CN104099267A - Construction and application of C.glutamicum subspecies lactofermentum with high L-methionine yield - Google Patents

Abstract

Belonging to the field of bioengineering, the invention relates to construction and application of C.glutamicum subspecies lactofermentum with high L-methionine yield. The methionine high-yielding recombinant strain C.glutamicum subspecies lactofermentum QW102/pJYW-4-hom<m>-lysC<m>-brnFE provided by the invention is preserved in China Center for Type Culture Collection with a preservation number of CCTCC M2014232. The recombinant strain has the genetic characteristics of: expression of transport protein BrnFE, expression of aspartokinase fragment lysC and homoserine dehydrogenase fragment hom, and deletion of thrB and mcbR genes on a genome at the same time. According to the invention, by a genetic engineering method, a recombinant strain with high methionine yield is obtained. The invention also provides a method for high yield of L-methionine by the recombinant strain.

Description

One plant height produces the construction and application of the Corynebacterium glutamicum of METHIONINE

Technical field

The present invention's one plant height produces the construction and application of the Corynebacterium glutamicum of METHIONINE, belongs to bioengineering field, is specifically related to a kind of method of high yield METHIONINE.

Background technology

METHIONINE is one of sulfur-bearing α sulphur amino acid.Its synthetic method is mainly two kinds of chemical synthesis and fermentation methods.At present suitability for industrialized production application be mainly chemosynthesis DL methionine(Met), and fermentation method does not still have industrial applications example at present.METHIONINE is widely used in feed industry, because fodder production does not need highly purified METHIONINE, still take at present chemical synthesis as main.But because chemical synthesis can produce a large amount of objectionable impuritiess, fermentative Production methionine(Met) more and more receives publicity.But because methionine(Met) route of synthesis energy consumption is higher, and sulfur assimilation approach is larger to cell self toxicity, and fermentative Production methionine(Met) does not reach technical scale.

The synthetic methionine(Met) of fermentation method mainly utilizes intestinal bacteria or Corynebacterium glutamicum as producing bacterial strain at present, but because intestinal bacteria self can produce intracellular toxin, is unfavorable for food safety fermentation.In intestinal bacteria, methionine metabolism approach is compared Corynebacterium glutamicum complexity simultaneously, is unfavorable for the metabolic engineering of bacterial strain.And Corynebacterium glutamicum produces bacterial strain as a strain aliment security level, it has higher L-glutamic acid and aspartate family amino acids production ability, also be used to produce METHIONINE, be widely used at present Methionin, Histidine, in the amino acid whose suitability for industrialized production such as Isoleucine, but methionine(Met) does not still have the example of industrial applications.

Summary of the invention

The object of the invention is to by engineered method, obtain the recombinant bacterial strain that a plant height produces methionine(Met).

According to one preferred example of the present invention, the recombinant bacterial strain of high yield methionine(Met) provided by the invention, comprises homoserine kinase encoding gene on genome thrBwith TetR type aporepressor mcbRknocking out of encoding gene.

According to one preferred example of the present invention, the recombinant bacterial strain of high yield methionine(Met) provided by the invention, also comprises ectogenic expression feedback resistance aspartokinase enzyme fragment, feedback resistance homoserine dehydrogenase fragment and translocator BrnFE fragment.

According to one preferred example of the present invention, the 311st Thr of the aspartokinase enzyme fragment of heterogenous expression sports Ile, and the 377th Gly of homoserine dehydrogenase fragment of heterogenous expression sports Glu.

According to one preferred example of the present invention, the exogenous fragment providing is present in carrier, and described carrier is plasmid pJYW-4.

Technical scheme of the present invention a: plant height produces the Corynebacterium glutamicum of METHIONINE, described strain classification called after Corynebacterium glutamicum QW102/pJYW-4- hom ^m -lysC ^m -brnFE,be preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC M 2014232.

Described bacterial strain QW102/pJYW-4- hom-lysC-brnFE,there is following genetics characteristics in it: express translocator simultaneously brnFE, express aspartokinase enzyme fragment lysCwith homoserine dehydrogenase fragment hom, on genome, lack simultaneously thrBwith mcbRgene.

The method of producing METHIONINE with described bacterial strain CCTCC M 2014232: with QW102/pJYW-4- hom ^m -lysC ^m -brnFEfor producing bacterium, choose single bacterium colony of activating in 50mL/500mL seed culture medium, 30 ℃, 200 rpm are cultivated 18 h; Then by initial OD ₅₆₂be 1 to be forwarded to 1.2L/3L fermentor tank; By Feeding ammonia water, regulate pH to 7.0, by associated mixing speed and aeration rate, controlling dissolved oxygen is 30%, by adding 50% glucose, controls residual sugar more than 20g/L; Every 4 h sampling and measuring cell concentrations, residual sugar and aminoacids content; Detected result demonstration, when fermentation 48 h, methionine(Met) output reaches the highest, and production peak is 3.1 g/L;

Seed culture medium: 25 g/L glucose, 20 g/L corn steep liquors, 1 g/L KH ₂pO ₄, 0.5 g/L MgSO ₄, 1.25 g/L urea;

Fermention medium: 100 g/L glucose, 20 g/L corn steep liquors, 20 g/L (NH ₄) ₂sO ₄, 1 g/L KH ₂pO ₄, 0.5 g/L MgSO ₄, 0.01 g/L MnSO ₄, 0.01 g/L FeSO ₄, 1 mg/L VB1,6 mg/L VB6,0.1 mg/L VH, 0.2 g/L VB12.

Beneficial effect of the present invention: the recombinant bacterial strain of high yield methionine(Met) provided by the invention ( c.glutamicum subspecies lactofermentum) QW102/pJYW-4- hom ^m -lysC ^m -brnFE,be preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC M 2014232.There is following genetics characteristics in this recombinant bacterial strain: expresses translocator brnFE, express aspartokinase enzyme fragment lysCwith homoserine dehydrogenase fragment hom, on genome, lack simultaneously thrBwith mcbRgene.The present invention, by engineered method, obtains the recombinant bacterial strain that a plant height produces methionine(Met); Method with this recombinant bacterial strain high yield METHIONINE is also provided.

Biological material specimens preservation a: plant height produces the Corynebacterium glutamicum of METHIONINE, and its Classification And Nomenclature is Corynebacterium glutamicum QW102/pJYW-4- hom ^m -lysC ^m -brnFECorynebacteriumglutamicumqW102/pJYW-4- hom ^m -lysC ^m -brnFE,be preserved in Chinese Typical Representative culture collection center, address: Wuhan, China, Wuhan University, preservation date on May 28th, 2014, preserving number is CCTCC NO:M 2014232.

Accompanying drawing explanation

Fig. 1, construction recombination plasmid collection of illustrative plates of the present invention.

Embodiment

Carry out by the following examples further to illustrate the present invention, the following example is for illustration purpose but not for limiting the scope of the invention.

In following embodiment of the present invention, plasmid extraction kit used, genome extraction test kit, glue reclaim test kit, e.colistraindh5α is purchased from Tian Gen biotech firm, restriction enzyme used, T4 DNA ligase, PCR reagent etc. are all purchased from TaKaRa biotech firm, and the primer is synthesized by JaRa biotech firm.

The structure of embodiment 1, restructuring Corynebacterium glutamicum

In the present embodiment, first build respectively the plasmid that knocks out and express genes involved for Corynebacterium glutamicum, then the plasmid having built has been transformed into respectively in corresponding Corynebacterium glutamicum, thereby realized knocking out and overexpression of genes involved.

1.1 homoserine kinase encoding genes thrBwith aporepressor McbR encoding gene mcbRknock out the structure of plasmid

1.1.1 knock out gene upstream and downstream homology arm mcbRU, mcbRD, thrBU, thrBD and loxp -kan -the amplification of loxp fragment

In the present embodiment, first designed the primer for increasing, extracting is for genome and the plasmid of amplification template, then by the PCR genes involved fragment that increased respectively.Specific as follows:

According to Genebank, report thrB, mcbRgene order, the fragment of each 1kb of Select gene upstream and downstream left and right size is as knocking out gene upstream and downstream homology arm, thrBU wherein, thrBD is used for thrBknocking out of gene, mcbRU, mcbRD is used for mcbRknocking out of gene.Following 8 primers of design, for cloning mcbRU, mcbRD, thrBU, thrBD sequence:

mcbRU-F ( PstI)： GCT CTGCAGT GAAGCTCGTG GCGCCGAACT

mcbRU-R ( BamHI)： TA GGATCCAT AGACAAACCG GTTCGTATAGT

mcbRD-F ( XbaI)： GCA TCTAGAT CTTGTTCGCG ATTTCTTTG

mcbRD-R ( XhoI)： GC CTCGAGGT GTGTTTTTAG ATCTTCGGTT

thrBU-F ( XhoI)： AT CTCGAGGG CAAGTCTGTT GTTA

thrBU-R ( XbaI)： ACC TCTAGAT TAGTCCCTTT CGAG

thrBD-F ( BamHI)： AC GGATCCCA GTCAAGGTTG AAGTT

thrBD-R ( PstI)： ATC CTGCAGC TACGTGGTCT ATCGC

McbRU-F and the mcbRU-R mcbRU fragment that is used for increasing wherein, mcbRD-F and the mcbRD-R mcbRD fragment that is used for increasing, thrBU-F and the thrBU-R thrBU fragment that is used for increasing, thrBD-F and the thrBD-R thrBD fragment that is used for increasing.Primer restriction enzyme site indicates with underscore, and the restriction enzyme that restriction enzyme site is corresponding is indicated in bracket.

The primer that is designed for amplification loxp-kan-loxp fragment according to plasmid pDTW-202, primer sequence is as follows:

kan-loxp-F (BamHI): AT GGATCCAA TACGACTCAC TATAGGGCG

kan-loxp-R (XbaI): ACC TCTAGAG CGCAATTAAC CCTCACTAAA G

Primer restriction enzyme site indicates with underscore, and the restriction enzyme that restriction enzyme site is corresponding is indicated in bracket.

Extracting obtains paddy rod ATCC13032 genome and pDTW-202 plasmid.

The paddy rod ATCC13032 genome of take is template, with mcbRU-F and mcbRU-R, and mcbRD-F and mcbRD-R, thrBU-F and thrBU-R, thrBD-F and thrBD-R are that primer pair carries out pcr amplification, obtain mcbRU, mcbRD, thrBU, thrBD fragment.Take pDTW-202 plasmid as template, take kan-loxp-F and kan-loxp-R as primer pair carries out pcr amplification, obtain loxp-kan-loxp fragment.PCR product is carried out respectively to detected through gel electrophoresis, and the product size of acquisition meets expection size.

Use glue to reclaim the mcbRU that test kit obtains purifying, mcbRD, thrBU, thrBD, loxp-kan-loxp fragment.

1.1.2 knock out the structure of plasmid pWTQ1 and pWTQ2

The fragment that purifying is crossed enzyme under 37 ℃ of conditions is cut and is processed 3h.Comprise pstIwith bamHIdouble digestion fragment mcbRU, xbaIwith xhoIdouble digestion fragment mcbRD, pstIwith bamHIdouble digestion fragment thrBD, xbaIwith xhoIdouble digestion fragment thrBU, bamHIwith xbaIdouble digestion fragment loxp-kan-loxp.Use the fragment after DNA product purification test kit is cut enzyme to carry out purifying.

With pstIwith xhoIdouble digestion plasmid pBluscriptII SK (+), obtains the linearization plasmid with sticky end.Use the plasmid after DNA product purification test kit is cut enzyme to carry out purifying.

Enzyme is cut to the plasmid that purifying crosses and use T4 DNA ligase to connect, linked system is as follows:

PBluscriptII SK (+) plasmid 50-100 ng

3 times of pBluscriptII SK of junction fragment (+) plasmid mol ratio

T4DNA ligase enzyme 1 μ L

T4 Buffer 2 μL

DdH ₂o adds to 20 μ L

Connect temperature: 16 ℃, tie-time 3-10 h

Utilize above linked system, junction fragment mcbRU, mcbRD, loxp-kan-loxp and plasmid pBluscriptII SK (+), to build mcbRknock out plasmid; Junction fragment thrBU, thrBD, loxp-kan-loxp and plasmid pBluscriptII SK (+), to build thrBknock out plasmid.

Above linked system is transformed to bacillus coli DH 5 alpha, and be coated with the LB flat board of the kantlex that contains 30mg/L, after cultivation, obtain transformant and identify.Final acquisition mcbRknock out plasmid, called after pWTQ1; Obtain thrBknock out plasmid called after pWTQ2 (accompanying drawing 1).

1.2 homoserine dehydrogenases, the structure of E.C. 2.7.2.4. and translocator BrnFE expression vector

1.2.1 Fixedpoint mutation modified homoserine dehydrogenase and E.C. 2.7.2.4.

In the present embodiment, select following mutational site to remove the feedback repression effect of homoserine dehydrogenase and E.C. 2.7.2.4..Wherein the Gly of the 377th of homoserine dehydrogenase is sported Glu, and the 311st Thr of E.C. 2.7.2.4. sported Ile.

According to genebank, report hom(SEQ ID NO.1) lysC(SEQ ID NO.2) sequence, following four primers of design are for clone hom, lysCsequence:

hom-F: GCGAATTCAT GACCTCAGCA TCT

hom-R: GATGAGCTCT TAGTCCCTTT CGAG

lysC-F: GCTGAGCTCG TGGCCCTGGT CGTAC

lysC-R: GCCTCGAGTT AGCGTCCGGT

Wherein hom-F and hom-R are for amplification homgene coding region, lysC-F and lysC-R are for amplification lysCcoding region.

Extracting obtains ATCC13032 genome.

Take ATCC13032 genome as template, and respectively with hom-F and hom-R, lysC-F and lysC-R are that primer pair carries out pcr amplification, obtain homwith lysCgene fragment.

Use the recovery of glue recovery test kit to obtain homwith lysCgene fragment.Fragment after recovery connects pMD18-T carrier, obtains respectively pMD18-T- homand pMD18-T- lysCplasmid.According to the mutational site design rite-directed mutagenesis primer of gene, sequence is as follows:

homint-F： TGGAAGATCG CGTGGG AGTT TTGGCTGAAT TGGC

homint-R： GCCAATTCAG CCAAAAC TCC CCACGCGATC TTCCA

lysCint-F： GCACCACCGA CATCA TCTTC ACCTGCCCTC

lysCint-R： GAGGGCAGGT GAAG ATGATG TCGGTGGTGC

Homint-F and the homint-R plasmid pMD18-T-that is used for increasing wherein hom, lysCint-F and the lysCint-R plasmid pMD18-T-that is used for increasing lysC.What in sequence, underscore indicated is the mutational site of gene.

With plasmid pMD18-T- homand pMD18-T- lysCfor template, respectively with homint-F and homint-R, lysCint-F and lysCint-R are that primer pair increases to whole plasmid.Amplified production is used dpnIprocess 1h for 37 ℃, the product of acquisition transforms bacillus coli DH 5 alpha, and is coated with the LB flat board of the penbritin that contains 30mg/L, obtains transformant and identify after cultivation.According to qualification result, finally obtain two plasmid pMD18-T- hom ^m and pMD18-T- lysC ^m .

Plasmid pMD18-T- hom ^m and pMD18-T- lysC ^m through sequence verification, contain respectively the mutational site of expection.

1.2.2 feed back the structure of resistance homoserine dehydrogenase and E.C. 2.7.2.4. and translocator BrnFE expression vector

According to genebank, report hom, lysC, brnFEgene order, following six primers of design are for clone hom ^m , lysC ^m , brnFE,( brnF; BrnEgene order is SEQ ID NO.3, SEQ ID NO.4);

hom-SD-F ( EcoRI)： GC GAATTCAG AAGGAGACTA GTAATGACCT CAGCATCT

hom-R ( SacI)： GAT GAGCTCT TAGTCCCTTT CGAG

lysC-SD-F ( SacI)： GCT GAGCTCA GAAGGAGAGA TGTTAGTGGC CCTGGTCGTA C

lysC-R ( XhoI)： GC CTCGAGTT AGCGTCCGGT

brnFE-SD-F ( BamHI)： TAA GGATCCA GAAGGAGATA TACCGTGCAA AAAACGCAAG

brnFE-R ( XbaI)： TAC TCTAGAT TAGAAAAGAT TCAC

Wherein hom-SD-F and hom-R are for amplification homgene coding region; LysC-SD-F and lysC-R are for amplification lysCgene coding region; BrnFE-SD-F and brnFE-R are for amplification brnFEgene coding region.Primer restriction enzyme site indicates with underscore, and the restriction enzyme that restriction enzyme site is corresponding is indicated in bracket.

With plasmid pMD18-T- hom ^m and pMD18-T- lysC ^m for template, respectively with primer hom-SD-F and hom-R, and primer lysC-SD-F and lysC-R be primer pair, carries out pcr amplification, obtains hom ^m with lysC ^m fragment.

Take ATCC13032 genome as template, take primer brnFE-SD-F and brnFE-R as primer pair carries out pcr amplification, obtain gene brnFEcoding region sequence.

With glue, reclaim test kit and reclaim acquisition hom ^m , lysC ^m with brnFEfragment.

Extracting obtains large intestine to paddy rod shuttle expression plasmid pJYW-4.

The fragment that purifying is crossed and plasmid enzyme under 37 ℃ of conditions is cut and is processed 3h, further uses glue to reclaim test kit and reclaims the fragment obtaining with sticky end hom ^m , lysC ^m with brnFE, and linearization plasmid pJYW-4.The plasmid of acquisition is connected with fragment, successfully obtains expression plasmid pJYW-4- hom ^m , pJYW-4- lysC ^m , pJYW-4- brnFE, pJYW-4- hom ^m - lysC ^m (accompanying drawing 1).

According to genebank, report hom, lysCgene order, following two primers of design are for clone hom ^m - lysC ^m gene fragment:

hom-lysC-F： TATGGGCCCA GAAGGAGACT AGTAATGACC TCAGCATCT

hom-lysC-R: TAGGGATCCT TAGCGTCCGG TGCCTG

With plasmid pJYW-4- hom ^m - lysC ^m for template, primer hom-lysC-F and hom-lysC-R are that primer pair carries out pcr amplification, obtain hom ^m - lysC ^m fragment.

With glue, reclaim test kit and reclaim acquisition hom ^m - lysC ^m fragment.

Purifying is crossed hom ^m - lysC ^m fragment and plasmid pJYW-4- brnFEunder 37 ℃ of conditions, enzyme is cut and is processed 3h, further uses glue to reclaim test kit and reclaims and obtain with sticky end hom ^m - lysC ^m fragment and linearization plasmid pJYW-4- brnFE.The plasmid of acquisition is connected with fragment, successfully obtains expression plasmid pJYW-4- hom ^m - lysC ^m - brnFE(accompanying drawing 1).

The structure of recombinant bacterial strain in 1.3 paddy rods

1.3.1 the structure of homoserine kinase and aporepressor McbR deletion mycopremna

Take Corynebacterium glutamicum ATCC13032 as starting strain, and electricity turns and knocks out plasmid pWTQ1 to ATCC13032 and lack to build homoserine kinase deletion mycopremna ATCC13032 thrB.Concrete operations flow process is as follows:

(1) from bacillus coli DH 5 alpha, extract plasmid pWTQ1, standby with the water elution of pH8.0.

(2) electric Pignus pignoris grain pWTQ1 is to paddy rod ATCC13032, and the LBHIS flat board that the coating of bacterium liquid contains kantlex is cultivated, and screening obtains correct transformant.

(3) electric Pignus pignoris grain pDTW-109 is to the middle transformant obtaining of step (2), and the LBHIS that coating contains paraxin is dull and stereotyped, and 25 ℃ are screened.The correct transformant called after WTQ101 obtaining.

Adopt aforesaid method, take WTQ101 as starting strain, electricity turns and knocks out plasmid pWTQ2 to WTQ101, obtains homoserine kinase and the common deletion mycopremna of McbR, called after WTQ102.

1.3.2 feed back resistance homoserine dehydrogenase, the heterogenous expression of E.C. 2.7.2.4. and translocator BrnFE

Extracting plasmid pJYW-4- hom ^m - lysC ^m and pJYW-4- hom ^m - lysC ^m - brnFE, electricity turns bacterial strain WTQ101 and WTQ102 respectively.The LBHIS that coating contains kantlex is dull and stereotyped, and screening transformant extracting plasmid carries out single endonuclease digestion checking, obtains following correct transformant:

WTQ101/pJYW-4- hom ^m - lysC ^m ；

WTQ102/pJYW-4- hom ^m - lysC ^m ；

WTQ102/pJYW-4- hom ^m - lysC ^m - brnFE。

Embodiment 2, restructuring Corynebacterium glutamicum shaking flask level are produced methionine(Met)

The culture medium prescription using in this example is as follows:

Seed culture medium: 25 g/L glucose, 20 g/L corn steep liquors, 1 g/L KH ₂pO ₄, 0.5 g/L MgSO ₄, 1.25 g/L urea.

Fermention medium: 100 g/L glucose, 20 g/L corn steep liquors, 20 g/L (NH ₄) ₂sO ₄, 1 g/L KH ₂pO ₄, 0.5 g/L MgSO ₄, 0.01 g/L MnSO ₄, 0.01 g/L FeSO ₄, 1 mg/L VB1,6 mg/L VB6,0.1 mg/L VH, 0.2 g/L VB12.

Choose single bacterium colony of activating in 30mL/250mL seed culture medium, 30 ℃, 200 rpm are cultivated 18 h.Then by initial OD ₅₆₂be 1 to be forwarded in 50mL/500mL fermention medium, wherein fermention medium adds 20 g/L calcium carbonate with balance pH.30 ℃, 200 rpm are cultivated 72 h.Sampling and measuring related amino acid output.

In the present embodiment, fermented liquid aminoacids content adopts HPLC to measure, and concrete grammar is as follows:

Get fermented sample 1mL, centrifugal 5 min of 1,2000 rpm, get supernatant and in a clean EP pipe, with 5% trichoroacetic acid(TCA), dilute 20 times, 4 ℃ of standing 6-9 h.Sample after standing is in centrifugal 10 min of 1,2000 rpm, and 0.22 nm water pin type filter is filtered to upper machine testing in amino acid sample bottle.

Each mutant strain methionine(Met) yield comparison is as follows:

Bacterial strain	Methionine(Met) output (g/L)
		ATCC13032	0
WTQ101	0.37
		WTQ102	0.6
WTQ101/pJYW-4- hom m - lysC m	0.56
		WTQ102/pJYW-4- hom m - lysC m	1.29
WTQ102/pJYW-4- hom m - lysC m - brnFE	1.79

Embodiment 3, best mutant strain WTQ102/pJYW-4- hom ^m - lysC ^m - brnFEin fermentor tank level, produce methionine(Met)

The seed culture medium and the fermention medium that in the present embodiment, use are in the same manner as in Example 2.

Choose single bacterium colony of activating in 50mL/500mL seed culture medium, 30 ℃, 200 rpm are cultivated 18 h.Then by initial OD ₅₆₂be 1 to be forwarded to 1.2L/3L fermentor tank.By Feeding ammonia water, regulate pH to 7.0, by associated mixing speed and aeration rate, controlling dissolved oxygen is 30%, by adding 50% glucose, controls residual sugar more than 20g/L.Every 4 h sampling and measuring cell concentrations, residual sugar and aminoacids content.Wherein amino acid detection method is identical with embodiment 2.Detected result demonstration, when fermentation 48 h, methionine(Met) output reaches the highest, and production peak is 3.1 g/L.

SEQ ID NO.1

According to genebank, report homsequence:

gtggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga 60

aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc 120

tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt 180

ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc 240

gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct 300

ggtgtgctca ccaccgagcg ccacggaaac gcacgcattg ttgatgtcac tccaggtcgt 360

gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat 420

aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg 480

ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat 540

accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa 600

atgctggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct 660

cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg 720

attgccggct ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc 780

gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg 840

aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc 900

tcttctgtag aagacggcac caccgacacc atcttcacct gccctcgttc cgacggccgc 960

cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac 1020

gacgaccagg tcggacaaag tctccctcgt gggtgctggc atgaagtctc acccaggtgt 1080

taccgcagag ttcatggaag ctctgcgcga tgtcaacgtg aacatcgaat tgatttccac 1140

ctctgagatt cgtatttccg tgctgatccg tgaagatgat ctggatgctg ctgcacgtgc 1200

attgcatgag cagttccagc tgggcggcga agacgaagcc gtcgtttatg caggcaccgg 1260

acgctaa 1267

SEQ ID NO.2

According to genebank, report lysCsequence:

atgacctcag catctgcccc aagctttaac cccggcaagg gtcccggctc agcagtcgga 60

attgcccttt taggattcgg aacagtcggc actgaggtga tgcgtctgat gaccgagtac 120

ggtgatgaac ttgcgcaccg cattggtggc ccactggagg ttcgtggcat tgctgtttct 180

gatatctcaa agccacgtga aggcgttgca cctgagctgc tcactgagga cgcttttgca 240

ctcatcgagc gcgaggatgt tgacatcgtc gttgaggtta tcggcggcat tgagtaccca 300

cgtgaggtag ttctcgcagc tctgaaggcc ggcaagtctg ttgttaccgc caataaggct 360

cttgttgcag ctcactctgc tgagcttgct gatgcagcgg aagccgcaaa cgttgacctg 420

tacttcgagg ctgctgttgc aggcgcaatt ccagtggttg gcccactgcg tcgctccctg 480

gctggcgatc agatccagtc tgtgatgggc atcgttaacg gcaccaccaa cttcatcttg 540

gacgccatgg attccaccgg cgctgactat gcagattctt tggctgaggc aactcgtttg 600

ggttacgccg aagctgatcc aactgcagac gtcgaaggcc atgacgccgc atccaaggct 660

gcaattttgg catccatcgc tttccacacc cgtgttaccg cggatgatgt gtactgcgaa 720

ggtatcagca acatcagcgc tgccgacatt gaggcagcac agcaggcagg ccacaccatc 780

aagttgttgg ccatctgtga gaagttcacc aacaaggaag gaaagtcggc tatttctgct 840

cgcgtgcacc cgactctatt acctgtgtcc cacccactgg cgtcggtaaa caagtccttt 900

aatgcaatct ttgttgaagc agaagcagct ggtcgcctga tgttctacgg aaacggtgca 960

ggtggcgcgc caaccgcgtc tgctgtgctt ggcgacgtcg ttggtgccgc acgaaacaag 1020

gtgcacggtg gccgtgctcc aggtgagtcc acctacgcta acctgccgat cgctgatttc 1080

ggtgagacca ccactcgtta ccacctcgac atggatgtgg aagatcgcgt gggggttttg 1140

gctgaattgg ctagcctgtt ctctgagcaa ggaatctccc tgcgtacaat ccgacaggaa 1200

gagcgcgatg atgatgcacg tctgatcgtg gtcacccact ctgcgctgga atctgatctt 1260

tcccgcaccg ttgaactgct gaaggctaag cctgttgtta aggcaatcaa cagtgtgatc 1320

cgcctcgaaa gggactaa 1338

SEQ ID NO.3

brnFgene order

gtgcaaaaaa cgcaagagat tcattcaagc ctggaggtgt cgccatccaa ggcagccctg 60

gaaccagatg ataaaggtta tcggcgctac gaaatcgcgc aaggtctaaa aacctccctt 120

gctgcaggtt tgggcatgta cccgattggt attgcgtttg gtctcttggt tattcaatac 180

ggctacgaat ggtgggcagc cccactgttt tccggcctga ttttcgcggg ctccaccgaa 240

atgctggtca tcgccctcgt tgtgggcgca gcgcccctgg gcgccatcgc gctcaccaca 300

ttgctggtga acttccgcca cgtattctat gcgttttcat tcccgctgca tgtggtcaaa 360

aaccccattg cccgtttcta ttcggttttc gcgcttatcg acgaagccta cgcagtcact 420

gcggccaggc ccgcaggctg gtcggcgtgg cgacttatct caatgcaaat agcgtttcac 480

tcctactggg tattcggcgg tctcaccgga gtggcgatcg cagagttgat tccttttgaa 540

attaagggcc tcgagttcgc cctttgctct ctctttgtca cgctgacttt ggattcctgc 600

cgaacgaaaa agcagatccc ttctctgctg ctcgcaggtt tgagcttcac cattgctctt 660

gtggtaattc caggtcaggc cctatttgcg gcgctgctga tcttcttggg tctgttgacc 720

atccggtact tcttcttggg aaaggctgct aa 752

SEQ ID NO.4

brnEgene order

atgacaactg atttctcctg tattctcctt gttgtcgcag tatgtgcagt cattactttt 60

gcgctccggg cggttccgtt cttaatcctt aagcccctac gtgaatcaca atttgtgggc 120

aaaatggcga tgtggatgcc agcaggaatc cttgccattt tgaccgcatc aacgtttcgc 180

agcaatgcga tagatctgaa gactctaacc tttggtctca ttgccgttgc gattacagtg 240

gtggcgcatc ttcttggcgg tcgacgcacc ttgttgagcg ttggcgctgg caccatcgtt 300

tttgttggac tggtgaatct tttctaa 327

Claims (4)

1. a plant height produces the Corynebacterium glutamicum of METHIONINE, it is characterized in that, described strain classification called after Corynebacterium glutamicum ( c.glutamicum subspecies lactofermentum) QW102/pJYW-4- hom ^m -lysC ^m -brnFE,be preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC M 2014232.

2. bacterial strain QW102/pJYW-4-described in claim 1 hom ^m -lysC ^m -brnFE,it is characterized in that it exists following genetics characteristics simultaneously: express translocator brnFE, express aspartokinase enzyme fragment lysCwith homoserine dehydrogenase fragment hom, on genome, lack simultaneously thrBwith mcbRgene.

3. the application of the Corynebacterium glutamicum of high yield METHIONINE claimed in claim 1, is characterized in that producing methionine(Met) for shaking flask level and fermentor tank level.

4. the method for producing METHIONINE with bacterial strain CCTCC M described in claim 1 2014232, is characterized in that: with QW102/pJYW-4- hom ^m -lysC ^m -brnFEfor producing bacterium, choose single bacterium colony of activating in 50mL/500mL seed culture medium, 30 ℃, 200 rpm are cultivated 18 h; Then by initial OD ₅₆₂be 1 to be forwarded to 1.2L/3L fermentor tank; By Feeding ammonia water, regulate pH to 7.0, by associated mixing speed and aeration rate, controlling dissolved oxygen is 30%, by adding 50% glucose, controls residual sugar more than 20g/L; Every 4 h sampling and measuring cell concentrations, residual sugar and aminoacids content; Detected result demonstration, when fermentation 48 h, methionine(Met) output reaches the highest, and production peak is 3.1 g/L;

Seed culture medium: 25 g/L glucose, 20 g/L corn steep liquors, 1 g/L KH ₂pO ₄, 0.5 g/L MgSO ₄, 1.25 g/L urea;

Fermention medium: 100 g/L glucose, 20 g/L corn steep liquors, 20 g/L (NH ₄) ₂sO ₄, 1 g/L KH ₂pO ₄, 0.5 g/L MgSO ₄, 0.01 g/L MnSO ₄, 0.01 g/L FeSO ₄, 1 mg/L VB1,6 mg/L VB6,0.1 mg/L VH, 0.2 g/L VB12.