GENETICALLY ENGINEERED STRAIN WITH HIGH YIELD OF L-VALINE AND METHOD FOR PRODUCING L-VALINE BY FERMENTATION

Abstract

A genetically engineered strain having high-yield of L-valine is disclosed. Starting from Escherichia coli W3110, an acetolactate synthase gene alsS of Bacillus subtilis is inserted into a genome thereof and overexpressed; a ppGpp 3′-pyrophosphate hydrolase mutant R290E/K292D gene spoTM of Escherichia coli is inserted into the genome and overexpressed; a lactate dehydrogenase gene ldhA, a pyruvate formate lyase I gene pflB, and genes frdA, frdB, frdC, frdD of four subunits of fumaric acid reductase are deleted from the genome; a leucine dehydrogenase gene bcd of Bacillus subtilis replaces a branched chain amino acid transaminase gene ilvE of Escherichia coli; and an acetohydroxy acid isomeroreductase mutant L67E/R68F/K75E gene ilvCM replaces the native acetohydroxy acid isomeroreductase gene ilvC of Escherichia coli. Furthermore, the L-valine fermentation method is improved by using a two-stage dissolved oxygen control. The L-valine titer and the sugar-acid conversion rate are increased.

Claims

1. A genetically engineered strain with high yield of L-valine, wherein E. coli W3110 is taken as a starting strain, a gene alsS encoding acetolactate synthase of Bacillus subtilis is inserted into a genome of the E. coli W3110 and subjected to overexpression; a mutant gene spoT.sup.M encoding ppGpp 3-pyrophosphate hydrolase mutant R290E/K292D of E. coli is inserted into the genome of the E. coli W3110 and subjected to overexpression; a gene ldhA encoding lactate dehydrogenase, a gene pflB encoding pyruvate formate lyase I, and genes frdA, frdB, frdC and frdD for four subunits of fumarate reductase are deleted from the genome of the E. coli W3110; a gene bcd encoding leucine dehydrogenase of Bacillus subtilis replaces a gene ilvE encoding branched-chain amino acid transaminase of the E. coli W3110; and a mutant gene ilvC.sup.M encoding acetohydroxy acid isomeroreductase mutant L67E/R68F/K75E replaces a gene ilvC encoding acetohydroxy acid isomeroreductase of the E. coli W3110.

2. The genetically engineered strain according to claim 1, wherein the gene alsS encoding the acetolactate synthase is inserted into a pseudogene ydeU site and controlled by a promoter P.sub.trc; and the mutant gene spoT.sup.M encoding the ppGpp 3′-pyrophosphate hydrolase mutant R290E/K292D is inserted into a pseudogene yeeP site and controlled by the promoter P.sub.trc.

3. The genetically engineered strain according to claim 1, wherein the mutant gene spoT.sup.M encoding the ppGpp 3′-pyrophosphate hydrolase mutant R290E/K292D has a nucleotide sequence as shown in SEQ ID NO: 1.

4. The genetically engineered strain according to claim 1, wherein the mutant gene ilvC.sup.M encoding the acetohydroxy acid isomeroreductase mutant L67E/R68F/K75E has a nucleotide sequence as shown in SEQ ID NO: 2.

5. Use of the genetically engineered strain according to claim 1 in a production of L-valine by a fermentation.

6. A method for producing L-valine by a fermentation using the genetically engineered strain according to claim 1, wherein the method is a two-stage dissolved oxygen control process, comprising: performing an aerobic fermentation in a first stage of fermentation, and then performing an anaerobic fermentation in a middle stage and later stage of fermentation.

7. The method according to claim 6, comprising: subjecting the genetically engineered strain to an activation to prepare a seed fermentation broth, inoculating the seed fermentation broth into a triangular flask filled with a fermentation medium with an inoculum size of 10-15%, and subjecting to a shake cultivation at 37° C. and 200 r/min to obtain a bacterial solution, after 12-16 h, transferring the bacterial solution to a 30 mL sealed flask isolated from air, and subjecting the 30 mL sealed flask to the shake cultivation at 37° C. and 200 r/min for 8-12 h, wherein the fermentation medium comprises 18 g/L glucose, 1 g/L yeast powder, 2 g/L peptone, 2 g/L KH.sub.2PO.sub.4, 1 g/L sodium citrate, 0.7 g/L MgSO.sub.4•7H.sub.2O, 100 mg/L FeSO.sub.4•7H.sub.2O, 100 mg/L MnSO.sub.4•H.sub.2O, 0.8 mg/L V.sub.B1, 0.3 mg/L V.sub.H, 20 mL/L phenol red, and two droplets of a defoaming agent, and the fermentation medium has an initial pH of 7.0-7.2.

8. The method according to claim 6, comprising: subjecting the genetically engineered strain to an activation on a slant for two generations, inoculating the genetically engineered strain activated into a fermentation medium with an inoculum size of 15-20% to perform a fermentation, wherein in the fermentation, pH is controlled at about 6.7, a temperature is maintained at 35° C., and dissolved oxygen is controlled at 25-30%; after glucose in the fermentation medium is depleted, feeding an 80% m/v glucose solution to maintain a glucose concentration in the fermentation medium at 0.1-5 g/L; 12-16 h later, stopping an introduction of sterile air and starting to carry out an anaerobic fermentation, wherein the fermentation medium comprises 30 g/L glucose, 2 g/L yeast powder, 7 g/L K.sub.2HPO.sub.4, 3 g/L (NH.sub.4).sub.2SO.sub.4, 2 g/L citric acid, 1 g/L MgSO.sub.4•7H.sub.2O, 30 mg/L FeSO.sub.4•7H.sub.2O, 10 mg/L MnSO.sub.4, 1 mg/L each of V.sub.B1, V.sub.B3, V.sub.B5, V.sub.B12, and V.sub.H, and the fermentation medium has an initial pH of 6.5-7.0.

9. A method for making a genetically engineered strain with high yield of L-valine, wherein the method adopts a CRISPR/Cas 9 mediated gene editing technology and the method comprises steps of: 1) taking E. coli W3110 as a starting strain, constructing a junction fragment P.sub.trc-alsS of a promoter P.sub.trc and a gene alsS encoding acetolactate synthase, and inserting the junction fragment P.sub.trc-alsS into a pseudogene ydeU site; 2) constructing a junction fragment P.sub.trc-spoT.sup.M of the promoter P.sub.trc and a mutant gene spoT.sup.M encoding ppGpp 3-pyrophosphate hydrolase mutant R290E/K292D, and inserting the junction fragment P.sub.trc-spoT.sup.M into a pseudogene yeeP site; 3) knocking out a gene ldhA encoding lactate dehydrogenase, a gene pflB encoding pyruvate formate lyase I, and genes frdA, frdB, frdC and frdD for four subunits of fumarate reductase from a genome of the E. coli W3110; 4) constructing a junction fragment of the promoter P.sub.trc and a gene bcd encoding leucine dehydrogenase to replace a gene ilvE on the genome of the E. coli W3110; and 5) constructing a junction fragment of the promoter P.sub.trc and a mutant gene ilvC.sup.M encoding acetohydroxy acid isomeroreductase mutant [L67E, R68F, K75E] to replace a gene ilvC on the genome of the E. coli W3110.

10. The use according to claim 5, wherein the gene alsS encoding the acetolactate synthase is inserted into a pseudogene ydeU site and controlled by a promoter P.sub.trc; and the mutant gene spoT.sup.M encoding the ppGpp 3′-pyrophosphate hydrolase mutant R290E/K292D is inserted into a pseudogene yeeP site and controlled by the promoter P.sub.trc.

11. The use according to claim 5, wherein the mutant gene spoT.sup.M encoding the ppGpp 3′-pyrophosphate hydrolase mutant R290E/K292D has a nucleotide sequence as shown in SEQ ID NO: 1.

12. The use according to claim 5, wherein the mutant gene ilvC.sup.M encoding the acetohydroxy acid isomeroreductase mutant L67E/R68F/K75E has a nucleotide sequence as shown in SEQ ID NO: 2.

13. The method according to claim 6, wherein the gene alsS encoding the acetolactate synthase is inserted into a pseudogene ydeU site and controlled by a promoter P.sub.trc; and the mutant gene spoT.sup.M encoding the ppGpp 3-pyrophosphate hydrolase mutant R290E/K292D is inserted into a pseudogene yeeP site and controlled by the promoter P.sub.trc.

14. The method according to claim 6, wherein the mutant gene spoT.sup.M encoding the ppGpp 3′-pyrophosphate hydrolase mutant R290E/K292D has a nucleotide sequence as shown in SEQ ID NO: 1.

15. The method according to claim 6, wherein the mutant gene ilvC.sup.M encoding the acetohydroxy acid isomeroreductase mutant L67E/R68F/K75E has a nucleotide sequence as shown in SEQ ID NO: 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1: Electrophoretogram for construction and verification of the junction fragment P.sub.trc-alsS. M: Marker, 1: ydeU upstream homologous arm, 2: alsS target fragment, 3: ydeU downstream homologous arm, 4: integrated target fragment, 5: negative control, 6: fragment identified after junction.

[0032] FIG. 2: Electrophoretogram for construction and verification of the junction fragment P.sub.trc-spoT.sup.M. M: Marker, 1: yeeP upstream homologous arm, 2: spoT.sup.M target fragment, 3: yeeP downstream homologous arm, 4: overlap fragment, 5: negative control, 6: fragment identified after junction.

[0033] FIG. 3: Electrophoretogram for construction and verification of the fragment with ldhA gene knockout. M: Marker, 1: ldhA upstream homologous arm, 2: ldhA downstream homologous arm, 3: overlap fragment, 4: positive control, 5: negative control, 6: fragment identified after knockout.

[0034] FIG. 4: Electrophoretogram for construction and verification of the fragment with pflB gene knockout. M: Marker, 1: pflB upstream homologous arm, 2: pflB downstream homologous arm, 3: overlap fragment, 4: negative control, 5: fragment identified after knockout.

[0035] FIG. 5: Electrophoretogram for construction and verification of the fragment with frdABCD gene knockout. M: Marker, 1: frdABCD upstream homologous arm, 2: frdABCD downstream homologous arm, 3: overlap fragment, 4: negative control, 5: fragments identified after knockout.

[0036] FIG. 6: Electrophoretogram for construction and verification of the junction fragment P.sub.trc-bcd. M: Marker, 1: bcd upstream homologous arm, 2: bcd fragment, 3: bcd downstream homologous arm, 4: overlap fragment, 5: negative control, 6: fragment identified after junction.

[0037] FIG. 7: Electrophoretogram for construction and verification of the junction fragment P.sub.trc-ilvC.sup.M. M: Marker, 1: ilvC.sup.M upstream homologous arm, 2: ilvC.sup.M fragment, 3: ilvC.sup.M downstream homologous arm, 4: overlap fragment, 5: negative control, 6: fragment identified after junction.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The disclosure is described below by specific embodiments. Unless otherwise specified, the technical means used in the disclosure are methods well known to those skilled in the art. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the disclosure, and the spirit and scope of the disclosure are limited only by the claims. It will be apparent to those skilled in that art that various changes or modifications that are made to the composition and amounts of materials in these embodiments without departing from the spirit and scope of the disclosure are within the protection scope of the disclosure.

Example 1

[0039] Strains VXR01, VXR02 and VXR03 with high yield of L-valine were constructed by a gene editing method as described in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia co/i using CRISPR-Cas9 meditated genome editing. Metabolic engineering, 2015, 31: 13-21.), which specifically comprises:

[0040] 1. Construction of Strain VXR01 by Integrating Acetolactate Synthase Encoding Gene alsS into Pseudogene ydeU Site

[0041] 1.1 Preparation of Recombinant DNA Fragments

[0042] The alsS gene was integrated into the ydeU pseudogene locus. The gene alsS (NCBI-Protein ID: NP_391482.2) was obtained by PCR amplification using the genome of B. subtilis 168 as the template. Using E. coli W3110 genome as the template, ydeU upstream and downstream homologous arm fragments (primers UP-ydeU-S, UP-ydeU-A; DN-ydeU-S, DN-ydeU-A) were amplified. The promoter P.sub.trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the alsS gene. Overlap PCR was carried out using the upstream and downstream homologous arms and the target gene alsS as templates respectively to obtain integrated fragment P.sub.trc-alsS (primers P.sub.trc-alsS-S and P.sub.trc-alsS-A). The electrophoretogram for construction of the integrated fragment P.sub.trc-alsS and PCR verification of the positive strain is shown in FIG. 1. PCR conditions (Takara PrimeSTAR HS enzyme): pre-denaturation (95° C.) for 5 min; 30 cycles: denaturation (98° C.) for 10 s and annealing for 15 s; elongation at 72° C. for 10 min; cooling (4° C.). The upstream homologous arm UP-ydeU, the E. coli P.sub.trc promoter, the target fragment alsS, and the downstream homologous arm DN-ydeU were connected by overlap PCR. The PCR amplification and overlap PCR systems are shown in the following tables.

TABLE-US-00001 TABLE 1 PCR amplification system Component Volume (50 μL) DNA template (200 ng/μL) 1 μL Upstream primer (10 μmol/L) 1 μL Downstream primer (10 μmol/L) 1 μL dNTP mixture(10 mmol/L) 4 μL 5 × Buffer 10 μL HS enzyme (5 U/μL) 0.5 μL ddH.sub.2O 32.5 μL

TABLE-US-00002 TABLE 2 Overlap PCR amplification system Component Volume (50 μL) Fragment template 2 μL Upstream homologous arm 1 μL upstream primer (10 μmol/L) Downstream homologous arm 1 μL downstream primer (10 μmol/L) dNTP mixture (10 mmol/L) 4 μL 5 × Buffer 10 μL HS enzyme (5 U/μL) 0.5 μL ddH.sub.2O 31.5 μL

[0043] 1.2 Construction of gRNA Plasmid

[0044] The purpose of constructing gRNA plasmid is to enable the complex of Cas9 protein and tracrRNA to identify the target site of the target gene through base pairing and PAM so as to break the target DNA double strands. The target sequence was designed using CRISPR RGEN Tools. The target sequence DNA fragment in the plasmid pGRB-ydeU was prepared by annealing the primers gRNA-ydeU-S and gRNA-ydeU-A. Reaction conditions: pre-denaturation at 95° C. for 5 min; annealing at 30-50° C. for 1 min. The annealing system is as follows:

TABLE-US-00003 TABLE 3 Annealing system Reaction system Volume (20 μL) Primer (10 μmol/L) 10 μL Reverse complement primer (10 μmol/L) 10 μL

[0045] The DNA fragment containing the target sequence was homologously recombined with the linearized vector pGRB. The recombinant system is shown in the following table. The recombinases used were those of CloneExpress® II One Step Cloning Kit series; recombination conditions: 37° C., 30 min.

TABLE-US-00004 TABLE 4 Recombinant system Reaction system Volume (10 μL) 5 × CE II Buffer 4 μL Linearized vector 1 μL Insert fragment 1 μL Exnase ® II 2 μL ddH.sub.2O 2 μL

[0046] The entire recombinant system was uniformly added into 100 μL of DH5a transformed competent cells, which were treated with ice bath for 30 min and incubate at 42° C. for 90 s, followed by the addition of 900 μL of resuscitation fluid for resuscitation at 37° C. for 1 h. After centrifuging at 8000 rmp for 2 min, part of the supernatant was discarded, and about 100 μL of bacterial cells was retained for resuspension, which was then uniformly coated on a plate containing ampicillin, and cultured overnight at 37° C. After single colonies grew out of the plate, positive recombinants were selected through colony PCR identification, plasmids were extracted and identified through enzyme digestion.

[0047] 1.3 Electro-Transformation of Plasmids and Recombinant DNA Fragments

[0048] The pGRB-ydeU plasmid and the donor P.sub.trc-alsS fragment were simultaneously electrotransformed to electro-competent cells containing pREDCas9. The cells resuscitated after electrotransformation were plated on LB plates containing ampicillin and spectinomycin and incubated overnight at 32° C. Colony PCR was performed using identification primers (ydeU-identification-S, ydeU-identification-A) to screen for positive recombinants.

[0049] 1.4 Elimination of Plasmids

[0050] Elimination of gRNA plasmid: The positive recombinants were cultured overnight in LB medium containing 0.2% arabinose, and then streaked in three regions on the LB plate containing spectinomycin resistance by the inoculation loop. The single colonies were selected and dispensed onto the LB plates containing ampicillin and spectinomycin respectively, and the single colonies that did not grow on the ampicillin plate but grew on the spectinomycin resistance plate were selected.

[0051] Elimination of pREDCas9 plasmid: The positive recombinants were transferred to LB liquid medium without resistance and cultured overnight at 42° C., and then streaked in three regions on the LB plate without resistance by the inoculation loop. The single colonies were selected and dispensed onto the LB plates containing spectinomycin and the LB plates without resistance respectively, and the single colonies that did not grow on the spectinomycin plate but grew on the plate without resistance were selected.

[0052] 2. Construction of Strain VXR02 by Integrating Gene spoT.sup.M into Pseudogene yeeP Site

[0053] 2.1 Preparation of Mutant Gene spoT.sup.M

[0054] Primer design software primer5 was used, the genome of E. coli W3110 was used as the template (NCBI-Protein ID: BAE77643), the mutation site was designed in the downstream primer of the first fragment, and the gene spoT.sup.M encoding mutant was divided into two fragments for amplification (primers UP-spoT.sup.M-S, up-spoT.sup.M-A; DN-spoT.sup.M-S, DN-spoT.sup.M-A). Then, overlap PCR was performed by using the two fragments as templates to obtain mutant gene spoT.sup.M (primers UP-spoT.sup.M-S and DN-spoT.sup.M-A), with the nucleotide sequence being as set forth in SEQ ID NO: 1.

[0055] 2.2 Preparation of Recombinant DNA Fragments

[0056] Using the genome of E. coli W3110 as the template, the yeeP upstream and downstream homologous arm fragments (primers UP-yeeP-S and UP-yeeP-A; DN-yeeP-S, DN-yeeP-A) were amplified. The promoter P.sub.trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the gene spoT.sup.f. Then, overlap PCR was carried out using the upstream and downstream homologous arms and the target gene spoT.sup.M as templates (primers P.sub.trc-spoT.sup.M-S and P.sub.trc-spoT.sup.M-A). The electrophoretogram for construction of the integrated fragment P.sub.trc-spoT.sup.M and PCR verification of the positive strain is shown in FIG. 2. The PCR amplification and overlap systems are the same as 1.1.

[0057] 2.3 Construction of gRNA Plasmid

[0058] The target sequence DNA fragment in plasmid pGRB-yeeP was prepared by annealing of primers gRNA-yeeP-S and gRNA-yeeP-A, and the construction method is the same as that in 1.2.

[0059] 2.4 Electro-Transformation of Plasmids and Recombinant DNA Fragments

[0060] The pGRB-yeeP plasmid and the donor P.sub.trc-spoT.sup.M fragment were simultaneously electrotransformed into electro-competent cells containing pREDCas9. The cells resuscitated after electrotransformation were plated on LB plates containing ampicillin and spectinomycin and incubated overnight at 32° C. Colony PCR was performed using the upstream primer of the upstream homologous arm and the downstream primer of the downstream homologous arm of the overlap fragments as identification primers to screen for positive recombinants.

[0061] 2.5 Elimination of Plasmids

[0062] The method is the same as described in 1.4.

[0063] 3. Construction of Strain VXR03 by Knockout of Genes ldhA, pflB, frdABCD

[0064] 3.1 Preparation of Recombinant DNA Fragments

[0065] With the genome of E. coli W3110 as the template, and based on the upstream and downstream sequences of the genes ldhA (NCBI-protein ID: NP_415898.1), pflB (NCBI-Protein ID: NP_415423.1) and frdABCD (NCBI-protein ID: NP_418578.1, NP_418577.1, NP_418576.1 and NP_418575.1), upstream homologous arm primers (U-ldhA-F, U-ldhA-R; U-pflB-F, U-pflB-R; U-frdcABCD-F, U-frdABCD-R) and downstream homologous arm primers (D-ldhA-F, D-ldhA-R; D-pflB-F, D-pflB-R; D-frdABCD-F, D-frdABCD-R) were designed respectively. Then, overlap PCR was performed using the upstream and downstream homologous arms as templates to obtain genes ldhA, pflB and frdABCD knock-out fragments (upstream homologous arm-downstream homologous arm). Fragment with gene ldhA knockout, fragment with gene pflB knockout, fragment with genes frdABCD knock-out. The electrophoretogram for construction of the knockout fragments and PCR verification of the positive strain is shown in FIGS. 3-5, respectively. The PCR amplification and overlap systems are the same as those in 1.1.

[0066] 3.2 Construction of gRNA Plasmid

[0067] Target sequence DNA fragments in plasmids pGRB-ldhA, pGRB-pflB, pGRB-frdABCD were prepared by annealing of primers (gRNA-ldhA-F, gRNA-ldhA-R; gRNA-pflB-F, gRNA-pflB-R; gRNA-frdABCD-F, gRNA-frdABCD-R), and the construction method is the same as in 1.2.

[0068] 3.3 Electro-Transformation of Plasmids and Recombinant DNA Fragments

[0069] The plasmid and DNA fragment were co-electrotransformed into competent cells. The cells resuscitated after electrotransformation were plated on LB plates containing ampicillin and spectinomycin and incubated overnight at 32° C. Colony PCR was performed using the upstream prim of the upstream homologous arm and the downstream primer of the downstream homologous arm of the overlap fragments identification primers to screen for positive recombinants, and the bacterial cells were preserved.

[0070] 3.4 Elimination of Plasmids

[0071] The method is the same as described in 1.4.

[0072] 4. Construction of Strain VXRO4 by Replacing the ilvE Gene of E. coli with the Leucine Dehydrogenase Encoding Gene Bcd of Bacillus subtilis

[0073] 4.1 Preparation of Recombinant DNA Fragments

[0074] The gene bcd (NCBI-protein ID: NP_390288.1) was obtained by PCR amplification using the genome of B. subtilis 168 as the template. Using the genome of E. coli W3110 as the template, upstream and downstream homologous arm fragments of gene ilvE (NCBI-Protein ID: YP_026247.1) were amplified (primers U-ilvE-F, U-ilvE-R; D-ilvE-F, D-ilvE-R). The promoter P.sub.trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the gene bcd. Overlap PCR was performed using the upstream and downstream homologous arms and the target gene bcd as templates, respectively, to obtain integration fragment P.sub.trc-bcd (primers P.sub.trc-bcd-F and P.sub.trc-bcd-R). The electrophoretogram for construction of the integration fragment P.sub.trc-bcd and PCR verification of the positive strain is shown in FIG. 6. The PCR amplification and overlap systems are the same as those in 1.1.

[0075] 4.2 Construction of gRNA Plasmid

[0076] The target sequence DNA fragment in the plasmid pGRB-ilvE was prepared by annealing the primers gRNA-ilvE-F and gRNA-ilvE-R, and the construction method was the same as that in 1.2.

[0077] 4.3 Electro-Transformation of Plasmids and Recombinant DNA Fragments

[0078] The pGRB-ilvE plasmid and the donor P.sub.trc-bcd fragment were simultaneously electrotransformed into electro-competent cells containing pREDCas9. The cells resuscitated after electrotransformation were plated on LB plates containing ampicillin and spectinomycin and incubated overnight at 32° C. Colony PCR was performed using primers (U-ilvE-F, D-ilvE-R) to screen for positive recombinants.

[0079] 4.4 Elimination of Plasmids

[0080] The method is the same as described in 1.4.

[0081] 5. Construction of Strain VXR05 by Replacing the Native ilvC Gene with Acetohydroxy Acid Isomeroreductase Mutant Gene ilvC.sup.M

[0082] 5.1 Preparation of Mutant Gene ilvC.sup.M

[0083] Primer design software primer5 was used, the genome of E. coli W3110 was used as the template (NCBI-Protein ID: NP_418222.1), mutation sites were designed in the downstream primer of the first fragment and the upstream primer of the second fragment, and the mutant gene to be amplified was divide into three fragments for amplification (primer 1-ilvC.sup.M-F, 1-ilvC.sup.M-R; 2-ilvC.sup.M-F, 2-ilvC.sup.M-R; 3-ilvC.sup.M-F, 3-ilvC.sup.M-R). Then, overlap PCR (primers 1-ilvC.sup.M-F, 3-ilvC.sup.M-R) was performed by using the two fragments as templates to obtain mutant gene ilvC.sup.M, wherein the nucleotide sequence is as shown in SEQ ID NO: 2.

[0084] 5.2 Preparation of Recombinant DNA Fragments

[0085] Using the genome of E. coli W3110 as the template, upstream and downstream homologous arm fragments of ilvC (NCBI-protein ID: NP_418222.1) were amplified (primers U-ilvC.sup.M-F, U-ilvC.sup.M-R; D-ilvC.sup.M-F, D-ilvC.sup.M-R). The promoter P.sub.trc was designed in the downstream primer of the upstream homologous arm and the upstream primer of the mutant gene. Then, overlap PCR was performed using the upstream and downstream homologous arms and the mutant gene ilvC.sup.M as templates (primers U-ilvC.sup.M-F, D-ilvC.sup.M-R). The electrophoretogram for construction of the integration fragment P.sub.trc-ilvC.sup.M and PCR verification of the positive strain is shown in FIG. 7. The PCR amplification and overlap systems are the same as 1.1.

[0086] 5.3 Construction of gRNA Plasmid

[0087] The target sequence DNA fragment in the plasmid pGRB-ilvC was prepared by annealing the primers gRNA-ilvC-F and gRNA-ilvC-R, and the construction method is the same as that in 1.2.

[0088] 5.4 Electro-Transformation of Plasmids and Recombinant DNA Segments

[0089] The pGRB-ilvC plasmid and the donor P.sub.trc-ilvC.sup.M fragment were simultaneously electrotransformed into electro-competent cells containing pREDCas9. The cells resuscitated after electrotransformation were plated on LB plates containing ampicillin and spectinomycin and incubated overnight at 32° C. Colony PCR was performed using the upstream primer of the upstream homologous arm and the downstream primer of the downstream homologous arm of the overlap fragment as identification primers to screen for positive recombinants.

[0090] 5.5 Elimination of Plasmids

[0091] The method is the same as described in 1.4.

[0092] 6. The primers involved in the construction of the above strains are shown in the following table.

TABLE-US-00005 TABLE 5 Primers Primer Sequence (5′-3′) SEQ ID NO: UP-spoT.sup.M-S TTGTATCTGTTTGAAAGCCTGAATC 3 UP-spoT.sup.M-A TCGCTTTTGGAATGGCGATATAGTCATCCACTTCGCCCG 4 DN-spoT.sup.M-S CGGGCGAAGTGGATGACTATATCGCCATTCCAAAAGCGA 5 DN-spoT.sup.M-A TTAATTTCGGTTTCGGGTGACT 6 UP-ydeU-S CTGCGTAATAGCATAAGCGGG 7 UP-ydeU-A AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCG 8 GATGATTAATTGTCAAGCTATTCATTTGAACCGTGCC P.sub.trc-alsS-S TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA 9 TTTCACACAGGAAACAGACCTTGACAAAAGCAACAAAA GAACA P.sub.trc-alsS-A ATTCCCCCACAGGCTAAGGTCTAGAGAGCTTTCGTTTTCA 10 TGAGT DN-ydeU-S ACTCATGAAAACGAAAGCTCTCTAGACCTTAGCCTGTGG 11 GGGAAT DN-ydeU-A ATGTCGTGAGCGTGGTATTGTC 12 gRNA-ydeU- AGTCCTAGGTATAATACTAGTGTTCGGGTTGATAACATTGG 13 S GTTTTAGAGCTAGAA gRNA-ydeU- TTCTAGCTCTAAAACCCAATGTTATCAACCCGAACACTAG 14 A TATTATACCTAGGACT ydeU- ACTGCGTAATAGCATAAGCGGG 15 identification- S ydeU- TGCTTGCCGACCCCTGAGA 16 identification- A UP-yeeP-S GGTCAGGAGGTAACTTATCAGCG 17 UP-yeeP-A AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGG 18 ATGATTAATTGTCAAATGGCAGGGCTCCGTTTT P.sub.trc-spoT.sup.M-S TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA 19 TTTCACACAGGAAACAGACCTTGTATCTGTTTGAAAGCCT GAATC P.sub.trc-spoT.sup.M-A AGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTAATT 20 TCGGTTTCGGGTGACT DN-yeeP-S TGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGGAACT 21 GGATTTTCTTCTGAACCTGT DN-yeeP-A ACGATGTCAGCAGCCAGCA 22 gRNA-yeeP-S AGTCCTAGGTATAATACTAGTACAGAATATTCGCGAAAAA 23 ACGGGTTTTAGAGCTAGAA gRNA-yeeP- TTCTAGCTCTAAAACCCGTTTTTTCGCGAATATTCTGTACT 24 A AGTATTATACCTAGGACT U-ilvE-F GGAACGATACAGCGAAACCAC 25 U-ilvE-R AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGG 26 ATGATTAATTGTCAAGCTTTCTTCGTGGTCATTTTTAT D-ilvE-F CCACAGTGTATTAAGCAGACGTTAAATACAAAAAATGGG 27 ACGGCAC D-ilvE-R TGGGAGTCAGATACTTTCGGGT 28 P.sub.trc-hed-F TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA 29 TTTCACACAGGAAACAGACCTCTGAAGAATACACACATT AGGAGGA P.sub.trc-bcd-R GTGCCGTCCCATTTTTTGTATTTAACGTCTGCTTAATACAC 30 TGTGG gRNA-ilvE-F AGTCCTAGGTATAATACTAGTCGCCAAAATCTATCGCTTCC 31 GTTTTAGAGCTAGAA gRNA-ilvE-R TTCTAGCTCTAAAACGGAAGCGATAGATTTTGGCGACTAG 32 TATTATACCTAGGACT U-ilvC.sup.M-F TCTACCGACACCTGATTACGCAC 33 U-ilvC.sup.M-R AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGG 34 ATGATTAATTGTCAAATGGTGATTCCTCGTGATGTTGT D-ilvC-F ACTTGATGACCGCCCTCTGTATTTTCGGTCTTCTCTCTCTG 35 ATTT D-ilvC-R ATAACAATGGGCAAAAATACGGT 36 l-ilvC.sup.M-F TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA 37 TTTCACACAGGAAACAGACCATGGCTAACTACTTCAATAC ACT l-ilvC.sup.M-R GCGCGCTCCTCGGCAATCGCTTCTTTAAACTCAGCGTAGG 38 AGATATCG 2-ilvC.sup.M-F TGGTCTCGATATCTCCTACGCTGAGTTTAAAGAAGCGATT 39 GCCGAGGAGCGCGC 2-ilvC.sup.M-R CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTT 40 CGACTGAGCCTTTCGTTTTATTTGTCGGGGTGAGGGCATC AG 3-ilvC.sup.M-F AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA 41 ACGCTCTCCTGAGTAGGACAAATCCCTCTCCCACAGGGA GAGG 3-ilvC.sup.M-R AAATCAGAGAGAGAAGACCGAAAATACAGAGGGCGGTC 42 ATCAAGT gRNA-ilvC-F AGTCCTAGGTATAATACTAGTTGGTCGCGAGTGGTTGATA 43 AGTTTTAGAGCTAGAA gRNA-ilvC-R TTCTAGCTCTAAAACTTATCAACCACTCGCGACCAACTAG 44 TATTATACCTAGGACT U-ldhA-F GCGGCTGGGATGTGAAAG 45 U-ldhA-R GAATACGCCAAAGGACTCGTTCACCT 46 D-ldhA-F TTTGGCGTATTCCGTCGCCTGTCTGC 47 D-ldhA-R ATGTCTGTTTTGCGGTCGC 48 gRNA-ldhA-F AGTCCTAGGTATAATACTAGTAAACGATGACGGCAGCCGC 49 CGTTTTAGAGCTAGAA gRNA-ldhA- TTCTAGCTCTAAAACGGCGGCTGCCGTCATCGTTTACTAG 50 R TATTATACCTAGGACT U-pflB-F TCGGCAACATTATCGGTGG 51 U-pflB-R GGTTCATTTACGGCAACGCAGGATG 52 V-pflB-V CGTAAATGAACCGTGAAATGCTGCTC 53 D-pflB-R GGCGATAGGTCACCACTTCC 54 gRNA-pflB-F AGTCCTAGGTATAATACTAGTTGTTCTCTGGCGACCCGATC 55 GTTTTAGAGCTAGAA gRNA-pflB-R TTCTAGCTCTAAAACGATCGGGTCGCCAGAGAACAACTA 56 GTATTATACCTAGGACT U-frdABCD-F GGTCTTCTTCGGTATCAGCAACA 57 U-frdABCD- TAGCAGCCAGACCGTAGAAAACCCTTCGTGCCCTTGTCA 58 R AAAACT D-frdABCD-F AGTTTTTGACAAGGGCACGAAGGGTTTTCTACGGTCTGG 59 CTGCTA D-frdABCD- ACGCCAGTAAATGTTTTGCTGAC 60 R gRNA- AGTCCTAGGTATAATACTAGTCCCAACCGAAATGACCCAA 61 frdABCD-R CGTTTTAGAGCTAGAA gRNA- TTCTAGCTCTAAAACGTTGGGTCATTTCGGTTGGGACTAG 62 frdABCD-R TATTATACCTAGGACT

Example 2

[0093] Shake-Flask Fermentation of Strains VXR02 and VXR05:

[0094] Slant culture: the strain preserved at −80° C. was streak-inoculated on the activation slant, cultured at 37° C. for 12 h, and passaged two times.

[0095] Shake-flask seed culture: two loops of the seed strain on the slant were scraped using an inoculation loop and inoculated into a 500 mL triangular flask containing 30 mL of a seed culture medium, and cultured at 37° C. and 200 rpm for 8-10 h.

[0096] Shake-flask aerobic fermentation: with an inoculum size of 10-15%, seed fermentation broth was inoculated into a 500 mL triangular flask (final volume, 30 mL) filled with a fermentation medium, and shake-cultured at 37° C. and 200 r/min; the pH was maintained at 6.7-7.0 by adding ammonia in the process of fermentation, and a 60% (m/v) glucose solution was added to maintain the fermentation; the fermentation cycle was 24 h.

[0097] Aerobic-anaerobic two-stage fermentation: with an inoculum size of 10-15%, the seed fermentation broth was inoculated into a 500 mL triangular flask filled with a fermentation medium (final volume, 30 mL), and shake-cultured at 37° C. and 200 r/min; 12-16 h later, the bacterial solution was transferred to a 30 mL sealed flask isolated from the air, and shake-cultured at 37° C. and 200 r/min for 8-12 h; the total fermentation cycle was 24 h.

[0098] The slant medium comprised 5 g/L glucose, 10 g/L peptone, 10 g/L beef extract, 5 g/L yeast powder, 2.5 g/L NaCl, and 21-25 g/L agar, pH 7.0-7.2.

[0099] The seed culture medium comprised 18 g/L glucose, 1% yeast powder, 0.6% peptone, 0.12% KH.sub.2PO.sub.4, 0.05% MgSO.sub.4•7H.sub.2O, 10 mg/L FeSO.sub.4•H.sub.2O, 10 mg/L MnSO.sub.4•H.sub.2O, 1.3 mg/L V.sub.B1, 0.3 mg/L V.sub.H, 20 ml/L phenol red, and two drops of a defoaming agent, initial pH 7.0-7.2.

[0100] The fermentation medium comprised 18 g/L glucose, 1 g/L yeast powder, 2 g/L peptone, 2 g/L KH.sub.2PO.sub.4, 1 g/L sodium citrate, 0.7 g/L MgSO.sub.4•7H.sub.2O, 100 mg/L FeSO.sub.4•7H.sub.2O, 100 mg/L MnSO.sub.4—H.sub.2O, 0.8 mg/L V.sub.B1, 0.3 mg/L V.sub.H, 20 mL/L phenol red, and two droplets of a defoaming agent, initial pH 7.0-7.2.

TABLE-US-00006 TABLE 6 Comparison of L-valine titer and conversion rate between shake-flask aerobic fermentation and aerobic-anaerobic two-stage fermentation Aerobic Aerobic-anaerobic fermentation two-stage fermentation Sugar-acid Sugar-acid L-valine titer conversion rate L-valine titer conversion rate Strain (g/L) (%) (g/L) (%) VXR02 40.1 29.2 25.1 27.5 VXR05 41.4 30.0 42.7 36.3

[0101] As shown in Table 6, or the Aerobic fermentation mode, the L-va ine titer an sugar-acid conversion rate of strain VXR05 were comparable to those of strain VXR02 in the Aerobic fermentation. For the aerobic-anaerobic two-stage fermentation mode, the titer of the strain VXR02 was remarkably reduced, indicating that the strain VXR02 cannot accumulate L-valine under the anaerobic condition. In contrast to the strain VXR02, the L-valine titer of VXR05 in the two-stage fermentation mode was higher than that in the aerobic fermentation mode, indicating that VXR05 can accumulate L-valine under anaerobic conditions. More beneficially, the sugar-acid conversion rate of VXR05 in the two-stage fermentation mode is remarkably increased by 21% compared with aerobic fermentation.

Example 3

[0102] Fermentation of VXR02 and VXR05 in 5 L Fermentor:

[0103] Aerobic fermentation: the strain was activated on the slant for two generations, and inoculated with an inoculum size of 15-20% into a fermentation medium to perform a fermentation. In the fermentation, the pH was controlled at about 6.7 and the temperature was maintained at 35° C., and the dissolved oxygen was controlled at 25-30%. After the glucose in the fermentation medium was depleted, an 80% (m/v) of glucose solution was fed to maintain the glucose concentration in the fermentation medium at 0.1-5 g/L; the fermentation cycle was 40 h.

[0104] Aerobic-anaerobic two-stage fermentation: the strain was activated on the slant for two generations, and inoculated with an inoculum size of 15-20% into a fermentation medium to perform a fermentation. In the fermentation, the pH was controlled at about 6.7, the temperature was maintained at 35° C., and the dissolved oxygen was controlled at 25-30%. After the glucose in the culture medium was depleted, an 80% (m/v) of glucose solution was fed to maintain the glucose concentration in the fermentation medium at 0.1-5 g/L; 12-16 h later, introduction of sterile air was stopped to carry out anaerobic fermentation; the total fermentation cycle was 40 h.

[0105] The seed culture medium comprised 60-90 g/L glucose, 5 g/L yeast powder, 4 g/L K.sub.2HPO.sub.4, 2.5 g/L (NH.sub.4).sub.2SO.sub.4, 2 g/L citric acid, 1-3 mg/L MgSO.sub.4•7H.sub.2O, 1-3 mg/L each of V.sub.B1, V.sub.B3, V.sub.B5, V.sub.B12, and V.sub.H, 2.8 mg/L/L FeSO.sub.4•7H.sub.2O and 1.2 mg/L MnSO.sub.4.

[0106] The fermentation medium consisted of 30 g/L glucose, 2 g/L yeast powder, 7 g/L K.sub.2HPO.sub.4, 3 g/L (NH.sub.4).sub.2SO.sub.4, 2 g/L citric acid, 1 g/L MgSO.sub.4•7H.sub.2O, 30 mg/L FeSO.sub.4•7H.sub.2O, 10 mg/L MnSO.sub.4, 1 mg/L each of V.sub.B1, V.sub.B3, V.sub.B5, V.sub.B12, and V.sub.H, pH 6.5-7.0.

TABLE-US-00007 TABLE 7 Comparison of parameters between aerobic fermentation and aerobic-anaerobic two-stage fermentation in fermentor Aerobic-anaerobic two- Aerobic fermentation stage fermentation L-valine Sugar-acid Produc- L-valine Sugar-acid Produc- titer conversion tivity titer conversion tivity Strain (g/L) rate (%) (g/L/h) (g/L) rate (%) (g/L/h) VXR05 80.2 30.6 2.0 80.8 40.7 2.0

[0107] As can be seen from Table 7, the L-valine titer reached 80.2 g/L and the sugar-acid conversion rate was 30.6% after the strain VXR05 underwent aerobic culture for 40 h in a 5 L fermentor. Adopting the Aerobic-anaerobic two-stage process, the L-valine titer reached 80.8 g/L, and the sugar-acid conversion rate was increased to 40.7%, 33% higher than that in aerobic fermentation.

[0108] Although the disclosure has been disclosed in terms of prefer embodiments, they are not intended to limit the disclosure, and various changes or modification may be made by those skilled in the art without departing from the spirit and scope of the disclosure. Therefore, the scope of the disclosure should be determined by the claims.