CN106520652A

CN106520652A - Corynebacterium glutamicum, and key tryptophan synthesis gene thereof

Info

Publication number: CN106520652A
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: 2017-03-22
Anticipated expiration: 2036-11-29
Also published as: CN106520652B

Abstract

The invention belongs to the fields of microorganism breeding and fermentation engineering, and concretely relates to a Corynebacterium glutamicum, and a key tryptophan synthesis gene thereof. The above high-yielding strain is obtained through editing the genome of Corynebacterium glutamicum CGMCC No.12302 through a gene engineering means, and integrating the trpDCB mutation site in a tryptophan operon to the genome of the CGMCC No.12302. The Corynebacterium glutamicum provides a new mutation site for tryptophan synthesis related gene, and provides a new research direction for biosynthesis of the tryptophan; and a shaking bottle fermentation result shows that the tryptophan output of the obtained strain is about 3.4 times original output, so the tryptophan production performance of the strain is greatly improved.

Description

One plant of Corynebacterium glutamicum and its key gene of synthesis tryptophan

Technical field：

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

Background technology：

Tryptophan is the aromatic amino acid for uniquely having indole side chain, and chemical name is 2- amino -3- indyl propanoic acid.Color Propylhomoserin is one of 8 kinds of aminoacid necessary to human body, and its source relies on food supply, and L-Tryptophan mainly has two outlets, one Bar is synthetic protein, and another is to enter catabolism.L-Tryptophan can produce 5- hydroxyl color ammonia in its catabolic process The several physiological active substances such as acid, nicotinic acid, pineal hormone and xanthurenic acid (4,8-Er Qiangjikuilinjiasuan), these bioactive substances have important pharmaceutical On the way；Additionally, L-Tryptophan can be used as food additive, flavoring agent and anti-oxidation and antisepsis agent；In recent years, made in feedstuff in a large number With methionine and lysine, tryptophan becomes the major limitation acidic amino acid in feedstuff, finds to add in feedstuff a small amount of Aminoacid just have good effect.Therefore L-Tryptophan has important industrial value at aspects such as medicine, food and feedstuffs.

The production method of tryptophan has Proteolytic enzyme extraction method, chemical synthesiss, enzyme catalysis method and microbe fermentation method.Egg Plain boiled water solution raw material sources are limited.Chemosynthesis need High Temperature High Pressure, synthesis be D-trp and L-Tryptophan mixture, Need other subtractive process.Used by enzyme catalysiss, substrate indole and serine 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 Strain to be produced is escherichia coli, and Escherichia coli fermentation process easily accumulates the by-products such as acetic acid, seriously suppresses thalli growth, produces The gentle saccharic acid conversion ratio of sour water；In addition, escherichia coli produce the application that endotoxin seriously limits 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 nearly 60 years by safety applications.At present, including glutamic acid, Most of aminoacid such as lysine, L-Valine, leucine, isoleucine, alanine, aspartic acid are all bar-shaped using glutamic acid Bacillus fermentation is produced.In Corynebacterium glutamicum, 4 enzymes of tryptophan are synthesized by 6 by the intermedium chorismic acid of shikimic acid pathway The trpEGDCBA tryptophan operons coding of individual genomic constitution.TrpE and trpG are separately encoded anthranilate synthase (ANS) Big small subunit, ANS catalysis chorismic acid to ortho-aminobenzoic acid the first step reaction.TrpD encoding anthranilate phosphoric acid cores Sugared transferring enzyme (PRT), trpC coding bifunctional enzyme ribose phosphate ortho-aminobenzoic acid isomerase/indole-3-glycerol-phosphate synthases, TrpB and trpA are separately encoded the β chains and α chains of tryptophan synthetase (TS).Strong inhibitions of the ANS by end-product tryptophan, suppresses The Tryptophan concentration of 50% enzyme activity is 0.0015mM (Sugimoto and Shiio (1983) .Agricultural and Biological Chemistry.47:2295–2305).0.15mM is respectively to the 50% enzyme activity inhibition concentration of PRT and TS (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 subunits by the Corynebacterium glutamicum AJ12036 of 5- fluorotryptophan resistant breedings A point mutation on gene causes serine codon (AGC) to be changed into arginine codon (CGC), corresponding polypeptide chain upper amino acid Change (Ser38Arg) cause feedback insensitive (Matsui etc. (1987) the .Journal of of ANS tryptophans Bacteriology.109:5330–5332).O ' GARA and Dunican have found the Corynebacterium glutamicum ATCC for producing tryptophan 21850 coding PRT trpD genes on two point mutation, bring two aminoacid on polypeptide chain change (Ser149Phe, Ala162Glu), cause PRT tryptophans and 5-methyl tryptophan that there is higher resistance (O ' GARA and Dunican (1995) .Applied and Environmental Microbiology.61(12):4477–4479)。

In sum, the enzyme of Corynebacterium glutamicum tryptophan operon coding is subject to the feedback suppression of end-product tryptophan, Become the key factor limited using Corynebacterium glutamicum high yield tryptophan, and at present about releasing the report that tryptophan feedback is adjusted Road is less, and the corresponding bacterial strain tryptophan level of production is also very low.Only Japanese researchers' report releases feedback using plasmid overexpression 3- deoxidations-D- Arab ketoheptose -7- the phosphate synthases of suppression, ANS and PRT, 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 afterwards There is the relevant report for seeing the bacterial strain, the mutational site of wherein key enzyme is also unclear.Therefore, continue selection-breeding releasing tryptophan anti- Feedback suppresses, obtains producing the Corynebacterium glutamicum of tryptophan just to seem very necessary.

In previous work, we obtain one plant of Corynebacterium glutamicum by mutation means and produce bacterial strain, and apply Patent.Additionally, in further mutation work, we have obtained one plant of Corynebacterium glutamicum, the tryptophan yield of the bacterial strain Further improve, be found that tryptophan operon trpD, trpC and trpB there occurs by the sequencing of tryptophan operator Gene mutation, thus it is speculated that trpDCB's is mutated possible past release feedback suppression of the tryptophan to enzyme coded by which, so as to improve The yield of tryptophan.In order to the raising for verifying tryptophan yield is caused by the mutation of trpDCB, the present invention with C.glutamicum TP607 be starting strain, edlin is entered to its genome, it may be found that trpDCB mutational site integrate To on the genome of C.glutamicum TP607, production bacterial strain C.glutamicum TP608 are obtained, shake flask fermentation is determined The yield of tryptophan, fermentation results show that the bacterial strain tryptophan yield is substantially improved, and further demonstrate carrying for tryptophan yield Height is to relieve feedback suppression of the tryptophan to enzyme coded by which due to the Mutational part of trpDCB.

The content of the invention：

It is an object of the invention to provide the Corynebacterium glutamicum superior strain 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 enter edlin, it may be found that tryptophan operon on the mutational site of trpDCB be incorporated into C.glutamicum TP Obtain 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 coded aminoacid becomes Phenylalanine (Ser149Phe) by serine；

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

(3) the 1076-1077 interdigits of tryptophan synthetase (TS) β chain encoding gene trpB be increased into three bases ACG, The C of the 1079th sports A, and the A of the 1080th sports G, causes alanine mutation into arginine (Ala360Arg), while Increased a glutamic acid；

Above-mentioned sequence pair answers the sequence number in sequence table to be shown in Table 1；

Adopt in the present invention and be defined as below：

1st, the nomenclature of aminoacid and DNA nucleotide sequences

Using the generally acknowledged IUPAC nomenclatures of amino acid residue, three-letter codes form is used.DNA nucleotide sequences are using generally acknowledged IUPAC nomenclatures.

2nd, the mark of mutant

The aminoacid being mutated in mutant is represented using " aminoacid that Original amino acid position is replaced ", such as Ser149Phe, represents that the aminoacid of position 149 is substituted for Phe by Ser, and the numbering of position is corresponding to aminoacid sequence before mutation The numbering of row；The base being mutated in mutant, such as C446T are represented using " base that original base positions are replaced ", position is represented The base for putting 116 is substituted for T by C, and the numbering of position is corresponding to the numbering for being mutated front nucleotide sequence.

Table 1

The construction method of the superior strain is as follows：

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

(2) overlapping fragmentses trp1, trp2 and trp3 are connected with carrier pK18mobsacB respectively；

(3) gained plasmid successively electricity is transformed into C.glutamicum TP607 electricity to turn in competence, to obtain superior strain.

Beneficial effect：

1st, according to the present invention, by genetic engineering means on the basis of bacterial strain C.glutamicumTP607, to trpD, TrpC and trpB genes carry out rite-directed mutagenesises, obtain new Corynebacterium glutamicum mutant C.glutamicum TP608.Push away Survey relieves feedback suppression of the tryptophan to these key enzymes.Found by shake flask fermentation, C.glutamicumTP608 tryptophans Yield is about 3.4 times of TP607；

2nd, the present invention has correlation gene to provide new mutational site for tryptophan synthesis, and the biosynthesiss for tryptophan are provided New research direction.

Description of the drawings：

Fig. 1 trpD mutant genes are compared with the DNA homolog that sets out；

Fig. 2 trpC mutant genes are compared with the DNA homolog that sets out；

Fig. 3 trpB mutant genes are compared with the DNA homolog that sets out；

The product acid amount of unit thalline in Fig. 4 fermented samples

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：

Embodiment 1：The structure of C.glutamicumTP608

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

Single bacterium colony is inoculated in LB to shake in pipe, after 32 DEG C of incubated overnight 12h, 30mL LB culture medium is accessed by 2% inoculum concentration In carry out shake-flask culture overnight.Extract genome step as follows：

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

(2) the TE buffer suspension thallines of 600 μ L pH=8.0,13000rpm centrifugation 2min is added to abandon supernatant.

(3) add 600 μ L TE suspension thallines again, add 6 μ lRNAase (10 μ g/mL), 30 μ l lysozyme (50 μ g/mL) Mix, 37 DEG C of insulation more than 30min.

(4) add 30 μ L 10%SDS and 10 μ L E.C. 3.4.21.64s, mix, 65 DEG C of insulation more than 15min.

(5) after adding 100 μ L 5mol/L NaCl solutions to mix, 80 μ L CTAB/NaCl solution are added, overturns and mix 50 times, 65 DEG C of water-bath 10min.

(6) 750 μ L phenol are added：Chloroform：Isoamyl alcohol (25：24：1) extract, overturn and mix 50 times, 13000rpm centrifugations 15min。

(7) supernatant is sucked in new 1.5mL EP pipes, adds isopyknic phenol：Chloroform：Isoamyl alcohol (25：24：1) take out Carry, overturn and mix 50 times, 13000rpm centrifugation 15min.

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

(9) 70% ethanol is added, flicks resuspended, standing 6-7min, 13000rpm centrifugation 2-5min, remove supernatant.

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

2. the preparation of Corynebacterium glutamicum electricity transformed competence colibacillus

(1) protect from -80 DEG C of glycerol and use in tube Inoculating needle picking Corynebacterium glutamicum, three zonings on LB plating mediums Line, is put in 32 DEG C of incubators, incubated overnight.

(2) the picking single bacterium colony in streak plate culture medium, is inoculated in the BHIS culture medium test tubes of 5mL, 30 DEG C, 200r/min cultivates 12-14h.

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

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

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

(6) bacterium is washed with 10% glycerol of 50mL pre-coolings is resuspended, 4 DEG C, 6500rpm is centrifuged 10min, abandons supernatant, and repetition washes three It is secondary.

(7) 10% glycerol with 2mL pre-coolings is resuspended by thalline, is managed with the EP of pre-cooling, 100 μ L/ pipe subpackages, remarks competence Title, Production Time, -80 DEG C save backup.

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

3.pK18mobsacB the structure of integration vector

(1) amplification of genes of interest

According to sequence (the Gene ID of trpD genes in the Corynebacterium glutamicum 13032 that GenBank is announced：1020974)、 Sequence (the Gene ID of trpC genes：1020975) sequence (the Gene ID with trpB genes：1020976) and they it is upper, Downstream sequence, design specific primer (5 ' -3 ') (table 1)：

1. primer sequence of table

Note：What " _ " marked is the sequence of restriction enzyme site, and what " " was marked is the mutational site for building

The amplification of trpD upstream and downstream homology arm：Genome with 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 trpC upstream and downstream homology arm：Genome with 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 trpB upstream and downstream homology arm：Genome with 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 is：95 DEG C of denaturations 5min, 98 DEG C of degeneration 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 30s, 30 circulations Afterwards, 72 DEG C of last extensions 10min (high-fidelity Prime STAR HS polymerases).Reaction takes 3 μ L amplified productions after terminating and uses 1% Agarose gel electrophoresiies detection clip size.PCR primer is reclaimed and is reclaimed using liquid phase recovery purifying DNA test kit.

(2) overlapping PCR primers mutational site

The construction method of trpD rite-directed mutagenesises (C446T)：It is same to divide upstream and downstream as separation with mutational site (C446T) Source arm, upstream homology arm are more than separation about 500bp, and downstream homology arm is below separation about 500bp, and mutational site is designed The base of labellings in table 1, is used () on trpD-up-A and trpD-down-S.With the trpD upstreams homology arm of recovery and downstream Homology arm is that (addition is mol ratio 1 to template:1), with trpD-up-S and trpD-down-A as primer, over-lap PCR is carried out, is obtained To the overlapping fragmentses trp1 for including rite-directed mutagenesises.

The construction method of trpC rite-directed mutagenesises (C193T)：It is same to divide upstream and downstream as separation with mutational site (C193T) Source arm, upstream homology arm are more than separation about 500bp, and downstream homology arm is below separation about 500bp, and mutational site is designed The base of labellings in table 1, is used () on trpC-up-A and trpC-down-S.With the trpC upstreams homology arm of recovery and downstream Homology arm is that (addition is mol ratio 1 to template:1), with trpC-up-S and trpC-down-A as primer, over-lap PCR is carried out, is obtained To the overlapping fragmentses trp2 for including rite-directed mutagenesises.

The construction method of trpB rite-directed mutagenesises：Upstream and downstream homology arm is divided with mutational site as separation, upstream is homologous Arm is more than separation about 500bp, and downstream homology arm is below separation about 500bp, mutational site design in trpB-up-S and TrpB-down-A (uses the base of labellings) in table 1.(added with the trpB upstreams homology arm and downstream homology arm of recovery as template Dosage is mol ratio 1:1), with trpB-up-S and trpB-down-A as primer, over-lap PCR is carried out, obtains including rite-directed mutagenesises Overlapping fragmentses trp3.

(3) PCR primer is reclaimed

PCR primer and enzyme action system are reclaimed using liquid phase recovery purifying DNA test kit.

(4) enzyme action and solutionI connections

Overlapping fragmentses trp1, trp2 and trp3 and carrier pK18mobsacB that double digestion is reclaimed, endonuclease reaction system are shown in Table 2, enzyme action time 0.5-1h, 37 DEG C of enzyme action temperature.Specifically restriction enzyme site is：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 concrete restriction enzyme site is used in table 1 Line out).After enzyme action is completed, reclaimed using liquid phase recovery purifying DNA test kit.The fragment and carrier of recovery are connected Connect：Trp1 (HindIII and PstI double digestions) and pK18mobsacB (HindIII and PstI double digestions) connects, and linked system is trp1-1；Trp2 (HindIII and PstI double digestions) and pK18mobsacB (HindIII and PstI double digestions) connections, connector It is for trp2-1；Trp3 (SalI and BamHI double digestions) and pK18mobsacB (SalI and BamHI double digestions) connections, connector It is for trp3-1.

10 μ L of coupled reaction system：After purpose fragment and carrier measure in addition system, mol ratio is 3:1-9:Between 1, 5 μ L of Solution I ligases, remaining deionized water polishing.Coupled reaction condition：16 DEG C, 4h.

2 double digestion system of table

(5) conversion of linked system and identification

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 positive transformant, and primer adopts 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 enzyme action 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 softwares to trpD overlapping geness sequencing result and sequence (Gene ID in GenBank：1020974) enter Row is compared, it is found that the 446th bit base C of mutant sports T (Fig. 1).The change of orresponding amino acid residue：149th serine Become Phenylalanine (Ser149Phe) (Fig. 1).It is consistent with the rite-directed mutagenesises for building.

The trpC overlapping geness for measuring are sequenced and sequence (Gene ID in GenBank using DNAMAN softwares： 1020975) sequence alignment is carried out, it is found that the 193rd bit base C of trpC is mutated into T so that coded aminoacid is by phenylpropyl alcohol ammonia Acid becomes serine (Phe65Ser).It is consistent with the rite-directed mutagenesises for building.

The trpB overlapping geness for measuring are sequenced and sequence (Gene ID in GenBank with DNAMAN softwares：1020976) Sequence alignment is carried out, it is found that the 1076-1077 positions of tryptophan synthetase (TS) the β chain encoding gene trpB of mutant increased three Individual base ACG, the C of the 1079th sport A, and the 1080th A for being sports G (Fig. 3).The change of orresponding amino acid residue is turned to： Alanine mutation is into arginine, while increased a glutaminic acid residue (Fig. 3).It is consistent with the rite-directed mutagenesises for building.

4. Corynebacterium glutamicum gene rite-directed mutagenesises integration process

(1) integrated plasmid pK18-trp1 electricity is gone to C.glutamicum TP607 electricity to turn in competence, to filter out generation There is the transformant that two wheels are exchanged in the transformant that one wheel is exchanged, further screening, obtain bacterial strain C.glutamicum TP607- Trp1 (bacterial strains of trpD rite-directed mutagenesises in integration)；

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

(3) on the basis of step (2) obtains bacterial strain, integrated plasmid pK18-trp3 electricity is gone to into C.glutamicum The electricity of TP607-trp1-trp2 turns in competence, filters out the transformant for occurring that a wheel is exchanged, and further two wheel of screening generation is handed over The transformant changed, obtains bacterial strain C.glutamicum TP608 (bacterial strains of trpD, trpC and trpB rite-directed mutagenesises in integration)

The screening step that specific electricity conversion and a wheel two take turns transformant is as follows：

1. take in appropriate recombiant plasmid electricity conversion C.glutamicum Electroporation-competent cells；

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

3., after recovery is completed, 8000rpm/min centrifugation 2min enrichment thalline are coated with LB resistant panel (Km^r10μg/ ML), 32 DEG C of 36～48h of culture, by the single bacterium colony grown on flat board to selecting 15% sucrose LB flat boards and LB kalamycin resistances Flat board (Km^r10 μ g/mL), 32 DEG C of 12～24h of culture；

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

5. recombinant bacterium shaking containing 15% sucrose pass in pipe twice (due to 15% sucrose exist in the case of, SacB gene expressions have lethal effect to Host Strains, therefore can be used to screen the bacterial strain that second homologous recombination occurs)；

6. appropriate dilution is carried out, is applied on the flat board of 15% sucrose, 32 DEG C of 24～36h of culture, by the single bacterium for growing Fall to o'clock to 15% sucrose plate and LB kalamycin resistance flat board (Km^r10 μ g/mL), 32 DEG C of 12～24h of culture are filtered out The growth of 15% sucrose plate is simultaneously in LB kalamycin resistance flat board (Km^r10 μ g/mL) no longer growth phenotype single bacterium colony；

7. above-mentioned phenotype correct single bacterium colony is transferred to LB to shake in pipe, 32 DEG C, 200rpm/min incubated overnight, extracts base Because of group, with genomic DNA as template, with the homologous recombination primer for designing, enter performing PCR amplification, PCR primer is entered using test kit Row is reclaimed, and the product of recovery is sequenced, and filters out the positive transformant that rite-directed mutagenesises occur, 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

Slant culture：- 80 DEG C of preservation of bacteria strain streak inoculations are taken in activated inclined plane, 32 DEG C of culture 24h, and pass on once, 32 DEG C culture 12h.

Activation medium (g/L)：Peptone 10, Carnis Bovis seu Bubali cream 10, yeast powder 5, NaCl 2.5, Semen Maydis pulp 15mL/L, agar 25, pH 7.0-7.2, sterilising conditions：121℃、20min.

3. seed culture

Seed culture：Using 500mL triangular flask culture seeds, liquid amount is 30mL, and strain is using secondary inclined-plane, inoculum concentration For the secondary inclined-plane of two rings, inoculation method is to scrape secondary inclined-plane seed with inoculating loop, is transferred in corresponding seed culture medium.Training 32 DEG C of foster temperature, pH 7.0 ± 0.2, rotating speed 200rpm, incubation time 12h, Fungal biodiversity OD600 reach 10 ± 2 or so, connect Plant in fermentation medium.

Seed culture medium (g/L)：Glucose 35, yeast powder 5, Semen Maydis 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

Fermentation culture：Using shake-flask culture, using 500mL baffle plate bottles, liquid amount 30mL, inoculum concentration are 10%, inoculation side Method is to draw the cultured seeds of 3ml using 5mL Sterile pipettes, is inoculated in fermentation medium.Condition of culture is culture temperature 32 DEG C of degree, rotating speed 200rpm, pH 7.0 ± 0.2 adjust pH using ammonia, and fermentation period is 36 hours.

Fermentation medium (g/L)：Glucose 100, Semen Maydis 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 is determined：

20 times or 100 times of mycelium dilution, using V1200 ultraviolet spectrophotometers, in the absorbance of 600nm determination samples. A certain amount of sample is taken, drying is surveyed dry weight, tries to achieve the corresponding relation of OD and dry cell weight.Thalline is calculated according to the OD values of fermentation liquid to do Weight.

(2) measure of tryptophan yield：

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

Tryptophan yield in fermented sample is determined according to said method, Fig. 4 is as a result seen.

In in Fig. 4, abscissa is four bacterial strains, and vertical coordinate is the content of tryptophan in shake flask fermentation 36h samples, and unit is The tryptophan production capacity of every gram of dry weight thalline.By figure it is observed that bacterial strain C.glutamicum TP607-trp1 and bacterial strain C.glutamicum TP607 are compared, and the yield of tryptophan is significantly improved, and illustrate that the integration in trpD mutational sites can improve color The yield of propylhomoserin, thus it is speculated that the possible past release tryptophan that introduces in trpD mutational sites presses down to 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 trpC mutational sites can improve the yield of tryptophan；Bacterial strain The yield that C.glutamicum TP608 compare tryptophan with bacterial strain C.glutamicum TP607-trp1-trp2 is significantly improved, Illustrate that the integration in trpB mutational sites can improve the yield of tryptophan, thus it is speculated that the introducing in trpB mutational sites may past release Feedback suppression 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

Flos Nelumbinis health industry limited company

<120>One plant of Corynebacterium glutamicum and its key gene of synthesis 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 carry out transformation by trpD, trpC, the trpB in genetic engineering means tryptophan operator at least one and obtain , it is characterised in that the bacterial strain contains at least one 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, it is characterised in that described Bacterial strain contains three sequences of sequence table SEQ ID No.4-6.

3. the Corynebacterium glutamicum superior strain of one plant of fermenting and producing tryptophan described in claim 1, it is characterised in that described The corresponding aminoacid sequences of SEQ ID No.4-6 are as shown in SEQ ID No.10-12.

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

(1) primer is designed, 3 overlapping fragmentses trp1 for including trpD, trpC, trpB mutational site is obtained by over-lap PCR, Trp2 and trp3；

(3) gained plasmid successively electricity is transformed into CGMCC No.12302 electricity to turn in competence, to obtain superior strain.

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

6. application of the bacterial strain described in claim 5 in fermenting and producing tryptophan, it is characterised in that specific as follows：

(1) seed culture

Seed culture：Using 500mL triangular flask culture seeds, liquid amount is 30mL, is inoculated with two ring slant strains, cultivation temperature 32 DEG C, pH 7.0 ± 0.2, rotating speed 200rpm, incubation time 12h, Fungal biodiversity OD600 reach 10 ± 2；

Seed culture medium is in terms of g/L：Glucose 35, yeast powder 5, Semen Maydis 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；

(2) fermentation culture

Fermentation culture：Inoculum concentration is 10%, 32 DEG C of cultivation temperature, rotating speed 200rpm, pH 7.0 ± 0.2, and fermentation period is 36 little When；

Fermentation medium is in terms of g/L：Glucose 100, Semen Maydis 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.