CN1185338C - Method of producing L-serine by fermentation - Google Patents

Abstract

Disclosed is a coryneform bacterium having resistance to azaserine or beta -(2-thienyl)-DL-alanine and having L-serine productivity. Also, disclosed are D-3-phosphoglycerate dehydrogenase derived from a coryneform bacterium, in which feedback inhibition by L-serine is desensitized; the D-3-phosphoglycerate dehydrogenase, which is obtainable from a coryneform bacterium having resistance to azaserine or beta -(2-thienyl)-DL-alanine and having L-serine productivity; the D-3-phosphoglycerate dehydrogenase having an amino acid sequence depicted in SEQ ID NO : 12 in Sequence Listing or the sequence including substitution, addition or deletion of one or more amino acids, wherein an amino acid residue corresponding to the 325th glutamic acid residue of the amino acid sequence in the SEQ ID NO : 12 is replaced with an amino acid other than glutamic acid; the D-3-phosphoglycerate dehydrogenase that has an amino acid sequence depicted in SEQ ID NO : 11 in Sequence Listing; a DNA coding for the D-3-phosphoglycerate dehydrogenase described above; the DNA that has a base sequence depicted in SEQ ID NO : 13 in Sequence Listing; a coryneform bacterium which harbors a recombinant DNA containing the DNA; and a method of producing L-serine, comprising the steps of cultivating the bacterium described above in a medium to allow accumulation of L-serine in the medium, and collecting the L-serine from the medium.

Description

Method by fermentative production L-Serine

The present invention relates to be used for prepare the DNA of the production method of the L-Serine of medicine, pharmaceutical chemicals and the used aminoacid mixture of cosmetic field, the excellent bacillus that constitutes present method, D-3-phosphoglycerate dehydrogenase (hereinafter being sometimes referred to as " 3-PGDH ") and this 3-PGDH that encodes.

As ordinary method by fermentative preparation L-Serine, reported a kind of like this method, promptly wherein glycine and the sugared bacterial isolates that changes into the L-Serine can be used to produce the L-Serine of 14g/L at the most in the substratum that contains the 30g/L glycine.By this method the output that glycine changes into the L-Serine is reached 46% (KubotaK. agricultural biochemistry, 49,7-12 (1985)).With the bacterial isolates that glycine and methyl alcohol can be converted to the L-Serine, can from the glycine of 100g/L, produce the L-Serine (79 roll up the 2nd phase, 181-183,1995 for T.Yoshida etc., fermentation and biotechnology magazine) of 53g/L.In the method for using the Nocardia bacterium, knownly can improve the productivity (the open No.57-1235 of Japanese Patent) of this bacterium L-Serine by cultivating those bacterial strains that hydroxamate, azaserine etc. is had a resistance.Yet these methods comprise that use is as the glycine of L-Serine precursor and comprise complicated operations and also be disadvantageous from the angle of expense.

As can be directly from sugar-fermenting L-Serine and need not substratum, to add the bacterial strain of L-Serine precursor, known have a Corynebacterium glutamicum, this bacterium to D-Serine, Alpha-Methyl Serine, O-methyl Serine, isoserine (isoserine) but, Serine hydroxamate and 3-chlorine L-Ala have low 0.8g/L (the Nogei Kagakukaishi that reaches of accumulation of resistance L-Serine, 48 volumes, the 3rd phase, p201-208,1974).Therefore, be necessary further to improve bacterial strain to be used for the direct fermentation of technical scale L-Serine.

On the other hand, about excellent bacillus, disclose can be in cell self-replicating and plasmid with drug resistance marker gene (with reference to United States Patent (USP) 4,514,502) and with gene import the method (disclosed Japanese patent application No.2-207791) in this cell and cultivate possibility (United States Patent (USP) 4,452,890 and 4 that produce L-Threonine or L-Isoleucine bacterium, 442,208).Equally, the relevant cultivation that produces L-Methionin bacterium, known a kind of to plasmid vector and in cell, increase technology (disclosed Japanese patent application No.56-160997) of this plasmid of the biosynthetic gene of L-Methionin that participates in of mixing that comprises.

In colibacillary situation, participate in the biosynthetic enzyme of L-Serine and comprise for L-Serine production in the wild-type the enzyme of feedback inhibition sensitivity and known a kind of like this example is arranged, thereby promptly wherein import the increase (Japanese Patent No.2584409) that mutator gene can cause the L-Serine to the feedback inhibition desensitization.As this gene, known particularly have a 3-PGDH gene (hereinafter, coding 3-PGDH proteic gene also will be called " SerA ").

In addition, in the situation of excellent bacillus, known have such example, and promptly wherein the amplification of 3-PGDH gene has influenced the productivity (disclosed Japanese patent application No.3-7591) of L-tryptophane.

The purpose of this invention is to provide and change sugar the microorganism of L-Serine into and the method for utilizing ability that microorganism is changed sugar into the L-Serine to accumulate the L-Serine in substratum, the i.e. method of superior production L-Serine in plant-scale practice are provided.

Owing in order to achieve the above object L-Serine production method has been carried out deep research, the result, the present inventor has now found that the excellent bacillus that screening has L-Serine productivity, particularly preferably be from coryneform bacterial strains, demonstrate azaserine or β-(2-thiophene)-DL-L-Ala are had resistance but L-Serine degrading activity has the mutant strain of defective carries out the accumulation that the fermentation of L-Serine will increase considerably the L-Serine as parent strain and with this bacterial strain that filters out.Finished the present invention according to this discovery.

That is, the present invention relates to azaserine or β-(2-thiophene)-DL-L-Ala are had resistance and have the excellent bacillus of L-Serine productivity.

In addition, the present invention relates to D-3-phosphoglycerate dehydrogenase from the excellent bacillus that wherein feedback inhibition of L-Serine is desensitized; Can be from azaserine or β-(2-thiophene)-DL-L-Ala being had resistance and having the above-mentioned D-3-phosphoglycerate dehydrogenase that the excellent bacillus of L-Serine productivity obtains; Have list aminoacid sequence in the sequence table among the SEQ ID:12 or comprise one or more amino acid replacement, interpolation or disappearance and wherein corresponding to the amino acid of the 325th glutaminic acid residue in the SEQ ID:12 aminoacid sequence by the D-3-phosphoglycerate dehydrogenase of the aminoacid sequence of the amino acid replacement of non-glutamic acid; And have the above-mentioned D-3-phosphoglycerate dehydrogenase of in sequence table SEQ ID NO:11, listing sequence.

Further, the present invention relates to encode above-mentioned D-3-phosphoglycerate dehydrogenase DNA and have the above-mentioned DNA that in sequence table, lists base sequence among the SEQ ID NO:13.

Further, the present invention relates to have the excellent bacillus of the recombinant DNA that contains above-mentioned DNA.

In addition, the present invention relates to produce the method for L-Serine, cultivate above-mentioned bacterium accumulates the L-Serine and collect the L-Serine from this substratum in substratum step in the substratum thereby be included in.

The specific examples that azaserine or β-(2-thiophene)-DL-L-Ala are had resistance and have an excellent bacillus of L-Serine productivity comprises brevibacterium flavum AJ13324 and AJ13327 or brevibacterium flavum AJ13325.

The invention provides the excellent bacillus that produces the L-Serine from sugar.This rod bacillus can be used for having in the L-Serine production method of industrial advantage.

Fig. 1 shows the feedback inhibition mode of L-Serine to various bacterial strain 3-PGDH.Transverse axis is illustrated in the concentration of L-Serine in this enzyme solution.Per-cent when the 3-PGDH activity did not exist the L-Serine when longitudinal axis represented that the L-Serine exists.Symbol ◆ show the feedback inhibition mode of L-Serine to ATCC14067 bacterial strain 3-PGDH.Symbol ■ shows the feedback inhibition mode of L-Serine to AJ13377 bacterial strain 3-PGDH.Symbol ▲ demonstration L-Serine is to the feedback inhibition mode of AJ13324 bacterial strain 3-PGDH.Symbol * demonstration L-Serine is to the feedback inhibition mode of AJ13325 bacterial strain 3-PGDH.Symbol ^*Show the feedback inhibition mode of L-Serine to AJ13327 bacterial strain 3-PGDH.

Fig. 2 shows the structure of plasmid pVK7 and pVK6.

The excellent bacillus of mentioning among the present invention isUncle Jie Shi Bacteria Identification handbook, the 8th edition, the 599th page (1974) One group of microorganism of middle definition, they are the aerobic Grams that do not have acid resistance and do not have the sporogenesis ability Positive bacillus. These excellent bacillus comprise Corynebacterium bacterium, belong to so far that brevibacterium is advanced in classification but at present Unified brevibacterium bacterium in Corynebacterium and closely-related with Corynebacterium and Microbacterium bacterium The bacterium of brevibacterium.

The present invention has resistance and has L-silk ammonia azaserine or β-(2-thiophene)-DL-Alanine The excellent bacillus of acid productivity, those coryneform bacterial strains of preferred Serine degrading activity deficiency are from wild type Or the excellent bacillus that produces Serine carries out artificial mutation as parent strain or induces.

For example, can obtain azaserine or β-(2-thiophene)-DL-Alanine tool by following method The excellent bacillus that resistance is arranged and have Serine degrading activity deficiency and have the Serine productivity. That is, by conventional method (contacting with N-methyl-N '-nitro-N-nitroso-guanidine etc.) to brevibacterium flavum ATCC 14067 suddenly change processing to obtain Serine degrading activity deficiency mutant strain and to do with this bacterial strain Be parent strain, obtain that azaserine or β-(2-thiophene)-DL-Alanine are had the bacterial strain of resistance. From the mutant strain that said method obtains, can obtain the bacterial strain with high concentration accumulation Serine.

Also can be prominent to parent's bacterial strain or Serine degrading activity deficiency by importing following mutant serA Become bacterial strain and obtain that azaserine or β-(2-thiophene)-DL-Alanine are had the bacterial strain of resistance.

Term " azaserine resistance " refers to that bacterium is thin than wild type in containing the culture medium of azaserine Bacteria growing is characteristic faster. For example, on the solid medium that contains the 0.25g/L azaserine 30 ℃ The bacterial strain that forms bacterium colony in 4-5 days is called and has the azaserine resistance.

Similarly, term " β-(2-thiophene)-DL-Alanine resistance " refers to that bacterium is containing β-(2-thiophene) In the culture medium of-DL-Alanine than the wild-type bacterium faster characteristic of growing. For example, contain 0.25g/L β-30 ℃ of bacterial strains that formed bacterium colony in 4-5 days are called on the solid medium of (2-thiophene)-DL-Alanine Has β-(2-thiophene)-DL-Alanine resistance.

The reaction that 3-PGDH catalysis is such, namely at NADH (NAD) as coenzyme When existing 3-phoshoglyceric acid is oxidized to the 3-phosphohydroxypyruvic acid.

For example, can be by coenzyme NAD H in the absorbance determination back reaction at 340nm place₂Decline (E. Sugimoto and L.I.Pizer, JBC, 243,2081,1968) or positive reaction in coenzyme NAD H₂Synthetic (Salach H.J. Enzymology method, 9 volumes, 216-220,1996) measure the activity of 3-PGDH.

The cell of collecting by collecting cell from the culture medium of excellent bacillus, Ultrasonic Pulverization and surpassing subsequently Speed centrifugal and from supernatant, separate the purpose enzyme by conventional method can purifying 3-PGDH. More particularly, Can analyse by ammonium sulfate precipitation, gel filtration, cation exchange resin layer, anion exchange resin layer is analysed, Reversed phase chromatographies etc. carry out series to the fraction with 3-PGDH activity and concentrate purifying 3-PGDH.

3-PGDH responsive and its active inhibition that existed by the 10mML-Serine from wild-type rod bacillus to the feedback inhibition of L-Serine.Term " wherein the feedback inhibition of L-Serine desensitized 3-PGDH " has no L-Serine when existing active 20% or more, preferred 40% or more even mean in the presence of the 10mML-Serine 3-PGDH, and more preferably 90% or more.3-PGDH from the brevibacterium flavum AJ13327 that describes among the following embodiment keeps 100% activity basically and therefore is one of most preferred 3-PGDHs in the presence of the 80mML-Serine.

Has the aminoacid sequence of listing among the SEQ ID NO:12 in the sequence table from the 3-PGDH of wild-type rod bacillus (below, the DNA of this enzyme of encoding is also referred to as " wild-type serA ").Wherein the specific examples of the 3-PGDH that desensitized of the feedback inhibition of L-Serine (below, the DNA of this enzyme of encoding is also referred to as " mutant serA ") comprise such D-3-phosphoglycerate dehydrogenase, it is characterized in that this D-3-phosphoglycerate dehydrogenase have the aminoacid sequence listed among the SEQ ID NO:12 in the sequence table or same as described above but have one or more amino acid replacement, interpolation or disappearance and corresponding to the amino-acid residue of the 325th glutaminic acid residue of aminoacid sequence among the SEQ ID NO:12 by other amino acid institute alternate aminoacid sequence.Most preferably this other amino acid is lysine residue.

For example, can according to the method for Saito and Miura (H.Saito and K.Miura, biochemistry. Acta Biophysica Sinica, 72,619 (1963)) etc. preparation chromosomal DNA and then by polymerase chain reaction method (PCR: polymerase chain reaction; With reference to White, T.J. etc., genetics progress 5,185 (1989)) and amplification serA gene separates the dna fragmentation that contains the serA gene from excellent bacillus.For example, contain the dna fragmentation of the SEQ ID NO:11ORF (172 to 1705) in the ordered list in order to increase, first base to the zone between the base that is close to the ATG front selects any 20 to 30 bases to obtain a primer from SEQ IN NO:11.In addition, the zone from the base of next-door neighbour terminator codon back to SEQ IN NO:11 between last base selects any 20 to 30 bases to obtain another primer.

When the wild type strain of 3-PGDH separates serA, obtain wild-type serA and from the mutant that has the 3-PGDH that the feedback inhibition of L-Serine wherein desensitized (3-PGDH mutant) separate the serA that serA obtains suddenling change.Specifically, wild-type serA has the sequence of listing among the SEQ ID NO:11 in the sequence table, and mutant serA has the sequence of listing among the SEQ ID NO:13 in the sequence table.

SerA that preferably will be by PCR method amplification with can be in intestinal bacteria and/or excellent bacilli-cell the carrier DNA of self-replicating be connected preparing recombinant DNA, and this recombinant DNA is imported in the aforesaid Bacillus coli cells.This prerequisite makes following operation become easy.Can in Bacillus coli cells, be preferably and to comprise by the carrier of self-replicating, pUC19 for example, pUC18, pBR322, pHSG299, pHSG399, the plasmid vector of self-replicating in the host cell of pHSG398 and RSF1010.

Can utilize transposon (international publication number WO 02/02627, international publication number WO 93/18151, disclosed european patent application No.0445385, disclosed Japanese patent application No.6-46867, Vertes, A.A. molecular microbiology., 11,739-746 (1994), Bonamy, C., Deng, molecular microbiology., 14,571-581 (1994) Vertes, A.A. etc., the molecule General Genetics., 245,397-405 (1994) Jagar, W. etc., the communication of FEMS microbiology, 126,1-6 (1995), disclosed Japanese patent application No.7-107976, disclosed Japanese patent application No.7-327680, etc.), phage vector, karyomit(e) reorganization (molecular genetics experiment, press of cold spring harbor laboratory (1972); Matsuyama, S. and Mizushima, S. bacteriology magazine., 162,1196 (1985)) etc. the preparation recombinant DNA.

Make plasmid can the dna fragmentation of the ability of self-replicating is inserted in these carriers in excellent bacillus the time when having, they can be used as in intestinal bacteria and excellent bacillus all can self-replicating so-called shuttle vectors.

Such shuttle vectors comprises following carrier.Having the microorganism of each carrier and the preserving number of international preservation mechanism is shown in parenthetic.

PHC4: intestinal bacteria AJ12617 (FERM BP-3532)

PAJ655: intestinal bacteria AJ11882 (FERM BP-136)

Corynebacterium glutamicum SR8201 (ATCC 39135)

PAJ1844: intestinal bacteria AJ11883 (FERM BP-137)

Corynebacterium glutamicum SR8202 (ATCC 39136)

PAJ611: intestinal bacteria AJ11884 (FERM BP-138)

PAJ3148: Corynebacterium glutamicum SR8203 (ATCC 39137)

PAJ440: subtilis AJ11901 (FERM BP-140)

These carriers can obtain from the microorganism of preservation as follows.The cell of collecting in logarithmic phase is with in addition cracking of N,O-Diacetylmuramidase and SDS, then in 30,000 * g is centrifugal to be separated to obtain supernatant and to wherein adding polyoxyethylene glycol, by CsCl-ethidium bromide equilibrium density gradient centrifugation method it to be carried out fractional separation and purifying then lysate.

Can basis, for example the D.M.Morrison method ( Enzymology method, 68, 326 (1979)) or recipient cell wherein with calcium chloride handle with the method that increases the DNA permeability (Mandel, M. and Higa, A., Molecule The biology magazine., 53, 159 (1970)) and import plasmid and transformed into escherichia coli.

The importing of plasmid in excellent bacillus can be passed through impulse method (Sugimoto etc., disclosed Japanese patent application No.2-207791) and carry out to cause conversion.

The example that is used to import the excellent bacillus of above-mentioned DNA comprises, for example, and following wild type strain:

Corynebacterium acctoacidophlum ATCC 13870;

Vinegar paddy rod bacillus ATCC 15806;

U.S. excellent bacillus ATCC 15991;

Corynebacterium glutamicum ATCC 13032;

(extension brevibacterium) ATCC 14020;

(brevibacterium) ATCC 13869;

(corynebacterium lilium) ATCC 15990;

(brevibacterium flavum) ATCC 14067;

Corynebacterium? melassecola?ATCC?17965；

Brevibacterium saccharolyticum ATCC 14066;

Brevibacterium? immariophilum?ATCC?14068；

Rose-colored tyrothricin ATCC 13825

Give birth to sulphur tyrothricin ATCC 19240;

Have a liking for ammonia microbacterium ATCC 15354;

Corynebacterium? thermoaminogenes?AJ12340(FERM?BP-1539)。

The 3-PGDH that the conversion bacterial strain generation L-Serine feedback inhibition wherein that the recombinant DNA that contains the serA that suddenlys change by importing obtains to the above-mentioned excellent bacillus is desensitized.The bacterial strain of this conversion has the resistance to β-(2-thiophene)-DL-L-Ala.

For carrying out the production of L-Serine, can make in the following method with bacterial strain of the present invention.As stand-by substratum, can use and contain carbon source, nitrogenous source, inorganic salt and if necessary, optional conventional liq substratum such as organic trace nutritive ingredients such as amino acid, VITAMIN.

As carbon source, can use as glucose, sucrose, fructose, semi-lactosi; The starch solution of saccharification, sweet potato syrup, sugar beet syrup and comprise the highest syrup organic acid such as the acetate of above-mentioned sugar; Alcohol is as ethanol; Glycerine etc.

As nitrogenous source, can use ammonia, water-soluble ammonia, ammonium salt, urea, nitrate etc.In addition, can use organic nitrogen source to be used for replenishing and use, for example, the casein of oiliness cake, soybean hydrolyzed solution, decomposition, other amino acid, corn steep liquor, yeast or yeast extract, peptide such as peptone etc.

As mineral ion, can choose adding phosphate ion, magnesium ion, calcium ion, iron ion and mn ion etc. wantonly.

When use needs nutrition such as amino acid to be used for the microorganism of the present invention of its growth, should replenish required nutrition.

Usually under the aerobic conditions of the temperature range of pH5 to 8 and 25 to 40 ℃, hatch microorganism.Whether the pH of substratum is controlled in the above-mentioned predetermined scope according to the existence of inorganic or organic acid, alkaline matter, urea, lime carbonate, ammonia etc.

For example, can be by separating and removing cell, carry out ion exchange resin treatment, concentrate crystallisation by cooling, membrane sepn and other known method and from fermented liquid, collect the L-Serine with any suitable combination.In order to remove impurity, can carry out purifying with activated carbon adsorption and recrystallization.

The description of preferred embodiment

Product L-Serine brevibacterium flavum AJ13324 that (embodiment 1) is new and the structure of AJ13327

Make up brevibacterium flavum AJ13324 and AJ13327 from the brevibacterium flavum AJ13377 available from wild-type brevibacterium flavum ATCC 14067 of L-Serine degrading activity defective.

In order to obtain mutant, will (contain 1g flesh of fish extract in 1 premium on currency, 1g gathers peptone, the 0.5g yeast extract at broth culture, with 0.5g sodium-chlor, transfer to the substratum of pH7.0) in 24 hours cell suspension of amplification (contain 10 in the phosphoric acid buffer (pH7.0) of 100mM ⁹To 10 ¹⁰In the individual cell/ml).NG (N-methyl-N '-nitro-N-nitrosoguanidine) is joined in the suspension to the concentration of 200 μ g/ml and in 30 ℃ of placements 30 minutes.With the cell handled like this with above-mentioned damping fluid thorough washing.

For screening from the cell that NG handles has the bacterial strain of no L-Serine degrading activity, the brevibacterium flavum ATCC14067 cell that the washing back is handled with NG is coated on the broth agar culture medium and in 30 ℃ to be cultivated 24 hours so that bacterium colony formation.Then, the bacterium colony on the broth agar culture medium being carried out replicon as negative bacterium colony and on minimum medium and the minimum medium that is used to screen forms.Then, screening grows on the minimum medium but does not grow in bacterial strain on the minimum medium that is used to screen.This minimum medium is that the substratum that contains following composition in every liter of distilled water comprises 20g glucose, 1g ammonium sulfate, the 1g potassium primary phosphate, 2.5g urea, 0.4g7 water magnesium sulfate, 0.01g7 water ferric sulfate (II), 0.01g4-to 5 water manganous sulfates, 50 μ g vitamin Hs, 200 μ g vitamin, 200ug niacinamide and 2.0g agar.The minimum medium that is used for screening is the substratum that every liter of distilled water contains following composition, comprise 1g ammonium sulfate, the 1g potassium primary phosphate, 2.5g urea, 0.4g7 water magnesium sulfate, 0.01g7 water ferric sulfate (II), 0.01g4-to 5 water manganous sulfates, 50 μ g vitamin Hs, 200 μ g vitamins, 200 μ g niacinamide, 0.5gL-Serine and 2.0g agar.Have the bacterial strain of no L-Serine degrading activity and obtain brevibacterium flavum AJ13377 by discovery in the mutant of this method acquisition is many as such bacterial strain.

For with the brevibacterium flavum AJ13377 bacterial strain as parent strain screening azaserine resistance from the bacterial strain that NG handles, the brevibacterium flavum AJ13377 cell inoculation that the NG after the washing is handled is in the minimum medium that is used to screen.The substratum that contains following composition on this minimum medium that is used for screening for every liter of distilled water, comprise 20g glucose, 1g ammonium sulfate, 1g potassium primary phosphate, 2.5g urea, 0.4g7 water magnesium sulfate, 0.01g7 water ferric sulfate (II), 0.01g4-to 5 water manganous sulfate, 50 μ g vitamin Hs, 200 μ g vitamins, 200 μ g niacinamide and 250mg azaserine.The mutant that NG handles was cultivated 5 to 10 days in above-mentioned substratum in 30 ℃.The cell culture that obtains like this is coated on the broth agar culture medium and cultivates and be used for bacterium colony in 24 hours and form in 30 ℃.From the bacterial strain that forms bacterium colony, obtain the azaserine resistant strain.The mutant of Huo Deing comprises many bacterial strains that accumulate the L-Serine with high yield in a large number like this.From these bacterial strains, obtain 2 bacterial strains, that is, and brevibacterium flavum AJ13324 and AJ13327.Confirmed that these bacterial strains can grow in the presence of the 0.25g/L azaserine.

The structure of the product L-Serine brevibacterium flavum AJ13325 that (embodiment 2) are new

Make up brevibacterium flavum AJ13325 from the brevibacterium flavum AJ13377 available from wild-type brevibacterium flavum ATCC14067 of L-Serine degrading activity defective.

For from as screening the bacterial strain of the NG processing of parent strain β-(2-thiophene)-DL-L-Ala being had the bacterial strain of resistance, brevibacterium flavum AJ13377 cell was handled and washing with NG before being inoculated on the minimum medium that is used to screen with brevibacterium flavum AJ13377.The substratum that contains following composition on this minimum medium that is used for screening for every liter of distilled water, comprise 20g glucose, 1g ammonium sulfate, 1g potassium primary phosphate, 2.5g urea, 0.4g7 water magnesium sulfate, 0.01g7 water ferric sulfate (II), 0.01g4-to 5 water manganous sulfate, 50 μ g vitamin Hs, 200 μ g vitamins, 200 μ g niacinamide and 250mg β-(2-thiophene)-DL-L-Ala.The mutant that NG handles was cultivated 5 to 10 days in above-mentioned substratum in 30 ℃.The cell culture that obtains like this is coated on the broth agar culture medium and cultivates and be used for bacterium colony in 24 hours and form in 30 ℃.Obtain that from these bacterial strains that form bacterium colony β-(2-thiophene)-DL-L-Ala is had the bacterial strain of resistance.The mutant of Huo Deing comprises many bacterial strains that accumulate the L-Serine with high yield in a large number like this.Obtained brevibacterium flavum AJ13325 as such bacterial strain.Confirmed that these bacterial strains can grow in the presence of 0.25g/L β-(2-thiophene)-DL-L-Ala.

(embodiment 3) by the brevibacterium flavum AJ13324 of new product L-Serine, AJ13325 and AJ13327 produce the L-Serine

With brevibacterium flavum AJ13324, AJ13325 and AJ13327 respectively at 30 ℃ cultivated on the broth culture 24 hours and with every kind of microbial inoculant of 1 loopful in the shaking in the bottle of 500ml, wherein this shakes bottle and contains the fermention medium that 20ml has component shown in the table 1.In contrast, inoculate parent strain brevibacterium flavum ATCC14067 and AJ13377 by above-mentioned identical mode.Regulate substratum to pH7.0 and in 115 ℃ of autoclavings 15 minutes with potassium hydroxide.After sterilization and the cooling, add the 5g/L amount in the lime carbonate of 180 ℃ of dry sterilizations.

Table 1

Component content/liter

Glucose 110.0g

Potassium primary phosphate 0.4g

7 water magnesium sulfate 0.4g

7 water ferric sulfate (II) 0.01g

4-to 5 water manganous sulfate 0.01g

Ammonium sulfate 25.0g

Vitamin 100 μ g

Vitamin H 100 μ g

Soybean protein hydrochloric acid lysate (" Mieki " (registered trademark)) 40ml

pH 7.0

With the mensuration demonstration brevibacterium flavum AJ13324 of high performance liquid chromatography (L-8500 of Hitachi amino acidanalyser) to the L-Serine, AJ13325 and AJ13327 accumulate the L-Serine with the amount of 15.2g/L, 14.3g/L and 15.4g/L respectively in substratum.On the other hand, brevibacterium flavum strains A TCC1406 that cultivates in contrast and AJ13377 accumulate the L-Serine with the amount of 0g/L and 5.0g/L respectively.

The substratum of centrifugal brevibacterium flavum AJ13324 and this supernatant is carried out desalting treatment with Zeo-karb, subsequently with anionite-exchange resin and anionite-exchange resin carry out chromatographic separation with remove byproduct and by crystallization purifying from 55% substratum output, to obtain the L-Serine crystallization of at least 99% purity.

The active mensuration of (embodiment 4) 3-PGDH

With brevibacterium flavum AJ13324, AJ13325 and AJ13327 respectively at 30 ℃ cultivated on the broth agar culture medium 24 hours and with every kind of microbial inoculant of 1 loopful in the shaking in the bottle of 500ml, wherein this shakes bottle and contains the fermention medium that 50ml has component shown in the table 2.In contrast, inoculate parent strain brevibacterium flavum ATCC14067 and AJ13377 by above-mentioned identical mode.Regulate the substratum be used to inoculate with sodium hydroxide to pH5.5 and in 115 ℃ of autoclavings 15 minutes.

Table 2

Component content/liter

Glucose 30.0g

Potassium primary phosphate 1.0g

7 water magnesium sulfate 0.4g

7 water ferric sulfate (II) 0.01g

4-to 5 water manganous sulfate 0.01g

Ammonium sulfate 3.0g

Soybean protein hydrochloric acid lysate (" Mieki " (registered trademark)) 3.0ml

Vitamin 200 μ g

Vitamin H 50 μ g

Urea 3.0g

Yeast extract 2.0g

pH 5.5

From the substratum of every kind of bacterial strain after the collecting cell, with physiological saline washed cell 2 times and be suspended in the sodium phosphate buffer (pH7.0) of the 50mM that contains the 2mM dithiothreitol (DTT).After ice-cold, with the liquid of suspension with ultrasonic apparatus smudge cells and ultracentrifugation gained.Ultracentrifugation is 45, and 000rpm carries out 1 hour to obtain thick enzyme solution.

Measure the enzymic activity of 3-PGDH with the method (Enzymology method, 9 volumes, 216-220 (1966)) of Salach H.J. etc.

More particularly, add 0.4ml 0.015M NAD, and 0.12ml 0.25M EDTA (pH9, NaOH), 0.1ml 0.05M glutathione (pH6, KOH), 0.5ml 1M hydrazine (pH9, acetate), 0.6ml 1MTris (pH9, Hcl), the L-Serine (0-40mM) of proper concn and water are heated to 25 ℃ in advance to make 2.3ml.Then, add the thick enzyme solution of 0.2ml and kept constant temperature 5 minutes.Afterwards, and adding 0.5ml0.1M 3-PGA (the glycerol 3-phosphate acid disodium salt, pH7, NaOH).After the stirring, in the light absorption ratio of 340nm place assaying reaction mixture 30 seconds.Reacting on 25 ℃ carries out.

In order to carry out determination of activity, use the U-2000A of Hitachi spectrophotometer.

Fig. 1 shows the result who obtains.Compare with wild type strain ATCC 14067, the L-Serine susceptibility of AJ13377 bacterial strain reduces.The L-Serine susceptibility of AJ13324 bacterial strain reduce manyly and the AJ13325 bacterial strain identical with AJ13324 bacterial strain level in this respect.The AJ13327 bacterial strain has reduced the susceptibility of L-Serine widely.And even in the presence of 80mM L-Serine, suppress to be desensitized fully.

Although reported L-Serine inhibition is desensitized to 3-PGDH some examples (Tosa and Pizer in the intestinal bacteria, bacteriology magazine .106:972-982 (1971) or disclosed Japanese patent application No.6-510911), but the known example that also in the presence of high density L-Serine like this, this inhibition is not desensitized fully.

(embodiment 5) are from the clone of excellent bacillus wild-type and mutant serA

As shown in Example 4, the feedback inhibition of L-Serine is desensitized fully in the AJ13327 bacterial strain.Therefore, in order to understand fully which type of variation and to confirm the expanding effect of 3-PGDH, attempt clones coding from ATCC14067 bacterial strain wild-type 3-PGDH with from the serA gene of AJ13327 bacterial strain mutant 3-PGDH.

For the serA that increases from brevibacterium flavum karyomit(e) with PCR method, it is necessary producing corresponding primer.Owing to do not have the report of relevant brevibacterium flavum serA clone and nucleotide sequence, used serA sequence from excellent bacillus.Extract plasmid from Corynebacterium glutamicum K82 (with reference to FERM BP-2444 and disclosed Japanese patent application No.3-7591), the serA fragment from excellent bacillus is wherein cloned with WizardMinipreps dna purification system (Promega productions) and with restriction enzyme BamHI (production of TakaraShuzo company limited) cutting contains the dna fragmentation of about 1.4kb of serA.

As being used for the segmental carrier of clone gene, be used for excellent bacillus with the cloning vector pVK7 that makes up recently.

By connecting (escherichia coli cloning carrier) pHSG299 (Kmr in the following manner; Takeshita, S. etc., gene, 61,63-74 (1987), disclosed Japanese patent application No.10-215883) and to pAM330, a kind of cryptic plasmid of Brevibacterium lactofermentus and make up pVK7.With the monospecific restriction enzyme AvaII (production of TakaraShuzo company limited) cutting pHSG299 and with T4 archaeal dna polymerase end-filling.It is connected with using HindIII (production of Takara Shuzo company limited) cutting and mend flat terminal pAM330 with the T4 archaeal dna polymerase.This 2 kinds of plasmid called after pVK6 and the pVK7 that will obtain with respect to the direction of insertion of pHSG299 according to pAM330.PVK7 is used for following experiment.PVK7 can be in intestinal bacteria and Brevibacterium lactofermentus self-replicating and keep multiple clone site and lacZ ' from pHSG299.Fig. 2 shows the building process of pVK6 and pVK7.

Go up dna fragmentation that contains about 1.4kb of serA of connection to the shuttle vectors pVK7 of such structure.PDTS9901 is with restriction enzyme BamHI (production of Takara Shuzo company limited) cuts and is connected to equally with restriction enzyme BamOn the pVK7 of HI cutting.Carry out the connection of DNA according to preceding method with dna ligation kit (production of Takara Shuzo company limited).

For sequencing reaction, use PCR thermal cycler MP type (production of Takara Shuzo company limited) and dyeing to stop cycle sequencing FS and promptly use reaction kit (Perkin-Elmer Corporations's production).As dna primer, use M13 (21), RV primer (production of Takara Shuzo company limited).SEQ ID NO:1 in the sequence table has shown the sequence that obtains like this.SEQ ID NO:2 has shown can be by the aminoacid sequence of this sequence encoding.

According to the base sequence synthetic primer of such mensuration and with the chromosomal DNA of mutant brevibacterium flavum AJ13327 as template by the PCR method serA that increases.SEQ ID NO:3 and 4 in the sequence table shows the synthetic terminal and C-end sequence of N-that is used for the dna primer of gene amplification respectively.

In the preparation of brevibacterium flavum chromosomal DNA, used genomic dna purification kit (bacteroidal) (production of pioneer's genetics technology company) and preparation method to carry out according to its appended scheme.

For the PCR reaction, PCP thermal cycler MP type (production of Takara Shuzo company limited) and TaKaRa Taq (production of Takara Shuzo company limited) have been used.

The PCR product is connected directly on the plasmid pCR2.1 carrier with Original TA clone test kit (Invitrogen production) and transforms with the competent cell of INV α F '.Cell transformed is coated on L substratum (the 10g/L bacto-tryptone that further contains 40 μ g/ml X-Gal (5-bromo-4-chloro-3-indoles-β-D-galactoside) and 25 μ g/ml kantlex, the 5g/L bacterium is used yeast extract, 15g/L NaCl and 15g/L agar) and overnight incubation.Collect the white colony that occurs and be separated to the bacterial strain of single bacterium colony to obtain to transform.

From the bacterial strain that transforms, extract plasmid and with restriction enzyme EcoRI handles through PCR method and confirms those plasmids of serA fragment insertion to be arranged and be connected among the shuttle vectors pVK.Base sequence mensuration to this product shows that the C-end does not contain full length sequence.The sequence that obtains like this is corresponding to the zone between 3 ' end, 1134 bases to the SEQ ID NO:13 of 5 ' end upstream 277 bases among the SEQ ID NO:13 in the sequence table.

In order to obtain to contain the fragment of total length serA gene, the clone of brevibacterium flavum AJ13327 strain chromosome DNA disappearance part carries out according to appended scheme with TaKaRa LA PGR body outer clone test kit (production of Takara Shuzo company limited).

At first, the chromosomal DNA with preparation like this is connected with various restriction enzyme complete digestiones and with the connecting box that wherein has respective limits enzyme site.Carry out the 1st and take turns PCR with boxlike primer (C1) (the SEQ ID NO:5 in the sequence table) with known DNA (S1) zone (the SEQ ID NO:6 in the sequence table) complementary primer.With the part of this reaction mixture, carry out the 2nd with inner primer C2 (the SEQ ID NO:7 in the sequence table) and S2 (the SEQ ID NO:8 in the sequence table) and take turns PCR with the target DNA that only increases.

When EcoRI (production of Takara Shuzo company limited) confirms the amplification of this target DNA and directly measures the base sequence of this PCR product when the restriction enzyme.According to the base sequence of such acquisition, produce the primer of the terminal side of coding C-and from as the brevibacterium flavum ATCC14067 of wild type strain with as collecting the fragment that contains total length serA the brevibacterium flavum AJ13327 of mutant strain.SEQ ID NOS:9 and 10 in the sequence table shows the sequence of N-end and C-end side chain DNA primer respectively.

With Original TA clone's test kit (Invitrogen production) gene fragment that contains total length wild-type serA and mutant serA respectively is connected to EcoAmong the shuttle vectors pVK7 of RI-cutting.Produce respectively and have the plasmid of corresponding gene fragment and measure its base sequence.SEQ ID NOS:11 and 13 represents the sequence of wild-type and mutant respectively.SEQ ID NOS:12 and 14 represents the aminoacid sequence of these sequence codifieds respectively.The base sequence of measuring confirms in mutant serA relatively like this, and the 1087th bases G is mutated into A and therefore the 325th amino acid, and L-glutamic acid becomes Methionin.

The plasmid that (embodiment 6) will contain the 3-PGDH gene imports in the brevibacterium flavum

The plasmid that will have wild-type serA or mutant serA imports respectively among the brevibacterium flavum AJ13377.These plasmids by impulse method (Sugimoto etc., disclosed Japanese plasmid application No.2-207791) import.In the perfect medium that contains 25 μ g/ml kantlex, screen cell transformed.

(embodiment 7) produce the L-Serine by cell transformed

Wherein imported transformant respectively and shaken in the bottle at 500ml and cultivate, and measured the L-Serine that produces according to embodiment 3 with the plasmid that contains total length wild-type serA or mutant serA gene fragment.In contrast, cultivate AJ13377 bacterial strain similarly as the host.

The transformant L-Serine productivity that observing its L-Serine productivity of transformant that wherein imports wild-type serA does not have to influence and confirm wherein to have imported mutant serA increases (table 3)

Table 3

The amount (g/L) of the L-Serine of the gene accumulation of bacterial strain amplification

AJ13377 - 5.0

serA 5.0

serA ^* 12.0

SerA ^*: mutant serA gene

Brevibacterium flavum AJ13377 has been preserved in Ministry of International Trade and Industry industrial science and technology institute bio-science and human technical institute (postcode: 305-8566 on October 15th, 1997,1 kind in ripple city east 1 fourth order is built No. 3 in the Ibaraki, Japan), preserving number is FERMP-16471, and be converted into international preservation on November 20th, 1998 by original preservation according to budapest treaty, and preserving number is FERM BP-6567.

In addition, the plasmid that contains mutant serA is included among the brevibacterium flavum ATCC14067.Have the bacterial strain called after brevibacterium flavum AJ1338 of this plasmid and be preserved in Ministry of International Trade and Industry industrial science and technology institute bio-science and human technical institute (postcode: 305-8566 on October 15th, 1997,1 kind in ripple city east 1 fourth order is built No. 3 in the Ibaraki, Japan), preserving number is FERM P-16472, and be converted into international preservation on November 20th, 1998 by original preservation according to budapest treaty, and preserving number is FERMBP-6577.

Sequence table

＜110〉Ajinomoto company limited

＜120〉method by fermentative production L-Serine

<130>OP812

<150>IP?10-3751

<151>1998-01-12

<150>JP?10-353513

<151>1998-12-11

<160>14

＜170〉patent is 2.0 editions

<210>1

<211>1432

<212>DNA

＜213〉Corynebacterium glutamicum

<220>

<221>CDS

<222>(398)..(1432)

<400>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?get 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?Gln?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?Va1?Asp

345

<210>2

<211>345

<212>PRT

＜213〉Corynebacterium glutamicum

<400>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

<210>3

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>3

ggacacacgt?gacaaaattg?tag 23

<210>4

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>4

gccagcaaga?agaccaagct?tgc 23

<210>5

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>5

gtacatattg?tcgttagaac?gcgtaatacg?actca 35

<210>6

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>6

tcatcaacgc?tgctcgtggt?ggc 23

<210>7

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>7

cgttagaacg?cgtaatacga?ctcactatag?ggaga 35

<210>8

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>8

gacgttgctg?attctgtgct?caa 23

<210>9

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<200>9

gggagggttt?agaatgtttc?tag 23

<210>10

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: primer

<400>10

ggttcaagca?aatggatctc?taa 23

<210>11

<211>1730

<212>DNA

＜213〉brevibacterium flavum

<220>

<221>CDS

<222>(115)..(1704)

<400>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?Sar?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

<210>12

<211>530

<212>PRT

＜213〉brevibacterium flavum

<400>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

<210>13

<211>1730

<212>DNA

＜213〉brevibacterium flavum

<220>

<221>CDS

<222>(115)..(1704)

<400>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

Set?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 170l

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

<210>14

<211>530

<212>PRT

＜213〉brevibacterium flavum

<400>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

Claims (8)

1. azaserine or β-(2-thienyl)-DL-L-Ala are had resistance and have the excellent bacillus of L-Serine productivity.

2.D-3-phosphoglycerate dehydrogenase, desensitized aspect the feedback inhibition that this enzyme causes at the L-Serine, this enzyme source is in the excellent bacillus of claim 1, and the aminoacid sequence that this enzyme had derives under appropriate condition with SEQ ID NO:6 and SEQ ID NO:8 as the primer and the polymerase chain reaction of carrying out as template with the chromosomal DNA of excellent bacillus, wherein corresponding to the amino-acid residue of the 325th glutaminic acid residue in the SEQ IDNO:12 aminoacid sequence by the amino acid replacement of non-glutamic acid residue.

3. the D-3-phosphoglycerate dehydrogenase in the claim 2, it is made up of the aminoacid sequence of SEQ IDNO:14 in the sequence table.

4. the DNA of the D-3-phosphoglycerate dehydrogenase in the claim 2 of encoding.

5. the DNA in the claim 4, wherein said DNA is made up of the base sequence of listing among the SEQ ID NO:13 in the sequence table.

6. carry the excellent bacillus of the recombinant DNA of the DNA that contains claim 4.

7. produce the method for L-Serine, cultivate the step that desired excellent bacillus accumulates the L-Serine and collect the L-Serine from this substratum in the claim 1 in this substratum in the substratum thereby be included in.

8. produce the method for L-Serine, cultivate the step that desired excellent bacillus accumulates the L-Serine and collect the L-Serine from this substratum in the claim 6 in this substratum in the substratum thereby be included in.

Images