Valine-producing strain and construction method and application thereof

Abstract

This invention provides a valine-producing strain, its construction method, and its applications. The strain is constructed using CRIPSR / Cas9 gene editing technology based on allele exchange. An artificial operon is designed and constructed at the ygaY site using the strong promoter P. trc control ilvIH (G14N S17F)‑ pykF Simultaneous multi-copy expression was performed, and an artificial operon was constructed at the ycgH site using the strong promoter P. trc control ilvC - ilvD Simultaneous multi-copy expression was performed, and the effects of valine on expression were simultaneously relieved. ilvIH Feedback suppression replaces the natural ilvIH promoter with the strong promoter P. trc This allows more pyruvate to flow to valine; the constructed strain is plasmid-free, defect-free, requires no induction, and has advantages such as good genetic stability and high fermentation yield, enabling efficient de novo synthesis of valine using glucose as a substrate.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering technology, and in particular to a valine-producing strain, its construction method, and its application. Background Technology

[0002] Valine, chemically known as 2-amino-3-methylbutyric acid, belongs to the branched-chain amino acid (BCAA) family. It is a crucial component of proteins, participating in various physiological and biochemical processes within the body, playing a key role in muscle metabolism, tissue repair, and energy supply. Furthermore, valine is an important pharmaceutical and chemical intermediate; due to its unique branched-chain structure, it is frequently used in the synthesis of antibiotics, nutritional supplements, and antihypertensive drugs. Currently, with the deepening of research into valine applications, the market has an urgent need for efficient and environmentally friendly production methods.

[0003] The main methods for producing valine include protein hydrolysis extraction, chemical synthesis, and microbial fermentation. Early valine production relied on protein hydrolysis or chemical synthesis, but these methods generally suffer from complex processes, high raw material costs, and environmental pollution. Therefore, microbial fermentation has become the primary direction for valine production. Microbial fermentation utilizes metabolic engineering to directionally modify microorganisms, enabling highly efficient biosynthesis of valine. This method uses glucose as a raw material to produce valine through fermentation, offering significant advantages such as mild conditions, environmental friendliness, and controllable costs.

[0004] In recent years, with the development of synthetic biology and systems metabolic engineering, constructing a high-valine-producing engineered strain with a clear genetic background, optimized metabolic network, and stable performance has become a core technical problem that urgently needs to be solved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a valine-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned valine-producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned valine-producing strain.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An artificial operon is constructed by reordering and linking genes related to valine synthesis, wherein the genes related to valine synthesis are selected from... ilvIH (G14N S17F) Gene, pykF Gene, ilvC Genes andilvD Two or more genes.

[0009] Preferably, in the above-mentioned artificial operon, the valine synthesis-related gene is: ilvIH (G14N S17F) Genes and pykF Genes, including promoters, RBS, and terminators, are listed in the following order: promoter, RBS, terminator, and terminator. ilvIH (G14N S17F) RBS pykF And the terminator, named the artificial cyborg. ilvIH (G14N S17F)- pykF .

[0010] Preferably, in the above-mentioned artificial manipulator, the promoter is connected to the... ilvH (G14N S17F) Genes are directly linked; the aforementioned ilvH (G14N S17F) Genes and the pykF The genes are linked using RBS, the nucleotide sequence of which is shown in SEQ ID NO. 25 of the sequence listing; pykF The gene is directly linked to the terminator at both ends.

[0011] Preferably, in the above-mentioned artificial manipulator, the promoter is a strong promoter, and the promoter is selected from any one of the following: TRC promoter, LAC promoter, TRP promoter, TAC promoter, LPL promoter or PGK100 promoter.

[0012] Preferably, in the above-mentioned artificial manipulator, the terminator is a strong terminator, and the terminator is selected from any one of the following: trc terminator, T7 terminator, rrnB T1 / 2 terminator, BBa_B1006 terminator or L3S2P21 / P55 terminator.

[0013] Preferably, the above-mentioned artificial operator is named artificial operator P. trc - ilvIH (G14N S17F)- pykF Its nucleotide sequence is shown in SEQ ID NO.26 of the sequence listing.

[0014] Preferably, in the above-mentioned artificial operon, the valine synthesis-related gene is: ilvC Genes and ilvD Genes, including promoters, RBS, and terminators, are listed in the following order: promoter, RBS, terminator, and terminator. ilvC RBS ilvD And the terminator, named the artificial cyborg. ilvC - ilvD .

[0015] Preferably, in the above-mentioned artificial manipulator, the promoter is a strong promoter, and the promoter is selected from any one of the following: TRC promoter, LAC promoter, TRP promoter, TAC promoter, LPL promoter or PGK100 promoter.

[0016] Preferably, in the above-mentioned artificial manipulator, the terminator is a strong terminator, and the terminator is selected from any one of the following: trc terminator, T7 terminator, rrnB T1 / 2 terminator, BBa_B1006 terminator or L3S2P21 / P55 terminator.

[0017] Preferably, the above-mentioned artificial operator is named artificial operator P. trc - ilvC - ilvD Its nucleotide sequence is shown in SEQ ID NO.27 of the sequence listing.

[0018] The above-mentioned method for constructing artificial operators is based on the same trc The promoter controls the artificial operon to begin transcription, and the same trc terminator controls the operon to terminate transcription. This is achieved by optimizing the gene connection method and the position of the gene in the operon. The nucleotide sequence of the trc promoter is shown in SEQ ID NO.23 of the sequence listing, and the nucleotide sequence of the trc terminator is shown in SEQ ID NO.24 of the sequence listing.

[0019] The above-mentioned artificial operon is used in the construction of valine-producing strains or in the fermentation production of valine.

[0020] A valine-producing strain, integrated with the aforementioned artificial operon ilvIH (G14N S17F)- pykF Or artificially manipulated P trc - ilvIH (G14N S17F)- pykF and / or the aforementioned artificial manipulators ilvC - ilvD Or artificially manipulated P trc - ilvC - ilvD .

[0021] Preferably, the above-mentioned valine-producing strain is produced by targeted modification of the starting strain. E.coli This was obtained through further modifications to the W3110, the modifications of which also include: E.coli Knockout of lactate dehydrogenase gene in the W3110 genome ldhA Overexpression of Escherichia coli on the genome E.coli The W3110-derived acetolactate synthase gene carrying the mutation site contains the gene with the mutation site. ilvIH (G14N S17F)and pyruvate kinase gene pykF Point mutation relieved valine feedback inhibition; overexpression of acetyl alcohol reductase (NADP) was achieved. + )Gene ilvC Dihydroxy acid dehydratase gene ilvD Branched-chain amino acid aminotransferase genes ilvE Overexpression of Corynebacterium glutamicum-derived Corynebacterium glutamicum ATCC 13032 branched-chain amino acid efflux protein gene brnFE .

[0022] Preferably, the valine-producing strain described above is modified using CRIPSR / Cas9 gene editing technology based on allele exchange.

[0023] Preferably, the above-mentioned valine-producing strain, wherein E.coli W3110 is E.coli W3110 ATCC 27325.

[0024] Preferably, the above-mentioned valine-producing strain, wherein ilvIH (G14N S17F) yes ilvIH It is obtained after point mutation of the gene, that is ilvH The change of G to A at the 41st base of the gene causes the 14th amino acid residue to change from glycine to aspartic acid, and the change of C to T at the 50th base causes the 17th amino acid residue to change from serine to phenylalanine.

[0025] Preferably, the above-mentioned valine-producing strain uses promoter P trc Overexpression of acetolactate synthase gene ilvI And ilvH (G14N S17F) Gene point mutations knock out branched-chain amino acid transport proteins brnQ, Heterologous introduction of glutamate-expressing Corynebacterium at this site Corynebacterium glutamicum Branched-chain amino acid efflux protein of ATCC 13032 brnFE .

[0026] Preferably, in the above-mentioned valine-producing strain, the acetolactate synthase contains a mutant gene. ilvIH (G14N S17F) and pyruvate kinase gene pykF Through the above artificial manipulator ilvIH (G14N S17F) - pykF Or artificially manipulated P trc - ilvIH (G14N S17F)- pykF Multiple copies were performed synchronously.

[0027] Preferably, the above-mentioned valine-producing strain, in ygaY The artificial operons mentioned above are sequentially integrated at the site. ilvIH (G14N S17F) - pykF Or artificially manipulated P trc - ilvIH (G14N S17F)- pykF and with promoter P trc Regulation.

[0028] Preferably, the above-mentioned valine-producing strain contains the acetyl alcohol reductase (NADP). + )Gene ilvC and dihydroxy acid dehydratase gene ilvD Through manual manipulation ilvC - ilvD Or artificially manipulated P trc - ilvC - ilvD Perform simultaneous multiple copies.

[0029] Preferably, the above-mentioned valine-producing strain, in ycgH Integrating the above artificial operons at the site ilvC - ilvD Or artificially manipulated P trc - ilvC - ilvD and with promoter P trc Regulation.

[0030] Preferably, the above-mentioned valine-producing strain will ilvIH Mutation ilvIH (G14N S17F) Replace the bootloader with a TRC bootloader; perform a double copy. ilvC and ilvD .

[0031] Preferably, the above-mentioned valine-producing strain, wherein ilvIH (G14N S17F) The nucleotide sequence is as shown in SEQ ID NO. 7 or is more than 95% identical to this sequence and originates from the same species. ilvI The amino acid sequence is shown in SEQ ID NO.4 (nucleotide sequence is shown in SEQ ID NO.3) or is more than 95% identical to this sequence and originates from the same species. Before the mutation... ilvH The amino acid sequence is shown in SEQ ID NO.6 (the nucleotide sequence is shown in SEQ ID NO.5) or is more than 95% identical to this sequence and originates from the same species. ilvCThe amino acid sequence is shown in SEQ ID NO.9 (the nucleotide sequence is shown in SEQ ID NO.8) or is more than 95% identical to this sequence and originates from the same species. ilvD The amino acid sequence is shown in SEQ ID NO. 11 (the nucleotide sequence is shown in SEQ ID NO. 10) or is more than 95% identical to this sequence and originates from the same species. ilvE The amino acid sequence is shown in SEQ ID NO. 13 (the nucleotide sequence is shown in SEQ ID NO. 12) or is more than 95% identical to this sequence and originates from the same species. pykF The amino acid sequence is as shown in SEQ ID NO. 15 (nucleotide sequence is as shown in SEQ ID NO. 14) or is more than 95% identical to this sequence and originates from the same species; brnFE The nucleotide sequence is as shown in SEQ ID NO. 22 of the sequence listing, or is more than 95% identical to this sequence and originates from the same species. brnF The amino acid sequence is shown in SEQ ID NO. 19 (the nucleotide sequence is shown in SEQ ID NO. 18) or is more than 95% identical to this sequence and originates from the same species. brnE The amino acid sequence is as shown in SEQ ID NO. 21 (nucleotide sequence is as shown in SEQ ID NO. 20) or is more than 95% identical to this sequence and originates from the same species; the promoter P trc The nucleotide sequence is shown in the sequence listing SEQ ID NO.23.

[0032] Preferably, the above-mentioned valine-producing strain, wherein ldhA The amino acid sequence is shown in SEQ ID NO.2 (the nucleotide sequence is shown in SEQ ID NO.1). brnQ The amino acid sequence is shown in SEQ ID NO.17 (the nucleotide sequence is shown in SEQ ID NO.16).

[0033] The specific steps for constructing the above-mentioned valine-producing strain are as follows: (1) Using the Escherichia coli W3110 genome as a template, the lactate dehydrogenase gene was knocked out. ldhA ; (2) Point mutation yields acetolactate synthase gene ilvIH (G14N S17F) Using the promoter P trc Replacement of acetolactate synthase gene ilvIH (G14N S17F) The natural promoter; (3) Ethyl ketone acid reductase (NADP) + )Gene ilvC A second copy, and using the promoter P trc Regulation; (4) Dihydroxy acid dehydratase gene ilvD A second copy, and using the promoter P trc Regulation; (5) Branched-chain amino acid aminotransferase gene ilvE A second copy, and using the promoter P trc Regulation; (6) Knockout of branched-chain amino acid transport proteins brnQ, Heterologous introduction of glutamate-expressing Corynebacterium at this site Corynebacterium glutamicum Branched-chain amino acid efflux protein of ATCC 13032 brnFE and with promoter P trc Regulation; (7) In ygaY Integrating the above artificial operons at the site ilvIH (G14N S17F) - pykF Or artificially manipulated P trc - ilvIH (G14N S17F)- pykF and with promoter P trc Regulation; (8) In ycgH Integrating the above artificial operons at the site ilvC - ilvD Or artificially manipulated P trc - ilvC - ilvD and with promoter P trc Regulation.

[0034] Application of the above-mentioned valine-producing strains in the fermentation production of valine.

[0035] Preferably, the above application follows these steps: (1) Seed activation and seed culture: The bacterial solution inoculated from the preservation tube is evenly spread on the activation slant for culture, and then transferred to the activation slant for further culture before being transferred to a shaker containing seed culture medium for seed culture. (2) Fermentation culture: Inoculate the seed liquid into the Erlenmeyer flask containing the fermentation culture medium at an inoculation rate of 10-15%, seal the flask with gauze, and shake to culture. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; glucose solution is added to maintain the fermentation.

[0036] Preferably, the above application follows these steps: (1) Seed activation and culture: The bacterial solution was inoculated from the preservation tube and evenly spread onto the activation slant. It was cultured at 32℃ for 12h, then transferred to the activation slant and cultured for another 10h. Finally, it was transferred to a shaker containing seed culture medium for seed culture. (2) Fermentation culture: Inoculate the seed liquid into the Erlenmeyer flask containing the fermentation medium at an inoculation rate of 10-15%, seal the flask with nine layers of gauze, and culture at 36℃ and 220 r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water. Add 60% (m / v) glucose solution to maintain the fermentation. The fermentation cycle is 30-32 h.

[0037] Preferably, in the above application, the slant culture medium used for seed activation is: glucose 1.0-3.0 g / L, peptone 7.0-13.0 g / L, yeast extract 4.0-6.0 g / L, sodium chloride 2.0-3.0 g / L, KH2PO4 0.5-1.5 g / L, MgSO4 0.1-0.3 g / L, agar powder 23-27%, with the remainder being water, pH 7.0-7.2.

[0038] Preferably, in the above application, the slant culture medium used for seed activation is: glucose 2.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the remainder being water, pH 7.0-7.2.

[0039] Preferably, in the above applications, the seed culture medium used in seed culture is: yeast extract 6.0-10.0 g / L, peptone 2.0-4.0 g / L, (NH4)2SO4 2.0-4.0 g / L, KH2PO4 2.0-4.0 g / L, V B1 V B2 V B3 V B5 V B12 1-3 mg / L each, V H 0.5-1.5 mg / L, MgSO4·7H2O 0.4-0.6 g / L, the remainder is water.

[0040] Preferably, in the above application, the seed culture medium used is: yeast extract 8.0 g / L, peptone 3.0 g / L, (NH4)2SO4 3.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0041] Preferably, in the above application, the fermentation medium used in the fermentation culture is: yeast powder 10.0 g / L, citric acid 2.0-4.0 g / L, glutamic acid 1.0-3.0 g / L, (NH4)2SO4 3.0-5.0 g / L, KH2PO4 7.0-9.0 g / L, MgSO4·7H2O 1.0-3.0 g / L, FeSO4·7H2O 35-45 mg / L, V B1 V B2 V B3 V B5 V B12 Each 0.5-1.5 mg / L, V H 0.07-0.13 mg / L, phenol red 1-3%, the remainder is water.

[0042] Preferably, in the above application, the fermentation medium used in the fermentation culture is: yeast extract 10.0 g / L, citric acid 3.0 g / L, glutamic acid 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 8.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0043] All of the above-mentioned culture media can be prepared using standard methods.

[0044] Beneficial effects: The aforementioned valine-producing strain first utilized CRIPSR / Cas9 gene editing technology based on allele exchange to remove the valine-dependent effects of valine production. ilvIH Feedback inhibition; at the same time ilvIH The natural promoter is replaced with the strong promoter P. trc This allows more pyruvate to flow to valine; an artificial operon is constructed at the ygaY site using the strong promoter P. trc control ilvIH (G14N S17F)- pykF Simultaneous multi-copy expression was performed, and an artificial operon was constructed at the ycgH site using the strong promoter P. trc control ilvC - ilvDSimultaneous multi-copy expression was performed; the constructed strain is plasmid-free, defect-free, requires no induction, and has advantages such as good genetic stability and high fermentation yield. It is an excellent strain that can stably produce valine. The strain can efficiently synthesize valine de novo using glucose as a substrate. After 32 h of shake-flask fermentation, the valine yield can reach up to 16.5 g / L. Attached Figure Description

[0045] Figure 1 A diagram illustrating the method for targeted modification of valine-producing strains. Detailed Implementation

[0046] 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.

[0047] Unless otherwise specified, the percentage sign "%" in the examples refers to volume percentage; the percentage of a solution "% (m / v)" refers to the number of grams of solute contained in 100 ml of solution.

[0048] The starting strain used in the examples was a wild-type Escherichia coli strain. E.coli W3110 ATCC 27325.

[0049] The gene editing method used was based on 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.). The engineered plasmid pGRB involved in this method uses pUC18 as its backbone and includes the promoter J23100, the gRNA-Cas9 binding region sequence, the terminator sequence, and ampicillin resistance (working concentration: 100 mg / L). The technical terms used in the following examples, such as gene integration and plasmid construction, are explained in this article. Primers used in the strain construction process are shown in Table 1.

[0050] Table 1 Primers used in strain construction

[0051]

[0052]

[0053] Example 1 like Figure 1 As shown, the specific process for constructing genetically engineered strains is as follows: Starting strain E.coli W3110 ATCC 27325 was prepared according to the operation mentioned in CN 202411087442.4. E.coli W3110 / pRed-Cas9 electrotransformation competent cells.

[0054] 1.1 Genes ldhA Knockout Using the Escherichia coli W3110 genome as a template, primer Up-Δ ldhA -F and Up-Δ ldhA -R, Down-Δ ldhA -F and Down-Δ ldhA -R were used for PCR amplification to obtain the upstream homologous arm Δ ldhA -Up and downstream homologous arms Δ ldhA -DW, using the recovered upstream and downstream homologous arms as templates, with primer Up-Δ ldhA -F and Down-Δ ldhA -R was used for overlap PCR to obtain the knockout gene. ldhA Required replacement fragment Δ ldhA Next, the primer pGRB-Δ ldhA -F and pGRB-Δ ldhA The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB-Δ ldhA Plasmid. Finally, pGRB-Δ ldhA Plasmids and overlapping fragments Δ ldhA Electric transfer to E.coli In W3110 / pRed-Cas9 electrocompetent cells; then with Up-Δ ldhA -F and Down-Δ ldhA -R is the identification primer, used to screen for positive transformants and obtain the bacterial strain. E.coli val-1.

[0055] 1.2 Genes ilvIH (G14N S17F) Point mutation integration ( yciQ ::P trc - ilvIH (G14N S17F) (integration) by E.coli Using the W3110 genome as a template, primers Up- yciQ -F and Up-P trc - yciQ -R, ilvIH -P trc -F1 and ilvIH -R1, ilvIH -F2 and ilvIH -Ptrc -R2, Down-P trc - yciQ -F, Down- yciQ PCR amplification was performed using -R, and the G41A C50T mutation was introduced using primers to obtain the upper homologous arm. yciQ -Up, lower homologous arm yciQ -DW and intermediate target fragment P trc - ilvIH Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primer Up- yciQ -F and Down- yciQ -R is integrated into the target fragment via overlap PCR. yciQ -P trc - ilvIH Then, the primer pGRB- yciQ -F and pGRB- yciQ The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- yciQ Plasmid. Finally, the purified plasmid... yciQ -P trc - ilvIH Integration fragment and plasmid pGRB- yciQ Simultaneously transferred via electroconversion E.coli In competent cells of val-1 / pRed-Cas9, and then with primer Up- yciQ -F and Down- yciQ -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E.coli val-2.

[0056] 1.3 Genes ilvC Second copy ( yghE ::P trc - ilvC (integration) Using the Escherichia coli W3110 genome as a template, primer Up- yghE -F and Up-P trc - yghE -R, ilvC -P trc -F and ilvC -P trc -R, Down-P trc - yghE -F and Down- yghE -R was used for PCR amplification to obtain the upper homologous arm. yghE -UP, lower homologous arm yghE -DW and intermediate target fragment P trc - ilvCUsing the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primer Up- yghE -F and Down- yghE -R is integrated into the target fragment via overlap PCR. yghE -P trc - ilvC Then, the primer pGRB- yghE -F and pGRB- yghE The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- yghE Plasmid. Finally, the purified plasmid... yghE -P trc - ilvC Integration fragment and plasmid pGRB- yghE Simultaneously transferred via electroconversion E.coli In competent cells of val-2 / pRed-Cas9, primer Up- yghE -F and Down- yghE -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E.coli val-3.

[0057] 1.4 Genes ilvD Second copy ( ylbE ::P trc - ilvD (integration) Using the Escherichia coli W3110 genome as a template, primer Up- ylbE -F and Up-P trc - ylbE -R, ilvD -P trc -F and ilvD -P trc -R, Down-P trc - ylbE -F and Down- ylbE -R was used for PCR amplification to obtain the upper homologous arm. ylbE -UP, lower homologous arm ylbE -DW and intermediate target fragment P trc - ilvD Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primer Up- ylbE -F and Down- ylbE -R is integrated into the target fragment via overlap PCR. ylbE -P trc - ilvD Then, the primer pGRB- ylbE -F and pGRB- ylbEThe DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- ylbE Plasmid. Finally, the purified plasmid... ylbE -P trc - ilvD Integration fragment and plasmid pGRB- ylbE Simultaneously transferred via electroconversion E. coli In competent cells of val-3 / pRed-Cas9, primer Up- ylbE -F and Down- ylbE -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E. coli val-4.

[0058] 1.5 genes ilvE Second copy ( yjgX ::P trc - ilvE (integration) Using the Escherichia coli W3110 genome as a template, primer Up- yjgX -F and Up-P trc - yjgX -R, ilvE -P trc -F and ilvE -P trc -R, Down-P trc - yjgX -F and Down- yjgX -R was used for PCR amplification to obtain the upper homologous arm. yjgX -UP, lower homologous arm yjgX -DW and intermediate target fragment P trc - ilvE Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primer Up- yjgX -F and Down- yjgX -R is integrated into the target fragment via overlap PCR. yjgX -P trc - ilvE Then, the primer pGRB- yjgX -F and pGRB- yjgX The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- yjgX Plasmid. Finally, the purified plasmid... yjgX -P trc - ilvE Integration fragment and plasmid pGRB- yjgX Simultaneously transferred via electroconversion E. coli In competent cells of val-4 / pRed-Cas9, primer Up- yjgX -F and Down- yjgX -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E. coli val-5.

[0059] 1.6 genes brnFE Integration (Δ) brnQ ::P trc - lrp -RBS- brnFE (integration) Using the Escherichia coli W3110 genome as a template, primer Up-Δ brnQ -F and Up-P trc -Δ brnQ -R, brnFE -F and brnFE -R, lrP -trc-F and lrP -RBS-R, brnFE -RBS-F and brnFE -trc-R,Down-P trc -Δ brnQ -F and Down-Δ brnQ -R was used for PCR amplification to obtain the upper homologous arm Δ brnQ -UP, lower homologous arm Δ brnQ -DW and intermediate target fragment P trc -lrp-RBS- brnFE Using the recovered upstream and downstream homologous arms and the intermediate target fragment as templates, primer Up-Δ brnQ -F and Down-Δ brnQ -R is integrated with the target fragment Δ via overlap PCR. brnQ -P trc -lrp-RBS- brnFE Then, the primer pGRB-Δ brnQ -F and pGRB-Δ brnQ The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB-Δ brnQ Plasmid. Finally, the purified Δ brnQ -P trc -lrp-RBS- brnFE Integration fragment and plasmid pGRB-Δ brnQ Simultaneously transferred via electroconversion E. coli In competent cells of val-5 / pRed-Cas9, the primer Up-Δ brnQ -F and Down-Δ brnQ -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E. coli val-6.

[0060] 1.7 Artificial operon gene Ptrc - ilvIH (G14N S17F)- pykF Integration ( ygaY ::P trc - ilvIH (G14N S17F)- pykF (integration) Using the Escherichia coli W3110 genome as a template, primer Up- ygaY -F and Up -P trc -ygaY- R, Down-P trc - ygaY -F and Down- ygaY -R, pykF -RBS-F and pykF -P trc -R was used for PCR amplification to obtain the upper homologous arm. ygaY -UP, lower homologous arm ygaY -DW, fragment P trc - pykF。 Using a usable concentration of E. coli val-2 genome as a template, primers were used... ilvIH -P trc -F1 and ilvIH -RBS-R2 was used for PCR amplification to directly obtain fragment P trc - ilvIH (G14N S17F) To recycle ygaY -UP、 ygaY -DW, P trc - ilvIH (G14N S17F) P trc - pykF Using the fragment as a template, and primer Up- ygaY -F and Down- ygaY -R fragments were obtained via overlap PCR. ygaY -P trc - ilvIH (G14N S17F)- pykF, Next, the primer pGRB- ygaY -F and pGRB- ygaY The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- ygaY Plasmid. Finally, the purified plasmid... ygaY -P trc - ilvIH (G14N S17F)- pykF Integration fragment and plasmid pGRB- ygaY Simultaneously transferred via electroconversion E. coliIn competent cells of val-6 / pRed-Cas9, and then with primer Up- ygaY -F and Down- ygaY -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E. coli val-7.

[0061] 1.8 ilvIH (G14N S17F) , pykF Gene integration ( ygaY ::P trc - ilvIH (G14N S17F) , yjiT ::P trc -pykF (integration) Using the Escherichia coli W3110 genome as a template, primer Up- ygaY -F and Up -P trc -ygaY- R, Down-P trc - ygaY -F and Down- ygaY -R, Up- yjiT -F and Up -P trc -yjiT- R, Down-P trc - yjiT -F and Down- yjiT -R, pykF -P trc -F and pykF -P trc -R was used for PCR amplification to obtain the upper homologous arms. ygaY -UP, yjiT -UP, lower homologous arm ygaY -DW, yjiT -DW, fragment P trc - pykF Using a usable concentration of E. coli val-2 genome as a template, primers were used... ilvIH -P trc -F1 and ilvIH -R2 was used for PCR amplification to directly obtain fragment P. trc - ilvIH (G14N S17F) To recycle ygaY -UP、 ygaY -DW, P trc - ilvIH (G14N S17F) , yjiT -UP、 yjiT -DW, P trc - pykF Using the fragment as a template, and primer Up- ygaY -F and Down- ygaY -R, Up- yjiT -F and Down- yjiT -R, fragment obtained by overlap PCR ygaY -P trc - ilvIH (G14N S17F) and yjiT ::P trc -pykF Then, the primer pGRB- ygaY -F and pGRB- ygaY -R, pGRB- yjiT -F and pGRB- yjiT The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- ygaY and pGRB- yjiT Plasmid. Finally, the purified plasmid... ygaY -P trc - ilvIH (G14N S17F) , yjiT ::P trc -pykF Integration fragment and plasmid pGRB- ygaY、 pGRB- yjiT Iterative transfer via electrical conversion E. coli In competent cells of val-6 / pRed-Cas9, and then with primer Up- ygaY -F and Down- ygaY -R, Up- yjiT -F and Down- yjiT -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E. coli val-8.

[0062] 1.9 Artificial Operator Genes ilvC - ilvD Integration ( ycgH ::P trc - ilvC - ilvD ) Using the Escherichia coli W3110 genome as a template, primer Up- ycgH -F and Up -P trc -ycgH- R, Down-P trc - ycgH -F and Down- ycgH -R, ilvC -P trc -F and ilvC-RBS-R, ilvD -RBS-F and ilvD -P trc -R was used for PCR amplification to obtain the upper homologous arms. ycgH -UP, lower homologous arm ycgH -DW, fragment P trc - ilvC、 P trc - ilvD . Recycled ycgH -UP、 ycgH -DW, P trc - ilvC、 P trc - ilvD Using the fragment as a template, and primer Up- ycgH -F and Down- ycgH -R, fragment obtained by overlap PCR ycgH -P trc - ilvC - ilvD Then, the primer pGRB- ycgH -F and pGRB- ycgH The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- ycgH Plasmid. Finally, the purified plasmid... ycgH -P trc - ilvC - ilvD Integration fragment and plasmid pGRB- ycgH Transmitted via electroconversion E. coli In competent cells of val-7 / pRed-Cas9, primer Up- ycgH -F and Down- ycgH -R indicates the primers used to screen for positive transformants, ultimately obtaining the strain. E. coli val-9.

[0063] 1.10 ilvC , ilvD Gene integration ( ycgH ::P trc - ilvC,yjiP ::P trc - ilvD ) Using the Escherichia coli W3110 genome as a template, primer Up- ycgH -F and Up -P trc -ycgH- R, Down-P trc - ycgH -F and Down- ycgH -R, Up- yjiP -F and Up-P trc -yjiP- R, Down-P trc - yjiP -F and Down- yjiP -R, ilvC -P trc -F and ilvC -P trc -R, ilvD -P trc -F and ilvD -P trc -R, perform PCR amplification, and obtain the upper homologous arms respectively. ycgH -UP, yjiP -UP, lower homologous arm ycgH -DW, yjiP -DW, fragment P trc - ilvC P trc - ilvD . Recycled ycgH -UP、 ycgH -DW, P trc - ilvC , yjiP -UP、 yjiP -DW, P trc - ilvD Using the fragment as a template, and primer Up- ycgH -F and Down- ycgH -R, Up- yjiP -F and Down- yjiP -R, fragment obtained by overlap PCR ycgH ::P trc - ilvC and yjiP ::P trc - ilvD Then, the primer pGRB- ycgH -F and pGRB- ycgH -R, pGRB- yjiP -F and pGRB- yjiP The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- ycgH and pGRB- yjiP Plasmid. Finally, the purified plasmid... ycgH ::P trc - ilvC , yjiP ::P trc - ilvD Integration fragment and plasmid pGRB- ycgH、 pGRB- yjiP Iterative transfer via electrical conversion E.coli In competent cells of val-7 / pRed-Cas9, primer Up- ycgH -F and Down- ycgH -R, Up- yjiP -F and Down- yjiP -R indicates that the primers are used to screen for positive transformants, ultimately yielding the strain. E.coli val-10.

[0064] 1.11 Artificial Operon Genes ilvC - ilvD Integration ( ycgH ::P trc - ilvC - ilvD ) The strain was constructed using the same method as in 1.9, and the purified strain was then... ycgH -P trc - ilvC - ilvD Integration fragment and plasmid pGRB- ycgH Iterative transfer via electroconversion E.coli The strain was eventually obtained from competent val-8 / pRed-Cas9 cells. E.coli val-11.

[0065] 1.12 ilvC , ilvD Gene integration ( ycgH ::P trc - ilvC,yjiP ::P trc - ilvD ) The strain was constructed using the same method as 1.10, by purifying the strain... ycgH ::P trc - ilvC , yjiP ::P trc - ilvD Integration fragment and plasmid pGRB- ycgH、 pGRB- yjiP Iterative transfer via electrical conversion E.coli The strain was eventually obtained from competent val-8 / pRed-Cas9 cells. E.coli val-12.

[0066] The strains involved in the above construction process are shown in Table 2.

[0067] The strains listed in Table 2

[0068] The above yciQ The nucleotide sequence is shown in SEQ ID NO.28 of the sequence listing. yghE The nucleotide sequence is shown in SEQ ID NO.29 of the sequence listing. ylbE The nucleotide sequence is shown in SEQ ID NO.30 of the sequence listing. yjgX The nucleotide sequence is shown in SEQ ID NO.31 of the sequence listing. ygaY The nucleotide sequence is shown in SEQ ID NO.32 of the sequence listing. yjiT The nucleotide sequence is shown in SEQ ID NO.33 of the sequence listing. ycgH The nucleotide sequence is shown in SEQ ID NO.34 of the sequence listing. yjiP The nucleotide sequence is shown in the sequence listing SEQ ID NO.35.

[0069] Example 2 Valine was produced by shake-flask fermentation using the valine-producing strain described in Example 1.

[0070] 2.1 Culture medium 2.1.1 Slant Culture Medium Glucose 2.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, dissolved in water, then adjusted to pH 7.0-7.2 with sodium hydroxide, and brought to a final volume of 500 mL. The solution was dispensed into test tubes (9 mL / tube) and flasks (45 mL / flask), and sterilized in an autoclave at 121℃ for 20 min.

[0071] 2.1.2 Seed Culture Medium Yeast extract 8.0 g / L, peptone 3.0 g / L, (NH4)2SO4 3.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0072] 2.1.3 Fermentation medium Yeast powder 10.0 g / L, citric acid 3.0 g / L, glutamic acid 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 8.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 V B2 V B3 V B5 VB12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0073] 2.2.1 Seed activation and culture: The bacterial culture was inoculated from the preservation tube and evenly spread onto the activation slant. It was then incubated at 37°C for 12 hours, transferred to the activation slant and incubated for another 10 hours. Finally, it was transferred to a shaker containing 5 mL of seed culture medium for seed culture.

[0074] 2.2.2 Fermentation culture: Inoculate the seed culture at a rate of 15% into a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL). Seal the flask with nine layers of gauze and incubate at 36°C with shaking at 220 rpm. During fermentation, maintain the pH at 7.0-7.2 by adding ammonia. Add 60% (m / v) glucose solution to maintain fermentation (using phenol red as an indicator; when the fermentation broth color no longer changes, it indicates a sugar deficiency; add 1-2 mL of 60% (m / v) glucose solution if a sugar deficiency is detected). The fermentation cycle is 32 h. No antibiotics or inducers are added during fermentation.

[0075] After 32 hours of shake-flask fermentation, the valine yield can reach up to 16.5 g / L.

[0076] 2.3 Fermentation Results: Table 3 shows the valine-producing strains described in Table 2 and their valine production via shake-flask fermentation. Table 3 Results of shake-flask fermentation of valine-producing strains

[0077] strain E.coli Val-1 through 6 indicate that the modification relieved the feedback inhibition of valine, thus opening up the valine production pathway. (Strain) E.coli Val-7, despite adding key genes for valine synthesis ilvIH Key genes for the supply of precursor pyruvate pykF However, the yield did not increase significantly; in contrast, using artificial manipulators for synchronized multiple copies... ilvIH、pykF The gene significantly increased yield. Subsequently, based on this strain, the key gene was... ilvC、ilvD Constructing artificial operons and performing simultaneous multiple copies further improved yield, enabling the strain to achieve higher productivity. E.coli Val-9 valine production can reach up to 16.5 g / L.

[0078] 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. A valine-producing strain, characterized in that: It utilizes targeted modification methods on the starting strain E. coli It was obtained by modifying the W3110, and the modification is as follows: E. coli Knockout of lactate dehydrogenase gene in the W3110 genome ldhA Overexpression of the acetolactate synthase gene carrying a point mutation ilvIH (G14N S17F) Using the promoter P trc Genes regulating acetolactate synthase ilvIH (G14N S17F) Expression; overexpression of acetyl alcohol reductase gene ilvC and with promoter P trc Regulation ilvC Expression; overexpression of dihydroxy acid dehydratase gene ilvD and with promoter P trc Regulation ilvD Expression; overexpression of branched-chain amino acid aminotransferase genes ilvE and with promoter P trc Regulation ilvE Expression; knockout of branched-chain amino acid transport proteins brnQ Overexpression of branched-chain amino acid efflux protein genes derived from Corynebacterium glutamicum brnFE and with promoter P trc Regulation; in ygaY Artificial operon gene P integrated at genomic locus trc - ilvIH (G14N S17F)- pykF ;exist ycgH Artificial operon gene P integrated at genomic locus trc - ilvC - ilvD ; wherein, the ilvIH (G14N S17F) The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing. ilvC The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing. ilvD The nucleotide sequence is shown in SEQ ID NO. 10 of the sequence listing. ilvE The nucleotide sequence is shown in SEQ ID NO.12 of the sequence listing. brnFE The nucleotide sequence is shown in SEQ ID NO.22 of the sequence listing, and the artificial operon gene P... trc - ilvIH (G14N S17F)- pykF The nucleotide sequence is shown in SEQ ID NO. 26 of the sequence listing, for the artificial operon gene P. trc - ilvC - ilvD The nucleotide sequence is shown in SEQ ID NO.27 of the sequence listing, and the promoter P trc The nucleotide sequence is shown in the sequence listing SEQ ID NO.

23.

2. The method for constructing the valine-producing strain according to claim 1, characterized in that: The specific steps are as follows: (1) Using the Escherichia coli W3110 genome as a template, the lactate dehydrogenase gene was knocked out. ldhA ; (2) Overexpression of the acetolactate synthase gene carrying a point mutation ilvIH (G14N S17F) Using the promoter P trc Genes regulating acetolactate synthase ilvIH (G14N S17F) The expression; (3) Overexpression of acetyl alcohol reductase gene ilvC and with promoter P trc Regulation ilvC The expression; (4) Overexpression of dihydroxy acid dehydratase gene ilvD and with promoter P trc Regulation ilvD The expression; (5) Overexpression of branched-chain amino acid aminotransferase genes ilvE and with promoter P trc Regulation ilvE The expression; (6) Knockout of branched-chain amino acid transport proteins brnQ, Heterologous introduction of branched-chain amino acid efflux proteins expressing Corynebacterium glutamicum at this site brnFE and with promoter P trc Regulation; (7) In ygaY Integrating artificial operon P at genomic loci trc - ilvIH (G14N S17F)- pykF Its nucleotide sequence is shown in SEQ ID NO.26 of the sequence listing. The artificial operon uses the promoter P. trc Regulation; (8) In ycgH Integrating artificial operon P at genomic loci trc - ilvC - ilvD Its nucleotide sequence is shown in SEQ ID NO.27 of the sequence listing. The artificial operon uses the promoter P. trc Regulation.

3. The application of the valine-producing strain of claim 1 in the fermentation production of valine.

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