CN108949661B - Recombinant escherichia coli for producing O-succinyl-L-homoserine and application thereof - Google Patents

Abstract

The invention discloses a recombinant escherichia coli transformed by means of metabolic engineering and a method for producing an O-succinyl-L-homoserine strain by using the recombinant escherichia coli. The invention firstly realizes the partial inactivation of MetD of an L-methionine import system by interrupting metI and overexpresses yjeH gene on a plasmid to promote the excretion of intracellular L-methionine, so that the homoserine O-succinyl transferase encoded by metA gene of the strain keeps high activity and stability. Secondly, the repression inhibition effect of the relevant genes (thrA, metL, lysC, asd, metA and the like) in the anabolic pathway of O-succinyl-L-homoserine is relieved by knocking out the negative regulatory transcription factor MetJ. Finally, the in situ promoter sequences of metL, thrA and sucA genes in the metabolic pathway were replaced by using Ptrc promoter, and metB gene in the metabolic pathway of O-succinyl-L-homoserine was blocked. Through the combination of the above transformation strategies, the Escherichia coli strain with high O-succinyl-L-homoserine yield is obtained.

Description

Recombinant escherichia coli for producing O-succinyl-L-homoserine and application thereof

Technical Field

The invention relates to recombinant escherichia coli for producing O-succinyl-L-homoserine and application of the recombinant escherichia coli in producing O-succinyl-L-homoserine by fermentation.

Background

O-succinyl-L-homoserine not only serves as an important intermediate for methionine synthesis in microorganisms, but also plays a very important role in the industrial production of the C4 compound. O-succinyl-L-homoserine biosynthesis in E.coli is specifically catalyzed by metA-encoded homoserine acetyltransferase, which includes homoserine- γ -hydroxy activation and succinyl-CoA acyl process. In the process of synthesizing O-succinyl-L-homoserine, metA gene is feedback-inhibited by L-methionine, which is an essential amino acid for growth and development of microorganism, so that it is important to obtain a mutant gene capable of resisting feedback inhibition for improving the production capacity of O-succinyl-L-homoserine. In the reported Patent (US Patent 7,851,180), feedback inhibition resistance of metA gene has been studied, and the improvement of O-succinyl-L-homoserine production has been shown to be effective. L-methionine is essential amino acid for biological growth, proteins related to L-methionine transport are distributed on cell membranes of organisms, two specific methionine internal transport systems including MetD and MetP exist in E. Whereas L-methionine auxotrophic strains activate transporters that transport L-methionine into the cell, resulting in an increase in the intracellular L-methionine content, metI was knocked out to effect partial inactivation of MetD of the L-methionine import system in order to lower the intracellular L-methionine content and thereby alleviate the feedback inhibition of metA gene, metD operon comprising metN, metI and metQ, ATPase, methionine permease, methionine binding protein encoding ABC transporter elements, respectively.

In order to decompose O-succinyl-L-homoserine into cysteine by cystathionine gamma synthase encoded by metB gene in microorganism, metB gene needs to be deleted in order to allow O-succinyl-L-homoserine to be accumulated in large amount.

Aspartic acid is the only precursor for synthesizing O-succinyl-L-homoserine by E coli, and is also the synthetic precursor of threonine, isoleucine, lysine and branched chain amino acid. Biosynthesis from aspartate to homoserine requires a four-step reaction in which thrA and metL encode homoserine dehydrogenase and aspartokinase II, which are bifunctional enzymes having aspartokinase activity and homoserine dehydrogenase activity, catalyzing the reduction of aspartate-semialdehyde to homoserine. the transcription levels of thrA, metL, lysC, asd, and metA genes are all regulated by the negative transcription factor MetJ, and thus, the metJ gene needs to be knocked out. L-threonine biosynthesis takes L-homoserine as a precursor, and is catalyzed by homoserine kinase and threonine synthase encoded by thrB and thrC genes, and O-succinyl-L-homoserine and L-threonine anabolism compete with each other for the precursor L-homoserine, so that it is necessary to block or reduce the anabolic flux of L-threonine. In addition, intracellular substrate levels during O-succinyl-L-homoserine biosynthesis also affect its production, and succinyl-CoA is an important substrate, so it is necessary to increase intracellular succinyl-CoA content by metabolic engineering means.

Disclosure of Invention

The object of the present invention is to transform E.coli by metabolic engineering means to obtain a recombinant E.coli strain with high O-succinyl-L-homoserine production.

The technical scheme adopted by the invention is as follows:

the invention provides a recombinant Escherichia coli for producing O-succinyl-L-homoserine, the recombinant Escherichia coli is obtained by sequentially knocking out metI gene encoding L-methionine transport protein, metJ gene encoding negative regulatory repressor, metB gene encoding cystathionine gamma synthetase, and thrB gene encoding homoserine kinase in Escherichia coli, then replacing the thrA gene coding homoserine dehydrogenase I, the metL gene coding homoserine dehydrogenase II and the sucA gene coding alpha-ketoglutarate decarboxylase with Ptrc promoter, finally introducing the metA mutant gene coding homoserine O-succinyl transferase and the yjeH gene coding L-methionine export protein, the metA mutant gene is obtained by replacing glutamic acid at position 64 of metA gene encoding protein (shown in SEQ ID NO. 16) with glutamine.

Further, said P_trcThe nucleotide sequence of the promoter is shown as SEQ ID NO. 21.

Further, the nucleotide sequence of the metI gene is shown as SEQ ID NO.1, the nucleotide sequence of the metJ gene is shown as SEQ ID NO.3, the nucleotide sequence of the metB gene is shown as SEQ ID NO.5, and the nucleotide sequence of the thrB gene is shown as SEQ ID NO. 7.

Further, the nucleotide sequence of the yjeH gene is shown as SEQ ID NO.17, the nucleotide sequence of the metA gene is shown as SEQ ID NO.15, and the amino acid sequence of the metA gene coding protein is shown as SEQ ID NO. 16.

The invention also provides application of the recombinant Escherichia coli for producing O-succinyl-L-homoserine in producing O-succinyl-L-homoserine by fermentation, wherein the application comprises the steps of inoculating the recombinant Escherichia coli to a fermentation culture medium, performing fermentation culture at 30 ℃ and 180-200rpm, and separating and purifying a culture solution to obtain O-succinyl-L-homoserine.

Further, the final concentration composition of the fermentation medium is 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, and vitamin B₁ 0.0001g/L、MgSO₄ 2g/L、FeSO₄ 0.005g/L、MnSO₄ 0.005g/L、ZnSO₄0.005g/L, deionized water as solvent, and pH 6.8.

Further, before the fermentation culture, slant activation and seed culture are carried out, and a seed solution is inoculated to the fermentation culture medium in an inoculation amount with a volume concentration of 5%, wherein the slant activation method comprises the following steps: inoculating the recombinant escherichia coli on an LB flat plate, and culturing overnight at 37 ℃ to obtain slant thalli; the seed culture method comprises the following steps: the single colony of the slant strain was inoculated into LB medium and cultured overnight at 37 ℃ and 200rpm to obtain a seed solution.

The fermentation culture is carried out in a fermentation tank, and the concentration of glucose in the fermentation tank is controlled to be 2-10g/L by adding a supplemented medium; the fermentation conditions are that the DO level is 30%, the stirring speed is 200-600rpm, and the aeration rate is controlled to be 1-2 vvm; and in the fermentation process, the culture temperature is controlled at 30 ℃, and the pH is adjusted to be 6.8-7.0 by using 50% ammonia water. The fermentation medium in the fermentation tank consists of: 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, deionized water as solvent, and pH 6.8; the composition of a supplemented medium is as follows: glucose 500g/L, phosphoric acid twoPotassium hydride 12.5g/L, L-threonine 2g/L, L-methionine 2g/L, L-isoleucine 2g/L, and the solvent is deionized water.

The recombinant Escherichia coli for producing O-succinyl-L-homoserine can relieve the repression inhibition effect on related genes (thrA, metL, lysC, asd, metA and the like) in an O-succinyl-L-homoserine anabolic pathway by knocking out a negative regulation transcription factor MetJ. First, the negative transcription factor gene metJ in Ecoli W3110 is knocked out, and the metI gene is knocked out to partially inactivate the L-methionine endoglin MetD, so that the intracellular methionine amount is reduced, and the substrate inhibition effect of methionine on homoserine O-succinyl transferase (MetA) can be reduced. By knocking out thrB gene, the precursor homoserine of O-succinyl-L-homoserine is reduced to be out of the way to branch amino acid metabolism. At the same time, the metB gene was knocked out to effect the degradation of the blocking product O-succinyl-L-homoserine. With a strong promoter P_trcReplacement of the in situ promoter sequence of the metL and thrA genes to increase the expression of aspartokinase II and homoserine dehydrogenase and use of a strong promoter P_trcThe sucA gene in situ promoter sequence was replaced to increase intracellular succinyl-CoA levels. Connecting yjeH gene on pTrc99A plasmid to promote the secretion of intracellular L-methionine, making the homoserine O-succinyl transferase encoded by metA gene of strain keep high activity and stability, and introducing the connecting plasmid into the above-mentioned modified Escherichia coli to make induction expression so as to make intracellular L-methionine only be required for growth of microorganism, and do not produce large influence on homoserine O-succinyl transferase encoded by metA gene. Intracellular homoserine O-succinyl transferase is transformed into O-succinyl-L-homoserine with minimal influence by L-methionine and secreted to the outside of the cell by overexpression of methionine desensitized homoserine O-succinyl transferase and methionine export protein.

The term "feedback inhibition" of the present invention means that homoserine O-succinyl transferase activity is inhibited by L-methionine. The present invention relates to a recombinant vector containing the YjeH encoding gene of the L-methionine transport system. The recombinant vector comprises a polynucleotide operably linked to control sequences suitable for directing expression in a host cell. Preferably, the expression vector is pTrc 99A. The term "enhance" refers to increasing the activity of an enzyme encoded by a corresponding polynucleotide. The expression regulatory sequence of the gene on the genome can be replaced by overexpression of the gene (promoter replacement, etc.).

The vector used in the present invention may not be particularly limited, and any vector known in the art may be used as long as the vector is replicable in the host.

The expression of the polypeptide of the present invention may be carried out by inserting a recombinant vector comprising a gene encoding the polypeptide or by inserting a polynucleotide encoding the polypeptide into a chromosome by means of transformation, but the method is not limited thereto.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for producing O-succinyl-L-homoserine with high yield by using escherichia coli modified by metabolic engineering. Compared with the wild type, the modified Escherichia coli can better utilize carbon source substances such as glucose and the like to produce O-succinyl-L-homoserine, and the yield of the modified Escherichia coli is improved from 0.14g/L to 24.25 g/L. The further modified L-methionine transport system of the invention can relieve the feedback inhibition effect of L-methionine substrate on MetA to achieve high yield of O-succinyl-L-homoserine, and produce L-methionine and other industrially important C4 compounds by the precursor.

Drawings

FIG. 1 construction process and map of pTrc9999A-metA11 recombinant vector in example 3.

FIG. 2 construction and map of the pA11y recombinant vector in example 4.

FIG. 3 structural formula of OSH in example 5.

FIG. 4 detection of OSH by High Performance Liquid Chromatography (HPLC) in example 5.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples are all conventional biochemical reagents unless otherwise specified.

Composition of LB medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride, deionized water as a solvent and natural pH value. LB plate is LB liquid medium with a final concentration of 2g/L agar.

Example 1 Metabolic engineering of strains based on wild-type E.coli

(1) Deletion of metI Gene

In order to block metI to achieve partial inactivation of the L-methionine import System MetD, which leads to a reduction in L-methionine uptake to reduce the feedback inhibition of the metA gene, the metI gene in wild-type strains has been knocked out, see Jiang Y, Chen B, Duan C, et al.

The metI gene encoding The L-methionine transport system in The genome of wild-type E.coli (Escherichia Coli) W3110 (purchased from The Coli Genetic Stock Center, E.coli Genetic Breeding Center) was edited by The CRISPR-Cas9 system. A pTarget- Δ metI mutation vector capable of expressing sgRNA targeting the target gene metI (nucleotide sequence shown in SEQ ID No. 1) was constructed by PCR using primer 1 and primer 2 and pTargetF vector as a template. The PCR reaction conditions were as follows: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 15s, 55 deg.C for 15s, and 72 deg.C for 2 min; extension was continued for 10min at 72 ℃. The PCR product was treated with Dpn I at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient cells, spread on LB solid plates containing a final concentration of 50mg/L spectinomycin hydrochloride resistance, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony, transferring the single colony into an LB liquid culture medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain a pTarget-delta metI vector.

The upstream homologous fragment of metI gene was amplified by PCR using primers 3 and 4 and Escherichia coli W3110 genome as template, and the PCR conditions were as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; extension was continued for 10min at 72 ℃. The downstream homologous fragment of metI gene was obtained by amplification using primer 5 and primer 6 in the same manner, PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by gel cutting. The two recovered DNA fragments were subjected to fusion PCR using primers 3 and 6 under the following conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1 min; the extension is continued for 10min at 72 ℃, the PCR product is detected by 1.0% agarose gel electrophoresis, and the fragment (the nucleotide sequence is shown as SEQ ID NO. 2) is recovered and purified by cutting gel. The pTarget-. DELTA.metI vector and the recovered DNA fragment were electroporated together into Escherichia coli W3110 strain containing pCas9 vector.

For electroporation, Escherichia coli W3110 strain transformed with pCas9 vector was cultured in LB medium containing 50mg/L kanamycin and 10mM L-arabinose at 30 ℃ until OD600 reached 0.6, and the bacterial suspension was centrifuged to obtain bacterial cells. The cells were washed twice with sterile distilled water and then once with 10% glycerol for use. Electroporation was performed at 2.5 KV.

The bacterial suspension after the electric transformation was applied to LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance and cultured overnight at 30 ℃. A single colony was picked as a template, PCR was performed with primer 7 and primer 8, and deletion of metI gene was confirmed by observing the presence of a 1000bp DNA band in 1.0% agarose gel. The strain confirmed by this was cultured overnight at 30 ℃ in LB medium containing 50mg/L kanamycin and 5mM IPTG to remove pTarget-. DELTA.metI vector. The strain from which pTarget- Δ metI vector had been removed was then cultured overnight in LB medium at 37 ℃ to remove pCas vector. The strain thus constructed was designated as W3110. DELTA.metI.

TABLE 1 primer sequences

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) Deletion of metJ Gene

In order to remove the repression inhibition effect of negative regulatory transcription factor MetJ on the transcription level of thrA, metL, lysC, asd, metA and other genes, the metJ gene in W3110 delta metI strain was knocked out.

The metJ gene encoding a negative regulatory transcription factor in the genome of strain W3110. delta. metI was edited by CRISPR-Cas9 system. A pTarget- Δ metJ mutation vector capable of expressing sgRNA targeting a target gene metJ (nucleotide sequence shown in SEQ ID No. 3) was constructed by PCR using primers 9 and 10 and pTargetF vector as a template. The PCR reaction conditions were as follows: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 15s, 55 deg.C for 15s, and 72 deg.C for 2 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 50mg/L spectinomycin hydrochloride resistance, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony, transferring the single colony into an LB liquid culture medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain a pTarget-delta metJ vector.

The upstream homologous fragment of metJ gene was amplified by PCR using primers 11 and 12 and E.coli Escherichia coli W3110 genome as template, and the PCR conditions were as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; extension was continued for 10min at 72 ℃. The downstream homologous fragment of metJ gene was amplified by the same method using primer 13 and primer 14, the PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by cutting the gel. The two recovered DNA fragments were subjected to fusion PCR using primers 11 and 14 under the following conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1 min; the extension is continued for 10min at 72 ℃, the PCR product is detected by 1.0% agarose gel electrophoresis, and the fragment (the nucleotide sequence is shown as SEQ ID NO. 4) is recovered and purified by cutting gel. The pTarget-. DELTA.metJ vector and the recovered DNA fragment were electroporated into W3110. DELTA.metI strain harboring pCas9 vector.

For electroporation, W3110. delta. metI strain transformed with pCas9 vector was cultured in LB medium containing 50mg/L kanamycin and 10mM L-arabinose at 30 ℃ until OD600 reached 0.6, and the bacterial suspension was centrifuged to obtain bacterial cells. The cells were washed twice with sterile distilled water and then once with 10% glycerol for use. Electroporation was performed at 2.5 KV.

The bacterial suspension after the electric transformation was applied to LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance and cultured overnight at 30 ℃. A single colony was picked as a template, PCR was performed with primer 15 and primer 16, and deletion of metJ gene was confirmed by observing the presence of a 1000bp DNA band in 1.0% agarose gel. The strain confirmed by this was cultured overnight at 30 ℃ in LB medium containing 50mg/L kanamycin and 5mM IPTG to remove pTarget-. DELTA.metJ vector. The strain from which pTarget- Δ metJ vector had been removed was then cultured overnight in LB medium at 37 ℃ to remove pCas vector. The strain thus constructed was designated as W3110. DELTA. metI. DELTA. metJ.

TABLE 2 primer sequences

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) Deletion of metB Gene

In order to block the catabolic pathway of O-succinyl-L-homoserine, the catabolic synthesis of cysteine from O-succinyl-L-homoserine was disrupted by deleting metB gene encoding cystathionine gamma synthase to obtain high concentration of O-succinyl-L-homoserine, so that metB gene was deleted in W3110. delta. metI. delta. metJ strain.

The metB gene encoding cystathionine γ synthase in the genome of W3110 Δ metI Δ metJ strain was edited by CRISPR-Cas9 system. A pTarget- Δ metB mutation vector capable of expressing sgRNA targeting a target gene metB (nucleotide sequence shown in SEQ ID No. 5) was constructed by PCR using primers 17 and 18, and pTargetF vector as a template. The PCR reaction conditions were as follows: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 15s, 55 deg.C for 15s, and 72 deg.C for 2 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 50mg/L spectinomycin hydrochloride resistance, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony, transferring the single colony into an LB liquid culture medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain a pTarget-delta metB vector.

The upstream homologous fragment of metB gene was amplified by PCR using primer 19 and primer 20 and genome of W3110. delta. metI. delta. metJ strain as template, and PCR reaction conditions were as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; extension was continued for 10min at 72 ℃. The downstream homologous fragment of metB gene was amplified by the same method using primer 21 and primer 22, the PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by gel cutting. The two recovered DNA fragments were subjected to fusion PCR using primers 19 and 22 under the following PCR conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1 min; the extension is continued for 10min at 72 ℃, the PCR product is detected by 1.0% agarose gel electrophoresis, and the fragment (the nucleotide sequence is shown as SEQ ID NO. 6) is recovered and purified by cutting gel. The pTarget-. DELTA.metB vector and the recovered DNA fragment were electroporated into W3110. DELTA.metI. DELTA.metJ strain harboring pCas9 vector.

For electroporation, W3110. delta. metI. delta. metJ strain transformed with pCas9 vector was cultured in LB medium containing 50mg/L kanamycin and 10mM L-arabinose at 30 ℃ until OD600 reached 0.6, and the bacterial suspension was centrifuged to obtain bacterial cells. The cells were washed twice with sterile distilled water and then once with 10% glycerol for use. Electroporation was performed at 2.5 KV. The bacterial suspension after the electric transformation was applied to LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance and cultured overnight at 30 ℃. A single colony was picked as a template, PCR was performed with the primers 23 and 24, and deletion of metB gene was confirmed by observing the presence of a 1000bp DNA band in 1.0% agarose gel. The strain confirmed by this was cultured overnight at 30 ℃ in LB medium containing 50mg/L kanamycin and 5mM IPTG to remove pTarget-. DELTA.metB vector. The strain from which pTarget- Δ metB vector had been removed was then cultured overnight in LB medium at 37 ℃ to remove pCas vector. The strain thus constructed was designated as W3110. DELTA. metI. DELTA. metJ. DELTA. metB.

TABLE 3 primer sequences

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) ThrB Gene knockout

In order to further accumulate the amount of L-homoserine, a precursor of intracellular O-succinyl-L-homoserine, the L-homoserine, is metabolized to synthesize L-threonine by thrB gene and thrC gene-encoded homoserine kinase and threonine synthase, which are required to block the L-homoserine metabolic pathway. Thus, the thrB gene was knocked out in the W3110. DELTA. metI. DELTA. metJ. DELTA. metB strain.

The thrB gene encoding homoserine kinase in the genome of W3110. delta. metI. delta. metJ. delta. metB strain was edited by CRISPR-Cas9 system. A pTarget- Δ thrB mutant vector capable of expressing sgRNA targeting a target gene thrB (nucleotide sequence shown in SEQ ID NO. 7) was constructed by PCR using the pTargetF vector as a template using primers 25 and 26. The PCR reaction conditions were as follows: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 15s, 55 deg.C for 15s, and 72 deg.C for 2 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 50mg/L spectinomycin hydrochloride resistance, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony, transferring the single colony into an LB liquid culture medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain a pTarget-delta thrB vector.

The upstream homologous fragment of thrB gene was amplified by PCR using primer 27 and primer 28, using the genome of W3110. delta. metI. delta. metJ. delta. metB strain as template, under the following conditions: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; extension was continued for 10min at 72 ℃. The downstream homologous fragment of thrB gene was obtained by amplification using primers 29 and 30 in the same manner, the PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by gel cutting. The two recovered DNA fragments were subjected to fusion PCR using primers 27 and 30 under the following PCR conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1 min; the extension is continued for 10min at 72 ℃, the PCR product is detected by 0.9% agarose gel electrophoresis, and the fragment (the nucleotide sequence is shown in SEQ ID NO. 8) is recovered and purified by cutting gel. The pTarget-. DELTA.thrB vector and the recovered DNA fragment were electroporated into W3110. DELTA.metI. DELTA.metJ. DELTA.metB strain with pCas9 vector.

For electroporation, W3110. delta. metI. delta. metJ. delta. metB strain transformed with pCas9 vector was cultured in LB medium containing 50mg/L kanamycin and 10mM L-arabinose at 30 ℃ until OD reached 0.6, and the bacterial suspension was centrifuged to obtain bacterial cells. The cells were washed twice with sterile distilled water and then once with 10% glycerol for use. Electroporation was performed at 2.5 KV.

The bacterial suspension after the electric transformation was applied to LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance and cultured overnight at 30 ℃. A single colony was picked as a template, PCR was performed with the primers 31 and 32, and deletion of thrB gene was confirmed by observing the presence of a 1000bp DNA band in 1.0% agarose gel. The strain confirmed by this was cultured overnight at 30 ℃ in LB medium containing 50mg/L kanamycin and 5mM IPTG to remove pTarget-. DELTA.thrB vector. The strain from which pTarget- Δ thrB vector had been removed was then cultured overnight in LB medium at 37 ℃ to remove pCas vector. The strain thus constructed was designated as W3110. DELTA. metI. DELTA. metJ. DELTA. metB. DELTA. thrB.

TABLE 4 primer sequences

Example 2 Metabolic engineering of strains based on W3110. DELTA. metI. DELTA. metJ. DELTA. metB. thrB

(1) Enhancement of metL Gene expression

The enzyme coded by metL gene has the double-function enzyme activity of aspartokinase II and homoserine dehydrogenase II, and in the metabolism of L-aspartic acid, metL and thrA genes play similar roles, so that the original promoter sequence shown in SEQ ID NO.9 of the metL gene nucleotide sequence is replaced by trc promoter sequence (shown in SEQ ID NO. 21) to achieve the purpose of enhancing the metL gene expression.

The promoter sequence of metL gene encoding homoserine dehydrogenase in the genome of W3110. delta. metI. delta. metJ. delta. metB. thrB strain was edited by CRISPR-Cas9 system. A pTarget-delta PmetL:: Ptrc mutation vector capable of expressing sgRNA targeting the metL promoter sequence of the gene of interest was constructed by PCR using primers 33 and 34, and the pTargetF vector as a template. The PCR reaction conditions were as follows: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 15s, 55 deg.C for 15s, and 72 deg.C for 2 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 50mg/L spectinomycin hydrochloride resistance, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony, transferring the single colony to an LB liquid culture medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain pTarget-delta PmetL:: Ptrc vector.

An upstream homologous fragment of the metL gene promoter sequence obtained by PCR using primers 35 and 36 and using the genome of W3110. delta. metI. delta. metJ. delta. metB. thrB strain as a template, the promoter sequence information of the metL gene was obtained based on the Ecocyc E.coli Database (EcoCyc gene accession number: EG10590, nucleotide sequence shown in SEQ ID NO. 18), and the PCR reaction conditions were as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; extension was continued for 10min at 72 ℃. The downstream homologous fragment of the metL gene promoter sequence was obtained by amplification using primer 37 and primer 38 in the same manner, PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by cutting the gel. The two recovered DNA fragments were subjected to fusion PCR using primers 35 and 38 under the following conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1 min; and continuing to extend for 10min at 72 ℃, detecting the PCR product by using 1.0% agarose gel electrophoresis, cutting the gel, recovering and purifying the fragment (the nucleotide sequence is shown as SEQ ID NO. 10), and inserting the Ptrc promoter sequence between the two homologous fragments in the gene band. Ptarget-. DELTA.PmetL:, Ptrc vector and recovered DNA fragment were electroporated together into W3110. DELTA.metI. DELTA.metJ. DELTA.metB. DELTA.thrB strain with pCas9 vector.

For electroporation, W3110. delta. metI. delta. metJ. delta. metB. thrB strain transformed with pCas9 vector was cultured in LB medium containing 50mg/L kanamycin and 10mM L-arabinose at 30 ℃ until OD600 reached 0.6, and the bacterial suspension was centrifuged to obtain bacterial cells. The cells were washed twice with sterile distilled water and then once with 10% glycerol for use. Electroporation was performed at 2.5 KV.

The bacterial suspension after the electric transformation was applied to LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance and cultured overnight at 30 ℃. A single colony was picked as a template, PCR was performed with the primers 39 and 40, and it was confirmed that the original promoter sequence of metL gene had been replaced with Ptrc promoter sequence by observing the presence of 700bp DNA band in 1.0% agarose gel. The strain thus confirmed was cultured overnight at 30 ℃ in LB medium containing 50mg/L of kanamycin and 5mM IPTG to remove pTarget-. DELTA.PmetL:: Ptrc vector. The strain from which pTarget-. DELTA.PmetL:. Ptrc vector had been removed was then cultured overnight in LB medium at 37 ℃ to remove pCas vector. The strain from which the pCas vector was removed was subjected to PCR amplification using primers 39 and 38 under the following PCR reaction conditions: 5min at 95 ℃; 30 cycles of 95 ℃ 30s, 55 ℃ 30s, 72 ℃ 1min15s were repeated; and continuing to extend for 10min at 72 ℃, carrying out sequencing verification on the PCR product, and confirming that the in-situ promoter sequence of the metL gene is successfully replaced by the Ptrc promoter through BLAST sequence alignment. The constructed strain was designated W3110. DELTA. metI. DELTA. metJ. DELTA. metB. thrB-metL (trc). The same procedure was used to construct strain W3110. DELTA. metI. DELTA. metJ. DELTA. metB-metL (trc) and the primers used to prepare this genotype strain were identical.

TABLE 5 primer sequences

(2) Enhancement of thrA Gene expression

the thrA gene coded enzyme has the activity of aspartokinase I and homoserine dehydrogenase I dual-function enzyme, in order to enable L-aspartic acid to flow more carbon source to precursor substance of O-succinyl-L-homoserine in the process of metabolizing the main path of L-homoserine, the trc promoter sequence is used for replacing the original promoter sequence in the thrA gene (the nucleotide sequence is shown in SEQ ID NO. 11) so as to achieve the purpose of enhancing the expression of the thrA gene.

The promoter sequence of thrA gene encoding homoserine dehydrogenase in the genome of W3110. delta. metI. delta. metJ. delta. metB. thrB-metL (trc) strain was edited by CRISPR-Cas9 system. A pTarget-delta PthrA:: Ptrc mutation vector capable of expressing sgRNA targeting the thrA promoter sequence of the gene of interest was constructed by PCR using primers 41 and 42, and the pTargetF vector as a template. The PCR reaction conditions were as follows: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 15s, 55 deg.C for 15s, and 72 deg.C for 2 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 50mg/L spectinomycin hydrochloride resistance, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony, transferring the single colony to an LB liquid culture medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain pTarget-delta PthrA and a Ptrc vector.

The upstream homologous fragment of the thrA gene promoter sequence was obtained by PCR using primer 43 and primer 44 and using the genome of W3110. delta. metI. delta. metJ. delta. metB. delta. thrB-metL (trc) strain as a template, the promoter sequence information of the thrA gene was obtained from Ecocyc E.coli Database (EcoCyc Gene accession No: EG10998), and the PCR conditions were as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; extension was continued for 10min at 72 ℃. The downstream homologous fragment of the thrA gene promoter sequence is obtained by amplification by using a primer 45 and a primer 46 according to the same method, a PCR product is detected by 1.0% agarose gel electrophoresis, and a purified fragment (the nucleotide sequence is shown as SEQ ID NO. 12) is recovered by cutting gel. The two recovered DNA fragments were subjected to fusion PCR using primers 43 and 46 under the following conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1 min; extension was continued at 72 ℃ for 10min, the PCR product was detected by 1.0% agarose gel electrophoresis and the fragment was recovered and purified by gel cutting, and the Ptrc promoter sequence had been inserted between the two homologous fragments in the gene band. Ptarget-. DELTA.PthrA vector and the recovered DNA fragment were electroporated into W3110. DELTA.metI. DELTA.metJ. DELTA.metB. thrB-metL (trc) strain with pCas9 vector.

For electroporation, W3110. delta. metI. delta. metJ. delta. metB. DELTA. thrB-metL (trc) strain transformed with pCas9 vector was cultured in LB medium containing 50mg/L of kanamycin and 10mM of L-arabinose at 30 ℃ until OD600 reached 0.6, and the bacterial suspension was centrifuged to obtain cells. The cells were washed twice with sterile distilled water and then once with 10% glycerol for use. Electroporation was performed at 2.5 KV.

The bacterial suspension after the electric transformation was applied to LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance and cultured overnight at 30 ℃. A single colony was picked as a template, PCR was performed with the primer 47 and the primer 48, and it was confirmed that the original promoter sequence of the thrA gene had been replaced with the Ptrc promoter sequence by observing the presence of a 700bp DNA band in 1.0% agarose gel. The strain thus confirmed was cultured overnight at 30 ℃ in LB medium containing 50mg/L of kanamycin and 5mM IPTG to remove pTarget-. DELTA.PthrA:: Ptrc vector. The strain from which pTarget-. DELTA.PthrA:wasremoved was then cultured overnight in LB medium at 37 ℃ to remove the pCas vector. The strain from which the pCas vector was removed was subjected to PCR amplification using primer 47 and primer 46 under the following PCR reaction conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1min for 15 s; and continuing to extend for 10min at 72 ℃, carrying out sequencing verification on the PCR product, and confirming that the in-situ promoter sequence (the nucleotide sequence is shown as SEQ ID NO. 19) of the thrA gene is successfully replaced by the Ptrc promoter (the nucleotide sequence is shown as SEQ ID NO. 21) through BLAST sequence comparison. The strain thus constructed was designated as W3110. DELTA. metI. DELTA. metJ. DELTA. metB. thrB-metL-thrA (trc).

TABLE 6 primer sequences

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) Enhancement of sucA Gene expression

Alpha-ketoglutarate decarboxylase coded by the sucA gene catalyzes alpha-ketoglutarate to synthesize succinyl-CoA, and in order to further improve the intracellular content of a substrate succinyl-CoA, the trc promoter sequence is used for replacing an original promoter sequence in the sucA gene (the nucleotide sequence is shown in SEQ ID NO. 13) so as to achieve the purpose of enhancing the expression of the sucA gene.

The promoter sequence of the sucA gene encoding homoserine dehydrogenase in the genome of W3110. delta. metI. delta. metJ. delta. metB. thrB-metL-thrA (trc) strain was edited by CRISPR-Cas9 system. A pTarget- Δ PsucA:: Ptrc mutation vector capable of expressing sgRNA targeting the promoter sequence of the desired gene sucA was constructed by PCR using the primers 49 and 50, and the pTargetF vector as a template. The PCR reaction conditions were as follows: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 15s, 55 deg.C for 15s, and 72 deg.C for 2 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing a final concentration of 50mg/L spectinomycin hydrochloride resistance, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony, transferring the single colony to an LB liquid culture medium containing spectinomycin hydrochloride resistance with the final concentration of 50mg/L, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain pTarget-delta PsucA vector.

The upstream homologous fragment of the sucA gene promoter sequence was obtained by PCR using primers 51 and 52 and using the genome of the W3110. delta. metI. delta. metJ. delta. metB. thrB-metL-thrA (trc) strain as a template, and the promoter sequence information of the sucA gene was obtained from the Ecocyc E.coli Database (EcoCyc Gene accession No: EG10998), and the PCR reaction conditions were as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 30 s; extension was continued for 10min at 72 ℃. A downstream homologous fragment of the sucA gene promoter sequence was obtained by amplification using the primer 53 and the primer 54 in the same manner, and the PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment (nucleotide sequence shown in SEQ ID NO. 14) was recovered by gel cutting. The two recovered DNA fragments were subjected to fusion PCR using primers for primer 51 and primer 54 under the following conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1 min; extension was continued at 72 ℃ for 10min, the PCR product was detected by 1.0% agarose gel electrophoresis and the fragment was recovered and purified by gel cutting, and the Ptrc promoter sequence had been inserted between the two homologous fragments in the gene band. The pTarget-. DELTA.PsucA vector and the recovered DNA fragment were electroporated together into the W3110. DELTA.metI. DELTA.metJ. DELTA.metB. thrB-metL-thrA (trc) strain with pCas9 vector.

For electroporation, W3110. delta. metI. delta. metJ. delta. metB. delta. thrB-metL-thrA (trc) strain transformed with pCas9 vector was cultured in LB medium containing 50mg/L kanamycin and 10mM L-arabinose at 30 ℃ until OD600 reached 0.6, and the bacterial suspension was centrifuged to obtain cells. The cells were washed twice with sterile distilled water and then once with 10% glycerol for use. Electroporation was performed at 2.5 KV.

The bacterial suspension after the electric transformation was applied to LB plates containing 50mg/L kanamycin and 50mg/L spectinomycin hydrochloride resistance and cultured overnight at 30 ℃. A single colony was picked as a template, PCR was performed with the primers 55 and 56, and it was confirmed that the original promoter sequence of the sucA gene had been replaced with the Ptrc promoter sequence by observing the presence of a 700bp DNA band in a 1.0% agarose gel. The strain thus confirmed was cultured overnight at 30 ℃ in LB medium containing 50mg/L of kanamycin and 5mM IPTG to remove pTarget-. DELTA.PsucA:: Ptrc vector. The strain from which pTarget-. DELTA.PsucA:wasremoved was then cultured overnight in LB medium at 37 ℃ to remove the pCas vector. The strain from which the pCas vector was removed was subjected to PCR amplification using primer 47 and primer 46 under the following PCR reaction conditions: 5min at 95 ℃; repeating 30 cycles at 95 deg.C for 30s, 55 deg.C for 30s, and 72 deg.C for 1min for 15 s; and continuing to extend for 10min at 72 ℃, carrying out sequencing verification on the PCR product, and confirming that the in-situ promoter sequence (the nucleotide sequence is shown as SEQ ID NO. 20) of the sucA gene is successfully replaced by the Ptrc promoter (the nucleotide sequence is shown as SEQ ID NO. 21) through BLAST sequence alignment. The strain thus constructed was designated as W3110. DELTA. metI. DELTA. metJ. DELTA. metB. thrB-metL-thrA-sucA (trc).

TABLE 7 primer sequences

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

EXAMPLE 3 construction of plasmid expressing metA Gene having resistance to feedback inhibition

PCR amplification was performed using The genome of wild-type E.coli Escherichia Coli W3110 (The Coli Genetic Stock Center, E.coli Genetic Breeding Center) as a template, together with primers 57 and 58. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 1min for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR product was detected by 1.0% agarose gel electrophoresis and the fragment was recovered and purified by cutting the gel, and base A was introduced into the 5' end of the fragment using Taq DNA polymerase. The fragment was ligated with pGEM-T vector under the action of T4DNA ligase to obtain the cloned recombinant plasmid pGEM-T-metA. Site-directed mutagenesis was performed on the metA gene (nucleotide sequence shown in SEQ ID NO.15 and amino acid sequence of encoded protein shown in SEQ ID NO. 16) on pGEM-T-metA plasmid to replace the 64 th amino acid glutamic acid with glutamine (E64Q), mutation was introduced by PCR using primer 59 and primer 60 and pGEM-T-metA plasmid as template, and the PCR reaction procedure was as follows: 3min at 98 ℃; repeating 30 cycles at 98 deg.C for 10s, 55 deg.C for 5s, and 72 deg.C for 3 min; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated and transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plate containing 50mg/L ampicillin sodium at final concentration, cultured at 37 ℃ for 12 hours, and then single colony was randomly picked up for sequencing analysis to obtain metA mutant gene. Based on the analysis results, an expression primer 61 and a primer 62 (having an NcoI restriction site and a SacI restriction site, respectively) were designed to obtain a 930bp metA11 gene sequence. The amplified fragment was digested with restriction enzymes NcoI and SacI, and the fragment was ligated with pTrc99A treated with the same restriction enzymes using T4DNA ligase to construct expression vector pTrc99A-metA 11.

TABLE 8 primer sequences

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

Example 4 construction of plasmid expressing yjeH Gene having L-methionine transporting function

PCR amplification was performed using The genome of wild-type E.coli Escherichia Coli W3110 (The Coli Genetic Stock Center, E.coli Genetic Breeding Center) as a template, together with primers 63 and 64. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, 95 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 1min for 30s, for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR product was detected by 1.0% agarose gel electrophoresis and the fragment was recovered and purified by cutting the gel, and base A was introduced into the 5' end of the fragment using Taq DNA polymerase. The fragment is connected with pGEM-T vector under the action of T4DNA ligase to obtain the cloned recombinant plasmid pGEM-T-yjeH. The recombinant plasmid was transformed into Escherichia coli JM109, plated on an LB plate containing ampicillin sodium at a concentration of 50. mu.g/mL, and positive clones were randomly picked up for sequencing analysis. Based on the analysis results, expression primers 65 and 66 (each having a Kpn I restriction site and a Hind III restriction site) were designed to obtain a 1257bp yjeH gene sequence (nucleotide sequence shown in SEQ ID NO. 17). The amplified fragment was digested with Kpn I and Hind III restriction enzymes, ligated with pTrc99A-metA11 treated with the same restriction enzymes using T4DNA ligase to construct expression vector pTrc99A-metA11-yjeH, and named pA11 y.

TABLE 9 primer sequences

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

EXAMPLE 5 construction of the test strains

(1) Construction of the Strain

Coli strains W3110, W3110. delta. metI. delta. metJ. metB. delta. metB, W3110. delta. metI. delta. metJ. delta. metB. delta. thrB, W3110. delta. metI. delta. metJ. metB. delta. thrB-metL (trc), W3110. delta. metI. delta. metJ. metB. delta. metB. thrB-metL-thrA (trc), W3110. delta. metI. delta. metJ. delta. metB. thrB-metL-thrA trc) were prepared as competent cells, and plasmid pA11y constructed in example 4 was transformed into the prepared competent cells by chemical transformation, spread on LB plates containing penicillin sodium at a concentration of 50. mu.g/mL, to obtain three strains of metabolic strain E.E.E. 11 y.

(2) Experiment of shaking flask fermentation

The above-mentioned cells were tested in fermentation experiments in shake flasks to compare the ability to produce O-succinyl-L-homoserine between the strains of the various genotypes. The shake flask fermentation experiment was performed as follows: each strain was streaked on LB plates containing 50. mu.g/mL ampicillin sodium resistance, cultured overnight in a 37 ℃ incubator, and a single colony was picked up and inoculated into 5mL of LB medium and cultured overnight in a 37 ℃ incubator at 200 rpm.

In a 500ml shake flask, 20ml of fermentation medium was added and 1ml of seed solution of each strain was inoculated into the shake flask medium. Then the shake flask was cultured in an incubator at 30 ℃ for 48 hours at 180rpm, 1ml of the liquid was taken from the shake flask, diluted 100-fold with ultrapure water, filtered, and analyzed by HPLC for O-succinyl-L-homoserine content in the fermentation broth, and finally the amount of O-succinyl-L-homoserine obtained was compared for each strain harboring the plasmid. The results are shown in Table 10 for the shake flask fermentation experiments with metabolically engineered strains.

The fermentation medium comprises the following components: 40/L glucose, 2g/L potassium dihydrogen phosphate, 17g/L ammonium sulfate, 4g/L yeast powder, 30g/L, L-threonine calcium carbonate, 0.2g/L vitamin B₁ 0.0001g/L、MgSO₄ 2g/L、FeSO₄ 0.005g/L、MnSO₄0.005g/L、ZnSO₄0.005g/L, deionized water as solvent, and pH 6.8.

TABLE 10 fermentation experiments with shake flask of metabolically engineered strains

As can be seen from Table 10, the 9 metabolically engineered and vector-carrying strains W3110. DELTA. metI. DELTA. metJ/pA11y, W3110. DELTA. metI. DELTA. metJ. DELTA. metB/pA11y, W3110. DELTA. metI. DELTA. metB. thrB/pA11y, W3110. DELTA. metI. DELTA. metJ. metB-metL (trc) and/pA 11y, W3110. DELTA. metI. metJ. metB. thrB-metL (trc) and/pA 11y, W3110. DELTA. metI. metJ. DELTA. metB. thrB-metB-metL-thrA-sur-E (31164) all had O-succinyl-homoserine producing ability and the feed back on methionine-L-methionine in the strain, and the feed back on the methionine-L-methionine-level in the strain, which did not significantly reduce the level of methionine-transporting system, thereby exerting excellent activity of the enzyme. The level of the strain with the best performance after modification for producing O-succinyl-L-homoserine by fermentation is increased from 0g/L to 21.8g/L compared with the wild type strain. Other modified strains produced O-succinyl-L-homoserine at a level that was improved to a different extent compared to the original strain.

(3) L-threonine addition amount test

L-threonine addition amount experiments were performed on L-threonine auxotrophic strain W3110. delta. metI. delta. metJ. delta. metB. delta. thrB-metL-thrA (trc)/pA11y, in which the amounts of L-threonine in the fermentation medium were 0g/L, 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L, and 1.6g/L, respectively, and 1ml of the seed solution of each strain was inoculated into the medium in a shake flask. The flasks were then incubated at 180rpm in an incubator at 30 ℃ for 48 hours. The W3110. DELTA. metI. DELTA. metJ. DELTA. metB. thrB-metL-thrA (trc)/pA11y strain was finally examined for its ability to produce O-succinyl-L-homoserine.

The production levels of O-succinyl-L-homoserine by the W3110. DELTA. metI. DELTA. metJ. DELTA. metB. thrB-metL-thrA (trc)/pA11y strain were shown in Table 11 by fermentation experiments with exogenous addition of L-threonine and glucose supplementation. In the case of L-threonine auxotrophic strains, the growth of the strain is limited without exogenous addition of L-threonine, and the ability to metabolically produce O-succinyl-L-homoserine is also limited. When L-threonine is excessively added, it not only does not promote the growth of the cells, but also adversely affects the accumulation of O-succinyl-L-homoserine.

TABLE 11 fermentation experiment with L-threonine addition

L-threonine addition amount (g/L)	OD600	Conversion ratio of sugar to acid (%)	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

Example 6 Large fermenter culture experiment

For large-scale production of L-homoserine, W3110. DELTA. metI. DELTA. metJ. DELTA. metB. thrB-metL-thrA-sucA (trc)/pA11y strain was used and cultured in a 5L fermentor. The strains were streaked on LB plates containing 50. mu.g/mL ampicillin sodium resistance and cultured overnight in a 37 ℃ incubator. Single colonies were picked and inoculated into 10mL LB tube medium, cultured at 37 ℃ for 8 hours, then 1mL of the tube culture was re-inoculated into a 500mL Erlenmeyer flask containing 50. mu.g/mL ampicillin sodium resistant 100mL of seed medium and cultured in a 37 ℃ incubator at 200rpm for 8-10 hours, and 200mL of the seed culture was inoculated into a 5L fermentor containing 50. mu.g/mL ampicillin sodium resistant 2L of fermentation medium, supplemented with a feed medium by the fed-batch method, and cultured for 50-100 hours. The glucose concentration is controlled at 2-10g/L by feeding during the fermentation process. Controlling the DO level to be about 30% in the fermentation process by a stirring coupled Dissolved Oxygen (DO) mode, controlling the stirring rotating speed to be 200-600rpm, controlling the aeration rate to be 1-2vvm, controlling the culture temperature to be 30 ℃ in the fermentation process, and adjusting the pH to be 6.8-7.0 by 50% ammonia water. The medium composition is shown in Table 11, and the concentration of O-succinyl-L-homoserine in the fermentation broth thus cultured was analyzed by amino acid analyzer to be 100 g/L.

TABLE 11 media formulation (deionized water as solvent, pH6.8)

Composition of	Fermentation medium	Supplementary culture 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 listing

<110> Zhejiang industrial university

<120> recombinant escherichia coli for producing O-succinyl-L-homoserine and application thereof

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 654

<212> DNA

<213> Unknown (Unknown)

<400> 1

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

<210> 2

<211> 1000

<212> DNA

<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 (8)

1. A recombinant Escherichia coli for producing O-succinyl-L-homoserine is characterized in that the recombinant Escherichia coli is obtained by encoding a metI gene encoding L-methionine transport protein in Escherichia coli Ecoli W3110 and encoding negative regulation repressionAfter the metJ gene of the factor, the metB gene of the cystathionine gamma synthetase and the thrB gene of the homoserine kinase are sequentially weakened, promoters of thrA gene of the homoserine dehydrogenase I, metL gene of the homoserine dehydrogenase II and sucA gene of the alpha-ketoglutarate decarboxylase are respectively replaced by Ptrc promoters, and finally metA mutant gene of the homoserine transsuccinylase and yjeH gene of the L-methionine export protein are introduced, wherein the metA mutant gene is obtained by replacing the 64 th glutamic acid of the metA gene encoding protein by glutamine; the P is_trcThe nucleotide sequence of the promoter is shown as SEQ ID NO. 21.

2. The recombinant Escherichia coli producing O-succinyl-L-homoserine as claimed in claim 1, wherein the metI gene nucleotide sequence is shown as SEQ ID No.1, the metJ gene nucleotide sequence is shown as SEQ ID No.3, the metB gene nucleotide sequence is shown as SEQ ID No.5, and the thrB gene nucleotide sequence is shown as SEQ ID No. 7.

3. The recombinant Escherichia coli producing O-succinyl-L-homoserine as claimed in claim 1, wherein the nucleotide sequence of yjeH gene is shown as SEQ ID NO.17, and the amino acid sequence of metA gene encoding protein is shown as SEQ ID NO. 16.

4. Use of the recombinant E.coli producing O-succinyl-L-homoserine according to claim 1 in the production of O-succinyl-L-homoserine by fermentation.

5. The use of claim 4, wherein the use is to inoculate the recombinant Escherichia coli into a fermentation medium, ferment and culture at 30 ℃ and 180-.

6. The use according to claim 5, characterized in that the fermentation medium has a final concentration composition of: glucose 40/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 17g/L, yeast powder 4g/L, carbonCalcium 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、MnSO₄0.005g/L、ZnSO₄0.005g/L, deionized water as solvent, and pH 6.8.

7. The use of claim 5, characterized in that the fermentation culture is preceded by slant activation and seed culture, and the seed solution is inoculated to the fermentation medium in an inoculum size of 5-10% by volume concentration, and the slant activation method comprises: inoculating the recombinant escherichia coli on an LB flat plate, and culturing overnight at 37 ℃ to obtain slant thalli; the seed culture method comprises the following steps: the single colony of the slant strain was inoculated into LB medium and cultured overnight at 37 ℃ and 200rpm to obtain a seed solution.

8. The use according to claim 5, characterized in that the fermentation culture is carried out in a fermenter, the glucose concentration in the fermenter being controlled by adding a feed medium to 2-10 g/L; the fermentation conditions are that the DO level is 30%, the stirring speed is 200-600rpm, and the aeration rate is controlled to be 1-2 vvm; controlling the culture temperature at 30 ℃ and adjusting the pH value to 6.8-7.0 by using 50% ammonia water in the fermentation process; the fermentation medium in the fermentation tank consists of: 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、FeSO₄0.005g/L、MnSO₄ 0.005g/L、ZnSO₄0.005g/L, vitamin B₁0.0001g/L, deionized water as solvent, and pH 6.8; the composition of a supplemented medium is as follows: 500g/L glucose, 12.5g/L, L g/potassium dihydrogen phosphate-threonine 2g/L, L-methionine 2g/L, L-isoleucine 2g/L, and deionized water as solvent.

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