Asp Ala Val Glu Val Arg Trp
Val 20 25 30 Asp Gly Pro Asn Arg Pro Glu Leu Leu Asp Thr Val Lys
Glu Ala Asp 35 40 45 Ala Leu Leu Val Arg Ser Ala Thr Thr Val Asp
Ala Glu Val Ile Ala 50 55 60 Ala Ala Pro Asn Leu Lys Ile Val Gly
Arg Ala Gly Val Gly Leu Asp 65 70 75 80 Asn Val Asp Ile Pro Ala Ala
Thr Glu Ala Gly Val Met Val Ala Asn 85 90 95 Ala Pro Thr Ser Asn
Ile His Ser Ala Cys Glu His Ala Ile Ser Leu 100 105 110 Leu Leu Ser
Thr Ala Arg Gln Ile Pro Ala Ala Asp Ala Thr Leu Arg 115 120 125 Glu
Gly Glu Trp Lys Arg Ser Ser Phe Asn Gly Val Glu Ile Phe Gly 130 135
140 Lys Thr Val Gly Ile Val Gly Phe Gly His Ile Gly Gln Leu Phe Ala
145 150 155 160 Gln Arg Leu Ala Ala Phe Glu Thr Thr Ile Val Ala Tyr
Asp Pro Tyr 165 170 175 Ala Asn Pro Ala Arg Ala Ala Gln Leu Asn Val
Glu Leu Val Glu Leu 180 185 190 Asp Glu Leu Met Ser Arg Ser Asp Phe
Val Thr Ile His Leu Pro Lys 195 200 205 Thr Lys Glu Thr Ala Gly Met
Phe Asp Ala Gln Leu Leu Ala Lys Ser 210 215 220 Lys Lys Gly Gln Ile
Ile Ile Asn Ala Ala Arg Gly Gly Leu Val Asp 225 230 235 240 Glu Gln
Ala Leu Ala Asp Ala Ile Glu Ser Gly His Ile Arg Gly Ala 245 250 255
Gly Phe Asp Val Tyr Ser Thr Glu Pro Cys Thr Asp Ser Pro Leu Phe 260
265 270 Lys Leu Pro Gln Val Val Val Thr Pro His Leu Gly Ala Ser Thr
Glu 275 280 285 Glu Ala Gln Asp Arg Ala Gly Thr Asp Val Ala Asp Ser
Val Leu Lys 290 295 300 Ala Leu Ala Gly Glu Phe Val Ala Asp Ala Val
Asn Val Ser Gly Gly 305 310 315 320 Arg Val Gly Glu Glu Val Ala Val
Trp Met Asp Leu Ala Arg Lys Leu 325 330 335 Gly Leu Leu Ala Gly Lys
Leu Val Asp Ala Ala Pro Val Ser Ile Glu 340 345 350 Val Glu Ala Arg
Gly Glu Leu Ser Ser Glu Gln Val Asp Ala Leu Gly 355 360 365 Leu Ser
Ala Val Arg Gly Leu Phe Ser Gly Ile Ile Glu Glu Ser Val 370 375 380
Thr Phe Val Asn Ala
Pro Arg Ile Ala Glu Glu Arg Gly Leu Asp Ile 385 390 395 400 Ser Val
Lys Thr Asn Ser Glu Ser Val Thr His Arg Ser Val Leu Gln 405 410 415
Val Lys Val Ile Thr Gly Ser Gly Ala Ser Ala Thr Val Val Gly Ala 420
425 430 Leu Thr Gly Leu Glu Arg Val Glu Lys Ile Thr Arg Ile Asn Gly
Arg 435 440 445 Gly Leu Asp Leu Arg Ala Glu Gly Leu Asn Leu Phe Leu
Gln Tyr Thr 450 455 460 Asp Ala Pro Gly Ala Leu Gly Thr Val Gly Thr
Lys Leu Gly Ala Ala 465 470 475 480 Gly Ile Asn Ile Glu Ala Ala Ala
Leu Thr Gln Ala Glu Lys Gly Asp 485 490 495 Gly Ala Val Leu Ile Leu
Arg Val Glu Ser Ala Val Ser Glu Glu Leu 500 505 510 Glu Ala Glu Ile
Asn Ala Glu Leu Gly Ala Thr Ser Phe Gln Val Asp 515 520 525 Leu Asp
530 13 1730 DNA Brevibacterium flavum CDS (115)..(1704) 13
gggagggttt agaatgtttc tagtcgcacg ccaaaacccg gcgtggacac gtctgcagcc
60 gacgcggtcg tgcctgttgt aggcggacat tcctagtttt tccaggagta actt gtg
117 Val 1 agc cag aat ggc cgt ccg gta gtc ctc atc gcc gat aag ctt
gcg cag 165 Ser Gln Asn Gly Arg Pro Val Val Leu Ile Ala Asp Lys Leu
Ala Gln 5 10 15 tcc act gtt gac gcg ctt gga gat gca gta gaa gtc cgt
tgg gtt gac 213 Ser Thr Val Asp Ala Leu Gly Asp Ala Val Glu Val Arg
Trp Val Asp 20 25 30 gga cct aac cgc cca gaa ctg ctt gat aca gtt
aag gaa gcg gac gca 261 Gly Pro Asn Arg Pro Glu Leu Leu Asp Thr Val
Lys Glu Ala Asp Ala 35 40 45 ctg ctc gtg cgt tct gct acc act gtc
gat gct gaa gtc atc gcc gct 309 Leu Leu Val Arg Ser Ala Thr Thr Val
Asp Ala Glu Val Ile Ala Ala 50 55 60 65 gcc cct aac ttg aag atc gtc
ggt cgt gcc ggc gtg ggc ttg gac aac 357 Ala Pro Asn Leu Lys Ile Val
Gly Arg Ala Gly Val Gly Leu Asp Asn 70 75 80 gtt gac atc cct gct
gcc act gaa gct ggc gtc atg gtt gct aac gca 405 Val Asp Ile Pro Ala
Ala Thr Glu Ala Gly Val Met Val Ala Asn Ala 85 90 95 ccg acc tct
aac att cac tct gct tgt gag cac gca att tct ttg ctg 453 Pro Thr Ser
Asn Ile His Ser Ala Cys Glu His Ala Ile Ser Leu Leu 100 105 110 ctg
tct act gct cgc cag atc cct gct gct gat gcg acg ctg cgt gag 501 Leu
Ser Thr Ala Arg Gln Ile Pro Ala Ala Asp Ala Thr Leu Arg Glu 115 120
125 ggc gag tgg aag cgg tct tct ttc aac ggt gtg gaa att ttc gga aaa
549 Gly Glu Trp Lys Arg Ser Ser Phe Asn Gly Val Glu Ile Phe Gly Lys
130 135 140 145 act gtc ggt atc gtc ggt ttt ggc cac att ggt cag ttg
ttt gct cag 597 Thr Val Gly Ile Val Gly Phe Gly His Ile Gly Gln Leu
Phe Ala Gln 150 155 160 cgt ctt gct gcg ttt gag acc acc att gtt gct
tac gat cct tac gct 645 Arg Leu Ala Ala Phe Glu Thr Thr Ile Val Ala
Tyr Asp Pro Tyr Ala 165 170 175 aac cct gct cgt gcg gct cag ctg aac
gtt gag ttg gtt gag ttg gat 693 Asn Pro Ala Arg Ala Ala Gln Leu Asn
Val Glu Leu Val Glu Leu Asp 180 185 190 gag ctg atg agc cgt tct gac
ttt gtc acc att cac ctt cct aag acc 741 Glu Leu Met Ser Arg Ser Asp
Phe Val Thr Ile His Leu Pro Lys Thr 195 200 205 aag gaa act gct ggc
atg ttt gat gcg cag ctc ctt gct aag tcc aag 789 Lys Glu Thr Ala Gly
Met Phe Asp Ala Gln Leu Leu Ala Lys Ser Lys 210 215 220 225 aag ggc
cag atc atc atc aac gct gct cgt ggt ggc ctt gtt gat gaa 837 Lys Gly
Gln Ile Ile Ile Asn Ala Ala Arg Gly Gly Leu Val Asp Glu 230 235 240
cag gct ttg gct gat gcg att gag tcc ggt cac att cgt ggc gct ggt 885
Gln Ala Leu Ala Asp Ala Ile Glu Ser Gly His Ile Arg Gly Ala Gly 245
250 255 ttc gat gtg tac tcc acc gag cct tgc act gat tct cct ttg ttc
aag 933 Phe Asp Val Tyr Ser Thr Glu Pro Cys Thr Asp Ser Pro Leu Phe
Lys 260 265 270 ttg cct cag gtt gtt gtg act cct cac ttg ggt gct tct
act gaa gag 981 Leu Pro Gln Val Val Val Thr Pro His Leu Gly Ala Ser
Thr Glu Glu 275 280 285 gct cag gat cgt gcg ggt act gac gtt gct gat
tct gtg ctc aag gcg 1029 Ala Gln Asp Arg Ala Gly Thr Asp Val Ala
Asp Ser Val Leu Lys Ala 290 295 300 305 ctg gct ggc gag ttc gtg gcg
gat gct gtg aac gtt tcc ggt ggt cgc 1077 Leu Ala Gly Glu Phe Val
Ala Asp Ala Val Asn Val Ser Gly Gly Arg 310 315 320 gtg ggc gaa aag
gtt gct gtg tgg atg gat ctg gct cgc aag ctt ggt 1125 Val Gly Glu
Lys Val Ala Val Trp Met Asp Leu Ala Arg Lys Leu Gly 325 330 335 ctt
ctt gct ggc aag ctt gtc gac gcc gcc cca gtc tcc att gag gtt 1173
Leu Leu Ala Gly Lys Leu Val Asp Ala Ala Pro Val Ser Ile Glu Val 340
345 350 gag gct cga ggc gag ctt tct tcc gag cag gtc gat gca ctt ggt
ttg 1221 Glu Ala Arg Gly Glu Leu Ser Ser Glu Gln Val Asp Ala Leu
Gly Leu 355 360 365 tcc gct gtt cgt ggt ttg ttc tcc gga att atc gaa
gag tcc gtt act 1269 Ser Ala Val Arg Gly Leu Phe Ser Gly Ile Ile
Glu Glu Ser Val Thr 370 375 380 385 ttc gtc aac gct cct cgc att gct
gaa gag cgt ggc ctg gac atc tcc 1317 Phe Val Asn Ala Pro Arg Ile
Ala Glu Glu Arg Gly Leu Asp Ile Ser 390 395 400 gtg aag acc aac tct
gag tct gtt act cac cgt tcc gtc ctg cag gtc 1365 Val Lys Thr Asn
Ser Glu Ser Val Thr His Arg Ser Val Leu Gln Val 405 410 415 aag gtc
att act ggc agc ggc gcg agc gca act gtt gtt ggt gcc ctg 1413 Lys
Val Ile Thr Gly Ser Gly Ala Ser Ala Thr Val Val Gly Ala Leu 420 425
430 act ggt ctt gag cgc gtt gag aag atc acc cgc atc aat ggc cgt ggc
1461 Thr Gly Leu Glu Arg Val Glu Lys Ile Thr Arg Ile Asn Gly Arg
Gly 435 440 445 ctg gat ctg cgc gca gag ggt ctg aac ctc ttc ctg cag
tac act gac 1509 Leu Asp Leu Arg Ala Glu Gly Leu Asn Leu Phe Leu
Gln Tyr Thr Asp 450 455 460 465 gct cct ggt gca ctg ggt acc gtt ggt
acc aag ctg ggt gct gct ggc 1557 Ala Pro Gly Ala Leu Gly Thr Val
Gly Thr Lys Leu Gly Ala Ala Gly 470 475 480 atc aac atc gag gct gct
gcg ttg act cag gct gag aag ggt gac ggc 1605 Ile Asn Ile Glu Ala
Ala Ala Leu Thr Gln Ala Glu Lys Gly Asp Gly 485 490 495 gct gtc ctg
atc ctg cgt gtt gag tcc gct gtc tcc gaa gag ctg gaa 1653 Ala Val
Leu Ile Leu Arg Val Glu Ser Ala Val Ser Glu Glu Leu Glu 500 505 510
gct gaa atc aac gct gag ttg ggt gct act tcc ttc cag gtt gat ctt
1701 Ala Glu Ile Asn Ala Glu Leu Gly Ala Thr Ser Phe Gln Val Asp
Leu 515 520 525 gac taattagaga tccattttct agaacc 1730 Asp 530 14
530 PRT Brevibacterium flavum 14 Val Ser Gln Asn Gly Arg Pro Val
Val Leu Ile Ala Asp Lys Leu Ala 1 5 10 15 Gln Ser Thr Val Asp Ala
Leu Gly Asp Ala Val Glu Val Arg Trp Val 20 25 30 Asp Gly Pro Asn
Arg Pro Glu Leu Leu Asp Thr Val Lys Glu Ala Asp 35 40 45 Ala Leu
Leu Val Arg Ser Ala Thr Thr Val Asp Ala Glu Val Ile Ala 50 55 60
Ala Ala Pro Asn Leu Lys Ile Val Gly Arg Ala Gly Val Gly Leu Asp 65
70 75 80 Asn Val Asp Ile Pro Ala Ala Thr Glu Ala Gly Val Met Val
Ala Asn 85 90 95 Ala Pro Thr Ser Asn Ile His Ser Ala Cys Glu His
Ala Ile Ser Leu 100 105 110 Leu Leu Ser Thr Ala Arg Gln Ile Pro Ala
Ala Asp Ala Thr Leu Arg 115 120 125 Glu Gly Glu Trp Lys Arg Ser Ser
Phe Asn Gly Val Glu Ile Phe Gly 130 135 140 Lys Thr Val Gly Ile Val
Gly Phe Gly His Ile Gly Gln Leu Phe Ala 145 150 155 160 Gln Arg Leu
Ala Ala Phe Glu Thr Thr Ile Val Ala Tyr Asp Pro Tyr 165 170 175 Ala
Asn Pro Ala Arg Ala Ala Gln Leu Asn Val Glu Leu Val Glu Leu 180 185
190 Asp Glu Leu Met Ser Arg Ser Asp Phe Val Thr Ile His Leu Pro Lys
195 200 205 Thr Lys Glu Thr Ala Gly Met Phe Asp Ala Gln Leu Leu Ala
Lys Ser 210 215 220 Lys Lys Gly Gln Ile Ile Ile Asn Ala Ala Arg Gly
Gly Leu Val Asp 225 230 235 240 Glu Gln Ala Leu Ala Asp Ala Ile Glu
Ser Gly His Ile Arg Gly Ala 245 250 255 Gly Phe Asp Val Tyr Ser Thr
Glu Pro Cys Thr Asp Ser Pro Leu Phe 260 265 270 Lys Leu Pro Gln Val
Val Val Thr Pro His Leu Gly Ala Ser Thr Glu 275 280 285 Glu Ala Gln
Asp Arg Ala Gly Thr Asp Val Ala Asp Ser Val Leu Lys 290 295 300 Ala
Leu Ala Gly Glu Phe Val Ala Asp Ala Val Asn Val Ser Gly Gly 305 310
315 320 Arg Val Gly Glu Lys Val Ala Val Trp Met Asp Leu Ala Arg Lys
Leu 325 330 335 Gly Leu Leu Ala Gly Lys Leu Val Asp Ala Ala Pro Val
Ser Ile Glu 340 345 350 Val Glu Ala Arg Gly Glu Leu Ser Ser Glu Gln
Val Asp Ala Leu Gly 355 360 365 Leu Ser Ala Val Arg Gly Leu Phe Ser
Gly Ile Ile Glu Glu Ser Val 370 375 380 Thr Phe Val Asn Ala Pro Arg
Ile Ala Glu Glu Arg Gly Leu Asp Ile 385 390 395 400 Ser Val Lys Thr
Asn Ser Glu Ser Val Thr His Arg Ser Val Leu Gln 405 410 415 Val Lys
Val Ile Thr Gly Ser Gly Ala Ser Ala Thr Val Val Gly Ala 420 425 430
Leu Thr Gly Leu Glu Arg Val Glu Lys Ile Thr Arg Ile Asn Gly Arg 435
440 445 Gly Leu Asp Leu Arg Ala Glu Gly Leu Asn Leu Phe Leu Gln Tyr
Thr 450 455 460 Asp Ala Pro Gly Ala Leu Gly Thr Val Gly Thr Lys Leu
Gly Ala Ala 465 470 475 480 Gly Ile Asn Ile Glu Ala Ala Ala Leu Thr
Gln Ala Glu Lys Gly Asp 485 490 495 Gly Ala Val Leu Ile Leu Arg Val
Glu Ser Ala Val Ser Glu Glu Leu 500 505 510 Glu Ala Glu Ile Asn Ala
Glu Leu Gly Ala Thr Ser Phe Gln Val Asp 515 520 525 Leu Asp 530 15
23 DNA Artificial Sequence Description of Artificial SequencePrimer
15 ggcaagacag aacaggacaa tca 23 16 23 DNA Artificial Sequence
Description of Artificial SequencePrimer 16 agcttttgcc acggtgtacc
tcg 23 17 23 DNA Artificial Sequence Description of Artificial
SequencePrimer 17 ccacattttt gccctcaacg gtt 23 18 23 DNA Artificial
Sequence Description of Artificial SequencePrimer 18 cggttagaaa
cgctcttgga acc 23
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