An L-homoserine-producing strain, its construction method and application

Abstract

This invention provides an L-homoserine-producing strain, its construction method, and its applications. The strain does not contain plasmids. 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 and the aspB gene derived from Bacillus subtilis were overexpressed, along with ppc, asd, and thrA. fbr ,rhtA,pntAB,spoT fbr Genes; the strain described above can effectively improve the L-homoserine synthesis efficiency by knocking out genes of the L-homoserine degradation pathway, enhancing the expression of key enzymes in the pathway, optimizing the L-homoserine transport system, increasing the reducing power NADPH content, and enhancing the ability to withstand amino acid starvation. It is genetically stable, requires no addition of resistance substances, has high acid production efficiency, and has good L-homoserine synthesis capacity.

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:

[0008] 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,spoTfbr Gene.

[0009] Preferably, the above-mentioned L-homoserine producing strain is obtained by modification using 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 was transcribed and integrated into the relA gene site in the genome; the trc promoter was used to enhance the expression of the gene derived from... B.subtilis The aspB gene at 168 was transcribed and integrated into the genomic thrB gene locus; the ppc gene transcription was enhanced using the trc promoter and integrated into the genomic yeeL pseudogene locus; the asd gene transcription intensity was enhanced using the trc promoter and integrated into the genomic ygaY pseudogene locus; the thrA gene transcription was enhanced using the trc promoter. fbr The transcriptional intensity of the rhtA gene was enhanced using the trc promoter and it was integrated into the ycgH pseudogene locus in the genome; the transcriptional intensity of the pntAB gene was enhanced using the trc promoter and it was integrated into the yghX gene locus in the genome; the transcriptional intensity of the spoT gene was enhanced using the trc promoter. fbr Gene transcription intensity and integration into the rph gene locus of the genome.

[0010] The above thrA fbr The gene has already been freed from the feedback inhibition of threonine.

[0011] Preferably, in the above-mentioned L-homoserine producing strain, 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.

[0012] Preferably, in the above-mentioned L-homoserine producing strain, 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.

[0013] Preferably, in the L-homoserine producing strain described above, the nucleotide sequence of the trc promoter is shown in SEQ ID NO. 11 of the sequence listing.

[0014] Preferably, the above-mentioned L-homoserine producing strain, wherein E.coli W3110 is E.coli W3110 ATCC27325.

[0015] 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:

[0016] (1) Blocking L-homoserine catabolism: The L-homoserine catabolism gene thrB was knocked out;

[0017] (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;

[0018] (3) Optimize the L-homoserine transport system: overexpress the rhtA gene using the trc promoter;

[0019] (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;

[0020] (5) Enhance amino acid starvation resistance: Knock out the relA gene and overexpress spoT using the trc promoter. fbr Gene.

[0021] Application of the above-mentioned L-homoserine producing strains in the fermentation production of L-homoserine.

[0022] 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.

[0023] Preferably, the application of the above-mentioned L-homoserine producing strains follows these specific steps:

[0024] ① Strain activation: L-homoserine producing strain was streaked onto activation slant, incubated at 37℃ for 12h, and passaged once;

[0025] ② Seed culture: Take solid slant culture and inoculate it into seed culture medium and culture at 37℃ and 200 r / min for 12h;

[0026] ③ Fermentation culture: Inoculate the seed culture 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 culture at 220 r / min. During the fermentation process, ammonia water is added to maintain the pH at 7.0-7.2; glucose solution is added to supplement the carbon source required by the cells. The fermentation cycle is 24h.

[0027] Preferably, in the application of the above-mentioned L-homoserine producing strain, the slant culture medium used in step ① is the general-purpose LB solid medium.

[0028] Preferably, in the application of the above-mentioned L-homoserine producing strain, 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.

[0029] Preferably, in the application of the above-mentioned L-homoserine producing strain, the fermentation medium used in step ③ is: MgSO4·7H2O 1.5g / L, yeast powder 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 10mg / L, VB1, VB3, and VB5 2mg / L each, with the remainder being water.

[0030] All of the above-mentioned culture media can be prepared using standard methods.

[0031] Beneficial effects:

[0032] The aforementioned L-homoserine-producing strains are plasmid-free, genetically stable, require no added resistance substances, possess excellent L-homoserine synthesis capabilities, exhibit low production costs, stable performance, and high acid production efficiency, resulting in significant economic benefits. These strains effectively improve L-homoserine synthesis efficiency and production levels by blocking genes involved in the L-homoserine degradation pathway, enhancing the expression of key enzymes in the pathway, optimizing the L-homoserine transport system, increasing NADPH content, and strengthening tolerance to amino acid starvation. This achieves highly efficient L-homoserine production and demonstrates excellent industrial application prospects. Specifically:

[0033] (1) The catabolism of L-homoserine was blocked by knocking out the thrB gene, and the L-homoserine transporter rhtA was overexpressed by the trc promoter to achieve efficient accumulation of L-homoserine.

[0034] (2) By increasing the transcription level of the pntAB gene, a gene derived from... Clostridium acetobutylicum The gapC gene encoding glyceraldehyde-3-phosphate dehydrogenase increases the supply of NADPH, providing a large amount of reducing power for cell production.

[0035] (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.

[0036] (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

[0037] Figure 1 A diagram illustrating the de novo synthesis pathway gene modification process for L-homoserine-producing strains. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Table 1 Primers used in strain construction

[0042] Primer Name Sequence Number 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

[0043] Example 1

[0044] 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.

[0045] 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:

[0046] (1) Blocking L-homoserine catabolism: The L-homoserine catabolism gene thrB was knocked out;

[0047] (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;

[0048] (3) Optimize the L-homoserine transport system: overexpress the rhtA gene using the trc promoter;

[0049] (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.

[0050] (5) Enhance amino acid starvation resistance: Knock out the relA gene and overexpress spoT using the trc promoter. fbr Gene.

[0051] 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:

[0052] ①Knockout of the relA gene and integration at this site originates from Clostridium acetobutylicum gapC: with E. coliUsing 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 acetobutylicum Using 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.

[0053] 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.

[0054] 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. coliW3110 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.

[0055] ④ Controlling asd gene overexpression at the ygaY 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 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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. coli Gene fragments were obtained from the W3110 genome using primers rph-US, rph-UA, rph-DS, rph-DA, spoT-S, spoT-TB-A, spoT-TB-S, spoT-A, rph-pGRB-S, and rph-pGRB-A. The competent cells were Hom-7, and strain Hom-8 was obtained.

[0060] Example 2

[0061] This embodiment aims to illustrate the application of the engineered strain Hom-8 in shake-flask fermentation. The specific steps are as follows:

[0062] ① Slant culture: Inoculate the bacterial strain onto a slant culture medium and incubate at 37℃ for 12 hours. The slant culture medium used is the general-purpose LB solid medium.

[0063] ② Shake-flask seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into an Erlenmeyer flask containing seed culture medium. Seal the flask with gauze, and culture at 37℃ for 12 hours. The shaking speed is 220 r / min, and the pH is 7.0. The seed culture medium used is: glucose 25 g / L, yeast powder 4 g / L, 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 remainder is water.

[0064] ③ Shake-flask fermentation culture: The inoculum size was 20%-25%, the culture temperature was 37℃, the pH was 7.0, the culture time was 24h, and the shaker speed was 220r / min. The fermentation medium used was: MgSO4·7H2O 1.5g / L, yeast powder 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 10mg / L, VB1, VB3, and VB5 2mg / L each, and the remainder was water.

[0065] The experiment used wild type E. coli W3110 served as the control group. After 24 hours of fermentation verification, the wild type was found to be... E. coli W3110 failed to accumulate L-homoserine, while the engineered strain Hom-8 described in Example 1 accumulated 18.2 g / L of L-homoserine, demonstrating the effectiveness of the strain.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

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 The genes, wherein 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.

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 2, characterized in that: The nucleotide sequence of the trc promoter is shown in SEQ ID NO.11 of the sequence listing.

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

6. The method for constructing the L-homoserine-producing strain according to any one of claims 1-5, 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 the reducing power of NADPH content: Overexpress the pntAB gene using the trc promoter, and simultaneously introduce NADPH from 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.

7. The use of the L-homoserine producing strain according to any one of claims 1-5 in the fermentation production of L-homoserine.

8. The application according to claim 7, 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.

9. The application according to claim 8, 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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