L-homoserine production strain as well as construction method and application thereof

Abstract

The invention provides an L-homoserine production strain as well as a construction method and application thereof. The strain does not contain plasmids, E.coli W3110 is taken as a chassis strain, relA and thrB genes are knocked out, a gapC gene from Clostridium acetobutylicum is heterologously expressed, an aspB gene from bacillus subtilis is expressed, and ppc, asd, thrAfbr, rhtA, pntAB and spoTfbr genes are overexpressed; by knocking out L-homoserine decomposition pathway genes, enhancing expression of pathway key enzymes, optimizing an L-homoserine transport system, improving the reducing power NADPH content and enhancing the amino acid hunger resistance, the strain can effectively improve the synthesis efficiency of L-homoserine, is stable in heredity, does not need to add resistant substances, is high in acid production efficiency, and is suitable for industrial production. And the L-homoserine has good L-homoserine synthesis capability.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering, and in particular to an L-homoserine producing strain, its construction method, and its application. Background Technology

[0002] L-homoserine is a naturally occurring non-protein amino acid and a key intermediate in the biosynthesis of threonine, methionine, and isoleucine. L-homoserine has broad application potential in the pharmaceutical, agricultural, and industrial fields. For example, L-homoserine and its derivatives are precursors for the synthesis of certain β-lactam antibiotics (such as cephalosporins), and L-homoserine can also be used to synthesize the herbicide L-glufosinate.

[0003] Currently, the main methods for producing L-homoserine include chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis suffers from drawbacks such as high cost, severe pollution, demanding reaction conditions, and easy generation of byproducts. Enzymatic catalysis often faces problems such as the use of toxic raw materials and expensive coenzymes. Microbial fermentation uses glucose as the energy source for microbial growth, allowing microorganisms to synthesize L-homoserine de novo without the need for substrate addition, reducing production costs. The fermentation process is also mild, making it a promising method for industrial production and gradually becoming the preferred method for L-homoserine production. However, strains for L-homoserine production via microbial fermentation still suffer from long fermentation cycles, low acid production and conversion rates, and high production costs. Therefore, constructing an L-homoserine production strain with high acid production efficiency and good genetic stability is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an L-homoserine producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-homoserine producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-homoserine producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An L-homoserine-producing strain, E. coli W3110, as a chassis strain, had the relA and thrB genes knocked out, and heterologously expressed genes derived from... Clostridium acetobutylicum The gapC gene, derived from Bacillus subtilis B. subtilis The aspB gene at level 168 overexpresses ppc, asd, and thrA. fbr ,rhtA,pntAB,spoT fbr Gene.

[0008] Preferably, the L-homoserine producing strain is obtained by the following method: using W3110 as the chassis strain, the transcription of the gapC gene derived from E. coli Clostridium acetobutylicum is strengthened by the trc promoter and integrated into the genomic relA gene site; the transcription of the aspB gene derived from B. subtilis 168 is strengthened by the trc promoter and integrated into the genomic thrB gene site; the transcription of the ppc gene is strengthened by the trc promoter and integrated into the genomic yeeL pseudogene site; the transcription of the asd gene is strengthened by the trc promoter and integrated into the genomic ygaY pseudogene site; the transcription of the thrA fbr gene is strengthened by the trc promoter and integrated into the genomic ycgH pseudogene site; the transcription of the rhtA gene is strengthened by the trc promoter and integrated into the genomic yeeP pseudogene site; the transcription of the pntAB gene is strengthened by the trc promoter and integrated into the genomic yghX gene site; the transcription of the spoT fbr gene is strengthened by the trc promoter and integrated into the genomic rph gene site.

[0009] The thrA fbr gene has been relieved of the feedback inhibition by threonine.

[0010] Preferably, the L-homoserine producing strain has the nucleotide sequence of the relA gene as shown in the sequence listing SEQ ID NO. 1, the nucleotide sequence of the thrB gene as shown in the sequence listing SEQ ID NO. 2.

[0011] Preferably, the L-homoserine producing strain has the nucleotide sequence of the gapC gene as shown in the sequence listing SEQ ID NO. 3, the nucleotide sequence of the aspB gene as shown in the sequence listing SEQ ID NO. 4, the nucleotide sequence of the ppc gene as shown in the sequence listing SEQ ID NO. 5, the nucleotide sequence of the asd gene as shown in the sequence listing SEQ ID NO. 6, the nucleotide sequence of the thrA fbr gene as shown in the sequence listing SEQ ID NO. 7, the nucleotide sequence of the rhtA gene as shown in the sequence listing SEQ ID NO. 8, the nucleotide sequence of the pntAB gene as shown in the sequence listing SEQ ID NO. 9, the nucleotide sequence of the spoT fbr gene as shown in the sequence listing SEQ ID NO. 10.

[0012] Preferably, the L-homoserine producing strain has the nucleotide sequence of the trc promoter as shown in the sequence listing SEQ ID NO. 11.​

[0013] Preferably, the above-mentioned L-homoserine producing strain, wherein E. coli W3110 is E. coli W3110 ATCC27325.

[0014] The above method for constructing L-homoserine-producing strains involves starting with the strain... E. coli The following are the specific steps for targeted modification based on the W3110: (1) Blocking L-homoserine catabolism: The L-homoserine catabolism gene thrB was knocked out; (2) Enhance the expression of key enzymes in the pathway: Heterologous introduction from Bacillus subtilis B. subtilis The aspB gene at 168 was overexpressed using the trc promoter, along with ppc, asd, and thrA. fbr Gene; (3) Optimize the L-homoserine transport system: overexpress the rhtA gene using the trc promoter; (4) Increase reducing NADPH content: Increase the transcription level of pntAB gene by overexpressing it using the trc promoter, and introduce NADPH from the trc promoter. Clostridium acetobutylicum The gapC gene; (5) Enhance amino acid starvation resistance: Knock out the relA gene and overexpress spoT using the trc promoter. fbr Gene.

[0015] Application of the above-mentioned L-homoserine producing strains in the fermentation production of L-homoserine.

[0016] The application of the aforementioned L-homoserine-producing strain involves fermenting the engineered bacteria in a culture medium under suitable fermentation conditions to obtain L-homoserine. The culture medium includes, but is not limited to, carbon sources, nitrogen sources, inorganic salts, and vitamins. Fermentation conditions include fermentation temperature, fermentation pH, dissolved oxygen levels, fermentation pressure, and fermentation time. All of the aforementioned culture media can be obtained through conventional methods and used for L-homoserine production. Fermentation conditions can be adjusted to suit the production characteristics of the strain.

[0017] Preferably, the application of the above-mentioned L-homoserine producing strains follows these specific steps: ① Strain activation: L-homoserine producing strain was streaked onto activation slant, incubated at 37℃ for 12h, and passaged once; ② Seed culture: Take solid slant culture and inoculate it into seed culture medium and culture at 37℃ and 200 r / min for 12h; ③ Fermentation culture: inoculate the seed liquid prepared after the activation of the strain with 10%-15% inoculation amount into the triangular flask containing the fermentation medium, seal with gauze, maintain the temperature at 37±0.2℃, and cultivate with 220 r / min oscillation, maintain the pH at 7.0-7.2 by supplementing ammonia water during the fermentation process, and supplement the carbon source required by the bacterial body by adding glucose solution, and the fermentation cycle is 24 h.

[0018] Preferably, in the application of the L-homoserine production strain, the slope culture medium used in step ① is general LB solid culture medium.

[0019] Preferably, in the application of the L-homoserine production strain, the seed culture medium used in step ② is: glucose 25 g / L, yeast powder 4 g / L, proteose peptone 1.5 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4·3H2O 3 g / L, ammonium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, and the rest is water.

[0020] Preferably, in the application of the L-homoserine production strain, the fermentation medium used in step ③ is: MgSO4·7H2O 1.5 g / L, yeast powder 4 g / L, proteose peptone 3 g / L, corn syrup 20 ml, ammonium sulfate 3 g / L, threonine 0.3 g / L, isoleucine 0.1 g / L, methionine 0.1 g / L, K2HPO4·3H2O 4 g / L, glutamic acid 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4 10 mg / L, VB1, VB3, VB5 each 2 mg / L, and the rest is water.

[0021] The above culture media can be prepared by standard methods.

[0022] Beneficial effects: The L-homoserine production strain does not contain plasmid, is genetically stable, does not need to add resistance substances, has good L-homoserine synthesis capacity, low production cost, stable performance, high acid production efficiency, and good economic benefits; the strain blocks the L-homoserine decomposition pathway gene, strengthens the expression of the key enzyme of the pathway, optimizes the L-homoserine transport system, increases the content of reducing power NADPH, and strengthens the amino acid starvation resistance, which can effectively improve the L-homoserine synthesis efficiency, improve the L-homoserine production level, realize the efficient production of L-homoserine, and has excellent industrial application prospect. Specifically: (1) The thrB gene is knocked out to block the L-homoserine decomposition metabolism, and the trc promoter is used to overexpress the L-homoserine transport protein rhtA to realize the efficient accumulation of L-homoserine; (2) The transcription level of the pntAB gene is improved, and the source of Clostridium acetobutylicumThe gapC gene encoding glyceraldehyde-3-phosphate dehydrogenase increases the supply of NADPH, providing a large amount of reducing power for cell production. (3) By introducing the thrA operon gene, which has been relieved of threonine feedback inhibition. fbr This leads to the efficient expression of key enzymes in the L-homoserine metabolic pathway, smooth metabolic pathway, and improved acid production and conversion rate, thereby greatly increasing the yield of L-homoserine. (4) Due to the knockout of the thrB gene, this strain exhibits a threonine auxotrophic effect. To minimize the burden of amino acid starvation on bacterial growth and production, in addition to the addition of threonine and isoleucine, the relA gene was knocked out and spoT was overexpressed using the trc promoter. fbr The gene reduced the production of ppGpp and promoted its degradation, weakening its inhibitory effect on bacterial growth and effectively enhancing the bacterial cell's ability to take up the substrate glucose. Attached Figure Description

[0023] Figure 1 A diagram illustrating the de novo synthesis pathway gene modification process for L-homoserine-producing strains. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0025] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0026] The starting strain used in the examples was wild-type. E. coli W3110 ATCC 27325, the corresponding promoter and gene are shown in the sequence listing. Primers used in the construction of the involved strains are shown in Table 1.

[0027] Table 1 Primers used in strain construction Primer name Sequence No. Primer sequence (5'-3') -pGRB-S SEQ ID NO. 12 AGTCCTAGGTATAATACTAGTaggattgctatgccgcactgGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO. 13 TTCTAGCTCTAAAACcagtgcggcatagcaatcctACTAGTATTATACCTAGGACT -U-S SEQ ID NO. 14 atggttgcggtaagaagtgcac -U-A SEQ ID NO. 15 gcgatgtggtgcatcaggttggcgcatcttttacttcgcg D-S SEQ ID NO. 16 cgcgaagtaaaagatgcgccaacctgatgcaccacatcgc -D-A SEQ ID NO. 17 ctaactcccgtgcaaccgac SEQ ID NO. 18 TATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCAAAGATAGCTATTAATGGTTTTGG SEQ ID NO. 19 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTATTTTGCTATTTTTGCAAAGTAAGC -pGRB-S SEQ ID NO. 20 AGTCCTAGGTATAATACTAGTATGGCGATGAATGAACACTGGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO. 21 TTCTAGCTCTAAAACCAGTGTTCATTCATCGCCATACTAGTATTATACCTAGGACT -U-S SEQ ID NO. 22 atggttaaagtttatgccccggc -U-A SEQ ID NO. 23 gctcaggctgacgggaaaacaggcactggagcctaag D-S SEQ ID NO. 24 cttaggctccagtgcctgttttcccgtcagcctgagc -D-A SEQ ID NO. 25 ttagttttccagtactcgtgcgc SEQ ID NO. 26 TATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCttgaaactggcaaaaagagtatccgc SEQ ID NO. 27 aacgaaaggcccagtctttcgactgagcctttcgttttatttgttagctatgtttttctacaaaacgcttgattctttca SEQ ID NO. 28 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA SEQ ID NO. 29 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT SEQ ID NO. 30 TTCATCGGGACGAGTGGAGA SEQ ID NO. 31 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCATAGCATCGCCAATCTGA SEQ ID NO. 32 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATAAAGCC SEQ ID NO. 33 CATTCCCTCTACAGAACTAGCCCT SEQ ID NO. 34 CTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAACGAACAATATTCCGCATTGC SEQ ID NO. 35 AACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGCCGGTATTACGCATACCTGC SEQ ID NO. 36 AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA SEQ ID NO. 37 TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT SEQ ID NO. 38 CCTACAAACCACATCGCACATT SEQ ID NO. 39 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCGAAGCAACCCAAAAG SEQ ID NO. 40 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACC SEQ ID NO. 41 GGAGTAGGGCTTTCCATAGAGTGT SEQ ID NO. 42 AATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAATGTTGGTTTTATCGGCTGG SEQ ID NO. 43 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGCCAGTTGACGAAGCATC -pGRB-S SEQ ID NO. 44 AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO. 45 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT -U-S SEQ ID NO. 46 TAAACTCGTCAGCGGCACAAC -U-A SEQ ID NO. 47 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTG -D-S SEQ ID NO. 48 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCG -D-A SEQ ID NO. 49 GATTCAGGTTGCCATTTACGC S SEQ ID NO. 50 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAGTGTTGAAGTTCGG -A SEQ ID NO. 51 agaagatcTctgagcaatggcg -S SEQ ID NO. 52 cgccattgctcagAgatcttct SEQ ID NO. 53 aacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgtcagactcctaacttccatgagaggg -pGRB-S SEQ ID NO.54 AGTCCTAGGTATAATACTAGTTGAACAGTTTACCGGTGCGGGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO.55 TTCTAGCTCTAAAACCCGCACCGGTAAACTGTTCAACTAGTATTATACCTAGGACT -U-S SEQ ID NO.56 GGTCAGGAGGTAACTTATCAGCG -U-A SEQ ID NO.57 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCAGGGCTCCGTTTTG -D-S SEQ ID NO.58 CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGTCG -D-A SEQ ID NO.59 ACGATGTCAGCAGCCAGC -S SEQ ID NO.60 GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCatgcctggttcattacgtaaaatgcc -A SEQ ID NO.61 taaaacgaaaggcccagtctttcgactgagcctttcgttttatttgttaattaatgtctaattcttttattttgctctctttgcgtac -pGRB-S SEQ ID NO.62 AGTCCTAGGTATAATACTAGTTATATCGCCCTGGCACCTGAGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO.63 TTCTAGCTCTAAAACTCAGGTGCCAGGGCGATATAACTAGTATTATACCTAGGACT -U-S SEQ ID NO.64 TCAAACGCTTTACGCAGGAT -U-A SEQ ID NO.65 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCTCATCTTTGCGGGCTT -D-S SEQ ID NO.66 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCCGCAAGCGACAGGC -D-A SEQ ID NO. 67 CGTTGATTCGGGTGTCCAG SEQ ID NO. 68 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG SEQ ID NO. 69 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC -pGRB-S SEQ ID NO. 70 AGTCCTAGGTATAATACTAGTGGCTGGATCACCGCAGAGTAGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO. 71 TTCTAGCTCTAAAACTACTCTGCGGTGATCCAGCCACTAGTATTATACCTAGGACT -U-S SEQ ID NO. 72 ATAGCGCAGGGTACATTCCACT -U-A SEQ ID NO. 73 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA -D-S SEQ ID NO. 74 TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGCCGCAGAGACCGACAT -D-A SEQ ID NO. 75 ACAGCGGTTGTGGTGGCA -S SEQ ID NO. 76 GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCttgtatctgtttgaaagcctgaatc -A SEQ ID NO. 77 tcgcttttggaatggcgatatagtcatccacttcgcccg -S SEQ ID NO. 78 cgggcgaagtggatgactatatcgccattccaaaagcga A SEQ ID NO. 79 caacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgttaatttcggtttcgggtgact

[0028] Example 1 This embodiment aims to illustrate the specific construction steps of strain Hom-8. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0029] like Figure 1 As shown, the strain was modified according to the metabolic pathway of the entire strain modification process to obtain the L-homoserine producing strain Hom-8. The modification process mainly includes the following 5 modules: (1) Blocking L-homoserine catabolism: The L-homoserine catabolism gene thrB was knocked out; (2) Enhance the expression of key enzymes in the pathway: Heterologous introduction from Bacillus subtilis B. subtilis The aspB gene at 168 was overexpressed using the trc promoter, along with ppc, asd, and thrA. fbr Gene; (3) Optimize the L-homoserine transport system: overexpress the rhtA gene using the trc promoter; (4) Increase reducing NADPH content: Increase the transcription level of pntAB gene by overexpressing it using the trc promoter, and introduce NADPH from the trc promoter. Clostridium acetobutylicum The gapC gene.

[0030] (5) Enhance amino acid starvation resistance: Knock out the relA gene and overexpress spoT using the trc promoter. fbr Gene.

[0031] The gene editing methods used in the above gene manipulations refer to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli (using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained within the context of this article. "Knockout" in this invention refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the engineered bacterial genome after ligation with a promoter and terminator. Specifically, the steps are as follows: ①Knockout of the relA gene and integration at this site originates from Clostridium acetobutylicum gapC: with E.coli Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using relA-US, relA-UA, relA-DS, and relA-DA, respectively. Clostridium acetobutylicumUsing the ATCC 824 genome as a template, the target gene fragment was amplified by PCR using gapC-S and gapC-A primers. Then, using upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were amplified by overlapping PCR using relA-US and relA-DA primers. The gRNA fragment was annealed using relA-pGRB-S and relA-pGRB-A primers and ligated into the pGRB vector to obtain relA-pGRB. E. coli W3110 electroporation competent cells were prepared, and the target fragment and relA-pGRB were electroporated into the competent cells. Positive transformants were screened to obtain strain Hom-1.

[0032] ②Knockout of the thrB gene and integration at this site originates from B.subtilis The aspB gene at 168: has the same operation as in ①, the difference being that it uses... E.coli W3110 B.subtilis Gene fragments were obtained from the 168 genome using primers thrB-US, thrB-UA, thrB-DS, thrB-DA, thrB-pGRB-S, thrB-pGRB-A, aspB-S, and aspB-A. Hom-1 was used as the competent cell line, and strain Hom-2 was obtained.

[0033] ③ Controlling ppc gene overexpression at the yeeL pseudogene site using the trc promoter: Using the E. coli W3110 genome as a template, yeeL-US, yeeL-UA, yeeL-DS, and yeeL-DA were used respectively to control ppc gene overexpression. ppc -S、 ppc -A is the primer. PCR amplification yields the upstream and downstream homologous arms and the target gene fragment. Using these as a template, overlap PCR is then performed to obtain P. trc - ppc (yeeL) gene integration fragment, wherein the gene integration fragment consists of the upstream homologous arm of yeeL, P trc - ppc The target gene and the downstream homologous arm of yeeL were combined; using yeeL-pGRB-S and yeeL-pGRB-A as primers, a gRNA fragment containing the target sequence was obtained through PCR annealing, and then ligated into the pGRB vector to obtain yeeL-pGRB; preparation E.coli W3110 was used to electrotransform competent cells, and overlapping fragments were co-electrotransformed with yeeL-pGRB into the competent cells. Positive transformants were then selected. The competent cells were Hom-2, and strain Hom-3 was obtained.

[0034] ④ Controlling asd gene overexpression at the ygaY pseudogene site using the trc promoter: This has the same operation as in ③, the difference being that it uses... E.coli Gene fragments were obtained from the W3110 genome using primers ygaY-US, ygaY-UA, ygaY-DS, and ygaY-DA. asd -S、 asd -A, ygaY-pGRB-S, and ygaY-pGRB-A. Competent cells were Hom-3, from which strain Hom-4 was obtained.

[0035] ⑤ Use the trc promoter to control thrA at the ycgH pseudogene site. fbr Gene overexpression: It has the same operation as ③, the difference being that it uses... E.coli Gene fragments were obtained from the W3110 genome using primers ycgH-US, ycgH-UA, ycgH-DS, and ycgH-DA. thrA -S、 thrA -TB-A、 thrA -TB-S、 thrA -A, ycgH-pGRB-S, and ycgH-pGRB-A. Competent cells were Hom-4, and strain Hom-5 was obtained.

[0036] ⑥ Controlling rhtA gene overexpression at the yeeP pseudogene site using the trc promoter: This has the same operation as in ③, except that it uses... E.coli Gene fragments were obtained from the W3110 genome using primers yeeP-US, yeeP-UA, yeeP-DS, yeeP-DA, rhtA-S, rhtA-A, yeeP-pGRB-S, and yeeP-pGRB-A. Hom-5 cells were used as competent cells to obtain strain Hom-6.

[0037] ⑦ Controlling pntAB gene overexpression at the yghX pseudogene site using the trc promoter: This has the same operation as in ③, except that it uses... E.coli Gene fragments were obtained from the W3110 genome using primers yghX-US, yghX-UA, yghX-DS, yghX-DA, pntAB-S, pntAB-A, yghX-pGRB-S, and yghX-pGRB-A. Hom-6 cells were used as competent cells to obtain strain Hom-7.

[0038] ⑧ Use the trc promoter to control spoT at the rph pseudogene site fbr Gene overexpression: It has the same operation as ③, the difference being that it uses... E.coliThe W3110 genome obtained gene fragments, and the primers were rph-U-S, rph-U-A, rph-D-S, rph-D-A, spoT-S, spoT-TB-A, spoT-TB-S, spoT-A, rph-pGRB-S and rph-pGRB-A respectively. The competent cell was Hom-7, and the obtained strain was Hom-8.

[0039] Example 2 This example aims to illustrate the shake flask fermentation application of the engineering bacteria Hom-8, and the specific steps are as follows: ①Inclined surface culture: the bacteria were inoculated on the inclined surface culture medium, and cultured at 37℃ for 12h. The inclined surface culture medium was selected as the general LB solid culture medium.

[0040] ②Shake flask seed culture: a ring of the inclined surface seed was scraped with an inoculation ring and inoculated into a triangular flask containing seed culture medium. The flask was sealed with gauze, the culture temperature was 37℃, the culture time was 12h, the rotation speed of the shaking table was 220r / min, and the pH was 7.0. The seed culture medium used was: glucose 25g / L, yeast powder 4g / L, proteose peptone 1.5g / L, MgSO4·7H2O 0.8g / L, KH2PO4·3H2O 3g / L, ammonium sulfate 1g / L, FeSO4·7H2O 10mg / L, and the rest was water.

[0041] ③Shake flask fermentation culture: the fermentation inoculation amount was 20%-25%, the culture temperature was 37℃, the pH was 7.0, the culture time was 24h, and the rotation speed of the shaking table was 220r / min. The fermentation culture medium used was: MgSO4·7H2O 1.5g / L, yeast powder 4g / L, proteose peptone 3g / L, corn syrup 20ml, ammonium sulfate 3g / L, threonine 0.3g / L, isoleucine 0.1g / L, methionine 0.1g / L, K2HPO4·3H2O 4g / L, glutamic acid 2g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, VB1, VB3, VB5 each 2mg / L, and the rest was water.

[0042] The wild type E.coli W3110 was used as the control group, and after 24h fermentation verification, the wild type E.coli W3110 failed to accumulate L-homoserine, and the engineering bacteria Hom-8 described in Example 1 accumulated 18.2g / L of L-homoserine, which proved the effectiveness of the strain.

[0043] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, some improvements and refinements can be made without departing from the principles of the present application, and the strain modification and other improvements made by the skilled person in the art based on the method of the present application are all considered to be within the protection scope of the present application.

Claims

1. An L-homoserine-producing strain, characterized in that: by E. coli W3110, as a chassis strain, had the relA and thrB genes knocked out, and heterologously expressed genes derived from... Clostridium acetobutylicum The gapC gene, derived from Bacillus subtilis B. subtilis The aspB gene at level 168 overexpresses ppc, asd, and thrA. fbr ,rhtA,pntAB,spoT fbr Gene.

2. The L-homoserine-producing strain according to claim 1, characterized in that: It was obtained by modifying the following method: E. coli W3110, as the chassis strain, was enhanced using the trc promoter to produce strains derived from... Clostridium acetobutylicum The gapC gene is transcribed and integrated into the relA gene locus in the genome; The TRC promoter was used to enhance the source B. subtilis The aspB gene at position 168 is transcribed and integrated into the thrB gene locus in the genome; The transcription of the ppc gene was enhanced using the trc promoter and integrated into the yeeL pseudogene locus in the genome; The transcriptional intensity of the asd gene was enhanced using the trc promoter, and it was integrated into the ygaY pseudogene locus in the genome; the thrA gene was enhanced using the trc promoter. fbr The transcriptional intensity of the rhtA gene was enhanced using the trc promoter and integrated into the ycgH pseudogene locus in the genome; the transcriptional intensity of the pntAB gene was enhanced using the trc promoter and integrated into the yghX gene locus in the genome. spoT was enhanced using the trc promoter. fbr Gene transcription intensity and integration into the rph gene locus of the genome.

3. The L-homoserine-producing strain according to claim 1 or 2, characterized in that: The nucleotide sequence of the relA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the thrB gene is shown in SEQ ID NO.

2.

4. The L-homoserine-producing strain according to claim 1 or 2, characterized in that: The nucleotide sequence of the gapC gene is shown in SEQ ID NO.3, the nucleotide sequence of the aspB gene is shown in SEQ ID NO.4, the nucleotide sequence of the ppc gene is shown in SEQ ID NO.5, the nucleotide sequence of the asd gene is shown in SEQ ID NO.6, and the thrA gene... fbr The nucleotide sequences of the genes are shown in SEQ ID NO.7, the rhtA gene in SEQ ID NO.8, and the pntAB gene in SEQ ID NO.

9. fbr The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing.

5. The L-homoserine-producing strain according to claim 2, characterized in that: The nucleotide sequence of the trc promoter is shown in SEQ ID NO.11 of the sequence listing.

6. The L-homoserine-producing strain according to claim 1 or 2, characterized in that: The E. coli W3110 is E. coli W3110 ATCC 27325.

7. The method for constructing the L-homoserine-producing strain according to any one of claims 1-6, characterized in that: In the originating strain E. coli The following are the specific steps for targeted modification based on the W3110: (1) Blocking L-homoserine catabolism: The L-homoserine catabolism gene thrB was knocked out; (2) Enhance the expression of key enzymes in the pathway: Heterologous introduction from Bacillus subtilis B. subtilis The aspB gene at 168 was overexpressed using the trc promoter, along with ppc, asd, and thrA. fbr Gene; (3) Optimize the L-homoserine transport system: overexpress the rhtA gene using the trc promoter; (4) Increase reducing NADPH content: Increase the transcription level of pntAB gene by overexpressing it using the trc promoter, and introduce NADPH from the trc promoter. Clostridium acetobutylicum The gapC gene; (5) Enhance amino acid starvation resistance: Knock out the relA gene and overexpress spoT using the trc promoter. fbr Gene.

8. The use of the L-homoserine producing strain according to any one of claims 1-6 in the fermentation production of L-homoserine.

9. The application according to claim 8, characterized in that: The specific steps are as follows: ① Strain activation: L-homoserine producing strain was streaked onto activation slant, incubated at 37℃ for 12h, and passaged once; ② Seed culture: Take solid slant culture and inoculate it into seed culture medium and culture at 37℃ and 200 r / min for 12 h; ③ Fermentation culture: Inoculate the seed liquid prepared after activation of the strain at an inoculation rate of 10%-15% into an Erlenmeyer flask containing fermentation culture medium, seal the flask with gauze, maintain the temperature at 37±0.2℃, and shake at 220 r / min. During the fermentation process, ammonia water is added to maintain the pH at 7.0-7.

2. Add glucose solution to supplement the carbon source required by the bacteria, and the fermentation cycle is 24 hours.

10. The application according to claim 9, characterized in that: The slant culture medium used in step ① is general-purpose LB solid medium; the seed culture medium used in step ② is: glucose 25g / L, yeast extract 4g / L, peptone 1.5g / L, MgSO4·7H2O 0.8g / L, KH2PO4·3H2O 3g / L, ammonium sulfate 1g / L, FeSO4·7H2O 10mg / L, with the remainder being water; the fermentation culture medium used in step ③ is: MgSO4·7H2O 1.5g / L, yeast extract 4g / L, peptone 3g / L, corn steep liquor 20ml, ammonium sulfate 3g / L, threonine 0.3g / L, isoleucine 0.1g / L, methionine 0.1g / L, K2HPO4·3H2O 4g / L, glutamic acid 2g / L, FeSO4·7H2O 20mg / L, MnSO4 10 mg / L, VB1, VB3, and VB5 each 2 mg / L, the remainder is water.

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