CN106520652B

CN106520652B - One plant of Corynebacterium glutamicum and its key gene for synthesizing tryptophan

Info

Publication number: CN106520652B
Application number: CN201611067957.3A
Authority: CN
Inventors: 李燕军; 陈宁; 韩洪军; 袁启发; 高立栋; 张顺棠; 李娟�; 谢希贤; 张成林; 徐庆阳; 范晓光; 马倩
Original assignee: Lotus Health Industry Group Ltd By Share Ltd; Tianjin University of Science and Technology
Current assignee: Lotus Health Industry Group Ltd By Share Ltd; Tianjin University of Science and Technology
Priority date: 2016-11-29
Filing date: 2016-11-29
Publication date: 2019-07-05
Anticipated expiration: 2036-11-29
Also published as: CN106520652A

Abstract

The invention belongs to Microbial Breeding and field of fermentation engineering, and in particular to one plant of Corynebacterium glutamicum and its key gene for synthesizing tryptophan.The superior strain be to pass through genetic engineering means on the basis of Corynebacterium glutamicum CGMCC No.12302, the genome of the bacterial strain is edited, the mutational site of trpDCB on tryptophan operon is integrated into and is obtained on the genome of CGMCC No.12302.One aspect of the present invention has correlation gene to provide new mutational site for tryptophan synthesis, provides new research direction for the biosynthesis of tryptophan；On the other hand, it is found by shake flask fermentation, the tryptophan yield of obtained strains is about original 3.4 times, greatly improves the performance of the chromogenic propylhomoserin of bacterial strain.

Description

One plant of Corynebacterium glutamicum and its key gene for synthesizing tryptophan

Technical field:

The invention belongs to Microbial Breeding and field of fermentation engineering, and in particular to the glutamic acid rod of fermenting and producing tryptophan The building of bacterium superior strain and its tryptophan synthesis have the mutational site of correlation gene trpD, trpC and trpB.

Background technique:

Tryptophan is the aromatic amino acid for uniquely having indoles branch, and chemical name is 2- amino -3- indyl propionic acid.Color Propylhomoserin is one of 8 kinds of amino acid necessary to human body, and source relies on food supply, and L-Trp mainly has two outlets, one Item is synthetic proteins matter, and another is to enter catabolism.L-Trp can generate 5- hydroxyl color ammonia in its catabolic process The several physiological active substances such as acid, niacin, epiphysin and xanthurenic acid (4,8-Er Qiangjikuilinjiasuan), these bioactive substances have important pharmaceutical On the way；In addition, L-Trp can be used as food additives, flavoring agent and anti-oxidation and antisepsis agent；In recent years, largely make in feed With methionine and lysine, tryptophan becomes the major limitation acidic amino acid in feed, and discovery is added a small amount of in feed Amino acid just have good effect.Therefore L-Trp medicine, food and in terms of have important industrial value.

The production method of tryptophan has proteolysis extraction method, chemical synthesis, enzyme catalysis method and microbe fermentation method.Egg Plain boiled water solution raw material sources are limited.Chemical synthesis needs high temperature and pressure, synthesis be D-trp and L-Trp mixture, Need other subtractive process.Substrate indoles and serine used in enzymatic are expensive, and enzyme itself is also dangerous.Therefore, First three methods are extremely restricted in the industrial production.Microbe fermentation method is the main method of tryptophan production at present.It is main Producing strain is Escherichia coli, and Escherichia coli fermentation process easily accumulates the by-products such as acetic acid, seriously inhibits thalli growth, produces The gentle saccharic acid conversion ratio of sour water；In addition, Escherichia coli, which produce endotoxin, seriously limits the application field of product.

Corynebacterium glutamicum is GRAS bio-safety bacterium, is potential tryptophan-producing Strain kind.Corynebacterium glutamicum exists Field of amino acid fermentation has very important status, so far by security application nearly 60 years.Currently, include glutamic acid, Most of amino acid such as lysine, valine, leucine, isoleucine, alanine, aspartic acid are all rodlike using glutamic acid Bacillus fermentation production.In corynebacterium glutamicum, 4 enzymes of tryptophan are synthesized by 6 by the intermediary chorismic acid of shikimic acid pathway The trpEGDCBA tryptophan operon coding of a gene composition.TrpE and trpG is separately encoded anthranilate synthase (ANS) Big small subunit, ANS be catalyzed chorismic acid to ortho-aminobenzoic acid the first step reaction.TrpD encoding anthranilate phosphoric acid core Sugared transferase (PRT), trpC encode bifunctional enzyme ribose phosphate ortho-aminobenzoic acid isomerase/indole-3-glycerol-phosphate synthase, TrpB and trpA is separately encoded the β chain and α chain of tryptophan synthetase (TS).ANS is inhibited by the strong inhibition of final product tryptophan The Tryptophan concentration of 50% enzyme activity is 0.0015mM (Sugimoto and Shiio (1983) .Agricultural and Biological Chemistry.47:2295–2305).The 50% enzyme activity inhibition concentration to PRT and TS is respectively 0.15mM (Sugimoto and Shiio (1983) .Agricultural and Biological Chemistry.47:2295-2305) and 7.7mM (Sugimoto and Shiio (1982) .Agricultural and Biological Chemistry.46:2711- 2718).Matsui etc. encodes the trpE of ANS large subunit by the Corynebacterium glutamicum AJ12036 of 5- fluorotryptophan resistant breeding A point mutation becomes serine codon (AGC) arginine codon (CGC) on gene, corresponding polypeptide chain upper amino acid Change (Ser38Arg) lead to feedback insensitive (Matsui etc. (1987) .Journal of of ANS tryptophan Bacteriology.109:5330–5332).O ' GARA and Dunican discovery generate the Corynebacterium glutamicum ATCC of tryptophan 21850 coding PRT trpD gene on two point mutation, bring two amino acid on polypeptide chain change (Ser149Phe, Ala162Glu), cause PRT tryptophan and 5-methyl tryptophan that there is higher resistance (O ' GARA and Dunican (1995) .Applied and Environmental Microbiology.61(12):4477–4479)。

In conclusion feedback inhibition of the enzyme of Corynebacterium glutamicum tryptophan operon coding by final product tryptophan, An important factor for as limitation using Corynebacterium glutamicum high yield tryptophan, and the report adjusted at present in relation to releasing tryptophan feedback Road is less, and corresponding bacterial strain tryptophan production level is also very low.Only Japanese researchers' report is overexpressed using plasmid releases feedback 3- deoxidation-D- Arab ketoheptose -7- phosphate synthase, ANS and the PRT of inhibition, obtain the glutamic acid rod of high yield tryptophan Bacillus (Katsumata and Ikeda (1993) .Nature Biotechnology.12:921-925；Ikeda and Katsumata (1999).Applied and Environmental Microbiology.65(6):2497–2502).However, not having again later There is the relevant report for seeing the bacterial strain, wherein the mutational site of key enzyme is also unclear.Therefore, it is anti-to continue breeding releasing tryptophan Feedback inhibits, obtains the Corynebacterium glutamicum of generation tryptophan just to seem very necessary.

In previous work, we obtain one plant of Corynebacterium glutamicum by mutagenesis means and produce bacterial strain, and apply Patent.In addition, we have obtained one plant of Corynebacterium glutamicum, the tryptophan yield of the bacterial strain in further mutagenesis work It further increases, has found that tryptophan operon trpD, trpC and trpB have occurred by the sequencing of tryptophan operon Gene mutation, thus it is speculated that the mutation of trpDCB may past release tryptophan to the feedback inhibition of enzyme coded by it, to improve The yield of tryptophan.In order to verify the raising of tryptophan yield be as caused by the mutation of trpDCB, the present invention with C.glutamicum TP607 be starting strain, its genome is edited, it may be found that trpDCB mutational site integration Onto the genome of C.glutamicum TP607, production bacterial strain C.glutamicum TP608, shake flask fermentation measurement have been obtained The yield of tryptophan, fermentation results show that the bacterial strain tryptophan yield is substantially improved, further demonstrate mentioning for tryptophan yield Height is that tryptophan is relieved as the Mutational part of trpDCB to the feedback inhibition of enzyme coded by it.

Summary of the invention:

The purpose of the present invention is to provide the Corynebacterium glutamicum superior strains and its color ammonia of one plant of fermenting and producing tryptophan Acid synthesis has the mutational site of correlation gene trpD, trpC and trpB.

The superior strain is Corynebacterium glutamicum (C.glutamicum) TP608, is in Corynebacterium glutamicum (C.glutamicum) by genetic engineering means on the basis of TP607 (deposit number CGMCC No.12302), to the bacterial strain Genome edited, it may be found that tryptophan operon on the mutational site of trpDCB be integrated into C.glutamicum TP It is obtained on 607 genome；

The mutational site of trpDCB is specific as follows:

(1) the 446th bit base C of the encoding gene trpD of ortho-aminobenzoic acid phosphoribosyltransferase (PRT) is mutated For T, so that encoded amino acid becomes phenylalanine (Ser149Phe) by serine；

(2) by bifunctional enzyme ribose phosphate ortho-aminobenzoic acid isomerase/indole-3-glycerol-phosphate synthase encoding gene The 193rd bit base C of trpC is mutated into T, so that encoded amino acid becomes serine (Phe 65Ser) by phenylalanine；

(3) the 1076-1077 interdigit of tryptophan synthetase (TS) β chain encoding gene trpB is increased into three base ACG, 1079th C sports A, and the 1080th A sports G, causes alanine mutation at arginine (Ala360Arg), simultaneously Increase a glutamic acid；

Sequence number in above-mentioned sequence corresponding sequence table is shown in Table 1；

It uses and such as gives a definition in the present invention:

1, the nomenclature of amino acid and DNA nucleic acid sequence

Using the generally acknowledged IUPAC nomenclature of amino acid residue, with three-letter codes form.DNA nucleic acid sequence is using generally acknowledged IUPAC nomenclature.

2, the mark of mutant

The amino acid being mutated in mutant is indicated using " amino acid of Original amino acid position replacement ", such as Ser149Phe indicates that the amino acid of position 149 is substituted for Phe by Ser, and the number of position, which corresponds to, is mutated preceding amino acid sequence The number of column；The base being mutated in mutant, such as C446T are indicated using " base of original base positions replacement ", indicate position It sets 116 bases and T is substituted for by C, the number of position corresponds to the number for being mutated preceding nucleotide sequence.

Table 1

The construction method of the superior strain is as follows:

(1) design primer, by over-lap PCR obtain include above-mentioned mutational site 3 overlapping fragments trp1, trp2 and trp3；

(2) overlapping fragments trp1, trp2 and trp3 are connect with carrier pK18mobsacB respectively；

(3) by gained plasmid, successively electrotransformation enters C.glutamicum TP607 electricity and turns in competence to obtain superior strain.

The utility model has the advantages that

1, according to the present invention, through genetic engineering means on the basis of bacterial strain C.glutamicumTP607, to trpD, TrpC and trpB gene carries out rite-directed mutagenesis, obtains new Corynebacterium glutamicum mutant C.glutamicum TP608.It pushes away Survey relieves tryptophan to the feedback inhibition of these key enzymes.It is found by shake flask fermentation, C.glutamicumTP608 tryptophan Yield is about 3.4 times of TP607；

2, the present invention has correlation gene to provide new mutational site for tryptophan synthesis, provides for the biosynthesis of tryptophan New research direction.

Detailed description of the invention:

Fig. 1 trpD mutated gene is compared with the DNA homolog that sets out；

Fig. 2 trpC mutated gene is compared with the DNA homolog that sets out；

Fig. 3 trpB mutated gene is compared with the DNA homolog that sets out；

The production acid amount of unit thallus in Fig. 4 fermented sample

Wherein, bacterial strain 1 is C.glutamicumTP607；Bacterial strain 2 is C.glutamicumTP607-trp1；

Bacterial strain 3 is C.glutamicum TP607-trp1-trp 2；Bacterial strain 4 is C.glutamicum TP608.

Specific embodiment:

The building of embodiment 1:C.glutamicumTP608

The extraction of 1.C.glutamicum TP607 (CGMCC No.12302) genomic DNA

Single colonie is inoculated in LB to shake in pipe, after 32 DEG C are incubated overnight 12h, accesses 30mL LB culture medium by 2% inoculum concentration Middle progress shaking flask overnight incubation.Extracting genome, steps are as follows:

(1) bacterium solution 13000rpm is centrifuged 2min, removes supernatant, is collected into the EP pipe of 1.5mL.

(2) plus the TE buffer suspension thalline of 600 μ L pH=8.0,13000rpm are centrifuged 2min, abandon supernatant.

(3) add 600 μ L TE suspension thalline again, be added 6 μ lRNAase (10 μ g/mL), 30 μ l lysozymes (50 μ g/mL) It mixes, 37 DEG C of heat preservation 30min or more.

(4) add 30 μ L 10%SDS and 10 μ L Proteinase Ks, mix, 65 DEG C of heat preservation 15min or more.

(5) after the mixing of 100 μ L 5mol/L NaCl solutions is added, 80 μ L CTAB/NaCl solution is added, are mixed by inversion 50 times, 65 DEG C of water-bath 10min.

(6) 750 μ L phenol: chloroform are added: isoamyl alcohol (25:24:1) extracting is mixed by inversion 50 times, 13000rpm centrifugation 15min。

(7) supernatant is sucked in new 1.5mL EP pipe, isometric phenol: chloroform is added: isoamyl alcohol (25:24:1) is taken out It mentions, is mixed by inversion 50 times, 13000rpm is centrifuged 15min.

(8) supernatant is sucked in new 1.5mL EP pipe, adds 1 times of volume isopropanol, overturn mixing, -80 DEG C of cold shocks 15min, 13000rpm are centrifuged 10min, abandon supernatant.

(9) 70% ethyl alcohol is added, flicks resuspension, stands 6-7min, 13000rpm is centrifuged 2-5min, removes supernatant.

(10) after oven drying (37 DEG C), 50 μ L deionized waters are added, dissolving DNA surveys DNA concentration, saves in -20 DEG C.

2. the preparation of Corynebacterium glutamicum electrotransformation competence

(1) it is protected from -80 DEG C of glycerol and uses transfer needle picking Corynebacterium glutamicum, three zonings on LB plating medium in tube Line is put in 32 DEG C of incubators, is incubated overnight.

(2) the picking single colonie on streak plate culture medium is inoculated into the BHIS culture medium test tube of 5mL, 30 DEG C, 200r/min cultivates 12-14h.

(3) being transferred to liquid amount by 1% inoculum concentration is that 100mL BHIS culture medium is (sweet containing 150 μ L Tween 80 and 2.5g Propylhomoserin) 500mL round-bottomed bottle in, under 18 DEG C of cryogenic conditions with 160rpm cultivate at least 18h start measure OD600 value.

(4) it is put in when OD600 is about between 0.5-0.7 and stands 20min on ice.

(5) in super-clean bench, bacterium solution is collected into 50mL centrifuge tube, 4 DEG C, 6500rpm, is centrifuged 10min, abandon supernatant.

(6) bacterium is washed with the 10% glycerol resuspension of 50mL pre-cooling, 4 DEG C, 6500rpm, is centrifuged 10min, abandon supernatant, repetition washes three It is secondary.

(7) thallus is resuspended with 10% glycerol that 2mL is pre-chilled, is managed with the EP of pre-cooling, the packing of 100 μ L/ pipes, remarks competence Title, Production Time, -80 DEG C save backup.

BHIS culture medium (g/L): Brain-Heart Infusion (brain heart extract) 37, Sorbital (sorbierite) 91, pH 7.0~7.2,115 DEG C of high pressure steam sterilization 15min.

3.pK18mobsacB the building of integration vector

(1) amplification of target gene

According to GenBank announce Corynebacterium glutamicum 13032 in trpD gene sequence (Gene ID:1020974), The sequence (Gene ID:1020975) of trpC gene and the sequence (Gene ID:1020976) of trpB gene and they it is upper, Downstream sequence designs specific primer (5 ' -3 ') (table 1):

1. primer sequence of table

Note: what " _ " marked is the sequence of restriction enzyme site, and " " mark is the mutational site constructed

The amplification of the upstream and downstream trpD homology arm: using the genome of C.glutamicum TP607 as template, with trpD-up- S and trpD-up-A is primer, carries out the upstream homology arm about 500bp of pcr amplified fragment trpD；With trpD-down-S and TrpD-down-A is primer, carries out the downstream homology arm about 500bp of pcr amplified fragment trpD.

The amplification of the upstream and downstream trpC homology arm: using the genome of C.glutamicum TP607 as template, with trpC-up- S and trpC-up-A is primer, carries out the upstream homology arm about 500bp of pcr amplified fragment trpC；With trpC-down-S and TrpC-down-A is primer, carries out the downstream homology arm about 500bp of pcr amplified fragment trpC.

The amplification of the upstream and downstream trpB homology arm: using the genome of C.glutamicum TP607 as template, with trpB-up- S and trpB-up-A is primer, carries out the upstream homology arm about 500bp of pcr amplified fragment trpB；With trpB-down-S and TrpB-down-A is primer, carries out the downstream homology arm about 500bp of pcr amplified fragment trpB.

Reaction condition are as follows: 95 DEG C of initial denaturation 5min, 98 DEG C of denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 30s, 30 recycle Afterwards, 72 DEG C of last extensions 10min (high-fidelity Prime STAR HS polymerase).Take 3 μ L amplified productions with 1% after reaction Agarose gel electrophoresis detect clip size.PCR product recycling is recycled using liquid phase recovery purifying DNA kit.

(2) overlapping PCR primers mutational site

The construction method of trpD rite-directed mutagenesis (C446T): being that separation is same to divide upstream and downstream with mutational site (C446T) Source arm, upstream homology arm are separation or more about 500bp, and downstream homology arm is separation or less about 500bp, mutational site design On trpD-up-A and trpD-down-S (base marked in table 1 with).With the upstream the trpD homology arm of recycling and downstream Homology arm is template (additive amount is molar ratio 1:1), using trpD-up-S and trpD-down-A as primer, carries out over-lap PCR, obtains To the overlapping fragments trp1 for including rite-directed mutagenesis.

The construction method of trpC rite-directed mutagenesis (C193T): being that separation is same to divide upstream and downstream with mutational site (C193T) Source arm, upstream homology arm are separation or more about 500bp, and downstream homology arm is separation or less about 500bp, mutational site design On trpC-up-A and trpC-down-S (base marked in table 1 with).With the upstream the trpC homology arm of recycling and downstream Homology arm is template (additive amount is molar ratio 1:1), using trpC-up-S and trpC-down-A as primer, carries out over-lap PCR, obtains To the overlapping fragments trp2 for including rite-directed mutagenesis.

The construction method of trpB rite-directed mutagenesis: upstream and downstream homology arm is divided as separation using mutational site, upstream is homologous Arm is separation or more about 500bp, and downstream homology arm is separation or less about 500bp, mutational site design in trpB-up-S and TrpB-down-A (base marked in table 1 with).(add using the upstream the trpB homology arm of recycling and downstream homology arm as template Dosage is molar ratio 1:1), using trpB-up-S and trpB-down-A as primer, over-lap PCR is carried out, obtains including rite-directed mutagenesis Overlapping fragments trp3.

(3) PCR product recycles

PCR product and digestion system are recycled using liquid phase recovery purifying DNA kit.

(4) digestion is connected with solutionI

The overlapping fragments trp1, trp2 and trp3 and carrier pK18mobsacB, endonuclease reaction system of double digestion recycling are shown in Table 2, digestion time 0.5-1h, 37 DEG C of digestion temperature.Specific restriction enzyme site are as follows: trp1 restriction enzyme site HindIII and PstI； The restriction enzyme site HindIII and PstI of trp2；The restriction enzyme site SalI and BamHI of trp3 is (under specific restriction enzyme site is used in table 1 It lines out).After digestion is completed, recycled using liquid phase recovery purifying DNA kit.The segment of recycling and carrier are connected Connect: trp1 (HindIII and PstI double digestion) and pK18mobsacB (HindIII and PstI double digestion) connection, linked system is trp1-1；Trp2 (HindIII and PstI double digestion) and pK18mobsacB (HindIII and PstI double digestion) connection, connector System is trp2-1；Trp3 (SalI and BamHI double digestion) and pK18mobsacB (SalI and BamHI double digestion) connection, connector System is trp3-1.

10 μ L of coupled reaction system: after target fragment and carrier measurement in addition system, molar ratio between 3:1-9:1, 5 μ L of Solution I ligase, remaining uses deionized water polishing.Connection reaction condition: 16 DEG C, 4h.

2 double digestion system of table

(5) conversion and identification of linked system

Use CaCl₂Linked system trp1-1, trp2-1 and trp3-1 are transformed into bacillus coli DH 5 alpha and turn competence by method Cell.Bacterium colony PCR identifies that positive transformant, primer use the identification primer (the identification primer that T is carried) of pK18mobsacB, wherein The corresponding positive transformant of trp1-1, trp2-1 and trp3-1 is respectively pK18-trp1, pK18-trp2 and pK18-trp3.Alkali Property cracking process extract plasmid pK18-trp1, pK18-trp2 and pK18-trp3DNA, carry out plasmid digestion identification.

(6) correct plasmid pK18-trp1, pK18-trp2 and pK18-trp3 are identified to above-mentioned (step 5), sends to gold only Intelligence company is sequenced.

(7) gene order cognate pair ratio

With DNAMAN software to sequence (Gene ID:1020974) in trpD overlapping genes sequencing result and GenBank into Row compares, and discovery the 446th bit base C of mutant strain sports T (Fig. 1).The variation of orresponding amino acid residue: the 149th serine Become phenylalanine (Ser149Phe) (Fig. 1).It is consistent with the rite-directed mutagenesis of building.

Using DNAMAN software to measure trpC overlapping genes sequencing with GenBank in sequence (Gene ID: 1020975) sequence alignment is carried out, it is found that the 193rd bit base C of trpC is mutated into T, so that encoded amino acid is by phenylpropyl alcohol ammonia Acid becomes serine (Phe65Ser).It is consistent with the rite-directed mutagenesis of building.

The sequencing of trpB overlapping genes and sequence (Gene ID:1020976) in GenBank with DNAMAN software to measuring Sequence alignment is carried out, it is found that 1076-1077 of tryptophan synthetase (TS) β chain encoding gene trpB of mutant strain increase three A base ACG, the 1079th C sport A, and the 1080th A for being sports G (Fig. 3).The variation of orresponding amino acid residue are as follows: Alanine mutation increases a glutaminic acid residue (Fig. 3) at arginine.It is consistent with the rite-directed mutagenesis of building.

4. Corynebacterium glutamicum gene rite-directed mutagenesis integration process

(1) integrated plasmid pK18-trp1 electricity C.glutamicum TP607 electricity is gone to turn to filter out generation in competence The transformant of one wheel exchange, further the transformant of two wheel exchanges occurs for screening, obtains bacterial strain C.glutamicum TP607- Trp1 (bacterial strain of trpD rite-directed mutagenesis in integration)；

(2) on the basis of step (1) obtains bacterial strain, integrated plasmid pK18-trp2 electricity is gone into C.glutamicum The electricity of TP607-trp1 turns in competence, filters out the transformant that a wheel exchange occurs, and further two wheel exchanges occur for screening Transformant obtains bacterial strain C.glutamicum TP607-trp1-trp2 (bacterial strain of trpD and trpC rite-directed mutagenesis in integration)；

(3) on the basis of step (2) obtains bacterial strain, integrated plasmid pK18-trp3 electricity is gone into C.glutamicum The electricity of TP607-trp1-trp2 turns in competence, filters out the transformant that a wheel exchange occurs, and further screening occurs two wheels and hands over The transformant changed obtains bacterial strain C.glutamicum TP608 (bacterial strain of trpD, trpC and trpB rite-directed mutagenesis in integration)

The screening step of two wheel transformant of specific electrotransformation and a wheel is as follows:

1. taking in appropriate recombinant plasmid electrotransformation C.glutamicum Electroporation-competent cells；

2. after electric shock, recovery medium BHIS, the rapid 46 DEG C of water-baths heat shock 6min of 46 DEG C of 1mL preheatings are added immediately, so 32 DEG C afterwards, 200rpm/min recovery 2h；

3. after recovery is completed, 8000rpm/min is centrifuged 2min and is enriched with thallus, it is coated with LB resistant panel (Km^r10μg/ ML), 32 DEG C of 36~48h of culture, by the single colonie grown on plate to selecting to 15% sucrose LB plate and LB kalamycin resistance Plate (Km^r10 μ g/mL), 32 DEG C of cultures 12~for 24 hours；

4. picking out in LB kalamycin resistance plate (Km^r10 μ g/mL) on grow, do not grown in 15% sucrose LB plate The bacterium colony of phenotype, switching LB shake pipe, 32 DEG C, 200rpm/min culture 12~for 24 hours, the genome of above-mentioned bacterium colony is extracted, with Kana- N and Kana-C is primer, and specific primer sequence is shown in Table 3, carries out Kana resistant gene PCR, and the weight of recombination for the first time occurs for verifying Group, protects bacterium；

5. in the case that recombinant bacterium shaking containing 15% sucrose passed in pipe twice (due to 15% sucrose there are, SacB gene expression has lethal effect to host strain, therefore can be used to screen the bacterial strain that second of homologous recombination occurs)；

6. carrying out dilution appropriate, it is applied on the plate of 15% sucrose, 32 DEG C of 24~36h of culture, the single bacterium that will be grown It falls to o'clock to 15% sucrose plate and LB kalamycin resistance plate (Km^r10 μ g/mL), 32 DEG C of cultures 12~for 24 hours, it filters out The growth of 15% sucrose plate is simultaneously in LB kalamycin resistance plate (Km^r10 μ g/mL) no longer grow the single colonie of phenotype；

7. the correct single colonie of above-mentioned phenotype is transferred to LB to shake in pipe, 32 DEG C, 200rpm/min be incubated overnight, extract base Because of group, using genomic DNA as template, with the homologous recombination primer of design, carry out PCR amplification, using kit to PCR product into Row recycling, the product of recycling is sequenced, and filters out the positive transformant that rite-directed mutagenesis occurs, and protects bacterium.

Table 3. identifies primer sequence

Embodiment 2: shaking flask carries out tryptophan fermentation

1. strain

Shake flask fermentation bacterial strain uses therefor is as follows:

C.glutamicumTP607；C.glutamicumTP607-trp1；

C.glutamicumTP607-trp1-trp2；C.glutamicum TP608；

2. actication of culture

Inclined-plane culture: taking -80 DEG C of preservation of bacteria strain streak inoculations in activated inclined plane, and 32 DEG C of cultures for 24 hours, and are passed on once, 32 DEG C culture 12h.

Activation medium (g/L): peptone 10, beef extract 10, yeast powder 5, NaCl 2.5, corn pulp 15mL/L, agar 25, pH 7.0-7.2, sterilising conditions: 121 DEG C, 20min.

3. seed culture

Seed culture: using 500mL triangular flask culture seed, liquid amount 30mL, and strain uses two generation inclined-planes, inoculum concentration For two rings, two generations inclined-plane, inoculation method is to scrape two generation inclined-plane seeds with oese, is transferred in corresponding seed culture medium.Training 32 DEG C, pH 7.0 ± 0.2, revolving speed 200rpm of temperature is supported, incubation time 12h, Fungal biodiversity OD600 reach 10 ± 2 or so, connect Kind is into fermentation medium.

Seed culture medium (g/L): glucose 35, yeast powder 5, corn pulp 60mL/L, soybean meal hydrolysate 20mL/L, KH₂PO₄ 1.5, MgSO₄0.5, FeSO₄·7H₂O 0.01, MnSO₄·H₂O 0.01, VB₁0.001, pH7.0-7.2, sterilising conditions: 121 ℃、20min。

4. shake flask fermentation culture

Fermented and cultured: shaking flask culture is used, 500mL baffle bottle, liquid amount 30mL, inoculum concentration 10%, inoculation side are used Method is to draw the cultured seed of 3ml using 5mL Sterile pipette, is inoculated into fermentation medium.Condition of culture is culture temperature 32 DEG C of degree, revolving speed 200rpm, pH 7.0 ± 0.2 adjust pH using ammonium hydroxide, and fermentation period is 36 hours.

Fermentation medium (g/L): glucose 100, corn pulp 25mL/L, soybean meal hydrolysate 25mL/L, (NH₄)₂SO₄3, KH₂PO₄2.2, MgSO₄0.7, FeSO₄·7H₂O 0.01, MnSO₄·H₂O 0.01, VB₁0.001, pH 7.0-7.2, sterilizing Condition: 121 DEG C, 20min.

5. analysis method

(1) Fungal biodiversity measures:

20 times or 100 times of mycelium dilution, using V1200 ultraviolet specrophotometer, in the absorbance of 600nm measurement sample. A certain amount of sample is taken, drying surveys dry weight, acquires the corresponding relationship of OD and dry cell weight.It is dry that thallus is calculated according to the OD value of fermentation liquid Weight.

(2) measurement of tryptophan yield:

The measurement of amino acid concentration content: it is measured using HPLC.Testing conditions are as follows: 1100 efficient liquid phase of instrument Agilent Chromatograph；Chromatographic column Phenomenex Gemini 5u C18 (150*4.6mm, U.S.'s phenomenex company)；Single mobile phase: 10% Acetonitrile；33 DEG C of column temperature；Detection wavelength 278nm；Flow velocity 1.0m L/min, gradient elution.

The tryptophan yield in fermented sample is measured according to the above method, as a result sees Fig. 4.

Middle abscissa is four bacterial strains in Fig. 4, and ordinate is the content of tryptophan in shake flask fermentation 36h sample, and unit is The tryptophan production capacity of every gram of dry weight thallus.By scheming it is observed that bacterial strain C.glutamicum TP607-trp1 and bacterial strain C.glutamicum TP607 is compared, and the yield of tryptophan significantly improves, and illustrates that the integration in the mutational site trpD can be improved color The yield of propylhomoserin, thus it is speculated that the possible past release tryptophan that introduces in the mutational site trpD presses down the feedback of the gene encoding enzyme System, improves the vigor of the enzyme；Bacterial strain C.glutamicum TP607-trp1-trp2 and bacterial strain C.glutamicum TP607- Trp1 is compared, the output increased of tryptophan, illustrates that the integration in the mutational site trpC can be improved the yield of tryptophan；Bacterial strain C.glutamicum TP608 is significantly improved with the bacterial strain C.glutamicum TP607-trp1-trp2 yield for comparing tryptophan, Illustrate that the integration in the mutational site trpB can be improved the yield of tryptophan, thus it is speculated that the introducing in the mutational site trpB may past release Feedback inhibition of the tryptophan to the gene encoding enzyme, improves the vigor of the enzyme.C.glutamicumTP608 tryptophan yield About the 3.4 of TP607 times.

SEQUENCE LISTING

<110>University Of Science and Technology Of Tianjin

Lotus flower health industry limited liability company

<120>one plants of Corynebacterium glutamicums and its key gene for synthesizing tryptophan

<130> 1

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 1047

<212> DNA

<213> CGMCC No.12302

<400> 1

atgacttctc cagcaacact gaaagttctc aacgcctact tggataaccc cactccaacc 60

ctggaggagg caattgaggt gttcaccccg ctgaccgtgg gtgaatacga tgacgtgcac 120

atcgcagcgc tgcttgccac catccgtact cgcggtgagc agttcgctga tattgccggc 180

gctgccaagg cgttcctcgc ggcggctcgt ccgttcccga ttactggcgc aggtttgcta 240

gattccgctg gtactggtgg cgacggtgcc aacaccatca acatcaccac cggcgcatcc 300

ctgatcgcag catccggtgg agtgaagctg gttaagcacg gcaaccgttc ggtgagctcc 360

aagtccggct ccgccgatgt gctggaagcg ctgaatattc ctttgggcct tgatgtggat 420

cgtgctgtga agtggttcga agcgtccaac ttcaccttcc tgttcgcacc tgcgtacaac 480

cctgcgattg cgcatgtgca gccggttcgc caggcgctga aattccccac catcttcaac 540

acgcttggac cattgctgtc cccggcgcgc ccggagcgtc agatcatggg cgtggccaat 600

gccaatcatg gacagctcat cgccgaggtc ttccgcgagt tgggccgtac acgcgcgctt 660

gttgtgcatg gcgcaggcac cgatgagatc gcagtccacg gcaccacctt ggtgtgggag 720

cttaaagaag acggcaccat cgagcattac accatcgagc ctgaggacct tggccttggc 780

cgctacaccc ttgaggatct cgtaggtggc ctcggcactg agaacgccga agctatgcgc 840

gctactttcg cgggcaccgg ccctgatgca caccgtgatg cgttggctgc gtccgcaggt 900

gcgatgttct acctcaacgg cgatgtcgac tccttgaaag atggtgcaca aaaggcgctt 960

tccttgcttg ccgacggcac cacccaggca tggttggcca agcacgaaga gatcgattac 1020

tcagaaaagg agtcttccaa tgactag 1047

<210> 2

<211> 1425

<212> DNA

<213> CGMCC No.12302

<400> 2

atgactagta ataatctgcc cacagtgttg gaaagcatcg tcgagggtcg tcgcggacac 60

ctggaggaaa ttcgcgctcg catcgctcac gtggatgtgg atgcgcttcc aaaatccacc 120

cgttctctgt ttgattccct caaccagggt aggggagggg cgcgtttcat catggagtgc 180

aagtccgcat cgccttcttt gggaatgatt cgtgagcact accagccggg tgaaatcgct 240

cgcgtgtact ctcgctacgc cagcggcatt tccgtgctgt gcgagccgga tcgttttggt 300

ggcgattacg atcacctcgc taccgttgcc gctacctctc atcttccggt gctgtgcaaa 360

gacttcatca ttgatcctgt ccaggtacac gcggcgcgtt actttggtgc tgatgccatc 420

ctgctcatgc tctctgtgct tgatgatgaa gagtacgcag cactcgctgc cgaggctgcg 480

cgttttgatc tggatatcct caccgaggtt attgatgagg aggaagtcgc ccgcgccatc 540

aagctgggtg cgaagatctt tggcgtcaac caccgcaacc tgcatgatct gtccattgat 600

ttggatcgtt cacgtcgcct gtccaagctc attccagcag atgccgtgct cgtgtctgag 660

tctggcgtgc gcgataccga aaccgtccgc cagctaggtg ggcactccaa tgcattcctc 720

gttggctccc agctgaccag ccaggaaaac gtcgatctgg cagcccgcga attagtctac 780

ggccccaaca aagtctgcgg actcacctca ccaagtgcag cacaaaccgc tcgcgcagcg 840

ggtgcggtct acggcgggct catcttcgaa gaggcatcgc cacgcaatgt ttcacgtgaa 900

acattgcaaa aaatcatcgc cgcagagccc aacctgcgct acgtcgcggt cagccgtcgc 960

acctccgggt acaaggattt gcttgtcgac ggcatcttcg ccgtacaaat ccacgcccca 1020

ctgcaggaca gcgtcgaagc agaaaaggca ttgatcgccg ccgttcgtga agaggttgga 1080

ccgcaggtcc aggtctggcg cgcgatctcg atgtccagcc ccttgggggc tgaagtggca 1140

gctgcggtgg agggtgacgt cgataagcta attcttgatg cccatgaagg tggcagcggg 1200

gaagtattcg actgggctac ggtgccggcc gctgtgaagg caaagtcttt gctcgcggga 1260

ggcatctctc cggacaacgc tgcgcaggca ctcgctgtgg gctgcgcagg tttggacatc 1320

aactctggcg tggaataccc cgccggtgca ggcacgtggg ctggggcgaa agacgccggc 1380

gcgctgctga aaattttcgc gaccatctcc acattccatt actaa 1425

<210> 3

<211> 1123

<212> DNA

<213> cgmcc no.12302

<400> 3

atgactgaaa aagaaaactt gggcggctcc acgctgctgc ctgcatactt cggtgaattc 60

ggcggccagt tcgtcgcgga atccctcctg cctgctctcg accagctgga gaaggccttc 120

gttgacgcga ccaacagccc agagttccgc gaagaactcg gcggctacct ccgcgattac 180

ctcggccgcc caaccccgct gaccgaatgc tccaacctgc cactcgcagg cgaaggcaaa 240

ggctttgcgc ggatcttcct caagcgcgaa gacctcgtcc acggcggtgc acacaaaact 300

aaccaggtga tcggccaggt gctgcttgcc aagcgcatgg gcaaaacccg catcatcgca 360

gagaccggcg caggccagca cggcaccgcc accgctctcg catgtgcgct catgggcctc 420

gagtgcgttg tctacatggg cgccaaggac gttgcccgcc agcagcccaa cgtctaccgc 480

atgcagctgc acggcgcgaa ggtcatcccc gtggaatctg gttccggcac cctgaaggac 540

gccgtgaatg aagcgctgcg cgattggacc gcaaccttcc acgagtccca ctaccttctc 600

ggcaccgccg ccggcccgca cccattccca accatcgtgc gtgaattcca caaggtgatc 660

tctgaggaag ccaaggcaca gatgctagag cgcaccggca agcttcccga cgttgtggtc 720

gcctgtgtcg gtggtggctc caacgccatc ggcatgttcg cagacttcat tgacgatgaa 780

ggtgtagagc tcgtcggcgc tgagccagcc ggtgaaggcc tcgactccgg caagcacggc 840

gcaaccatca ccaacggtca gatcggcatc ctgcacggca cccgttccta cctgatgcgc 900

aactccgacg gccaagtgga agagtcctac tccatctccg ccggacttga ttacccaggc 960

gtcggcccac agcacgcaca cctgcacgcc accggccgcg ccacctacgt tggtatcacc 1020

gacgccgaag ccctccaagc attccagtac ctcgcccgct acgaaggcat catccccgca 1080

ctggaatcct cacacgcgtt cgcctacgca ctcaagcgcg cca 1123

<210> 4

<211> 1047

<212> DNA

<213>artificial sequence

<400> 4

atgacttctc cagcaacact gaaagttctc aacgcctact tggataaccc cactccaacc 60

ctggaggagg caattgaggt gttcaccccg ctgaccgtgg gtgaatacga tgacgtgcac 120

atcgcagcgc tgcttgccac catccgtact cgcggtgagc agttcgctga tattgccggc 180

gctgccaagg cgttcctcgc ggcggctcgt ccgttcccga ttactggcgc aggtttgcta 240

gattccgctg gtactggtgg cgacggtgcc aacaccatca acatcaccac cggcgcatcc 300

ctgatcgcag catccggtgg agtgaagctg gttaagcacg gcaaccgttc ggtgagctcc 360

aagtccggct ccgccgatgt gctggaagcg ctgaatattc ctttgggcct tgatgtggat 420

cgtgctgtga agtggttcga agcgttcaac ttcaccttcc tgttcgcacc tgcgtacaac 480

cctgcgattg cgcatgtgca gccggttcgc caggcgctga aattccccac catcttcaac 540

acgcttggac cattgctgtc cccggcgcgc ccggagcgtc agatcatggg cgtggccaat 600

gccaatcatg gacagctcat cgccgaggtc ttccgcgagt tgggccgtac acgcgcgctt 660

gttgtgcatg gcgcaggcac cgatgagatc gcagtccacg gcaccacctt ggtgtgggag 720

cttaaagaag acggcaccat cgagcattac accatcgagc ctgaggacct tggccttggc 780

cgctacaccc ttgaggatct cgtaggtggc ctcggcactg agaacgccga agctatgcgc 840

gctactttcg cgggcaccgg ccctgatgca caccgtgatg cgttggctgc gtccgcaggt 900

gcgatgttct acctcaacgg cgatgtcgac tccttgaaag atggtgcaca aaaggcgctt 960

tccttgcttg ccgacggcac cacccaggca tggttggcca agcacgaaga gatcgattac 1020

tcagaaaagg agtcttccaa tgactag 1047

<210> 5

<211> 1425

<212> DNA

<213>artificial sequence

<400> 5

atgactagta ataatctgcc cacagtgttg gaaagcatcg tcgagggtcg tcgcggacac 60

ctggaggaaa ttcgcgctcg catcgctcac gtggatgtgg atgcgcttcc aaaatccacc 120

cgttctctgt ttgattccct caaccagggt aggggagggg cgcgtttcat catggagtgc 180

aagtccgcat cgtcttcttt gggaatgatt cgtgagcact accagccggg tgaaatcgct 240

cgcgtgtact ctcgctacgc cagcggcatt tccgtgctgt gcgagccgga tcgttttggt 300

ggcgattacg atcacctcgc taccgttgcc gctacctctc atcttccggt gctgtgcaaa 360

gacttcatca ttgatcctgt ccaggtacac gcggcgcgtt actttggtgc tgatgccatc 420

ctgctcatgc tctctgtgct tgatgatgaa gagtacgcag cactcgctgc cgaggctgcg 480

cgttttgatc tggatatcct caccgaggtt attgatgagg aggaagtcgc ccgcgccatc 540

aagctgggtg cgaagatctt tggcgtcaac caccgcaacc tgcatgatct gtccattgat 600

ttggatcgtt cacgtcgcct gtccaagctc attccagcag atgccgtgct cgtgtctgag 660

tctggcgtgc gcgataccga aaccgtccgc cagctaggtg ggcactccaa tgcattcctc 720

gttggctccc agctgaccag ccaggaaaac gtcgatctgg cagcccgcga attagtctac 780

ggccccaaca aagtctgcgg actcacctca ccaagtgcag cacaaaccgc tcgcgcagcg 840

ggtgcggtct acggcgggct catcttcgaa gaggcatcgc cacgcaatgt ttcacgtgaa 900

acattgcaaa aaatcatcgc cgcagagccc aacctgcgct acgtcgcggt cagccgtcgc 960

acctccgggt acaaggattt gcttgtcgac ggcatcttcg ccgtacaaat ccacgcccca 1020

ctgcaggaca gcgtcgaagc agaaaaggca ttgatcgccg ccgttcgtga agaggttgga 1080

ccgcaggtcc aggtctggcg cgcgatctcg atgtccagcc ccttgggggc tgaagtggca 1140

gctgcggtgg agggtgacgt cgataagcta attcttgatg cccatgaagg tggcagcggg 1200

gaagtattcg actgggctac ggtgccggcc gctgtgaagg caaagtcttt gctcgcggga 1260

ggcatctctc cggacaacgc tgcgcaggca ctcgctgtgg gctgcgcagg tttggacatc 1320

aactctggcg tggaataccc cgccggtgca ggcacgtggg ctggggcgaa agacgccggc 1380

gcgctgctga aaattttcgc gaccatctcc acattccatt actaa 1425

<210> 6

<211> 1126

<212> DNA

<213>artificial sequence

<400> 6

atgactgaaa aagaaaactt gggcggctcc acgctgctgc ctgcatactt cggtgaattc 60

ggcggccagt tcgtcgcgga atccctcctg cctgctctcg accagctgga gaaggccttc 120

gttgacgcga ccaacagccc agagttccgc gaagaactcg gcggctacct ccgcgattac 180

ctcggccgcc caaccccgct gaccgaatgc tccaacctgc cactcgcagg cgaaggcaaa 240

ggctttgcgc ggatcttcct caagcgcgaa gacctcgtcc acggcggtgc acacaaaact 300

aaccaggtga tcggccaggt gctgcttgcc aagcgcatgg gcaaaacccg catcatcgca 360

gagaccggcg caggccagca cggcaccgcc accgctctcg catgtgcgct catgggcctc 420

gagtgcgttg tctacatggg cgccaaggac gttgcccgcc agcagcccaa cgtctaccgc 480

atgcagctgc acggcgcgaa ggtcatcccc gtggaatctg gttccggcac cctgaaggac 540

gccgtgaatg aagcgctgcg cgattggacc gcaaccttcc acgagtccca ctaccttctc 600

ggcaccgccg ccggcccgca cccattccca accatcgtgc gtgaattcca caaggtgatc 660

tctgaggaag ccaaggcaca gatgctagag cgcaccggca agcttcccga cgttgtggtc 720

gcctgtgtcg gtggtggctc caacgccatc ggcatgttcg cagacttcat tgacgatgaa 780

ggtgtagagc tcgtcggcgc tgagccagcc ggtgaaggcc tcgactccgg caagcacggc 840

gcaaccatca ccaacggtca gatcggcatc ctgcacggca cccgttccta cctgatgcgc 900

aactccgacg gccaagtgga agagtcctac tccatctccg ccggacttga ttacccaggc 960

gtcggcccac agcacgcaca cctgcacgcc accggccgcg ccacctacgt tggtatcacc 1020

gacgccgaag ccctccaagc attccagtac ctcgcccgct acgaaggcat catcccacgc 1080

gagctggaat cctcacacgc gttcgcctac gcactcaagc gcgcca 1126

<210> 7

<211> 348

<212> PRT

<213> CGMCC No.12302

<400> 7

Met Thr Ser Pro Ala Thr Leu Lys Val Leu Asn Ala Tyr Leu Asp Asn

1 5 10 15

Pro Thr Pro Thr Leu Glu Glu Ala Ile Glu Val Phe Thr Pro Leu Thr

20 25 30

Val Gly Glu Tyr Asp Asp Val His Ile Ala Ala Leu Leu Ala Thr Ile

35 40 45

Arg Thr Arg Gly Glu Gln Phe Ala Asp Ile Ala Gly Ala Ala Lys Ala

50 55 60

Phe Leu Ala Ala Ala Arg Pro Phe Pro Ile Thr Gly Ala Gly Leu Leu

65 70 75 80

Asp Ser Ala Gly Thr Gly Gly Asp Gly Ala Asn Thr Ile Asn Ile Thr

85 90 95

Thr Gly Ala Ser Leu Ile Ala Ala Ser Gly Gly Val Lys Leu Val Lys

100 105 110

His Gly Asn Arg Ser Val Ser Ser Lys Ser Gly Ser Ala Asp Val Leu

115 120 125

Glu Ala Leu Asn Ile Pro Leu Gly Leu Asp Val Asp Arg Ala Val Lys

130 135 140

Trp Phe Glu Ala Ser Asn Phe Thr Phe Leu Phe Ala Pro Ala Tyr Asn

145 150 155 160

Pro Ala Ile Ala His Val Gln Pro Val Arg Gln Ala Leu Lys Phe Pro

165 170 175

Thr Ile Phe Asn Thr Leu Gly Pro Leu Leu Ser Pro Ala Arg Pro Glu

180 185 190

Arg Gln Ile Met Gly Val Ala Asn Ala Asn His Gly Gln Leu Ile Ala

195 200 205

Glu Val Phe Arg Glu Leu Gly Arg Thr Arg Ala Leu Val Val His Gly

210 215 220

Ala Gly Thr Asp Glu Ile Ala Val His Gly Thr Thr Leu Val Trp Glu

225 230 235 240

Leu Lys Glu Asp Gly Thr Ile Glu His Tyr Thr Ile Glu Pro Glu Asp

245 250 255

Leu Gly Leu Gly Arg Tyr Thr Leu Glu Asp Leu Val Gly Gly Leu Gly

260 265 270

Thr Glu Asn Ala Glu Ala Met Arg Ala Thr Phe Ala Gly Thr Gly Pro

275 280 285

Asp Ala His Arg Asp Ala Leu Ala Ala Ser Ala Gly Ala Met Phe Tyr

290 295 300

Leu Asn Gly Asp Val Asp Ser Leu Lys Asp Gly Ala Gln Lys Ala Leu

305 310 315 320

Ser Leu Leu Ala Asp Gly Thr Thr Gln Ala Trp Leu Ala Lys His Glu

325 330 335

Glu Ile Asp Tyr Ser Glu Lys Glu Ser Ser Asn Asp

340 345

<210> 8

<211> 474

<212> PRT

<213> CGMCC No.12302

<400> 8

Met Thr Ser Asn Asn Leu Pro Thr Val Leu Glu Ser Ile Val Glu Gly

1 5 10 15

Arg Arg Gly His Leu Glu Glu Ile Arg Ala Arg Ile Ala His Val Asp

20 25 30

Val Asp Ala Leu Pro Lys Ser Thr Arg Ser Leu Phe Asp Ser Leu Asn

35 40 45

Gln Gly Arg Gly Gly Ala Arg Phe Ile Met Glu Cys Lys Ser Ala Ser

50 55 60

Pro Ser Leu Gly Met Ile Arg Glu His Tyr Gln Pro Gly Glu Ile Ala

65 70 75 80

Arg Val Tyr Ser Arg Tyr Ala Ser Gly Ile Ser Val Leu Cys Glu Pro

85 90 95

Asp Arg Phe Gly Gly Asp Tyr Asp His Leu Ala Thr Val Ala Ala Thr

100 105 110

Ser His Leu Pro Val Leu Cys Lys Asp Phe Ile Ile Asp Pro Val Gln

115 120 125

Val His Ala Ala Arg Tyr Phe Gly Ala Asp Ala Ile Leu Leu Met Leu

130 135 140

Ser Val Leu Asp Asp Glu Glu Tyr Ala Ala Leu Ala Ala Glu Ala Ala

145 150 155 160

Arg Phe Asp Leu Asp Ile Leu Thr Glu Val Ile Asp Glu Glu Glu Val

165 170 175

Ala Arg Ala Ile Lys Leu Gly Ala Lys Ile Phe Gly Val Asn His Arg

180 185 190

Asn Leu His Asp Leu Ser Ile Asp Leu Asp Arg Ser Arg Arg Leu Ser

195 200 205

Lys Leu Ile Pro Ala Asp Ala Val Leu Val Ser Glu Ser Gly Val Arg

210 215 220

Asp Thr Glu Thr Val Arg Gln Leu Gly Gly His Ser Asn Ala Phe Leu

225 230 235 240

Val Gly Ser Gln Leu Thr Ser Gln Glu Asn Val Asp Leu Ala Ala Arg

245 250 255

Glu Leu Val Tyr Gly Pro Asn Lys Val Cys Gly Leu Thr Ser Pro Ser

260 265 270

Ala Ala Gln Thr Ala Arg Ala Ala Gly Ala Val Tyr Gly Gly Leu Ile

275 280 285

Phe Glu Glu Ala Ser Pro Arg Asn Val Ser Arg Glu Thr Leu Gln Lys

290 295 300

Ile Ile Ala Ala Glu Pro Asn Leu Arg Tyr Val Ala Val Ser Arg Arg

305 310 315 320

Thr Ser Gly Tyr Lys Asp Leu Leu Val Asp Gly Ile Phe Ala Val Gln

325 330 335

Ile His Ala Pro Leu Gln Asp Ser Val Glu Ala Glu Lys Ala Leu Ile

340 345 350

Ala Ala Val Arg Glu Glu Val Gly Pro Gln Val Gln Val Trp Arg Ala

355 360 365

Ile Ser Met Ser Ser Pro Leu Gly Ala Glu Val Ala Ala Ala Val Glu

370 375 380

Gly Asp Val Asp Lys Leu Ile Leu Asp Ala His Glu Gly Gly Ser Gly

385 390 395 400

Glu Val Phe Asp Trp Ala Thr Val Pro Ala Ala Val Lys Ala Lys Ser

405 410 415

Leu Leu Ala Gly Gly Ile Ser Pro Asp Asn Ala Ala Gln Ala Leu Ala

420 425 430

Val Gly Cys Ala Gly Leu Asp Ile Asn Ser Gly Val Glu Tyr Pro Ala

435 440 445

Gly Ala Gly Thr Trp Ala Gly Ala Lys Asp Ala Gly Ala Leu Leu Lys

450 455 460

Ile Phe Ala Thr Ile Ser Thr Phe His Tyr

465 470

<210> 9

<211> 374

<212> PRT

<213> CGMCC No.12302

<400> 9

Met Thr Glu Lys Glu Asn Leu Gly Gly Ser Thr Leu Leu Pro Ala Tyr

1 5 10 15

Phe Gly Glu Phe Gly Gly Gln Phe Val Ala Glu Ser Leu Leu Pro Ala

20 25 30

Leu Asp Gln Leu Glu Lys Ala Phe Val Asp Ala Thr Asn Ser Pro Glu

35 40 45

Phe Arg Glu Glu Leu Gly Gly Tyr Leu Arg Asp Tyr Leu Gly Arg Pro

50 55 60

Thr Pro Leu Thr Glu Cys Ser Asn Leu Pro Leu Ala Gly Glu Gly Lys

65 70 75 80

Gly Phe Ala Arg Ile Phe Leu Lys Arg Glu Asp Leu Val His Gly Gly

85 90 95

Ala His Lys Thr Asn Gln Val Ile Gly Gln Val Leu Leu Ala Lys Arg

100 105 110

Met Gly Lys Thr Arg Ile Ile Ala Glu Thr Gly Ala Gly Gln His Gly

115 120 125

Thr Ala Thr Ala Leu Ala Cys Ala Leu Met Gly Leu Glu Cys Val Val

130 135 140

Tyr Met Gly Ala Lys Asp Val Ala Arg Gln Gln Pro Asn Val Tyr Arg

145 150 155 160

Met Gln Leu His Gly Ala Lys Val Ile Pro Val Glu Ser Gly Ser Gly

165 170 175

Thr Leu Lys Asp Ala Val Asn Glu Ala Leu Arg Asp Trp Thr Ala Thr

180 185 190

Phe His Glu Ser His Tyr Leu Leu Gly Thr Ala Ala Gly Pro His Pro

195 200 205

Phe Pro Thr Ile Val Arg Glu Phe His Lys Val Ile Ser Glu Glu Ala

210 215 220

Lys Ala Gln Met Leu Glu Arg Thr Gly Lys Leu Pro Asp Val Val Val

225 230 235 240

Ala Cys Val Gly Gly Gly Ser Asn Ala Ile Gly Met Phe Ala Asp Phe

245 250 255

Ile Asp Asp Glu Gly Val Glu Leu Val Gly Ala Glu Pro Ala Gly Glu

260 265 270

Gly Leu Asp Ser Gly Lys His Gly Ala Thr Ile Thr Asn Gly Gln Ile

275 280 285

Gly Ile Leu His Gly Thr Arg Ser Tyr Leu Met Arg Asn Ser Asp Gly

290 295 300

Gln Val Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu Asp Tyr Pro Gly

305 310 315 320

Val Gly Pro Gln His Ala His Leu His Ala Thr Gly Arg Ala Thr Tyr

325 330 335

Val Gly Ile Thr Asp Ala Glu Ala Leu Gln Ala Phe Gln Tyr Leu Ala

340 345 350

Arg Tyr Glu Gly Ile Ile Pro Ala Leu Glu Ser Ser His Ala Phe Ala

355 360 365

Tyr Ala Leu Lys Arg Ala

370

<210> 10

<211> 348

<212> PRT

<213>artificial sequence

<400> 10

Met Thr Ser Pro Ala Thr Leu Lys Val Leu Asn Ala Tyr Leu Asp Asn

1 5 10 15

Pro Thr Pro Thr Leu Glu Glu Ala Ile Glu Val Phe Thr Pro Leu Thr

20 25 30

Val Gly Glu Tyr Asp Asp Val His Ile Ala Ala Leu Leu Ala Thr Ile

35 40 45

Arg Thr Arg Gly Glu Gln Phe Ala Asp Ile Ala Gly Ala Ala Lys Ala

50 55 60

Phe Leu Ala Ala Ala Arg Pro Phe Pro Ile Thr Gly Ala Gly Leu Leu

65 70 75 80

Asp Ser Ala Gly Thr Gly Gly Asp Gly Ala Asn Thr Ile Asn Ile Thr

85 90 95

Thr Gly Ala Ser Leu Ile Ala Ala Ser Gly Gly Val Lys Leu Val Lys

100 105 110

His Gly Asn Arg Ser Val Ser Ser Lys Ser Gly Ser Ala Asp Val Leu

115 120 125

Glu Ala Leu Asn Ile Pro Leu Gly Leu Asp Val Asp Arg Ala Val Lys

130 135 140

Trp Phe Glu Ala Phe Asn Phe Thr Phe Leu Phe Ala Pro Ala Tyr Asn

145 150 155 160

Pro Ala Ile Ala His Val Gln Pro Val Arg Gln Ala Leu Lys Phe Pro

165 170 175

Thr Ile Phe Asn Thr Leu Gly Pro Leu Leu Ser Pro Ala Arg Pro Glu

180 185 190

Arg Gln Ile Met Gly Val Ala Asn Ala Asn His Gly Gln Leu Ile Ala

195 200 205

Glu Val Phe Arg Glu Leu Gly Arg Thr Arg Ala Leu Val Val His Gly

210 215 220

Ala Gly Thr Asp Glu Ile Ala Val His Gly Thr Thr Leu Val Trp Glu

225 230 235 240

Leu Lys Glu Asp Gly Thr Ile Glu His Tyr Thr Ile Glu Pro Glu Asp

245 250 255

Leu Gly Leu Gly Arg Tyr Thr Leu Glu Asp Leu Val Gly Gly Leu Gly

260 265 270

Thr Glu Asn Ala Glu Ala Met Arg Ala Thr Phe Ala Gly Thr Gly Pro

275 280 285

Asp Ala His Arg Asp Ala Leu Ala Ala Ser Ala Gly Ala Met Phe Tyr

290 295 300

Leu Asn Gly Asp Val Asp Ser Leu Lys Asp Gly Ala Gln Lys Ala Leu

305 310 315 320

Ser Leu Leu Ala Asp Gly Thr Thr Gln Ala Trp Leu Ala Lys His Glu

325 330 335

Glu Ile Asp Tyr Ser Glu Lys Glu Ser Ser Asn Asp

340 345

<210> 11

<211> 474

<212> PRT

<213>artificial sequence

<400> 11

Met Thr Ser Asn Asn Leu Pro Thr Val Leu Glu Ser Ile Val Glu Gly

1 5 10 15

Arg Arg Gly His Leu Glu Glu Ile Arg Ala Arg Ile Ala His Val Asp

20 25 30

Val Asp Ala Leu Pro Lys Ser Thr Arg Ser Leu Phe Asp Ser Leu Asn

35 40 45

Gln Gly Arg Gly Gly Ala Arg Phe Ile Met Glu Cys Lys Ser Ala Ser

50 55 60

Ser Ser Leu Gly Met Ile Arg Glu His Tyr Gln Pro Gly Glu Ile Ala

65 70 75 80

Arg Val Tyr Ser Arg Tyr Ala Ser Gly Ile Ser Val Leu Cys Glu Pro

85 90 95

Asp Arg Phe Gly Gly Asp Tyr Asp His Leu Ala Thr Val Ala Ala Thr

100 105 110

Ser His Leu Pro Val Leu Cys Lys Asp Phe Ile Ile Asp Pro Val Gln

115 120 125

Val His Ala Ala Arg Tyr Phe Gly Ala Asp Ala Ile Leu Leu Met Leu

130 135 140

Ser Val Leu Asp Asp Glu Glu Tyr Ala Ala Leu Ala Ala Glu Ala Ala

145 150 155 160

Arg Phe Asp Leu Asp Ile Leu Thr Glu Val Ile Asp Glu Glu Glu Val

165 170 175

Ala Arg Ala Ile Lys Leu Gly Ala Lys Ile Phe Gly Val Asn His Arg

180 185 190

Asn Leu His Asp Leu Ser Ile Asp Leu Asp Arg Ser Arg Arg Leu Ser

195 200 205

Lys Leu Ile Pro Ala Asp Ala Val Leu Val Ser Glu Ser Gly Val Arg

210 215 220

Asp Thr Glu Thr Val Arg Gln Leu Gly Gly His Ser Asn Ala Phe Leu

225 230 235 240

Val Gly Ser Gln Leu Thr Ser Gln Glu Asn Val Asp Leu Ala Ala Arg

245 250 255

Glu Leu Val Tyr Gly Pro Asn Lys Val Cys Gly Leu Thr Ser Pro Ser

260 265 270

Ala Ala Gln Thr Ala Arg Ala Ala Gly Ala Val Tyr Gly Gly Leu Ile

275 280 285

Phe Glu Glu Ala Ser Pro Arg Asn Val Ser Arg Glu Thr Leu Gln Lys

290 295 300

Ile Ile Ala Ala Glu Pro Asn Leu Arg Tyr Val Ala Val Ser Arg Arg

305 310 315 320

Thr Ser Gly Tyr Lys Asp Leu Leu Val Asp Gly Ile Phe Ala Val Gln

325 330 335

Ile His Ala Pro Leu Gln Asp Ser Val Glu Ala Glu Lys Ala Leu Ile

340 345 350

Ala Ala Val Arg Glu Glu Val Gly Pro Gln Val Gln Val Trp Arg Ala

355 360 365

Ile Ser Met Ser Ser Pro Leu Gly Ala Glu Val Ala Ala Ala Val Glu

370 375 380

Gly Asp Val Asp Lys Leu Ile Leu Asp Ala His Glu Gly Gly Ser Gly

385 390 395 400

Glu Val Phe Asp Trp Ala Thr Val Pro Ala Ala Val Lys Ala Lys Ser

405 410 415

Leu Leu Ala Gly Gly Ile Ser Pro Asp Asn Ala Ala Gln Ala Leu Ala

420 425 430

Val Gly Cys Ala Gly Leu Asp Ile Asn Ser Gly Val Glu Tyr Pro Ala

435 440 445

Gly Ala Gly Thr Trp Ala Gly Ala Lys Asp Ala Gly Ala Leu Leu Lys

450 455 460

Ile Phe Ala Thr Ile Ser Thr Phe His Tyr

465 470

<210> 12

<211> 375

<212> PRT

<213>artificial sequence

<400> 12

Met Thr Glu Lys Glu Asn Leu Gly Gly Ser Thr Leu Leu Pro Ala Tyr

1 5 10 15

Phe Gly Glu Phe Gly Gly Gln Phe Val Ala Glu Ser Leu Leu Pro Ala

20 25 30

Leu Asp Gln Leu Glu Lys Ala Phe Val Asp Ala Thr Asn Ser Pro Glu

35 40 45

Phe Arg Glu Glu Leu Gly Gly Tyr Leu Arg Asp Tyr Leu Gly Arg Pro

50 55 60

Thr Pro Leu Thr Glu Cys Ser Asn Leu Pro Leu Ala Gly Glu Gly Lys

65 70 75 80

Gly Phe Ala Arg Ile Phe Leu Lys Arg Glu Asp Leu Val His Gly Gly

85 90 95

Ala His Lys Thr Asn Gln Val Ile Gly Gln Val Leu Leu Ala Lys Arg

100 105 110

Met Gly Lys Thr Arg Ile Ile Ala Glu Thr Gly Ala Gly Gln His Gly

115 120 125

Thr Ala Thr Ala Leu Ala Cys Ala Leu Met Gly Leu Glu Cys Val Val

130 135 140

Tyr Met Gly Ala Lys Asp Val Ala Arg Gln Gln Pro Asn Val Tyr Arg

145 150 155 160

Met Gln Leu His Gly Ala Lys Val Ile Pro Val Glu Ser Gly Ser Gly

165 170 175

Thr Leu Lys Asp Ala Val Asn Glu Ala Leu Arg Asp Trp Thr Ala Thr

180 185 190

Phe His Glu Ser His Tyr Leu Leu Gly Thr Ala Ala Gly Pro His Pro

195 200 205

Phe Pro Thr Ile Val Arg Glu Phe His Lys Val Ile Ser Glu Glu Ala

210 215 220

Lys Ala Gln Met Leu Glu Arg Thr Gly Lys Leu Pro Asp Val Val Val

225 230 235 240

Ala Cys Val Gly Gly Gly Ser Asn Ala Ile Gly Met Phe Ala Asp Phe

245 250 255

Ile Asp Asp Glu Gly Val Glu Leu Val Gly Ala Glu Pro Ala Gly Glu

260 265 270

Gly Leu Asp Ser Gly Lys His Gly Ala Thr Ile Thr Asn Gly Gln Ile

275 280 285

Gly Ile Leu His Gly Thr Arg Ser Tyr Leu Met Arg Asn Ser Asp Gly

290 295 300

Gln Val Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu Asp Tyr Pro Gly

305 310 315 320

Val Gly Pro Gln His Ala His Leu His Ala Thr Gly Arg Ala Thr Tyr

325 330 335

Val Gly Ile Thr Asp Ala Glu Ala Leu Gln Ala Phe Gln Tyr Leu Ala

340 345 350

Arg Tyr Glu Gly Ile Ile Pro Arg Glu Leu Glu Ser Ser His Ala Phe

355 360 365

Ala Tyr Ala Leu Lys Arg Ala

370 375

<210> 13

<211> 34

<212> DNA

<213>artificial sequence

<400> 13

cccaagctta tgacttctcc agcaacactg aaag 34

<210> 14

<211> 42

<212> DNA

<213>artificial sequence

<400> 14

cgaacaggaa ggtgaagttg aacgcttcga accacttcac ag 42

<210> 15

<211> 42

<212> DNA

<213>artificial sequence

<400> 15

ctgtgaagtg gttcgaagcg ttcaacttca ccttcctgtt cg 42

<210> 16

<211> 28

<212> DNA

<213>artificial sequence

<400> 16

aaactgcagc aagcaaggaa agcgcctt 28

<210> 17

<211> 31

<212> DNA

<213>artificial sequence

<400> 17

cccaagcttc gctacaccct tgaggatctc g 31

<210> 18

<211> 43

<212> DNA

<213>artificial sequence

<400> 18

ctcacgaatc attcccaaag aagacgatgc ggacttgcac tcc 43

<210> 19

<211> 43

<212> DNA

<213>artificial sequence

<400> 19

ggagtgcaag tccgcatcgt cttctttggg aatgattcgt gag 43

<210> 20

<211> 28

<212> DNA

<213>artificial sequence

<400> 20

aaactgcaga cctagctggc ggacggtt 28

<210> 21

<211> 27

<212> DNA

<213>artificial sequence

<400> 21

cgcgtcgaca tgaagcgctg cgcgatt 27

<210> 22

<211> 40

<212> DNA

<213>artificial sequence

<400> 22

gtgaggattc cagctcgcgt gggatgatgc cttcgtagcg 40

<210> 23

<211> 40

<212> DNA

<213>artificial sequence

<400> 23

cgctacgaag gcatcatccc acgcgagctg gaatcctcac 40

<210> 24

<211> 28

<212> DNA

<213>artificial sequence

<400> 24

cgcggatcct ccaagccacg ggtgaaag 28

Claims

1. the Corynebacterium glutamicum superior strain of one plant of fermenting and producing tryptophan is in Corynebacterium glutamicum CGMCC No.12302 On the basis of acquisition is transformed by trpD, trpC, trpB in genetic engineering means tryptophan operon, feature exists In the bacterial strain contains three sequences of sequence table SEQ ID No.4-6.

2. the Corynebacterium glutamicum superior strain of one plant of fermenting and producing tryptophan described in claim 1, which is characterized in that described The corresponding amino acid sequence of SEQ ID No.4-6 is as shown in SEQ ID No.10-12.

3. the Corynebacterium glutamicum superior strain of one plant of fermenting and producing tryptophan described in claim 1, which is characterized in that described The construction method of superior strain is as follows:

(1) design primer obtains 3 overlapping fragments trp1 for including the mutational site trpD, trpC, trpB by over-lap PCR, Trp2 and trp3；

(3) by gained plasmid, successively electrotransformation enters CGMCC No.12302 electricity and turns in competence to obtain superior strain.

4. application of the bacterial strain described in claim 1 in fermenting and producing tryptophan.

5. application of the bacterial strain as claimed in claim 4 in fermenting and producing tryptophan, which is characterized in that specific as follows:

(1) seed culture

Seed culture: using 500mL triangular flask culture seed, and liquid amount 30mL is inoculated with two ring slant strains, cultivation temperature 32 DEG C, pH7.0 ± 0.2, revolving speed 200rpm, incubation time 12h, Fungal biodiversity OD600 reaches 10 ± 2；

Seed culture medium is in terms of g/L: glucose 35, yeast powder 5, corn pulp 60mL/L, soybean meal hydrolysate 20mL/L, KH₂PO₄ 1.5, MgSO₄0.5, FeSO₄·7 H₂O 0.01, MnSO₄·H₂O 0.01, VB₁0.001, pH7.0-7.2, sterilising conditions: 121 ℃,20min；

(2) fermented and cultured

Fermented and cultured: inoculum concentration 10%, 32 DEG C of cultivation temperature, revolving speed 200rpm, pH7.0 ± 0.2, fermentation period is 36 small When；

Fermentation medium is in terms of g/L: glucose 100, corn pulp 25mL/L, soybean meal hydrolysate 25mL/L, (NH₄)₂SO₄3, KH₂PO₄2.2, MgSO₄0.7, FeSO₄·7 H₂O 0.01, MnSO₄·H₂O 0.01, VB₁0.001, pH7.0-7.2, sterilizing Condition: 121 DEG C, 20min.