CN108949661A - A kind of production O- succinyl-L- homoserine recombination bacillus coli and its application - Google Patents

Abstract

A kind of method the invention discloses recombination bacillus coli being transformed by metabolic engineering means and its for producing O- succinyl-L- homoserine bacterial strain.The present invention passes through first by interrupting metI to realize the partial inactivation of L-Methionine input system MetD; and yjeH gene is overexpressed on plasmid to promote the excretion of L-Methionine intracellular, so that the homoserine O-succinyl transferase that the metA gene of bacterial strain encodes keeps high vigor and stability.Inhibiting effect is checked secondly by negative regulation transcription factor MetJ is knocked out to release it to related gene (thrA, metL, lysC, asd, metA etc.) in O- succinyl-L- homoserine anabolism path.Finally by use Ptrc promoter replacement metabolic pathway in metL, thrA and sucA gene original position promoter sequence, and blocked O- succinyl-L- homoserine metabolism outlet in metB gene.By the combination of the above Reconstruc-tion policy, the coli strain of high yield O- succinyl-L- homoserine is obtained.

Description

A kind of production O- succinyl-L- homoserine recombination bacillus coli and its application

Technical field

The present invention relates to a kind of production O- succinyl-L- homoserine recombination bacillus colis and its fermentation to produce O- succinyl-L- The application of homoserine.

Background technique

O- succinyl-L- homoserine serves not only as the important intermediate of synthetic methionine in microbial body, and in industry Very important status is occupied during production C4 compound.The biology of O- succinyl-L- homoserine closes in Escherichia coli At being catalyzed and synthesized by the homoserine transacetylase specificity of metA coding, including the activation of homoserine-γ-hydroxyl Acyl group process is provided with succinyl-CoA.During synthesizing O- succinyl-L- homoserine, metA gene is by L-Methionine Feedback inhibition, and L-Methionine is used as amino acid necessary to microorganism growth and development, therefore obtaining can be with feedback-inhibition resistance Mutated gene for improve O- succinyl-L- homoserine production capacity be of great significance.In reported patent (US Patent 7,851,180) in had to metA gene carry out feedback-inhibition resistance research, to O- succinyl-L- homoserine The promotion of yield plays certain effect.L-Methionine is the essential amino acid of biological growth, is distributed on biological cell film With L-Methionine transport associated protein, there are transportation system in two class specificity methionine of MetD and MetP, MetD in E.coli High to the affinity of methionine, MetP affinity is relatively low.And L-Methionine auxotrophic strain can be activated to fortune intracellular The transport protein of defeated L-Methionine increases L-Methionine content intracellular, in order to reduce the content of L-Methionine intracellular to Mitigating the feedback inhibition to metA gene, knockout metI transports the partial inactivation of system MetD to realize in L-Methionine, MetD operon includes metN, metI and metQ, is separately encoded ATPase, the methionine permeases, egg of abc transport protein component Propylhomoserin binding protein.

The cystathionine Gamma synzyme encoded in microbial body by metB gene can decompose O- succinyl-L- homoserine Cysteine is generated to need to knock out metB gene to enable O- succinyl-L- homoserine largely to accumulate.

Aspartic acid be E coli synthesis O- succinyl-L- homoserine unique precursor substance, while be also threonine, The synthesis precursor of isoleucine, lysine and branched-chain amino acid.Biosynthesis from aspartic acid to homoserine need by Four-step reaction, wherein the homoserine dehydrogenase of thrA and metL coding and aspartokinase II belong to bifunctional enzyme, in addition to With aspartokinase enzymatic activity, while there are also homoserine dehydrogenase activity, catalysis aspartate-semialdehyde is reduced into Kosé ammonia Acid.The gene transcription levels such as thrA, metL, lysC, asd, metA are all by the adjusting of negative regulation transcription factor MetJ, therefore, need MetJ gene is knocked out.The biosynthesis of L-threonine is using L- homoserine as precursor substance, by thrB and thrC The homoserine kinase and threonine synthetase of gene coding are catalyzed to obtain, and O- succinyl-L- homoserine and L-threonine close Precursor substance L- homoserine is competed with one another at metabolism, it is therefore necessary to block or reduce the anabolism flux of L-threonine. In addition, in O- succinyl-L- homoserine biosynthetic process, substrate-level intracellular also will affect its yield, and amber Acyl-CoA is a wherein important substrate, it is therefore necessary to increase containing for succinyl-CoA intracellular by metabolic engineering means Amount.

Summary of the invention

The purpose of the present invention is Escherichia coli are transformed to obtain high yield O- succinyl-L- Kosé ammonia by metabolic engineering means The recombinant escherichia coli strain of acid.

The technical solution adopted by the present invention is that:

The present invention provides a kind of recombination bacillus coli for producing O- succinyl-L- homoserine, and the recombination bacillus coli is MetI gene, the metJ gene for encoding negative regulation repressor, coding of L-Methionine transport protein will be encoded in Escherichia coli After the metB gene of cystathionine Gamma synzyme, the thrB gene of encoded homoserine kinase successively knock out, then respectively will coding height The thrA gene of serine dehydrogenase I, the metL gene of encoded homoserine dehydrogenase II and coding α-ketoglutaric acid decarboxylase The promoter of sucA gene replace with Ptrc promoter, finally import encoded homoserine O- succinyl transferase again The yjeH gene of metA mutated gene and coding L-Methionine outward transport albumen, the metA mutated gene is to encode metA gene The 64th glutamic acid of albumen (shown in SEQ ID NO.16) replaces with glutamine acquisition.

Further, the P_trcPromoter nucleotides sequence is classified as shown in SEQ ID NO.21.

Further, the metI gene nucleotide series are shown in SEQ ID NO.1, metJ gene nucleotide series are Shown in SEQ ID NO.3, metB gene nucleotide series are shown in SEQ ID NO.5, thrB gene nucleotide series are SEQ Shown in ID NO.7.

Further, the yjeH gene nucleotide series are shown in SEQ ID NO.17, metA gene nucleotide series are Shown in SEQ ID NO.15, shown in metA gene coded protein amino acid sequence SEQ ID NO.16.

The present invention also provides a kind of recombination bacillus colis for producing O- succinyl-L- homoserine to produce O- amber in fermentation Application in acyl-L- homoserine, the application are that the recombination bacillus coli is seeded to fermentation medium, 30 DEG C, Fermented and cultured under the conditions of 180-200rpm, culture solution is isolated and purified, and obtains O- succinyl-L- homoserine.

Further, the fermentation medium final concentration composition are as follows: glucose 40g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L, yeast powder 4g/L, calcium carbonate 30g/L (for adjusting pH), L-threonine 0.2g/L, vitamin B₁ 0.0001g/L、 MgSO₄ 2g/L、FeSO₄ 0.005g/L、MnSO₄ 0.005g/L、ZnSO₄0.005g/L, solvent are deionized water, pH value 6.8。

Further, slant activation and seed culture are first carried out before the fermented and cultured, by seed liquor with volumetric concentration 5% Inoculum concentration is seeded to fermentation medium, the slant activation method are as follows: on LB plate by recombination bacillus coli inoculation, at 37 DEG C Overnight incubation obtains inclined-plane thalline；The seed culture method are as follows: picking inclined-plane thalline single colonie is seeded in LB culture medium, Overnight incubation under conditions of 37 DEG C, 200rpm obtains seed liquor.

The fermented and cultured carries out in the fermenter, controls concentration of glucose 2- in fermentor by addition supplemented medium 10g/L；Fermentation condition is DO level 30%, and speed of agitator 200-600rpm, Ventilation Rate is controlled in 1-2vvm；Fermentation process Middle control cultivation temperature adjusts range of the pH 6.8~7.0 at 30 DEG C and with 50% ammonium hydroxide.Fermentation cylinder for fermentation culture medium group At: glucose 40g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L, yeast powder 4g/L, L-threonine 0.2g/L, L-Methionine 0.2g/L, l-Isoleucine 0.2g/L, MgSO₄ 2g/L、FeSO₄ 0.005g/L、MnSO₄ 0.005g/L、ZnSO₄ 0.005g/ L, vitamin B₁0.0001g/L, solvent are deionized water, pH value 6.8；Supplemented medium composition: glucose 500g/L, phosphoric acid Potassium dihydrogen 12.5g/L, L-threonine 2g/L, L-Methionine 2g/L, l-Isoleucine 2g/L, solvent are deionized water.

The present invention produces the recombination bacillus coli of O- succinyl-L- homoserine, by knocking out negative regulation transcription factor MetJ To release it to related gene (thrA, metL, lysC, asd, metA in O- succinyl-L- homoserine anabolism path Deng) check inhibiting effect.The negative regulation transcription factor gene metJ in Ecoli W3110 has been knocked out first, and has been knocked out MetI gene transports the partial inactivation of albumen MetD to realize in l-methionine, to reduce methionine amount intracellular, and then can be with Methionine is reduced to the substrate inhibition of homoserine O-succinyl transferase (MetA).By knocking out thrB gene, with The precursor homoserine of O- succinyl-L- homoserine is reduced to the metabolism outlet of branch amino acid.At the same time, metB is knocked out Gene realizes the degradation for blocking product O- succinyl-L- homoserine.With strong promoter P_trcIt is in situ to replace metL and thrA gene Promoter sequence, to improve the expression of aspartokinase II and homoserine dehydrogenase, and with strong promoter P_trcReplacement SucA gene original position promoter sequence is to improve the content of succinyl-CoA intracellular.YjeH gene is connected on pTrc99A plasmid, To promote the excretion of L-Methionine intracellular, so that the homoserine O-succinyl transferase that the metA gene of bacterial strain encodes is kept High vigor and stability, and inducing expression is carried out by changing to turn to import the connection plasmid in above-mentioned improved Escherichia coli, So that L-Methionine intracellular is only for that will not turn the homoserine O-succinyl that metA gene encodes needed for microorganism growth It moves enzyme and generates large effect.The homoserine O-succinyl transferase and egg ammonia that desensitize to methionine are overexpressed by plasmid Acid outward transport albumen, realizes homoserine O-succinyl transferase intracellular in the case where being influenced minimum situation by L-Methionine by Kosé ammonia Acid is converted into O- succinyl-L- homoserine and secretes to extracellular.

Term " feedback inhibition " of the present invention refers to homoserine O-succinyl transferase active by the suppression of L-Methionine System.The present invention relates to the recombinant vectors containing the L-Methionine transportation system YjeH encoding gene.The recombinant vector include with The polynucleotides that the control sequence for being suitble to guidance to express in host cell is operably connected.It is preferred that the expression vector is pTrc99A.Term " enhancing " refers to increasing the activity of the enzyme by corresponding polynucleotide encoding.Table can be crossed by gene Reach or replacement gene group on the gene expression regulation sequence (promoter replacement etc.).

Carrier used in the present invention can not be limited specifically, if carrier be in host it is reproducible, can make With any carrier as known in the art.

The expression of polypeptide can will include the recombinant vector for the gene for encoding the polypeptide by way of conversion in the present invention Or in the insertion chromosome of the polynucleotides by that will encode the polypeptide, but method is without being limited thereto.

Compared with prior art, the beneficial effects are mainly reflected as follows:

The present invention provides a kind of side of Escherichia coli high yield O- succinyl-L- homoserine using metabolic engineering Method.By improved Escherichia coli O- amber preferably can be carried out using carbon source materials such as glucose compared to wild type The production of acyl-L- homoserine, and its yield is increased to 24.25g/L from 0.14g/L.The L- egg ammonia that the present invention is further transformed The substrate that acid transporter system can mitigate L-Methionine reaches high yield O- succinyl-L- Kosé ammonia to the feedback inhibition of MetA Acid, and with precursor production L-Methionine and other industrial important C4 compounds.

Detailed description of the invention

PTrc9999A-metA11 construction of recombinant vector process and map in Fig. 1 embodiment 3.

PA11y construction of recombinant vector process and map in Fig. 2 embodiment 4.

The structural formula of OSH in Fig. 3 embodiment 5.

High performance liquid chromatography (HPLC) detects OSH in Fig. 4 embodiment 5.

Specific embodiment

Below with reference to specific example, the present invention will be further described in detail, but the present invention is not limited to following implementations Example.

Experimental method in following embodiments is unless otherwise specified conventional method.

Test material as used in the following examples is unless otherwise specified conventional biochemical reagent.

LB culture medium composition: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, solvent are deionized water, and pH value is certainly So.LB plate is the addition final concentration 2g/L agar in LB liquid medium.

Embodiment 1 is based on the Metabolically engineered of Wild type E. coli strain

(1) knockout of metI gene

For the partial inactivation for blocking metI to realize L-Methionine input system MetD, it is made to reduce taking the photograph for L-Methionine Enter to reduce the feedback inhibition to metA gene, therefore the metI gene in wild-type strain is knocked out, referring to Jiang Y,Chen B,Duan C,et al.Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System[J].Applied&Environmental Microbiology,2015,81(7): 2506.。

(large intestine is purchased from by CRISPR-Cas9 system editor wild-type e. coli (Escherichia coli) W3110 Bacillus genetic breeding center The Coli Genetic Stock Center) L-Methionine transportation system is encoded in genome MetI gene.Primer 1 and primer 2 are used by PCR, building, which can be expressed, using pTargetF carrier as template targets purpose base Because of the pTarget- Δ metI mutational vector of the sgRNA of metI (nucleotides sequence is classified as shown in SEQ ID NO.1).PCR reacts item Part is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C are continued to extend 10min.By PCR Product is with Dpn I in 37 DEG C of processing 3h, and conversion is coated on into E.coli BL21 (DE3) recipient bacterium containing final concentration after inactivation On the LB solid plate of 50mg/L spectinomycin hydrochloride resistance, 37 DEG C of culture 12h.Random picking single colonie is forwarded to containing final concentration In the LB liquid medium of 50mg/L spectinomycin hydrochloride resistance, 37 DEG C of culture 12h collect thallus and extract plasmid acquisition PTarget- Δ metI carrier.

Primer 3 and primer 4 are used by PCR, using Escherichia coli Escherichia coli W3110 genome as template Amplification obtains metI upstream region of gene homologous fragment, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 30s repeats 30 circulations；72 DEG C are continued to extend 10min.MetI base is obtained using primer 5 and the amplification of primer 6 in the same way Because of downstream homologous fragment, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purified fragments.By the two of recycling A DNA fragmentation carries out fusion DNA vaccine using primer 3 and primer 6, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend 10min, and PCR product is detected with 1.0% agarose gel electrophoresis And gel extraction purifies the segment (nucleotides sequence is classified as shown in SEQ ID NO.2).By pTarget- Δ metI carrier and recycling DNA fragmentation together electrotransformation to the Escherichia coli W3110 bacterial strain containing pCas9 carrier.

For electroporation, conversion has the Escherichia coli W3110 bacterial strain of pCas9 carrier containing 50mg/L's Cultivated at 30 DEG C in the LB culture medium of kanamycins and 10mM L-arabinose, until OD600 reaches 0.6, bacterium solution pass through from Gains in depth of comprehension are to thallus.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to use.Electricity is worn Hole is carried out at 2.5KV.

Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the spectinomycin hydrochloride resistance of 50mg/L On LB plate, 30 DEG C of overnight incubations.Picking single colonie carries out PCR as template, with primer 7 and primer 8, and by observing There are the missings of the DNA band of 1000bp confirmation metI gene in 1.0% Ago-Gel.The bacterial strain confirmed by this is existed In the LB culture medium of kanamycins containing 50mg/L and 5mM IPTG at 30 DEG C overnight incubation to remove pTarget- Δ MetI carrier.Then will have been removed the bacterial strain of pTarget- Δ metI carrier in LB culture medium at 37 DEG C overnight incubation with Remove pCas carrier.The bacterial strain so constructed is denoted as W3110 Δ metI.

1 primer sequence of table

Primer 1	TAATACTAGTCTACATCGGCTATAACGCGAGTTTTAGAGCTAGAAATAGC
		Primer 2	GCTCTAAAACTCGCGTTATAGCCGATGTAGACTAGTATTATACCTAGGAC
Primer 3	GACACGTTCTATTCTCGAAC
		Primer 4	GTGTTGAACGAACCCAGTACCTCTACTTTT
Primer 5	GTACTGGGTTCGTTCAACACAACATAAATA
		Primer 6	AAGCCCACTTTTTGCAGCAG
Primer 7	TACTGTTTTTGGCAACGTGG
		Primer 8	TGGACGAATTTCTTCACGTT

(2) knockout of metJ gene

In order to remove resistance of the negative regulation transcription factor MetJ to gene transcription levels such as thrA, metL, lysC, asd, metA Hold back inhibiting effect, the metJ gene in W3110 Δ metI bacterial strain is knocked out.

By encoding negative regulation transcription factor in CRISPR-Cas9 system editor's W3110 Δ metI strain gene group MetJ gene.Primer 9 and primer 10 are used by PCR, building, which can be expressed, using pTargetF carrier as template targets purpose The pTarget- Δ metJ mutational vector of the sgRNA of gene metJ (nucleotides sequence is classified as shown in SEQ ID NO.3).PCR reaction Condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C are continued to extend 10min.It will PCR product is with DpnI in 37 DEG C of processing 3h, and conversion is coated on into E.coli BL21 (DE3) recipient bacterium containing final concentration after inactivation On the LB solid plate of 50mg/L spectinomycin hydrochloride resistance, 37 DEG C of culture 12h.Random picking single colonie is forwarded to containing final concentration In the LB liquid medium of 50mg/L spectinomycin hydrochloride resistance, 37 DEG C of culture 12h collect thallus and extract plasmid acquisition PTarget- Δ metJ carrier.

Primer 11 and primer 12 are used by PCR, using Escherichia coli Escherichia coli W3110 genome as mould Plate expands to obtain metJ upstream region of gene homologous fragment, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 30s repeats 30 circulations；72 DEG C are continued to extend 10min.MetJ is obtained using primer 13 and the amplification of primer 14 in the same way Downstream of gene homologous fragment, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purified fragments.By recycling Two DNA fragmentations carry out fusion DNA vaccine using primer 11 and primer 14, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend 10min, and PCR product is examined with 1.0% agarose gel electrophoresis It surveys and gel extraction purifies the segment (nucleotides sequence is classified as shown in SEQ ID NO.4).By pTarget- Δ metJ carrier and return The DNA fragmentation of receipts together electrotransformation to the W3110 Δ metI bacterial strain for having pCas9 carrier.

For electroporation, conversion have the W3110 Δ metI bacterial strain of pCas9 carrier in the kanamycins containing 50mg/L and It is cultivated at 30 DEG C in the LB culture medium of 10mM L-arabinose, until OD600 reaches 0.6, bacterium solution obtains bacterium by centrifugation Body.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to use.Electroporation exists It is carried out under 2.5KV.

Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the spectinomycin hydrochloride resistance of 50mg/L On LB plate, 30 DEG C of overnight incubations.Picking single colonie carries out PCR as template, with primer 15 and primer 16, and passes through observation To in 1.0% Ago-Gel, there are the missings of the DNA band of 1000bp confirmation metJ gene.The bacterial strain that will be confirmed by this In the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG at 30 DEG C overnight incubation to remove pTarget- Δ MetJ carrier.Then will have been removed the bacterial strain of pTarget- Δ metJ carrier in LB culture medium at 37 DEG C overnight incubation with Remove pCas carrier.The bacterial strain so constructed is denoted as W3110 Δ metI Δ metJ.

2 primer sequence of table

Primer 9	TAATACTAGTATCTGCGTAAAGAGCGCAGCGTTTTAGAGCTAGAAATAGC
		Primer 10	GCTCTAAAACGCTGCGCTCTTTACGCAGATACTAGTATTATACCTAGGAC
Primer 11	ATGCCGGTATTAGTAAGTAC
		Primer 12	CTTTTTTGCTGAGATACTTAATCCTCTTCG
Primer 13	TAAGTATCTCAGCAAAAAAGAGCGGCGCGG
		Primer 14	TTTTGCCGTTTGCGCCAGTT
Primer 15	GTACCAGTTTGGGTTTTTCT
		Primer 16	GAATATTCTTGCCGTAACGT

(3) knockout of metB gene

In order to block the katabolism path of O- succinyl-L- homoserine, the cystathionine Gamma by knocking out coding is synthesized The metB gene of enzyme destroys the process of O- succinyl-L- homoserine catabolism synthesis cysteine, to obtain high concentration O- succinyl-L- homoserine, therefore in W3110 Δ metI Δ metJ strain to metB gene implement knock out.

It is synthesized by encoding cystathionine γ in CRISPR-Cas9 system editor's W3110 Δ metI Δ metJ strain gene group The metB gene of enzyme.Target can be expressed as template building using primer 17 and primer 18 and pTargetF carrier by PCR Determine the pTarget- Δ metB mutational vector of the sgRNA of target gene metB (nucleotides sequence is classified as shown in SEQ ID NO.5). PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C are continued to extend 10min.By PCR product with DpnI in 37 DEG C of processing 3h, conversion is coated on into E.coli BL21 (DE3) recipient bacterium after inactivation On the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h.Random picking single colonie is forwarded to In the LB liquid medium of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h collect thallus and extract plasmid Obtain pTarget- Δ metB carrier.

Primer 19 and primer 20 are used by PCR, is obtained using the genome of W3110 Δ metI Δ metJ bacterial strain as template amplification To metB upstream region of gene homologous fragment, PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s are repeated 30 circulations；72 DEG C are continued to extend 10min.MetB downstream of gene is obtained using primer 21 and the amplification of primer 22 in the same way Homologous fragment, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purified fragments.By two DNA pieces of recycling Section carries out fusion DNA vaccine using primer 19 and primer 22, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 1min repeats 30 circulations；72 DEG C are continued to extend 10min, and PCR product is detected with 1.0% agarose gel electrophoresis and cuts glue and returned It receives and purifies the segment (nucleotides sequence is classified as shown in SEQ ID NO.6).By the DNA fragmentation of pTarget- Δ metB carrier and recycling Electrotransformation is to the W3110 Δ metI Δ metJ bacterial strain for having pCas9 carrier together.

For electroporation, conversion has the W3110 Δ metI Δ metJ bacterial strain of pCas9 carrier, and in the card containing 50mg/L, that is mould It is cultivated in the LB culture medium of element and 10mM L-arabinose at 30 DEG C, until OD600 reaches 0.6, bacterium solution obtains bacterium by centrifugation Body.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to use.Electroporation exists It is carried out under 2.5KV.The spectinomycin hydrochloride that bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and 50mg/L is resisted On the LB plate of property, 30 DEG C of overnight incubations.Picking single colonie carries out PCR as template, with primer 23 and primer 24, and passes through Observe that there are the missings of the DNA band of 1000bp confirmation metB gene in 1.0% Ago-Gel.By what is confirmed by this Bacterial strain in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG at 30 DEG C overnight incubation to remove PTarget- Δ metB carrier.Then the bacterial strain of pTarget- Δ metB carrier will be had been removed in LB culture medium at 37 DEG C Overnight incubation is to remove pCas carrier.The bacterial strain so constructed is denoted as W3110 Δ metI Δ metJ Δ metB.

3 primer sequence of table

Primer 17	TAATACTAGTTTCGACAGTCTGGCGAAACGGTTTTAGAGCTAGAAATAGC
		Primer 18	GCTCTAAAACCGTTTCGCCAGACTGTCGAAACTAGTATTATACCTAGGAC
Primer 19	GCTTTACTTTGCGATGAGCG
		Primer 20	ACACTCATTTGTGATGAAGTTCCCTGGGCT
Primer 21	ACTTCATCACAAATGAGTGTGATTGCGCAG
		Primer 22	CAGCTGTTGCAGCAACGGGT
Primer 23	TGAGCGGGGTGTATTTCACC
		Primer 24	ATTTGTGTCGCGGAATAGTC

(4) knockout of thrB gene

In order to accumulate the measuring for precursor substance L- homoserine of O- succinyl-L- homoserine intracellular further It is tired, need to block the metabolism branch of L- homoserine, and L- homoserine intracellular is encoded by thrB gene and thrC gene Homoserine kinase and threonine synthetase metabolism synthesis L-threonine.Therefore, to W3110 Δ metI Δ metJ Δ metB bacterium ThrB gene in strain is knocked out.

By CRISPR-Cas9 system to encoded homoserine in W3110 Δ metI Δ metJ Δ metB strain gene group The thrB gene of kinases is edited.Primer 25 and primer 26 are used by PCR, energy is constructed using pTargetF carrier as template The pTarget- Δ thrB that enough expression target the sgRNA of target gene thrB (nucleotides sequence is classified as shown in SEQ ID NO.7) is prominent Variable load body.PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations；72 DEG C after It renews and stretches 10min.PCR product is converted after inactivation into E.coli BL21 (DE3) recipient bacterium with DpnI in 37 DEG C of processing 3h, It is coated on the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h.Random picking single colonie It is forwarded in the LB liquid medium of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h, collects thallus and mentions Plasmid is taken to obtain pTarget- Δ thrB carrier.

Primer 27 and primer 28 are used by PCR, using the genome of W3110 Δ metI Δ metJ Δ metB bacterial strain as template Amplification obtains thrB upstream region of gene homologous fragment, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 30s repeats 30 circulations；72 DEG C are continued to extend 10min.ThrB is obtained using primer 29 and the amplification of primer 30 in the same way Downstream of gene homologous fragment, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purified fragments.By recycling Two DNA fragmentations carry out fusion DNA vaccine using primer 27 and primer 30, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend 10min, and PCR product is examined with 0.9% agarose gel electrophoresis It surveys and gel extraction purifies the segment (nucleotides sequence is classified as shown in SEQ ID NO.8).By pTarget- Δ thrB carrier and return The DNA fragmentation of receipts together electrotransformation to the W3110 Δ metI Δ metJ Δ metB bacterial strain for having pCas9 carrier.

For electroporation, conversion has the W3110 Δ metI Δ metJ Δ metB bacterial strain of pCas9 carrier containing 50mg/L's It is cultivated at 30 DEG C in the LB culture medium of kanamycins and 10mM L-arabinose, until OD reaches 0.6, bacterium solution is by centrifugation Obtain thallus.Thallus is washed twice using sterile distilled water, then washed once using 10% glycerol to use.Electroporation It is carried out at 2.5KV.

Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the spectinomycin hydrochloride resistance of 50mg/L On LB plate, 30 DEG C of overnight incubations.Picking single colonie carries out PCR as template, with primer 31 and primer 32, and passes through observation To in 1.0% Ago-Gel, there are the missings of the DNA band of 1000bp confirmation thrB gene.The bacterial strain that will be confirmed by this In the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG at 30 DEG C overnight incubation to remove pTarget- Δ ThrB carrier.Then will have been removed the bacterial strain of pTarget- Δ thrB carrier in LB culture medium at 37 DEG C overnight incubation with Remove pCas carrier.The bacterial strain so constructed is denoted as W3110 Δ metI Δ metJ Δ metB Δ thrB.

4 primer sequence of table

Embodiment 2 is Metabolically engineered based on W3110 Δ metI Δ metJ Δ metB Δ thrB bacterial strain

(1) enhancing of metL gene expression

The enzyme of metL gene coding be also with aspartokinase II and homoserine dehydrogenase II bifunctional enzyme activity, In the metabolism of L-Aspartic acid, metL and thrA gene plays similar effect, therefore will also use trc promoter sequence (core Nucleotide sequence is shown in SEQ ID NO.21) replacement metL gene nucleotide series be that original in SEQ ID NO.9 shown is opened Promoter sequences are to achieve the purpose that enhance metL gene expression.

By being encoded in CRISPR-Cas9 system editor's W3110 Δ metI Δ metJ Δ metB Δ thrB strain gene group The promoter sequence of the metL gene of homoserine dehydrogenase.It is carried by PCR using primer 33 and primer 34 and pTargetF Body can express the pTarget- Δ PmetL: for the sgRNA for targeting target gene metL promoter sequence as template building: Ptrc mutational vector.PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 circulations； 72 DEG C are continued to extend 10min.By PCR product with DpnI in 37 DEG C of processing 3h, after inactivation conversion to E.coli BL21 (DE3) by It in body bacterium, is coated on the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h.It chooses at random Single colonie is taken to be forwarded in the LB liquid medium of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h are collected Thallus simultaneously extracts plasmid acquisition pTarget- Δ PmetL::Ptrc carrier.

Primer 35 and primer 36 are used by PCR, with the genome of W3110 Δ metI Δ metJ Δ metB Δ thrB bacterial strain MetL gene promoter sequence upstream homologous fragment is obtained for template amplification, the promoter sequence information of metL gene is to be based on Obtained in Ecocyc E.coli Database database (EcoCyc gene accession number: EG10590, nucleotides sequence are classified as SEQ Shown in ID NO.18), PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations； 72 DEG C are continued to extend 10min.It is obtained under metL gene promoter sequence using primer 37 and the amplification of primer 38 in the same way Homologous fragment is swum, PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purified fragments.By two DNA of recycling Segment carries out fusion DNA vaccine using the primer of primer 35 and primer 38, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend 10min, and PCR product is detected with 1.0% agarose gel electrophoresis And gel extraction purifies the segment (nucleotides sequence is classified as shown in SEQ ID NO.10), by Ptrc promoter in the gene band Sequence is inserted between two homologous fragments.By pTarget- Δ PmetL::Ptrc carrier and the DNA fragmentation of recycling electrotransformation together To the W3110 Δ metI Δ metJ Δ metB Δ thrB bacterial strain for having pCas9 carrier.

For electroporation, conversion has the W3110 Δ metI Δ metJ Δ metB Δ thrB bacterial strain of pCas9 carrier containing It is cultivated at 30 DEG C in the kanamycins of 50mg/L and the LB culture medium of 10mM L-arabinose, until OD600 reaches 0.6, bacterium Liquid obtains thallus by centrifugation.Thallus is washed twice using sterile distilled water, then using 10% glycerol washed once so as to It uses.Electroporation is carried out at 2.5KV.

Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the spectinomycin hydrochloride resistance of 50mg/L On LB plate, 30 DEG C of overnight incubations.Picking single colonie carries out PCR as template, with primer 39 and primer 40, and passes through observation To there are the original promoter sequences of the DNA band of 700bp confirmation metL gene have been opened by Ptrc in 1.0% Ago-Gel Promoter sequences replacement.By the bacterial strain confirmed by this in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG Overnight incubation is at 30 DEG C to remove pTarget- Δ PmetL::Ptrc carrier.Then pTarget- Δ PmetL: will be had been removed: The bacterial strain of Ptrc carrier in LB culture medium at 37 DEG C overnight incubation to remove pCas carrier.By the bacterium after removal pCas carrier Strain carries out PCR amplification using primer 39 and primer 38, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 1min15s repeats 30 circulations；72 DEG C are continued to extend 10min, PCR product are carried out sequence verification, sequencing result passes through The promoter sequence in situ of BLAST sequence alignment confirmation metL gene is successfully replaced by Ptrc promoter.The bacterial strain of building is remembered For W3110 Δ metI Δ metJ Δ metB Δ thrB-metL (trc).Same method constructs bacterial strain W3110 Δ metI Δ metJ Δ MetB-metL (trc), it is also consistent to prepare primer used in the genotype strain.

5 primer sequence of table

(2) enhancing of thrA gene expression

The enzyme of thrA gene coding has aspartokinase I and homoserine dehydrogenase I bifunctional enzyme activity, in order to make More carbon sources are flowed to O- succinyl-L- homoserine into the main path metabolic process of L- homoserine by L-Aspartic acid Precursor substance will be replaced original in thrA gene (nucleotides sequence is classified as shown in SEQ ID NO.11) with trc promoter sequence Promoter sequence is to achieve the purpose that enhance thrA gene expression.

Pass through CRISPR-Cas9 system editor W3110 Δ metI Δ metJ Δ metB Δ thrB-metL (trc) bacterial strain base Because of the promoter sequence of the thrA gene of encoded homoserine dehydrogenase in group.Primer 41 and primer 42 are used by PCR, and PTargetF carrier can express the pTarget- Δ for the sgRNA for targeting target gene thrA promoter sequence as template building PthrA::Ptrc mutational vector.PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 2min repeat 30 A circulation；72 DEG C are continued to extend 10min.PCR product is converted after inactivation to E.coli BL21 with DpnI in 37 DEG C of processing 3h (DE3) it in recipient bacterium, is coated on the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h. Random picking single colonie is forwarded in the LB liquid medium of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of cultures 12h collects thallus and extracts plasmid acquisition pTarget- Δ PthrA::Ptrc carrier.

Primer 43 and primer 44 are used by PCR, with W3110 Δ metI Δ metJ Δ metB Δ thrB-metL (trc) bacterium The genome of strain is that template amplification obtains thrA gene promoter sequence upstream homologous fragment, the promoter sequence letter of thrA gene Breath is based on (the EcoCyc gene accession number: EG10998) obtained in Ecocyc E.coli Database database, and PCR is anti- Answer condition as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations；72 DEG C are continued to extend 10min.It presses Same method obtains thrA gene promoter sequence downstream homologous fragment using primer 45 and the amplification of primer 46, and PCR product is used The detection of 1.0% agarose gel electrophoresis and gel extraction purified fragments (nucleotides sequence is classified as shown in SEQ ID NO.12).It will return Two DNA fragmentations received carry out fusion DNA vaccine using the primer of primer 43 and primer 46, and PCR reaction condition is as follows: 95 DEG C of 5min； 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend 10min, 1.0% agarose of PCR product Simultaneously gel extraction purifies the segment to detected through gel electrophoresis, and Ptrc promoter sequence is inserted into two homologous in the gene band Between section.By pTarget- Δ PthrA::Ptrc carrier and the DNA fragmentation of recycling together electrotransformation to there is pCas9 carrier W3110 Δ metI Δ metJ Δ metB Δ thrB-metL (trc) bacterial strain.

For electroporation, conversion has W3110 Δ metI Δ metJ Δ metB Δ thrB-metL (trc) bacterium of pCas9 carrier Strain is cultivated at 30 DEG C in the LB culture medium of the kanamycins containing 50mg/L and 10mM L-arabinose, until OD600 reaches To 0.6, bacterium solution obtains thallus by centrifugation.Thallus is washed twice using sterile distilled water, is then washed using 10% glycerol Once to use.Electroporation is carried out at 2.5KV.

Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the spectinomycin hydrochloride resistance of 50mg/L On LB plate, 30 DEG C of overnight incubations.Picking single colonie carries out PCR as template, with primer 47 and primer 48, and passes through observation To there are the original promoter sequences of the DNA band of 700bp confirmation thrA gene have been opened by Ptrc in 1.0% Ago-Gel Promoter sequences replacement.By the bacterial strain confirmed by this in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG Overnight incubation is at 30 DEG C to remove pTarget- Δ PthrA::Ptrc carrier.Then pTarget- Δ PthrA: will be had been removed: The bacterial strain of Ptrc carrier in LB culture medium at 37 DEG C overnight incubation to remove pCas carrier.By the bacterium after removal pCas carrier Strain carries out PCR amplification using primer 47 and primer 46, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 1min 15s repeats 30 circulations；72 DEG C are continued to extend 10min, PCR product are carried out sequence verification, sequencing result passes through BLAST sequence alignment confirm thrA gene promoter sequence in situ (nucleotides sequence is classified as shown in SEQ ID NO.19) by Ptrc promoter (nucleotides sequence is classified as shown in SEQ ID NO.21) is successfully replaced.The bacterial strain so constructed is denoted as W3110 Δ metIΔmetJΔmetBΔthrB-metL-thrA(trc)。

6 primer sequence of table

Primer 41	GCTCTAAAACCGTTACCTTTGGTCGAAAAAACTAGTATTATACCTAGGAC
		Primer 42	GCTCTAAAACCGTTACCTTTGGTCGAAAAAACTAGTATTATACCTAGGAC
Primer 43	TTAAAGTTTTCCCGACATTG
		Primer 44	ACCACACATTATACGAGCCGGATGATTAATTGTCAATAAAATTTTAATTTACTCAC
Primer 45	CGGCTCGTATAATGTGTGGTCACAAAGGAGATATACATGCGAGTGTTGAAGTTCGG
		Primer 46	CACCACCAGTTCGCCTTTTT
Primer 47	TTCGCGGTTATTTGATCAAG
		Primer 48	GTATATCTCCTTTGTGACCA

(2) enhancing of sucA gene expression

α-ketoglutaric acid is catalyzed and synthesized succinyl-CoA by the α-ketoglutaric acid decarboxylase of sucA gene coding, in order into one Step improves substrate succinyl CoA in content intracellular, and replacing sucA gene with trc promoter sequence, (nucleotides sequence is classified as SEQ Shown in ID NO.13) in original promoter sequence with achieve the purpose that enhance sucA gene expression.

Pass through CRISPR-Cas9 system editor W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA (trc) bacterium The promoter sequence of the sucA gene of encoded homoserine dehydrogenase in pnca gene group.Primer 49 and primer 50 are used by PCR, And pTargetF carrier can express the sgRNA's for targeting target gene sucA promoter sequence as template building PTarget- Δ PsucA::Ptrc mutational vector.PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 15s, 55 DEG C of 15s, 72 DEG C 2min repeats 30 circulations；72 DEG C are continued to extend 10min.By PCR product with DpnI in 37 DEG C of processing 3h, converted after inactivation to In E.coli BL21 (DE3) recipient bacterium, it is coated on the LB solid plate of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration, 37 DEG C of culture 12h.Random picking single colonie is forwarded to the LB liquid medium of the spectinomycin hydrochloride resistance of 50mg/L containing final concentration In, 37 DEG C of culture 12h collect thallus and extract plasmid acquisition pTarget- Δ PsucA::Ptrc carrier.

Primer 51 and primer 52 are used by PCR, with W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA (trc) genome of bacterial strain is that template amplification obtains sucA gene promoter sequence upstream homologous fragment, the starting of sucA gene Sub-sequence information be based on obtained in Ecocyc E.coli Database database (EcoCyc gene accession number: EG10998), PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s repeat 30 circulations；72 DEG C after It renews and stretches 10min.It is homologous that sucA gene promoter sequence downstream is obtained using primer 53 and the amplification of primer 54 in the same way Segment, with the detection of 1.0% agarose gel electrophoresis, simultaneously (nucleotides sequence is classified as SEQ ID to gel extraction purified fragments to PCR product Shown in NO.14).Two DNA fragmentations of recycling are subjected to fusion DNA vaccine, PCR reaction condition using the primer of primer 51 and primer 54 It is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min repeat 30 circulations；72 DEG C are continued to extend 10min, PCR product With the detection of 1.0% agarose gel electrophoresis and gel extraction purifies the segment, by Ptrc promoter sequence in the gene band It is inserted between two homologous fragments.By pTarget- Δ PsucA::Ptrc carrier and the DNA fragmentation of recycling together electrotransformation to having W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA (trc) bacterial strain of pCas9 carrier.

For electroporation, conversion has the W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA of pCas9 carrier (trc) bacterial strain is cultivated at 30 DEG C in the LB culture medium of the kanamycins containing 50mg/L and 10mM L-arabinose, until OD600 reaches 0.6, and bacterium solution obtains thallus by centrifugation.Thallus is washed twice using sterile distilled water, then using 10% it is sweet Oil wash is once to use.Electroporation is carried out at 2.5KV.

Bacterium solution after electrotransformation is applied to the kanamycins containing 50mg/L and the spectinomycin hydrochloride resistance of 50mg/L On LB plate, 30 DEG C of overnight incubations.Picking single colonie carries out PCR as template, with primer 55 and primer 56, and passes through observation To there are the original promoter sequences of the DNA band of 700bp confirmation sucA gene have been opened by Ptrc in 1.0% Ago-Gel Promoter sequences replacement.By the bacterial strain confirmed by this in the LB culture medium of the kanamycins containing 50mg/L and 5mM IPTG Overnight incubation is at 30 DEG C to remove pTarget- Δ PsucA::Ptrc carrier.Then pTarget- Δ PsucA: will be had been removed: The bacterial strain of Ptrc carrier in LB culture medium at 37 DEG C overnight incubation to remove pCas carrier.By the bacterium after removal pCas carrier Strain carries out PCR amplification using primer 47 and primer 46, and PCR reaction condition is as follows: 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 1min 15s repeats 30 circulations；72 DEG C are continued to extend 10min, PCR product are carried out sequence verification, sequencing result passes through BLAST sequence alignment confirm sucA gene promoter sequence in situ (nucleotides sequence is classified as shown in SEQ ID NO.20) by Ptrc promoter (nucleotides sequence is classified as shown in SEQ ID NO.21) is successfully replaced.The bacterial strain so constructed is denoted as W3110 Δ metIΔmetJΔmetBΔthrB-metL-thrA-sucA(trc)。

7 primer sequence of table

Primer 49	TAATACTAGTAAGAGCTCGCAAGTGAACCCGTTTTAGAGCTAGAAATAGC
		Primer 50	GCTCTAAAACGGGTTCACTTGCGAGCTCTTACTAGTATTATACCTAGGAC
Primer 51	AAGAAGATTGTGATTCGCCC
		Primer 52	ACCACACATTATACGAGCCGGATGATTAATTGTCAAGATTTCCTGCATTTCTTTGT
Primer 53	CGGCTCGTATAATGTGTGGTCACAAAGGAGATATACATGCAGAACAGCGCTTTGAA
		Primer 54	TCAGTTCGCTTAAGAAGCGT
Primer 55	TAAAAGAGAAAGATCCCAGC
		Primer 56	GTATATCTCCTTTGTGACCA

The expression of embodiment 3 has the building of the plasmid of the metA gene of feedback-inhibition resistance

With wild-type e. coli Escherichia coli W3110 (Escherichia coli genetic breeding center The Coli Genetic Stock Center) genome is as template, together with primer 57 and the progress PCR amplification of primer 58.PCR reacts item Part: initial denaturation 95 DEG C of 5min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, totally 30 recycle, last 72 DEG C of extensions 10min.PCR is produced Object is detected with 1.0% agarose gel electrophoresis and gel extraction purifies the segment, is drawn using Taq archaeal dna polymerase to the end of segment 5 ' Enter base A.The segment is attached with pGEM-T carrier under T4DNA connection enzyme effect, obtains cloning recombinant plasmids pGEM- T-metA.To metA gene, (nucleotides sequence is classified as shown in SEQ ID NO.15, encodes albumen ammonia on pGEM-T-metA plasmid Base acid sequence is shown in SEQ ID NO.16) rite-directed mutagenesis is carried out, by 64 amino acids -- glutamic acid replaces with glutamine (E64Q), it using primer 59 and primer 60, and using pGEM-T-metA plasmid as template, is introduced and is mutated by PCR, PCR reaction interval Sequence is as follows: 98 DEG C of 3min；98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 3min repeat 30 circulations；72 DEG C are continued to extend 10min.By PCR Product is with DpnI in 37 DEG C of processing 3h, and conversion is coated on into E.coli BL21 (DE3) recipient bacterium containing final concentration after inactivation On the LB solid plate of 50mg/L ampicillin sodium resistance, after 37 DEG C of culture 12h, random picking single colonie carries out sequencing point Analysis obtains metA mutated gene.Expression primer 61 is designed based on the analysis results and primer 62 (is respectively provided with NcoI limitation in primer Property restriction enzyme site and SacI restriction enzyme site), obtain the metA11 gene order of 930bp.It is limited using NcoI and SacI Property restriction endonuclease to amplified fragments carry out digestion processing, will be at segment restriction enzyme identical as use using T4DNA ligase The pTrc99A of reason is attached, construction of expression vector pTrc99A-metA11.

8 primer sequence of table

Primer 57	ATGCCGATTCGTGTGCCGGA
		Primer 58	TTAATCCAGCGTTGGATTCA
Primer 59	CTTTGCAGGTCGATATTCAG
		Primer 60	CTGAATATCGACCTGCAAAG
Primer 61	ccatggATGCCGATTCGTGTGCCGGA
		Primer 62	gagctcTTAATCCAGCGTTGGATTCA

The building of the plasmid of the yjeH gene of the expression tool L-Methionine transportation function of embodiment 4

With wild-type e. coli Escherichia coli W3110 (Escherichia coli genetic breeding center The Coli Genetic Stock Center) genome is as template, together with primer 63 and the progress PCR amplification of primer 64.PCR reacts item Part: initial denaturation 95 DEG C of 5min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min30s, totally 30 recycle, last 72 DEG C of extensions 10min. PCR product is detected with 1.0% agarose gel electrophoresis and gel extraction purifies the segment, using Taq archaeal dna polymerase to segment 5 ' ends introduce base A.The segment is attached with pGEM-T carrier under T4DNA connection enzyme effect, obtains clone's recombination matter Grain pGEM-T-yjeH.By the recombinant plasmid transformed into e. coli jm109, being coated on containing concentration is that 50 μ g/mL ammonia benzyls are green The LB plate of mycin sodium resistance, random picking positive colony carry out sequencing analysis.65 He of expression primer is designed based on the analysis results Primer 66 (is respectively provided with Kpn I restriction enzyme site and Hind III restriction enzyme site) in primer, obtain 1257bp YjeH gene order (nucleotides sequence is classified as shown in SEQ ID NO.17).Utilize Kpn I and Hind III restriction enzyme Digestion processing is carried out to amplified fragments, using T4DNA ligase by segment restriction enzyme enzymatic treatment identical as use PTrc99A-metA11 is attached, construction of expression vector pTrc99A-metA11-yjeH, and is named as pA11y.

9 primer sequence of table

Primer 63	ATGAGTGGACTCAAACAAGA
		Primer 64	TTATGTGGTTATGCCATTTT
Primer 65	ccatgg ATGAGTGGACTCAAACAAGA
		Primer 66	gagctc TTATGTGGTTATGCCATTTT

The building of 5 experimental strain of embodiment

(1) building of bacterial strain

By coli strain W3110, W3110 Δ metI, W3110 Δ metI Δ metJ, W3110 Δ metI Δ metJ Δ metB、W3110ΔmetIΔmetJΔmetBΔthrB、W3110ΔmetIΔmetJΔmetBΔthrB-metL(trc)、 W3110ΔmetIΔmetJΔmetB-metL(trc)、W3110ΔmetIΔmetJΔmetBΔthrB-metL-thrA (trc), W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA-sucA (trc) is prepared into competent cell, and The plasmid pA11y constructed in embodiment 4 is converted into the competent cell of above-mentioned preparation by chemical transformation, is coated on and contains Have concentration be 50 μ g/mL ampicillin sodium resistances LB plate, obtain three kinds containing pA11y plasmid it is Metabolically engineered after bacterium Strain.

(2) shake flask fermentation is tested

Fermenting experiment test is carried out in shaking flask to above-mentioned thallus, to produce O- amber between more each genotype strain The ability of acyl-L- homoserine.Shake flask fermentation experiment is carried out by following scheme: each bacterial strain streak inoculation is being contained 50 μ g/ On the LB plate of mL ampicillin sodium resistance, the overnight incubation in 37 DEG C of incubators, picking single colonie is seeded to the LB training of 5ml Support base in, and with the revolving speed of 200rpm in 37 DEG C of incubators overnight incubation.

The fermentation medium of 20ml is added in the shaking flask of 500ml, and the seed liquor of every kind of bacterial strain of 1ml is seeded to shaking flask Culture medium in.Then shaking flask is cultivated 48 hours in 30 DEG C of incubator with the revolving speed of 180rpm, and 1ml liquid is taken from shaking flask Body dilutes 100 times with ultrapure water, analyzes the content of O- succinyl-L- homoserine in fermentation liquid after filter membrane by HPLC, most Every kind of bacterial strain for relatively carrying plasmid eventually obtains the amount of O- succinyl-L- homoserine.As a result as the Metabolically engineered bacterial strain of table 10 shakes Shown in bottle fermenting experiment.

Fermentation medium composition: glucose 40/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L, yeast powder 4g/L, carbonic acid Calcium 30g/L, L-threonine 0.2g/L, vitamin B₁ 0.0001g/L、MgSO₄ 2g/L、FeSO₄ 0.005g/L、MnSO₄ 0.005g/L、ZnSO₄0.005g/L, solvent are deionized water, pH value 6.8.

The experiment of the Metabolically engineered bacterial strain shake flask fermentation of table 10

According to table 10 it is found that 9 plants are passed through Metabolically engineered and carrying carrier W3110 Δ metI/pA11y, W3110 Δ metI ΔmetJ/pA11y、W3110ΔmetIΔmetJΔmetB/pA11y、W3110ΔmetIΔmetJΔmetBΔthrB/ pA11y、W3110ΔmetIΔmetJΔmetB-metL(trc)/pA11y、W3110ΔmetIΔmetJΔmetBΔthrB- metL(trc)/pA11y、W3110ΔmetIΔmetJΔmetBΔthrB-metL-thrA(trc)/pA11y、W3110Δ MetI Δ metJ Δ metB Δ thrB-metL-thrA-sucA (trc)/pA11y bacterial strain all has production O- succinyl-L- Kosé The ability of propylhomoserin, and the transformation of the part of L-Methionine transportation system is not caused significantly to influence on the growth of bacterial strain, lead to The control to bacterial strain L-Methionine content intracellular is crossed, its feedback inhibition to metA gene can be weakened, it should to play The good activity of enzyme.The bacterial strain of best performance is compared in the level of fermenting and producing O- succinyl-L- homoserine after transformation 21.8g/L is increased to from 0g/L in the bacterial strain of wild type.Other are by improved bacterial strain in production O- succinyl-L- Kosé ammonia The level of acid has different degrees of raising compared to original strain.

(3) L-threonine additive amount is tested

To L-threonine auxotrophic strain W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA (trc)/ PA11y has carried out the experiment of L-threonine additive amount, by L-threonine amount in fermentation medium be respectively 0g/L, 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L, 1.6g/L, and the seed liquor of every kind of bacterial strain of 1ml is seeded in the culture medium of shaking flask.Then Shaking flask is cultivated 48 hours in 30 DEG C of incubator with the revolving speed of 180rpm.Final examination W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA (trc)/pA11y bacterial strain production O- succinyl-L- homoserine ability.

L-threonine is added by external source and adds the fermenting experiment of glucose, W3110 Δ metI Δ metJ Δ metB Δ ThrB-metL-thrA (trc)/pA11y bacterial strain production O- succinyl-L- homoserine yield level is as shown in table 11.For For L-threonine auxotrophic strain, when no external source adds L-threonine, the growth of bacterial strain is restricted, The ability of metabolism production O- succinyl-L- homoserine is also restricted therewith.When L-threonine is excessively added, not only cannot Promote thalli growth, can also play negative impact to the accumulation of O- succinyl-L- homoserine.

Table 11L- threonine additive amount fermenting experiment

L-threonine additive amount (g/L)	OD600	Saccharic acid conversion ratio (%)	O- succinyl-L- homoserine (g/L)
				0	7.33	16.14	6.456235
0.1	10.17	46.62	18.64797
				0.2	9.96	49.10	19.64033
0.4	9.5	50.79	20.31498
				0.8	9.67	49.15	19.65896
1.6	9.36	46.56	18.62247

6 large fermentation tank culture experiment of embodiment

In order to which L- homoserine is mass produced, W3110 Δ metI Δ metJ Δ metB Δ thrB-metL-thrA- is used SucA (trc)/pA11y bacterial strain, is cultivated in 5L fermentor.Bacterial strain streak inoculation is being contained into 50 μ g/mL ammonia benzyl moulds On the LB plate of plain sodium resistance, the overnight incubation in 37 DEG C of incubators.Picking single colonie is seeded to the LB Tube propagation base of 10ml In, 8h is cultivated at 37 DEG C, then inoculates 1ml tube culture into containing 50 μ g/mL ampicillin sodium resistances In the 500ml triangular flask of the seed culture medium of 100ml, and 8-10h is cultivated in 37 DEG C of incubators with the revolving speed of 200rpm, and And the inoculum of 200ml is inoculated with the 5L fermentor into the 2L fermentation medium containing 50 μ g/mL ampicillin sodium resistances In, supplemented medium is added by fed-batch process, and cultivate 50-100h.Pass through feed supplement in fermentation process for glucose Concentration is controlled in 2-10g/L.By DO level in stirring coupling dissolved oxygen (DO) scheme control fermentation process 30% or so, stir Revolving speed control controls cultivation temperature in 1-2vvm, fermentation process and is used in combination at 30 DEG C in 200-600rpm, Ventilation Rate control 50% ammonium hydroxide adjusts range of the pH 6.8~7.0.Medium component is as shown in table 11, and is analyzed by amino-acid analyzer The concentration of O- succinyl-L- homoserine in the fermentation liquid so cultivated is 100g/L.

11 culture medium prescription of table (solvent is deionized water, pH6.8)

Composition	Fermentation medium	Supplemented medium
			Glucose (g/L)	40	500
Yeast powder (g/L)	4
			KH2PO4(g/L)	2	12.5
MgSO4(g/L)	2
			(NH4)2SO4(g/L)	17
L-Methionine (g/L)	0.2	2
			L-threonine (g/L)	0.2	2
L-Isoleucine (g/L)	0.2	2
			FeSO4(g/L)	0.005
MnSO4(g/L)	0.005
			ZnSO4(g/L)	0.005
Vitamin B1 (g/L)	0.0001

Sequence table

<110>Zhejiang Polytechnical University

<120>a kind of production O- succinyl-L- homoserine recombination bacillus coli and its application

<160> 21

<170> SIPOSequenceListing 1.0

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atgtctgagc cgatgatgtg gctgctggtt cgtggcgtat gggaaacgct ggcaatgacc 60

ttcgtatccg gtttttttgg ctttgtgatt ggtctgccgg ttggcgttct gctttatgtc 120

acgcgtccgg ggcaaattat tgctaacgcg aagctgtatc gtaccgtttc tgcgattgtg 180

aacattttcc gttccatccc gttcattatc ttgcttgtat ggatgattcc gtttacccgc 240

gttattgtcg gtacatcgat tggtttgcag gcagcgattg ttccgttaac cgttggtgca 300

gcaccgttta ttgcccgtat ggtcgagaac gctctgctgg agatcccaac cgggttaatt 360

gaagcttccc gcgcaatggg tgccacgccg atgcagatcg tccgtaaggt gctgttaccg 420

gaagcgctgc cgggtctggt gaatgcggca actatcaccc tgattaccct ggtcggttat 480

tccgcgatgg gtggtgcagt cggtgccggt ggtttaggtc agattggcta tcagtatggc 540

tacatcggct ataacgcgac ggtgatgaat acggtactgg tattgctggt cattctggtt 600

tatttaattc agttcgcagg cgaccgcatc gtccgggctg tcactcgcaa gtaa 654

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<213>unknown (Unknown)

<400> 2

gacacgttct attctcgaac tgctgaaaga catcaaccgc cgtctggggt tgacgattct 60

gttgatcacc cacgaaatgg acgttgtgaa gcgcatttgt gattgcgtgg cggtcatcag 120

caatggagaa ctgatcgagc aggacacggt aagtgaagtg ttctcgcatc cgaaaacgcc 180

gctggcgcag aagtttattc agtcgaccct gcatctggat atcccggaag attaccagga 240

acgtctgcaa gcggagccat ttactgactg cgtgccgatg ctgcgtctgg agtttaccgg 300

tcaatcggtc gatgccccac tgctttctga aaccgcgcgt cgtttcaacg tcaacaacaa 360

cattattagc gcgcagatgg attacgccgg tggcgttaag ttcggcatca tgctgactga 420

aatgcacggc acacaacaag atacgcaagc cgccattgcc tggctgcagg aacaccatgt 480

aaaagtagag gtactgggtt cgttcaacac aacataaata attgaagaag gaataaggta 540

tggcgttcaa attcaaaacc tttgcggcag tgggagccct gatcggatca ctggcactgg 600

taggctgcgg tcaggatgaa aaagatccaa accacattaa agtcggcgtg attgttggtg 660

ccgaacagca ggttgcagaa gtcgcgcaga aagttgcgaa agacaaatat ggcctggacg 720

ttgagctggt aaccttcaac gactatgttc tgccaaacga agcattgagc aaaggcgata 780

tcgacgccaa cgccttccag cataaaccgt accttgatca gcaactgaaa gatcgtggct 840

acaaactggt cgcagtaggc aacacttttg tttatccgat tgctggttac tccaagaaaa 900

tcaaatcact ggatgaactg caggatggtt cgcaggttgc cgtgccaaac gacccaacta 960

accttggtcg ttcactgctg ctgctgcaaa aagtgggctt 1000

<210> 3

<211> 318

<212> DNA

<213>unknown (Unknown)

<400> 3

atggctgaat ggagcggcga atatatcagc ccatacgctg agcacggcaa gaagagtgaa 60

caagtcaaaa agattacggt ttccattcct cttaaggtgt taaaaatcct caccgatgaa 120

cgcacgcgtc gtcaggtgaa caacctgcgt cacgctacca acagcgagct gctgtgcgaa 180

gcgtttctgc atgcctttac cgggcaacct ttgccggatg atgccgatct gcgtaaagag 240

cgcagcgacg aaatcccgga agcggcaaaa gagatcatgc gtgagatggg gattaacccg 300

gagacgtggg aatactaa 318

<210> 4

<211> 1013

<212> DNA

<213>unknown (Unknown)

<400> 4

atgccggtat tagtaagtac tgcaccagca ccaccttcca gttctgccag cgcacgctga 60

accacatcgc gcgttgggtt gccgcgacgc gagtaatcat gcgcgcgcgg ttcattaaat 120

ccggtaaagt tataggtgct ggaaagatgg atcggtggga caacgcaacc atactgttcg 180

tcgtcattta acccgctacg cactgcgatg gtggcctgtt tacgcgtcat gtgatgaagt 240

tccctgggct ttgtcggtga aatgtcaggc accagagtaa acattgtgtt aatggacgtc 300

aatacatctg gacatctaaa cttctttgcg tatagattga gcaaatccca aatagccgtt 360

aaaattatat gcattatcac gccgacaggt gcattacacg atgtcacggt aacgcctgta 420

cggtaaacta tgcgggttta cggtcagtac ccacatcaac tgtgtggtct ggtctcaatt 480

tattgacgaa gaggattaag tatctcagca aaaaagagcg gcgcggagtg gaatcgcctg 540

atgcgctacg cttatcaggc ctacgtcata ttgcaattta ttgaatttgc acgaacttgt 600

aggccggata aggcgttcac gccgcatccg gcataaacaa cgagcacgtt gtctgcgacc 660

caccgctttt tatacatgga cgtttaacta tgaaaaacag gctgctgatc ctcagcctgc 720

tggtttctgt acctgccttt gcctggcagc cacaaaccgg cgacatcatc tttcagatct 780

ctcgctcatc gcaaagtaaa gcgatccaac tggcgaccca taccgattat agccacaccg 840

gtatgctggt gatacgcaac aaaaagccct acgtttttga agcagtcggc ccggtgaaat 900

acaccccgct caagcagtgg atcgcccatg gtgaaaaggg caaatacgtt gttcgccgcg 960

ttgaaggcgg actgagtgtt gaacaacagc aaaaactggc gcaaacggca aaa 1013

<210> 5

<211> 1160

<212> DNA

<213>unknown (Unknown)

<400> 5

atgacgcgta aacaggccac catcgcagtg cgtagcgggt taaatgacga cgaacagtat 60

ggttgcgttg tcccaccgat ccatctttcc agcacctata actttaccgg atttaatgaa 120

ccgcgcgcgc atgattactc gcgtcgcggc aacccaacgc gcgatgtggt tcagcgtgcg 180

ctggcagaac tggaaggtgg tgctggtgca gtacttacta ataccggcat gtccgcgatt 240

cacctggtaa cgaccgtctt tttgaaacct ggcgatctgc tggttgcgcc gcacgactgc 300

tacggcggta gctatcgcct gttcgacagt ctggcgaaac gcggttgcta tcgcgtgttg 360

tttgttgatc aaggcgatga acaggcatta cgggcagcgc tggcagaaaa acccaaactg 420

gtactggtag aaagcccaag taatccattg ttacgcgtcg tggatattgc gaaaatctgc 480

catctggcaa gggaagtcgg ggcggtgagc gtggtggata acaccttctt aagcccggca 540

ttacaaaatc cgctggcatt aggtgccgat ctggtgttgc attcatgcac gaaatatctg 600

aacggtcact cagacgtagt ggccggcgtg gtgattgcta aagacccgga cgttgtcact 660

gaactggcct ggtgggcaaa caatattggc gtgacgggcg gcgcgtttga cagctatctg 720

ctgctacgtg ggttgcgaac gctggtgccg cgtatggagc tggcgcagcg caacgcgcag 780

gcgattgtga aatacctgca aacccagccg ttggtgaaaa aactgtatca cccgtcgttg 840

ccggaaaatc aggggcatga aattgccgcg cgccagcaaa aaggctttgg cgcaatgttg 900

agttttgaac tggatggcga tgagcagacg ctgcgtcgtt tcctgggcgg gctgtcgttg 960

tttacgctgg cggaatcatt agggggagtg gaaagtttaa tctctcacgc cgcaaccatg 1020

acacatgcag gcatggcacc agaagcgcgt gctgccgccg ggatctccga gacgctgctg 1080

cgtatctcca ccggtattga agatggcgaa gatttaattg ccgacctgga aaatggcttc 1140

cgggctgcaa acaaggggta 1160

<210> 6

<211> 1388

<212> DNA

<213>unknown (Unknown)

<400> 6

gctttacttt gcgatgagcg agagatctga aagatgatgt cgccggtttg tggctgccag 60

gcaaaggcag gtacagaaac cagcaggctg aggatcagca gcctgttttt catagttaaa 120

cgtccatgta taaaaagcgg tgggtcgcag acaacgtgct cgttgtttat gccggatgcg 180

gcgtgaacgc cttatccggc ctacaagttc gtgcaaattc aataaattgc aatatgacgt 240

aggcctgata agcgtagcgc atcaggcgat tccactccgc gccgctcttt tttgctgaga 300

tacttaatcc tcttcgtcaa taaattgaga ccagaccaca cagttgatgt gggtactgac 360

cgtaaacccg catagtttac cgtacaggcg ttaccgtgac atcgtgtaat gcacctgtcg 420

gcgtgataat gcatataatt ttaacggcta tttgggattt gctcaatcta tacgcaaaga 480

agtttagatg tccagatgta ttgacgtcca ttaacacaat gtttactctg gtgcctgaca 540

tttcaccgac aaagcccagg gaacttcatc acgagatact taatcctctt cgtcaataaa 600

ttgagaccag accacacagt tgatgtgggt actgaccgta aacccgcata gtttaccgta 660

caggcgttac cgtgacatcg tgtaatgcac ctgtcggcgt gataatgcat ataattttaa 720

cggctatttg ggatttgctc aatctatacg caaagaagtt tagatgtcca gatgtattga 780

cgtccattaa cacaatgttt actctggtgc ctgacatttc accgacaaag cccagggaac 840

ttcatcacaa aatgagtgtg attgcgcagg caggggcgaa aggtcgtcag ctgcataaat 900

ttggtggcag tagtctggct gatgtgaagt gttatttgcg tgtcgcgggc attatggcgg 960

agtactctca gcctgacgat atgatggtgg tttccgccgc cggtagcacc actaaccagt 1020

tgattaactg gttgaaacta agccagaccg atcgtctctc tgcgcatcag gttcaacaaa 1080

cgctgcgtcg ctatcagtgc gatctgatta gcggtctgct acccgctgaa gaagccgata 1140

gcctcattag cgcttttgtc agcgaccttg agcgcctggc ggcgctgctc gacagcggta 1200

ttaacgacgc agtgtatgcg gaagtggtgg gccacgggga agtatggtcg gcacgtctga 1260

tgtctgcggt acttaatcaa caagggctgc cagcggcctg gcttgatgcc cgcgagtttt 1320

tacgcgctga acgcgccgca caaccgcagg ttgatgaagg gctttcttac ccgttgctgc 1380

aacagctg 1388

<210> 7

<211> 933

<212> DNA

<213>unknown (Unknown)

<400> 7

atggttaaag tttatgcccc ggcttccagt gccaatatga gcgtcgggtt tgatgtgctc 60

ggggcggcgg tgacacctgt tgatggtgca ttgctcggag atgtagtcac ggttgaggcg 120

gcagagacat tcagtctcaa caacctcgga cgctttgccg ataagctgcc gtcagaacca 180

cgggaaaata tcgtttatca gtgctgggag cgtttttgcc aggaactggg taagcaaatt 240

ccagtggcga tgaccctgga aaagaatatg ccgatcggtt cgggcttagg ctccagtgcc 300

tgttcggtgg tcgcggcgct gatggcgatg aatgaacact gcggcaagcc gcttaatgac 360

actcgtttgc tggctttgat gggcgagctg gaaggccgta tctccggcag cattcattac 420

gacaacgtgg caccgtgttt tctcggtggt atgcagttga tgatcgaaga aaacgacatc 480

atcagccagc aagtgccagg gtttgatgag tggctgtggg tgctggcgta tccggggatt 540

aaagtctcga cggcagaagc cagggctatt ttaccggcgc agtatcgccg ccaggattgc 600

attgcgcacg ggcgacatct ggcaggcttc attcacgcct gctattcccg tcagcctgag 660

cttgccgcga agctgatgaa agatgttatc gctgaaccct accgtgaacg gttactgcca 720

ggcttccggc aggcgcggca ggcggtcgcg gaaatcggcg cggtagcgag cggtatctcc 780

ggctccggcc cgaccttgtt cgctctgtgt gacaagccgg aaaccgccca gcgcgttgcc 840

gactggttgg gtaagaacta cctgcaaaat caggaaggtt ttgttcatat ttgccggctg 900

gatacggcgg gcgcacgagt actggaaaac taa 933

<210> 8

<211> 1137

<212> DNA

<213>unknown (Unknown)

<400> 8

taaattcctc tatgacacca acgttggggc tggattaccg gttattgaga acctgcaaaa 60

tctgctcaat gcaggtgatg aattgatgaa gttctccggc attctttctg gttcgctttc 120

ttatatcttc ggcaagttag acgaaggcat gagtttctcc gaggcgacca cgctggcgcg 180

ggaaatgggt tataccgaac cggacccgcg agatgatctt tctggtatgg atgtggcgcg 240

taaactattg attctcgctc gtgaaacggg acgtgaactg gagctggcgg atattgaaat 300

tgaacctgtg ctgcccgcag agtttaacgc cgagggtgat gttgccgctt ttatggcgaa 360

tctgtcacaa ctcgacgatc tctttgccgc gcgcgtggcg aaggcccgtg atgaaggaaa 420

agttttgcgc tatgttggca atattgatga agatggcgtc tgccgcgtga agattgccga 480

agtggatggt aatgatccgc tgttcaaagt gaaaaatggc gaaaacgccc tggccttcta 540

tagccactat tatcagccgc tgccgttggt actgcgcgga tatggtgcgg gcaatgacgt 600

tacagctgcc ggtgtctttg ctgatctgct acgtaccctc tcatggaagt taggagtctg 660

aatgaaactc tacaatctga aagatcacaa cgagcaggtc agctttgcgc aagccgtaac 720

ccaggggttg ggcaaaaatc aggggctgtt ttttccgcac gacctgccgg aattcagcct 780

gactgaaatt gatgagatgc tgaagctgga ttttgtcacc cgcagtgcga agatcctctc 840

ggcgtttatt ggtgatgaaa tcccacagga aatcctggaa gagcgcgtgc gcgcggcgtt 900

tgccttcccg gctccggtcg ccaatgttga aagcgatgtc ggttgtctgg aattgttcca 960

cgggccaacg ctggcattta aagatttcgg cggtcgcttt atggcacaaa tgctgaccca 1020

tattgcgggt gataagccag tgaccattct gaccgcgacc tccggtgata ccggagcggc 1080

agtggctcat gctttctacg gtttaccgaa tgtgaaagtg gttatcctct atccacg 1137

<210> 9

<211> 2433

<212> DNA

<213>unknown (Unknown)

<400> 9

atgagtgtga ttgcgcaggc aggggcgaaa ggtcgtcagc tgcataaatt tggtggcagt 60

agtctggctg atgtgaagtg ttatttgcgt gtcgcgggca ttatggcgga gtactctcag 120

cctgacgata tgatggtggt ttccgccgcc ggtagcacca ctaaccagtt gattaactgg 180

ttgaaactaa gccagaccga tcgtctctct gcgcatcagg ttcaacaaac gctgcgtcgc 240

tatcagtgcg atctgattag cggtctgcta cccgctgaag aagccgatag cctcattagc 300

gcttttgtca gcgaccttga gcgcctggcg gcgctgctcg acagcggtat taacgacgca 360

gtgtatgcgg aagtggtggg ccacggggaa gtatggtcgg cacgtctgat gtctgcggta 420

cttaatcaac aagggctgcc agcggcctgg cttgatgccc gcgagttttt acgcgctgaa 480

cgcgccgcac aaccgcaggt tgatgaaggg ctttcttacc cgttgctgca acagctgctg 540

gtgcaacatc cgggcaaacg tctggtggtg accggattta tcagccgcaa caacgccggt 600

gaaacggtgc tgctggggcg taacggttcc gactattccg cgacacaaat cggtgcgctg 660

gcgggtgttt ctcgcgtaac catctggagc gacgtcgccg gggtatacag tgccgacccg 720

cgtaaagtga aagatgcctg cctgctgccg ttgctgcgtc tggatgaggc cagcgaactg 780

gcgcgcctgg cggctcccgt tcttcacgcc cgtactttac agccggtttc tggcagcgaa 840

atcgacctgc aactgcgctg tagctacacg ccggatcaag gttccacgcg cattgaacgc 900

gtgctggcct ccggtactgg tgcgcgtatt gtcaccagcc acgatgatgt ctgtttgatt 960

gagtttcagg tgcccgccag tcaggatttc aaactggcgc ataaagagat cgaccaaatc 1020

ctgaaacgcg cgcaggtacg cccgctggcg gttggcgtac ataacgatcg ccagttgctg 1080

caattttgct acacctcaga agtggccgac agtgcgctga aaatcctcga cgaagcggga 1140

ttacctggcg aactgcgcct gcgtcagggg ctggcgctgg tggcgatggt cggtgcaggc 1200

gtcacccgta acccgctgca ttgccaccgc ttctggcagc aactgaaagg ccagccggtc 1260

gaatttacct ggcagtccga tgacggcatc agcctggtgg cagtactgcg caccggcccg 1320

accgaaagcc tgattcaggg gctgcatcag tccgtcttcc gcgcagaaaa acgcatcggc 1380

ctggtattgt tcggtaaggg caatatcggt tcccgttggc tggaactgtt cgcccgtgag 1440

cagagcacgc tttcggcacg taccggcttt gagtttgtgc tggcaggtgt ggtggacagc 1500

cgccgcagcc tgttgagcta tgacgggctg gacgccagcc gcgcgttagc cttcttcaac 1560

gatgaagcgg ttgagcagga tgaagagtcg ttgttcctgt ggatgcgcgc ccatccgtat 1620

gatgatttag tggtgctgga cgttaccgcc agccagcagc ttgctgatca gtatcttgat 1680

ttcgccagcc acggtttcca cgttatcagc gccaacaaac tggcgggagc cagcgacagc 1740

aataaatatc gccagatcca cgacgccttc gaaaaaaccg ggcgtcactg gctgtacaat 1800

gccaccgtcg gtgcgggctt gccgatcaac cacaccgtgc gcgatctgat cgacagcggc 1860

gatactattt tgtcgatcag cgggatcttc tccggcacgc tctcctggct gttcctgcaa 1920

ttcgacggta gcgtgccgtt taccgagctg gtggatcagg cgtggcagca gggcttaacc 1980

gaacctgacc cgcgtgacga tctctctggc aaagacgtga tgcgcaagct ggtgattctg 2040

gcgcgtgaag caggttacaa catcgaaccg gatcaggtac gtgtggaatc gctggtgcct 2100

gctcattgcg aaggcggcag catcgaccat ttctttgaaa atggcgatga actgaacgag 2160

cagatggtgc aacggctgga agcggcccgc gaaatggggc tggtgctgcg ctacgtggcg 2220

cgtttcgatg ccaacggtaa agcgcgtgta ggcgtggaag cggtgcgtga agatcatccg 2280

ttggcatcac tgctgccgtg cgataacgtc tttgccatcg aaagccgctg gtatcgcgat 2340

aaccctctgg tgatccgcgg acctggcgct gggcgcgacg tcaccgccgg ggcgattcag 2400

tcggatatca accggctggc acagttgttg taa 2433

<210> 10

<211> 1069

<212> DNA

<213>unknown (Unknown)

<400> 10

cagttggatc gctttacttt gcgatgagcg agagatctga aagatgatgt cgccggtttg 60

tggctgccag gcaaaggcag gtacagaaac cagcaggctg aggatcagca gcctgttttt 120

catagttaaa cgtccatgta taaaaagcgg tgggtcgcag acaacgtgct cgttgtttat 180

gccggatgcg gcgtgaacgc cttatccggc ctacaagttc gtgcaaattc aataaattgc 240

aatatgacgt aggcctgata agcgtagcgc atcaggcgat tccactccgc gccgctcttt 300

tttgctgaga tacttaatcc tcttcgtcaa taaattgaga ccagaccaca cagttgatgt 360

gggtactgac cgtaaacccg catagtttac cgtacaggcg ttaccgtgac atcgtgtaat 420

gcacctgtcg gcgtgataat gcatataatt ttaacggcta tttgggattt gctcaatcta 480

tacgcaaaga agtttagatg tccagatgta ttgacaatta atcatccggc tcgtataatg 540

tgtggtcaca aaggagatat acatgagtgt gattgcgcag gcaggggcga aaggtcgtca 600

gctgcataaa tttggtggca gtagtctggc tgatgtgaag tgttatttgc gtgtcgcggg 660

cattatggcg gagtactctc agcctgacga tatgatggtg gtttccgccg ccggtagcac 720

cactaaccag ttgattaact ggttgaaact aagccagacc gatcgtctct ctgcgcatca 780

ggttcaacaa acgctgcgtc gctatcagtg cgatctgatt agcggtctgc tacccgctga 840

agaagccgat agcctcatta gcgcttttgt cagcgacctt gagcgcctgg cggcgctgct 900

cgacagcggt attaacgacg cagtgtatgc ggaagtggtg ggccacgggg aagtatggtc 960

ggcacgtctg atgtctgcgg tacttaatca acaagggctg ccagcggcct ggcttgatgc 1020

ccgcgagttt ttacgcgctg aacgcgccgc acaaccgcag gttgatgaa 1069

<210> 11

<211> 2463

<212> DNA

<213>unknown (Unknown)

<400> 11

atgcgagtgt tgaagttcgg cggtacatca gtggcaaatg cagaacgttt tctgcgtgtt 60

gccgatattc tggaaagcaa tgccaggcag gggcaggtgg ccaccgtcct ctctgccccc 120

gccaaaatca ccaaccacct ggtggcgatg attgaaaaaa ccattagcgg ccaggatgct 180

ttacccaata tcagcgatgc cgaacgtatt tttgccgaac ttttgacggg actcgccgcc 240

gcccagccgg ggttcccgct ggcgcaattg aaaactttcg tcgatcagga atttgcccaa 300

ataaaacatg tcctgcatgg cattagtttg ttggggcagt gcccggatag catcaacgct 360

gcgctgattt gccgtggcga gaaaatgtcg atcgccatta tggccggcgt attagaagcg 420

cgcggtcaca acgttactgt tatcgatccg gtcgaaaaac tgctggcagt ggggcattac 480

ctcgaatcta ccgtcgatat tgctgagtcc acccgccgta ttgcggcaag ccgcattccg 540

gctgatcaca tggtgctgat ggcaggtttc accgccggta atgaaaaagg cgaactggtg 600

gtgcttggac gcaacggttc cgactactct gctgcggtgc tggctgcctg tttacgcgcc 660

gattgttgcg agatttggac ggacgttgac ggggtctata cctgcgaccc gcgtcaggtg 720

cccgatgcga ggttgttgaa gtcgatgtcc taccaggaag cgatggagct ttcctacttc 780

ggcgctaaag ttcttcaccc ccgcaccatt acccccatcg cccagttcca gatcccttgc 840

ctgattaaaa ataccggaaa tcctcaagca ccaggtacgc tcattggtgc cagccgtgat 900

gaagacgaat taccggtcaa gggcatttcc aatctgaata acatggcaat gttcagcgtt 960

tctggtccgg ggatgaaagg gatggtcggc atggcggcgc gcgtctttgc agcgatgtca 1020

cgcgcccgta tttccgtggt gctgattacg caatcatctt ccgaatacag catcagtttc 1080

tgcgttccac aaagcgactg tgtgcgagct gaacgggcaa tgcaggaaga gttctacctg 1140

gaactgaaag aaggcttact ggagccgctg gcagtgacgg aacggctggc cattatctcg 1200

gtggtaggtg atggtatgcg caccttgcgt gggatctcgg cgaaattctt tgccgcactg 1260

gcccgcgcca atatcaacat tgtcgccatt gctcagggat cttctgaacg ctcaatctct 1320

gtcgtggtaa ataacgatga tgcgaccact ggcgtgcgcg ttactcatca gatgctgttc 1380

aataccgatc aggttatcga agtgtttgtg attggcgtcg gtggcgttgg cggtgcgctg 1440

ctggagcaac tgaagcgtca gcaaagctgg ctgaagaata aacatatcga cttacgtgtc 1500

tgcggtgttg ccaactcgaa ggctctgctc accaatgtac atggccttaa tctggaaaac 1560

tggcaggaag aactggcgca agccaaagag ccgtttaatc tcgggcgctt aattcgcctc 1620

gtgaaagaat atcatctgct gaacccggtc attgttgact gcacttccag ccaggcagtg 1680

gcggatcaat atgccgactt cctgcgcgaa ggtttccacg ttgtcacgcc gaacaaaaag 1740

gccaacacct cgtcgatgga ttactaccat cagttgcgtt atgcggcgga aaaatcgcgg 1800

cgtaaattcc tctatgacac caacgttggg gctggattac cggttattga gaacctgcaa 1860

aatctgctca atgcaggtga tgaattgatg aagttctccg gcattctttc tggttcgctt 1920

tcttatatct tcggcaagtt agacgaaggc atgagtttct ccgaggcgac cacgctggcg 1980

cgggaaatgg gttataccga accggacccg cgagatgatc tttctggtat ggatgtggcg 2040

cgtaaactat tgattctcgc tcgtgaaacg ggacgtgaac tggagctggc ggatattgaa 2100

attgaacctg tgctgcccgc agagtttaac gccgagggtg atgttgccgc ttttatggcg 2160

aatctgtcac aactcgacga tctctttgcc gcgcgcgtgg cgaaggcccg tgatgaagga 2220

aaagttttgc gctatgttgg caatattgat gaagatggcg tctgccgcgt gaagattgcc 2280

gaagtggatg gtaatgatcc gctgttcaaa gtgaaaaatg gcgaaaacgc cctggccttc 2340

tatagccact attatcagcc gctgccgttg gtactgcgcg gatatggtgc gggcaatgac 2400

gttacagctg ccggtgtctt tgctgatctg ctacgtaccc tctcatggaa gttaggagtc 2460

tga 2463

<210> 12

<211> 1304

<212> DNA

<213>unknown (Unknown)

<400> 12

ttaaagtttt cccgacattg gctgaatcgt tacacgatgt cgatttcact gtcgccacca 60

ctgcgcgcag tcgggcgaaa tatcattact acgccacgcc agttgaactg gtgccgctgt 120

tagaggaaaa atcttcatgg atgagccatg ccgcgctggt gtttggtcgc gaagattccg 180

ggttgactaa cgaagagtta gcgttggctg acgttcttac tggtgtgccg atggtggcgg 240

attatccttc gctcaatctg gggcaggcgg tgatggtcta ttgctatcaa ttagcaacat 300

taatacaaca accggcgaaa agtgatgcaa cggcagacca acatcaactg caagctttac 360

gcgaacgagc catgacattg ctgacgactc tggcagtggc agatgacata aaactggtcg 420

actggttaca acaacgcctg gggcttttag agcaacgaga cacggcaatg ttgcaccgtt 480

tgctgcatga tattgaaaaa aatatcacca aataaaaaac gccttagtaa gtatttttca 540

gcttttcatt ctgactgcaa cgggcaatat gtctctgtgt ggattaaaaa aagagtgtct 600

gatagcagct tctgaactgg ttacctgccg tgagtaaatt aaaattttat tgacaattaa 660

tcatccggct cgtataatgt gtggtcacaa aggagatata catgcgagtg ttgaagttcg 720

gcggtacatc agtggcaaat gcagaacgtt ttctgcgtgt tgccgatatt ctggaaagca 780

atgccaggca ggggcaggtg gccaccgtcc tctctgcccc cgccaaaatc accaaccacc 840

tggtggcgat gattgaaaaa accattagcg gccaggatgc tttacccaat atcagcgatg 900

ccgaacgtat ttttgccgaa cttttgacgg gactcgccgc cgcccagccg gggttcccgc 960

tggcgcaatt gaaaactttc gtcgatcagg aatttgccca aataaaacat gtcctgcatg 1020

gcattagttt gttggggcag tgcccggata gcatcaacgc tgcgctgatt tgccgtggcg 1080

agaaaatgtc gatcgccatt atggccggcg tattagaagc gcgcggtcac aacgttactg 1140

ttatcgatcc ggtcgaaaaa ctgctggcag tggggcatta cctcgaatct accgtcgata 1200

ttgctgagtc cacccgccgt attgcggcaa gccgcattcc ggctgatcac atggtgctga 1260

tggcaggttt caccgccggt aatgaaaaag gcgaactggt ggtg 1304

<210> 13

<211> 2802

<212> DNA

<213>unknown (Unknown)

<400> 13

atgcagaaca gcgctttgaa agcctggttg gactcttctt acctctctgg cgcaaaccag 60

agctggatag aacagctcta tgaagacttc ttaaccgatc ctgactcggt tgacgctaac 120

tggcgttcga cgttccagca gttacctggt acgggagtca aaccggatca attccactct 180

caaacgcgtg aatatttccg ccgcctggcg aaagacgctt cacgttactc ttcaacgatc 240

tccgaccctg acaccaatgt gaagcaggtt aaagtcctgc agctcattaa cgcataccgc 300

ttccgtggtc accagcatgc gaatctcgat ccgctgggac tgtggcagca agataaagtg 360

gccgatctgg atccgtcttt ccacgatctg accgaagcag acttccagga gaccttcaac 420

gtcggttcat ttgccagcgg caaagaaacc atgaaactcg gcgagctgct ggaagccctc 480

aagcaaacct actgcggccc gattggtgcc gagtatatgc acattaccag caccgaagaa 540

aaacgctgga tccaacagcg tatcgagtct ggtcgcgcga ctttcaatag cgaagagaaa 600

aaacgcttct taagcgaact gaccgccgct gaaggtcttg aacgttacct cggcgcaaaa 660

ttccctggcg caaaacgctt ctcgctggaa ggcggtgacg cgttaatccc gatgcttaaa 720

gagatgatcc gccacgctgg caacagcggc acccgcgaag tggttctcgg gatggcgcac 780

cgtggtcgtc tgaacgtgct ggtgaacgtg ctgggtaaaa aaccgcaaga cttgttcgac 840

gagttcgccg gtaaacataa agaacacctc ggcacgggtg acgtgaaata ccacatgggc 900

ttctcgtctg acttccagac cgatggcggc ctggtgcacc tggcgctggc gtttaacccg 960

tctcaccttg agattgtaag cccggtagtt atcggttctg ttcgtgcccg tctggacaga 1020

cttgatgagc cgagcagcaa caaagtgctg ccaatcacca tccacggtga cgccgcagtg 1080

accgggcagg gcgtggttca ggaaaccctg aacatgtcga aagcgcgtgg ttatgaagtt 1140

ggcggtacgg tacgtatcgt tatcaacaac caggttggtt tcaccacctc taatccgctg 1200

gatgcccgtt ctacgccgta ctgtactgat atcggtaaga tggttcaggc cccgattttc 1260

cacgttaacg cggacgatcc ggaagccgtt gcctttgtga cccgtctggc gctcgatttc 1320

cgtaacacct ttaaacgtga tgtcttcatc gacctggtgt gctaccgccg tcacggccac 1380

aacgaagccg acgagccgag cgcaacccag ccgctgatgt atcagaaaat caaaaaacat 1440

ccgacaccgc gcaaaatcta cgctgacaag ctggagcagg aaaaagtggc gacgctggaa 1500

gatgccaccg agatggttaa cctgtaccgc gatgcgctgg atgctggcga ttgcgtagtg 1560

gcagagtggc gtccgatgaa catgcactct ttcacctggt cgccgtacct caaccacgaa 1620

tgggacgaag agtacccgaa caaagttgag atgaagcgcc tgcaggagct ggcgaaacgc 1680

atcagcacgg tgccggaagc agttgaaatg cagtctcgcg ttgccaagat ttatggcgat 1740

cgccaggcga tggctgccgg tgagaaactg ttcgactggg gcggtgcgga aaacctcgct 1800

tacgccacgc tggttgatga aggcattccg gttcgcctgt cgggtgaaga ctccggtcgc 1860

ggtaccttct tccaccgcca cgcggtgatc cacaaccagt ctaacggttc cacttacacg 1920

ccgctgcaac atatccataa cgggcagggc gcgttccgtg tctgggactc cgtactgtct 1980

gaagaagcag tgctggcgtt tgaatatggt tatgccaccg cagaaccacg cactctgacc 2040

atctgggaag cgcagttcgg tgacttcgcc aacggtgcgc aggtggttat cgaccagttc 2100

atctcctctg gcgaacagaa atggggccgg atgtgtggtc tggtgatgtt gctgccgcac 2160

ggttacgaag ggcaggggcc ggagcactcc tccgcgcgtc tggaacgtta tctgcaactt 2220

tgtgctgagc aaaacatgca ggtttgcgta ccgtctaccc cggcacaggt ttaccacatg 2280

ctgcgtcgtc aggcgctgcg cgggatgcgt cgtccgctgg tcgtgatgtc gccgaaatcc 2340

ctgctgcgtc atccgctggc ggtttccagc ctcgaagaac tggcgaacgg caccttcctg 2400

ccagccatcg gtgaaatcga cgagcttgat ccgaagggcg tgaagcgcgt agtgatgtgt 2460

tctggtaagg tttattacga cctgctggaa cagcgtcgta agaacaatca acacgatgtc 2520

gccattgtgc gtatcgagca actctacccg ttcccgcata aagcgatgca ggaagtgttg 2580

cagcagtttg ctcacgtcaa ggattttgtc tggtgccagg aagagccgct caaccagggc 2640

gcatggtact gcagccagca tcatttccgt gaagtgattc cgtttggggc ttctctgcgt 2700

tatgcaggcc gcccggcctc cgcctctccg gcggtagggt atatgtccgt tcaccagaaa 2760

cagcaacaag atctggttaa tgacgcgctg aacgtcgaat aa 2802

<210> 14

<211> 1277

<212> DNA

<213>unknown (Unknown)

<400> 14

aagaagattg tgattcgccc gctgccaggt ttaccggtga tccgcgattt ggtggtagac 60

atgggacaat tctatgcgca atatgagaaa attaagcctt acctgttgaa taatggacaa 120

aatccgccag ctcgcgagca tttacagatg ccagagcagc gcgaaaaact cgacgggctg 180

tatgaatgta ttctctgcgc atgttgttca acctcttgtc cgtctttctg gtggaatccc 240

gataagttta tcggcccggc aggcttgtta gcggcatatc gtttcctgat tgatagccgt 300

gataccgaga ctgacagccg cctcgacggt ttgagtgatg cattcagcgt attccgctgt 360

cacagcatca tgaactgcgt cagtgtatgt ccgaaggggc tgaacccgac gcgcgccatc 420

ggccatatca agtcgatgtt gttgcaacgt aatgcgtaaa ccgtaggcct gataagacgc 480

gcaagcgtcg catcaggcaa ccagtgccgg atgcggcgtg aacgccttat ccggcctaca 540

agtcattacc cgtaggcctg ataagcgcag cgcatcaggc gtaacaaaga aatgcaggaa 600

atcttgacaa ttaatcatcc ggctcgtata atgtgtggtc acaaaggaga tatacatgca 660

gaacagcgct ttgaaagcct ggttggactc ttcttacctc tctggcgcaa accagagctg 720

gatagaacag ctctatgaag acttcttaac cgatcctgac tcggttgacg ctaactggcg 780

ttcgacgttc cagcagttac ctggtacggg agtcaaaccg gatcaattcc actctcaaac 840

gcgtgaatat ttccgccgcc tggcgaaaga cgcttcacgt tactcttcaa cgatctccga 900

ccctgacacc aatgtgaagc aggttaaagt cctgcagctc attaacgcat accgcttccg 960

tggtcaccag catgcgaatc tcgatccgct gggactgtgg cagcaagata aagtggccga 1020

tctggatccg tctttccacg atctgaccga agcagacttc caggagacct tcaacgtcgg 1080

ttcatttgcc agcggcaaag aaaccatgaa actcggcgag ctgctggaag ccctcaagca 1140

aacctactgc ggcccgattg gtgccgagta tatgcacatt accagcaccg aagaaaaacg 1200

ctggatccaa cagcgtatcg agtctggtcg cgcgactttc aatagcgaag agaaaaaacg 1260

cttcttaagc gaactga 1277

<210> 15

<211> 930

<212> DNA

<213>unknown (Unknown)

<400> 15

atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60

tttgtgatga caacttctcg tgcgtctggt caggaaattc gtccacttaa ggttctgatc 120

cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180

tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg 240

cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt 300

gacggtttga ttgtaactgg tgcgccgctg ggcctggtgg agtttaatga tgtcgcttac 360

tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgttt 420

gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa gcaaactcgc 480

accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg 540

cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt tccggcagcg 600

ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660

ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720

caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780

tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac 840

ggtaatttac tgtttaccaa ctggctcaac tattacgtct accagatcac gccatacgat 900

ctacggcaca tgaatccaac gctggattaa 930

<210> 16

<211> 309

<212> PRT

<213>unknown (Unknown)

<400> 16

Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg

1 5 10 15

Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu

20 25 30

Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile

35 40 45

Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln

50 55 60

Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr

65 70 75 80

Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln

85 90 95

Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu

100 105 110

Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu

115 120 125

Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala

130 135 140

Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg

145 150 155 160

Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His

165 170 175

Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser

180 185 190

Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu

195 200 205

Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser

210 215 220

Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala

225 230 235 240

Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp

245 250 255

Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr

260 265 270

Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp

275 280 285

Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met

290 295 300

Asn Pro Thr Leu Asp

305

<210> 17

<211> 1257

<212> DNA

<213>unknown (Unknown)

<400> 17

atgagtggac tcaaacaaga actggggctg gcccagggca ttggcctgct atcgacgtca 60

ttattaggca ctggcgtgtt tgccgttcct gcgttagctg cgctggtagc gggcaataac 120

agcctgtggg cgtggcccgt tttgattatc ttagtgttcc cgattgcgat tgtgtttgcg 180

attctgggtc gccactatcc cagcgcaggc ggcgtcgcgc acttcgtcgg tatggcgttt 240

ggttcgcggc ttgagcgagt caccggctgg ctgtttttat cggtcattcc cgtgggtttg 300

cctgccgcac tacaaattgc cgccgggttc ggccaggcga tgtttggctg gcatagctgg 360

caactgttgt tggcagaact cggtacgctg gcgctggtgt ggtatatcgg tactcgcggt 420

gccagttcca gtgctaatct acaaaccgtt attgccggac ttatcgtcgc gctgattgtc 480

gctatctggt gggcgggcga tatcaaacct gcgaatatcc cctttccggc acctggtaat 540

atcgaactta ccgggttatt tgctgcgtta tcagtgatgt tctggtgttt tgtcggtctg 600

gaggcatttg cccatctcgc ctcggaattt aaaaatccag agcgtgattt tcctcgtgct 660

ttgatgattg gtctgctgct ggcaggatta gtctactggg gctgtacggt agtcgtctta 720

cacttcgacg cctatggtga aaaaatggcg gcggcagcat cgcttccaaa aattgtagtg 780

cagttgttcg gtgtaggagc gttatggatt gcctgcgtga ttggctatct ggcctgcttt 840

gccagtctca acatttatat acagagcttc gcccgcctgg tctggtcgca ggcgcaacat 900

aatcctgacc actacctggc acgcctctct tctcgccata tcccgaataa tgccctcaat 960

gcggtgctcg gctgctgtgt ggtgagcact ttggtgattc atgctttaga gatcaatctg 1020

gacgctctta ttatttatgc caatggcatc tttattatga tttatctgtt atgcatgctg 1080

gcaggctgta aattattgca aggacgttat cgactactgg cggtggttgg cgggctgtta 1140

tgcgttctgt tactggcaat ggtcggctgg aaaagtctct atgcgctgat catgctggcg 1200

gggttatggc tgttgctgcc aaaacgaaaa acgccggaaa atggcataac cacataa 1257

<210> 18

<211> 72

<212> DNA

<213>unknown (Unknown)

<400> 18

ttgacgtcca ttaacacaat gtttactctg gtgcctgaca tttcaccgac aaagcccagg 60

gaacttcatc ac 72

<210> 19

<211> 226

<212> DNA

<213>unknown (Unknown)

<400> 19

ttgacttagg tcactaaata ctttaaccaa tataggcata gcgcacagac agataaaaat 60

tacagagtac acaacatcca tgaaacgcat tagcaccacc attaccacca ccatcaccat 120

taccacaggt aacggtgcgg gctgacgcgt acaggaaaca cagaaaaaag cccgcacctg 180

acagtgcggg cttttttttt cgaccaaagg taacgaggta acaacc 226

<210> 20

<211> 156

<212> DNA

<213>unknown (Unknown)

<400> 20

tttaaaaact gcccctgaca ctaagacagt ttttaaaggt tccttcgcga gccactacgt 60

agacaagagc tcgcaagtga accccggcac gcacatcact gtgcgtggta gtatccacgg 120

cgaagtaagc ataaaaaaga tgcttaaggg atcacg 156

<210> 21

<211> 52

<212> DNA

<213>unknown (Unknown)

<400> 21

ttgacaatta atcatccggc tcgtataatg tgtggtcaca aaggagatat ac 52

Claims (9)

1. it is a kind of produce O- succinyl-L- homoserine recombination bacillus coli, it is characterised in that the recombination bacillus coli be by The metI gene that L-Methionine transport protein is encoded in Escherichia coli, the metJ gene for encoding negative regulation repressor, coding Guang After the metB gene of thioether γ synzyme, the thrB gene of encoded homoserine kinase successively knock out, then Kosé will be encoded respectively The thrA gene of propylhomoserin dehydrogenase I, the metL gene of encoded homoserine dehydrogenase II and coding α-ketoglutaric acid decarboxylase The promoter of sucA gene replaces with Ptrc promoter, and the metA for finally importing encoded homoserine transsuccinylase again is prominent Become gene and encode the yjeH gene of L-Methionine outward transport albumen, the metA mutated gene is by metA gene coded protein the 64 glutamic acid replace with glutamine acquisition.

2. producing the recombination bacillus coli of O- succinyl-L- homoserine as described in claim 1, it is characterised in that the P_trcIt opens Mover nucleotides sequence is classified as shown in SEQ ID NO.21.

3. producing the recombination bacillus coli of O- succinyl-L- homoserine as described in claim 1, it is characterised in that the metI base Because nucleotides sequence is classified as shown in SEQ ID NO.1, metJ gene nucleotide series be SEQ ID NO.3 shown in, metB gene core Nucleotide sequence is shown in SEQ ID NO.5, thrB gene nucleotide series are shown in SEQ ID NO.7.

4. producing the recombination bacillus coli of O- succinyl-L- homoserine as described in claim 1, it is characterised in that the yjeH base Because nucleotides sequence is classified as shown in SEQ ID NO.17, the amino acid sequence of metA gene coded protein is SEQ ID NO.16 institute Show.

5. the recombination bacillus coli for producing O- succinyl-L- homoserine described in a kind of claim 1 produces O- succinyl-L- in fermentation Application in homoserine.

6. application as claimed in claim 5, it is characterised in that the application is that the recombination bacillus coli is seeded to hair Ferment culture medium, fermented and cultured under the conditions of 30 DEG C, 180-200rpm, culture solution is isolated and purified, and obtains O- succinyl-L- Kosé Propylhomoserin.

7. application as claimed in claim 6, it is characterised in that the fermentation medium final concentration composition are as follows: glucose 40/L, Potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L, yeast powder 4g/L, calcium carbonate 30g/L, L-threonine 0.2g/L, L-Methionine 0.2g/L, l-Isoleucine 0.2g/L, vitamin B₁ 0.0001g/L、MgSO₄ 2g/L、FeSO₄ 0.005g/L、MnSO₄ 0.005g/L、ZnSO₄0.005g/L, solvent are deionized water, pH value 6.8.

8. application as claimed in claim 6, it is characterised in that slant activation and seed culture are first carried out before the fermented and cultured, Seed liquor is seeded to fermentation medium, the slant activation method with the inoculum concentration of volumetric concentration 5-10% are as follows: recombination is big Enterobacteria is inoculated on LB plate, in 37 DEG C of overnight incubations, obtains inclined-plane thalline；The seed culture method are as follows: picking inclined-plane Thallus single colonie is seeded in LB culture medium, overnight incubation under conditions of 37 DEG C, 200rpm, obtains seed liquor.

9. application as claimed in claim 6, it is characterised in that the fermented and cultured carries out in the fermenter, by adding feed supplement Culture medium controls concentration of glucose 2-10g/L in fermentor；Fermentation condition is DO level 30%, speed of agitator 200- 600rpm, Ventilation Rate are controlled in 1-2vvm；Cultivation temperature is controlled in fermentation process to adjust at 30 DEG C and with 50% ammonium hydroxide PH6.8~7.0；Fermentation cylinder for fermentation culture medium composition: glucose 40g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L, yeast Powder 4g/L, L-threonine 0.2g/L, L-Methionine 0.2g/L, l-Isoleucine 0.2g/L, MgSO₄ 2g/L、FeSO₄ 0.005g/ L、MnSO₄ 0.005g/L、ZnSO₄0.005g/L, vitamin B₁0.0001g/L, solvent are deionized water, pH value 6.8；Feed supplement Culture medium composition: glucose 500g/L, potassium dihydrogen phosphate 12.5g/L, L-threonine 2g/L, the different bright ammonia of L-Methionine 2g/L, L- Sour 2g/L, solvent are deionized water.