Ketoalkyd reductoisomerase mutant, high-yield D-pantothenic acid bacteria and application

Abstract

The invention belongs to the technical field of genetically engineered bacteria and microbial fermentation, and particularly relates to a keto-alcohol acid reductoisomerase mutant, high-yield D-pantothenic acid bacteria and application, keto-alcohol acid reductoisomerase mutant is obtained by performing site-specific mutagenesis on 71st, 110th and / or 417th sites of keto-alcohol acid reductoisomerase, and the site-specific mutagenesis mode is that the 71st site is mutated into N, Q or Y from A, the 110th site is mutated into Y or R from Q, and the 71st site is mutated into N, Q or Y from R; the 417th site is mutated from A to Q, Y or R; the sequence of the keto-alcohol acid reductoisomerase is SEQ ID NO. 47; the production capacity of D-pantothenic acid is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering bacteria and microbial fermentation, and specifically relates to a ketol-acid reductoisomerase mutant, a high-yield D-pantothenate bacterium and uses thereof. Background Art

[0002] D-pantothenic acid (DPA), also known as vitamin B5, is an essential acidic substance commonly found in organisms. It is also an important precursor for the biosynthesis of coenzyme A and plays an important role in promoting the growth of organisms. In recent years, DPA has gradually been developed and applied in the food, feed, cosmetics and pharmaceutical industries due to its good market prospects and value. Among them, more than 70% of downstream applications are used as feed additives.

[0003] There are three methods for synthesizing D-pantothenic acid: chemical synthesis, enzymatic method and full biosynthesis. At present, the chemical synthesis method for producing D-pantothenic acid is relatively mature and has been widely used in industry. Feed-grade DPA is obtained by heating two precursors of DPA (D-pantothenic acid lactone and β-alanine) in a methanol or ethanol environment, of which calcium D-pantothenate is the final product. Today, the industrial production of pantothenic acid still mainly uses chemical synthesis methods. The chemical synthesis process has many limitations. For example, hydrocyanic acid and sodium cyanide cause serious environmental pollution and human harm. Due to the increasingly serious environmental pollution problem and the growing environmental awareness, experiments have begun to use microbial fermentation technology to produce DPA. At present, the existing engineered strains have the problem of slow growth of Escherichia coli and low D-pantothenic acid production.

[0004] The biosynthesis of D-pantothenic acid involves multiple metabolic pathways and genes. Among them, the ilvC gene is involved in the synthesis of branched-chain amino acids and is in the biosynthetic pathway of isoleucine, valine, leucine, and pantothenic acid. The ilvC gene catalyzes the alkyl migration and ketoacid reduction of (S)-2-acetolactate (S2AL) to produce (R)-2,3-dihydroxyisovalerate. In the isomerase reaction, S2AL is converted to (R)-2,3-dihydroxyisovalerate by Mg. 2+ The 2-ketoacid undergoes metal-dependent reduction with NADPH to generate (R)-2,3-dihydroxyisovalerate. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a ketol-acid reductoisomerase mutant, a high-yielding D-pantothenic acid bacterium and a use thereof, so as to improve the production capacity of D-pantothenic acid.

[0006] The embodiment of the present invention provides a ketol-acid reductoisomerase mutant, which is characterized in that it is obtained by performing site-directed mutagenesis on the ketol-acid reductoisomerase at positions 71, 110 and / or 417, wherein the site-directed mutagenesis is performed in such a way that position 71 is mutated from A to N (asparagine), Q (glutamine) or Y (tyrosine), position 110 is mutated from Q to Y or R (arginine), and position 417 is mutated from A to Q, Y or R; the sequence of the ketol-acid reductoisomerase is SEQ ID NO.47.

[0007] Preferably, position 71 mutates from A to N, or position 110 mutates from Q to R, or position 417 mutates from A to Q. More preferably, position 71 mutates from A to N, and position 417 mutates from A to Q.

[0008] The embodiment of the present invention provides a ketol-acid reductoisomerase mutant gene, encoding the ketol-acid reductoisomerase mutant.

[0009] The embodiment of the present invention provides a high-yield D-pantothenic acid bacteria, which contains the ketol-acid reductoisomerase mutant gene.

[0010] In one embodiment, the high-yield D-pantothenic acid bacteria is a host cell of Escherichia coli, which is deposited in the China Center for Type Culture Collection and has a deposit number of CCTCC No.M 20231938. The ketol-acid reductoisomerase mutant of the present invention can be expressed in any bacteria or cells containing a ketol-acid reductoisomerase mutant gene, can be expressed after site-directed mutation in the original host cell containing the ketol-acid reductoisomerase gene, or can be inserted into any vector for expression. The host cell of the present invention can also be a gene-edited Escherichia coli (deposited in the China Center for Type Culture Collection and has a deposit number of CCTCC No.M 20231938).

[0011] Preferably, the host cell has a glk gene knocked out, and the ketol-acid reductoisomerase mutant gene is inserted into the knockout site of the glk gene.

[0012] The embodiment of the present invention provides a use of the high-yield D-pantothenic acid bacteria in preparing D-pantothenic acid by microbial fermentation.

[0013] In one embodiment, the high-yield D-pantothenic acid bacteria are inoculated into a fermentation medium containing ampicillin resistance, and fermentation culture is carried out at 25-37° C. and 100-220 rpm. After the fermentation is completed, the fermentation supernatant is collected and separated and purified to obtain D-pantothenic acid.

[0014] In one embodiment, fermentation ends when OD600 is 10-20 and pH is 5.5-8.5; the fermentation medium comprises: 15-30 g / L glucose monohydrate, 10-15 g / L ammonium sulfate, 0.5-1.0 g / L dipotassium hydrogen phosphate, 0.2-0.4 g / L magnesium sulfate heptahydrate, 10-25 g / L calcium carbonate, 2-5 g / L yeast powder, and 2-6 g / L β-alanine.

[0015] Before the high-yield D-pantothenic acid bacteria are used for fermentation preparation, a single clone colony is first selected and inoculated into SOB medium, cultured at 37° C. and 220 rpm for 12 hours, and then inoculated into fermentation medium at a volume concentration of 5% for fermentation culture.

[0016] The present invention provides a method for detecting changes in NADH utilization efficiency of ilvC, comprising the steps of synthesizing a mutant nucleic acid sequence, loading the mutant nucleic acid sequence into an expression plasmid pet28a, transforming the mutant nucleic acid sequence into a strain (such as a host cell with a deposit number of CCTCC No. M20231938), obtaining a mutant expression strain, and after inducing expression, detecting changes in the NADH / NAD+ ratio in the mutant expression strain to determine changes in its NADH utilization efficiency.

[0017] The beneficial effect of the present invention is that ilvC utilizes 2-ketopantoic acid, 2-ketoisovaleric acid, 2-ketovaleric acid, 2-ketobutyric acid, 3-hydroxypyruvic acid, 3-hydroxy-2-ketobutyric acid and pyruvic acid. At the same time, in the pantothenic acid synthesis branch, in addition to ilvC, panE also utilizes NADPH as a reducing power supply to catalyze 2-ketopantoic acid to generate (R)-pantoic acid. Therefore, in the pantothenic acid anabolism process, the cofactor NADPH plays a role in multiple enzyme catalysis systems, which may lead to an imbalance in the supply of redox power in this pathway within the cell, thereby causing a decrease in pantothenic acid synthesis.

[0018] Specific mutants of ilvC are used to increase its affinity for the cofactor NADH and the number of substrate turnovers, thereby enhancing its utilization of NADH in the intracellular environment, helping to alleviate the competitive utilization of NADPH in the pantothenate synthesis pathway, solving the problem of insufficient NADPH supply, balancing the redox power supply in the metabolic pathway, and improving the catalytic efficiency of ketol-acid reductoisomerase, thereby enhancing the efficiency of the multi-step reactions in the pantothenate synthesis pathway and obtaining a genetically engineered strain with high pantothenate production.

[0019] The present invention uses metabolic engineering to finely regulate the balance of metabolic pathways and protein engineering to rationally transform efficient and specific catalytic enzymes to construct a genetically engineered strain with high D-pantothenic acid production. The specific mutant of ketol-acid reductoisomerase ilvC is used to improve its affinity for the cofactor NADH and the number of substrate conversions, thereby improving the catalytic efficiency of the enzyme, thereby enhancing the efficiency of the multi-step reactions of the pantothenic acid synthesis pathway, and obtaining a genetically engineered strain with high pantothenic acid production, and finally increasing the D-pantothenic acid titer to 3.116 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Intracellular NAD(H) levels and NADH / NAD+ ratios of wild-type and mutant expression strains.

[0021] Figure 1 (a) is the NAD(H) level before and after mutation at site 71. Figure 1 (b) is the NADH / NAD+ ratio before and after mutation at site 71;

[0022] Figure 1 (c) is the NAD(H) level before and after the mutation at site 110. Figure 1 (d) is the NADH / NAD+ ratio before and after the mutation of site 110;

[0023] Figure 1 (e) is the NAD(H) level before and after the mutation of site 417. Figure 1 (f) is the NADH / NAD+ ratio before and after the mutation at site 71.

[0024] Figure 2 The intracellular NAD(H) levels and NADH / NAD+ ratios of the wild-type and multi-site mutant expression strains, as well as the conformational changes and hydrogen bond formation of multi-site mutations.

[0025] Figure 2 (a) is the intracellular NAD(H) level of wild-type and multi-site mutant expression strains, Figure 2 (b) is the NADH / NAD+ ratio of the wild-type and multi-site mutant expression strains, Figure 2 (c) Conformational changes and hydrogen bond formation caused by multi-site mutations.

[0026] Figure 3 D-pantothenic acid titer and OD600 value of XHX-DPA-11 shake flask fermentation. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0028] The wild-type strain XHX-DAP-01 described in the present invention originates from this laboratory and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No.M 20231938. The starting strain is XHX-DPA-05-02 (disclosed in patent application number CN202410827812.7 and patent publication number CN118460582A).

[0029] In the following examples, the final concentrations of ampicillin and kanamycin in the culture medium were 0.1 mg / L and 0.05 mg / L, respectively.

[0030] The primer sequence information used in the examples is shown in Table 1.

[0031] Table 1 Primer sequences used for site-directed mutagenesis and gene editing

[0032]

[0033]

[0034] Example 1: Analysis and detection method of D-pantothenic acid Chromatographic conditions:

[0035] Chromatographic column: C18-028 Welch Ultimate, Polar-RP 4.6mm×250mm, 5μm, flow rate: 1.0ml / min, wavelength: 210nm, column temperature: 30℃, injection volume: 10μl, running time: more than 30min. Mobile phase: acetonitrile-water-phosphoric acid (49:950:1), test sample: filter the sample solution and take the filtrate. Reference substance: take an appropriate amount of calcium pantothenate reference substance, accurately weigh, dissolve in ultrapure water and quantitatively dilute to make a solution containing about 0.5mg per 1ml, filter. Result calculation: external standard method.

[0036] Example 2: ilvC mutant plasmid construction and protein expression

[0037] (1) The wild-type nucleic acid sequence of E. coliilvC was designed, and HindⅢ and BamHI restriction enzyme sites were introduced, and loaded into the pUC19 plasmid. The wild-type nucleic acid sequence of E. coliilvC is shown in SEQ ID NO.45.

[0038] (2) Design of ilvC single point mutation sequence

[0039] The 71st position of the ilvC protein sequence was mutated from A to N, Q, T, Y, and R, respectively, and the single point mutation primer sequences are shown in SEQ ID NOs. 3 to 12;

[0040] The 110th position of the ilvC protein sequence was mutated from Q to N, T, Y, and R, respectively, and the single point mutation primer sequences are shown in SEQ ID NOs. 13 to 20;

[0041] The 417th position of the ilvC protein sequence was mutated from A to N, Q, T, Y, and R, respectively, and the single point mutation primer sequences are shown in SEQ ID NOs. 21 to 30.

[0042] (3) Construction of ilvC mutant and wild-type expression plasmids

[0043] The sequence was amplified using primers SEQ ID NO.1 and SEQ ID NO.4, SEQ ID NO.2 and SEQ ID NO.3, and template SEQID NO.45; after obtaining two nucleic acid sequences, the fragments were purified and recovered using a gel recovery kit; the pet28a plasmid was double-digested with EcoRⅠ and NotⅠ restriction enzyme sites to obtain a linearized fragment; the three fragments were spliced ​​using the Gibson assembly method and transformed into Escherichia coli competent cells Top 10; 100 μl of the transformed bacterial solution was spread on an LB solid culture medium plate (containing kanamycin resistance), and after overnight culture, a single clone was picked and verified by colony PCR using primers SEQ ID NO.1 and SEQ ID NO.2; it was further sent for sequencing verification, and the plasmid with correct sequencing was extracted using a plasmid extraction kit to obtain the pet28a-ilvC:p.A71N mutant.

[0044] The wild-type and other mutant expression plasmids were constructed as described above using primers SEQ ID NO.1 and SEQ ID NO.2, and primers SEQ ID NO.5-30.

[0045] The wild-type expression plasmid was constructed as described above using primers SEQ ID NO.1 and SEQ ID NO.2.

[0046] (4) Protein induction expression

[0047] Transform the pet28a-ilvC:p.A71N plasmid into the XHX-DPA-01 strain, take 100 μl of the transformed bacterial solution and spread it on an LB solid medium plate (containing kanamycin resistance), culture overnight, pick a single clone; inoculate a single clone into 100 ml LB liquid medium (containing kanamycin resistance), and culture at 37°C and 220 rpm until OD 600 Reach 0.2-0.4; add IPTG to a final concentration of 0.5 mM and culture at 25°C, 220 rpm for 16 h.

[0048] The remaining mutants and wild-type expression plasmids were expressed in the same manner as above.

[0049] Example 3: NADH / NAD+ Detection Method

[0050] (1) Sample preparation: Take 20 ml of culture medium, centrifuge at 6000 rpm for 10 min to collect bacteria; add 10 ml of PBS buffer and resuspend by pipetting, and wash twice; add 5 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 mM NaCl, 0.1% Triton X-100) and thoroughly resuspend the bacteria by pipetting; ultrasonically disrupt the cells in an ice bath at 400 w, 2 s / 2 s, 20 min; then centrifuge at 12,000 × g, 4°C for 10 min, and take the supernatant as the sample to be tested.

[0051] (2) Preparation of NADH / NAD+ detection kit: aspirate 655 μl of NADH preparation solution and fully dissolve 5 mg of NADH provided by the detection kit to obtain 10 mM NADH standard; dilute the 10 mM NADH standard with lysis buffer to a concentration gradient of 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 μM, and add 20 μl of the standard to each well of a 96-well plate, which is equivalent to 0, 5, 10, 20, 40, 80, 120, 160, and 200 pmol of NADH per well; dilute alcohol dehydrogenase 45 times with reaction buffer, add 2 μl of alcohol dehydrogenase to 88 μl of reaction buffer to obtain 90 μl of alcohol dehydrogenase working solution.

[0052] (3) Sample determination:

[0053] a. Determination of the total amount of NAD+ and NADH in the sample: dilute the sample to be tested 10 times, pipette 2 μl of the sample to be tested into 18 μl of lysis buffer to obtain 20 μl of the diluted sample to be tested, then add 20 μl of the diluted sample to each well of a 96-well plate. In order to reduce experimental errors, set up three replicate wells for the samples.

[0054] b. Determination of NAD+, NADH content and NADH / NAD+ ratio in samples: Pipette 100 μl of the sample to be tested into a centrifuge tube, heat in a 60°C water bath for 30 min to decompose NAD+; centrifuge at 10,000×g, 4°C for 5 min; dilute the supernatant 10 times, pipette 2 μl of the supernatant into 18 μl of lysis buffer to obtain 20 μl of diluted supernatant; pipette 20 μl of the diluted supernatant as the sample to be tested into a 96-well plate, and set up three replicate wells for samples to reduce experimental errors.

[0055] c. Add 90 μl / well of ethanol dehydrogenase working solution to the blank control well, standard well and sample well on the 96-well plate, mix well, and incubate at 37°C in the dark for 10 min. Then add 10 μl of color development solution to each well, mix well, and incubate at 37°C in the dark for 30 min. At this time, orange-yellow formazan will be formed. Measure the absorbance at 450 nm.

[0056] (4) Calculation of samples: Prepare a standard curve with NADH concentration as the horizontal axis and the absorbance value of the standard group (standard absorbance value - blank absorbance value) as the vertical axis; calculate the concentration of the sample using the standard curve, and measure the concentration of the sample heated at 60°C as the total NAD(H) concentration, and the concentration of the sample after treatment as the NADH concentration; calculate the NAD+ concentration in the sample using the formula NAD+=NAD(H)-NADH; calculate the NADH / NAD+ ratio using the formula NADH / NAD+=NADH / (NAD(H)-NADH).

[0057] Example 4: Detection of changes in NADH / NAD+ ratio of ilvC single point mutants

[0058] Inducible expression of XHX-DPA-01 / pet28a-ilvC WT (ilvC wild type), XHX-DPA-01 / pet28a-ilvC Mute After the ilvC mutant, the NADH / NAD+ ratio was detected by the method described in Example 3. The results are as follows Figure 1 As shown, when the ilvC protein 71 site mutated to asparagine, 110 site mutated to arginine, and 417 site mutated to glutamine, the NADH / NAD+ ratio decreased the most, indicating that the conversion of intracellular NADH to NAD+ was enhanced and the utilization efficiency of NADH was improved. The data are presented as the mean ± SD of three independent biological replicates (n = 3).

[0059] Example 5: Detection of changes in NADH / NAD+ ratios of ilvC multi-point mutants

[0060] The ilvC multi-point mutation expression strain XHX-DPA-01 / pet28a-ilvC:p.A71N / Q110R, XHX-DPA-01 / pet28a-ilvC:p.A71N / Q110R / A417Q was constructed and induced to express by the method described in Example 2. The NADH / NAD+ ratio was detected by the method described in Example 3. The results are as follows: Figure 2As shown in (a)-(b), the mutant M1 (i.e., ilvC: p.A71N / Q110R) composed of ilvC A71 mutation to N and Q110 mutation to R showed no significant difference in NADH / NAD+ ratio compared with the wild type; the mutant M2 (i.e., ilvC: p.A71N / Q110R / A417Q) composed of ilvC A71 mutation to N, Q110 mutation to R and A417 mutation to Q showed a significant change in NADH / NAD+ ratio compared with the wild type, decreasing by about 66.9%, which showed a significant difference. Data are expressed as mean ± SD of three independent biological replicates (n = 3). The significance was tested by one-way ANOVA method, *P < 0.05.

[0061] Example 6: Comparison of conformational changes and non-bonded interactions at mutation sites

[0062] Pymol was used to perform amino acid replacement after site-directed mutagenesis of the docking complex, compare the conformational changes of the amino acid residues at the replacement site, search for newly generated non-bonded interactions and calculate the distances between hydrogen-bonded atoms. The results are as follows Figure 2 As shown in (c), when A71 mutates to N and A417 mutates to Q, the bond distances are and The hydrogen bonds strengthened the interaction between the ligand NADH and the amino acid residues in the binding pocket; however, when Q110 mutated to R, no new non-bonded interactions were formed due to the large distance between atoms, indicating that the mutation at position 110 had no contribution to the change in binding free energy, which was consistent with the NADH / NAD+ ratio detection result.

[0063] Example 7: Editing the genomic ilvC nucleic acid sequence based on the CRISPR-Cas9 gene editing system

[0064] Taking strain XHX-DPA-05-02 (XHX-DPA-01, ΔavtA / Δglk / Δpta-ackA::alsS / ΔlacI::panE) as the starting strain, gene editing technology was used to site-directedly mutate the corresponding codon nucleic acid sequences at the 71st and 417th positions of the amino acid sequence of the ilvC gene on the genome, so that A71 mutated to N71, and A417 mutated to Q417, and the ilvC:p.A71N / A417Q nucleotides were obtained, with the sequence of SEQ ID NO.46.

[0065] The steps are:

[0066] (1) Design and construct Target plasmid:

[0067] At the upstream and downstream of the N20 position of the vector plasmid pTarget, the 20bp sequence before NGG at the mutation site of the ilvC gene was combined to synthesize sgRNAs targeting the mutation site; the vector plasmid pTarget and sgRNAs were digested with NdeⅠ and SalⅠ restriction endonucleases, treated at 37℃ for 30min, to obtain linearized pTarget and sgRNAs with sticky ends, and the digested fragments were purified and recovered using a gel recovery kit; the linearized vector and sgRNAs fragments were connected at a molar ratio of 1:10, and reacted at 37℃ for 1h; the connection product was transformed into E.coli JM109 competent cells, ice bathed for 45min, heat shocked at 42℃ for 90s', ice bathed for 2min, added with 700μL LB culture medium, and revived and cultured at 37℃ and 220rpm for 1h; 100μL of the revived bacterial solution was spread on an LB plate (containing 0.05mg / mL SD resistance) and cultured at 37℃ for 16h; a single clone was picked and inoculated in 4ml The plasmid was extracted after culturing at 37°C and 220 rpm for 16 h in LB liquid medium and sent for sequencing to obtain the pT-ilvC plasmid.

[0068] (2) Design and construction of Donor plasmid:

[0069] The pet28a-ilvC:p.A71N / A417Q expression plasmid was constructed by the method described in Example 2, and used as a template, SEQ ID NO.31 and SEQ ID NO.32 as primers, and 98°C denaturation, 55°C annealing, and 72°C extension were used as PCR amplification reaction conditions to amplify the ilvC gene mutant nucleic acid sequence; after 2% agarose electrophoresis for 30 minutes, the corresponding position gel strip was cut off, and the DNA fragment was recovered using a gel recovery kit; the aforementioned constructed targeting plasmid pT-ilvC was extracted using a plasmid mini-extraction kit; 1 μg of pT-ilvC plasmid was taken, incubated with XbaⅠ and HindⅢ restriction endonucleases at 37°C for 30 minutes, and the linearized plasmid fragment was purified and recovered using a PCR product purification kit; Infusion connection was performed with a molar ratio of the amplified sequence to the linearized vector fragment of 10:1, and the reaction was carried out at 50°C for 15 minutes to obtain a recombinant vector; the recombinant vector was transformed into competent cells E.coli JM109 was spread on LB plates (containing 0.05 mg / mL SD resistance) after recovery and cultured at 37°C for 16 h; a single clone of the strain was picked and inoculated into 4 ml LB liquid culture medium (containing 0.05 mg / mL SD resistance) for expansion, and colony PCR detection was performed with the primer pair of SEQ ID NO.31 and SEQ ID NO.32, and sequencing verification was performed at the same time to obtain the pTD-ilvC plasmid with the correct sequence.

[0070] (3) Preparation of electrocompetent cells containing pEcCas

[0071] The pEcCas plasmid was extracted using a plasmid extraction kit; the plasmid was transformed into the XHX-DPA-05-02 (plasmid-free) strain and spread on an LB plate (containing 0.05 mg / mL of Kana resistance) to obtain the XHX-DPA-05-02 / pEcCas strain; a monoclonal positive colony was picked and inoculated into 10 ml of LB liquid culture medium (containing 0.05 mg / mL of Kana resistance), and 100 μL of sterile L-Ara mother solution (1 M) was added for expansion, and cultured overnight at 37°C and 220 rpm as seed solution; 1 ml of seed culture solution was inoculated into 50 ml of LB culture medium (Kana resistance), and 500 μL of L-Ara mother solution, culture at 37°C, 220rpm for 2h, until OD600 is 0.4-0.6; pre-cool shake flasks and centrifuge tubes in an ice bath, collect the bacterial solution, and centrifuge at 4°C, 4000rpm for 5min; discard the supernatant, use 40ml pre-cooled sterile ultrapure water in an ice bath to gently resuspend the bacteria, and centrifuge at 4°C, 4000rpm for 8min; repeat the steps of discarding the supernatant and resuspending; discard the supernatant, use 20ml pre-cooled 10% glycerol in an ice bath to gently resuspend the bacteria, and centrifuge at 4°C, 5000rpm for 12min; discard the supernatant, add 1ml pre-cooled 10% glycerol in an ice bath to gently resuspend the bacteria, and dispense into 1.5ml EP tubes at 100μL / tube and store at -80°C for later use.

[0072] (4) Electric shock transformation

[0073] Take the electroporation competent XHX-DPA-05-02 / pEcCas strain and melt it in an ice bath. When it is in a molten state, add 2μL Donor plasmid pTD-ilvC; transfer it to a 2mm electroporation cup pre-cooled in an ice bath after 2 minutes of ice bath; absorb the condensed water on the outer wall of the electroporation cup with filter paper, place it in the electroporator, and electroporate in ECO 2 mode; add 1ml of pre-cooled LB culture medium to the electroporation cup, gently resuspend the bacteria, and quickly transfer the bacterial solution to a 1.5ml EP tube; place it in a shaker and resuscitate and culture at 30℃ and 220rpm for 1h; take 500μL of bacterial solution and spread it on an LB plate (containing 0.05mg / mL SD and Kana resistance) and culture it at 37℃ overnight.

[0074] (5) Positive clone verification

[0075] A pair of verification primers SEQ ID NO.33 and SEQ ID NO.34 were designed at 100bp outside the homology arm, and single clones on the double-antibody LB medium plate were selected as templates for colony PCR verification. At the same time, the wild-type genome (XHX-DPA-01) was used as a template as a negative control; the PCR band size was correct and significantly different from the negative control as a positive clone. The mutant PCR band size was consistent with the negative control, and its positive clone was based on the sequencing results.

[0076] (6) Elimination of gene editing plasmids

[0077] The positive monoclonal clone was inoculated into 10 ml LB medium (Kana resistance), and 100 μL rhamnose mother solution (1 M) was added, and cultured at 37°C, 220 rpm shaking for 16 h; streaked onto LB plates (Kana resistance) and cultured at 37°C overnight; 20 partitions of the monoclonal clone were picked and streaked onto LB plates (SD resistance) and numbered, and cultured at 37°C overnight; the colonies that could not grow in the corresponding areas of the LB plates (SD resistance) were monoclonal clones in which the pTD-ilvC plasmid was eliminated.

[0078] The single clone that successfully eliminated the pTD-ilvC plasmid was inoculated into 10 ml LB medium (containing 5 g / L glucose, no resistance), and cultured at 37°C, 220 rpm shaking for 16 h; streaked on LB plates (containing 5 g / L glucose, 10 g / L sucrose, no resistance), and cultured at 37°C overnight; 20 single clones were picked and streaked on LB plates (Kana resistance) and LB plates (no resistance) in corresponding areas and numbered, and cultured at 37°C overnight; the colonies that could not grow in the corresponding areas of the LB plate (Kana resistance) were single clones with pEcCas plasmid eliminated.

[0079] (7) Colony PCR and sequencing verification

[0080] Using the single clone that successfully eliminated the gene editing plasmid as a template, colony PCR was performed using verification primers SEQ ID NO.33 and SEQ ID NO.34; the single clone with the correct PCR band size was selected for sequencing verification, and if the sequencing result was correct, the strain XHX-DPA-05-02 [ilvC: p.A71N / A417Q] with the ilvC gene mutation was obtained.

[0081] Example 8: Insertion of ilvC mutant nucleic acid sequence based on CRISPR-Cas9 gene editing system, referring to the gene editing method in Example 7, the steps are:

[0082] (1) Designing an N20 primer pair targeting the glk gene knockout site (SEQ ID NO.35 and SEQ ID NO.36) and amplifying the pTarget plasmid with the primer pair to obtain the pT-Δglk plasmid;

[0083] (2) Using XHX-DPA-05-02 as a template, SEQ ID NO.37, SEQ ID NO.38 and SEQ ID NO.39, SEQ ID NO.40 as primers to amplify the 500 bp homology arm sequences upstream and downstream of the glk gene knockout site, namely Δglk_UP_seg and Δglk_DOWN_seg, respectively; using pet28a-ilvC:p.A71N / A417Q plasmid as a template, primers SEQ ID NO.41 and SEQ ID NO.42 as primers to amplify the ilvC mutant sequence, namely ilvC_Mute_seg; using XbaⅠ and HindⅢ restriction endonucleases to linearize the pT-Δglk plasmid, namely pT-Δglk_linear; purifying and recovering the above four nucleic acid fragments using a gel recovery kit;

[0084] (3) The nucleic acid fragments Δglk_UP_seg:Δglk_DOWN_seg:ilvC_Mute_seg:pT-Δglk_linear were infused at a molar ratio of 10:10:10:1. The ligation product was transformed into E. coli JM109 competent cells, diluted and coated on LB plates, and single clones were picked for colony PCR and sequencing verification to obtain the pTD-Δglk::ilvC_Mute plasmid.

[0085] Continue to prepare the pEcCas electroporation competent state of strain XHX-DPA-05-02 [ilvC: p.A71N / A417Q] by the method described in Example 7. After electroporation of the cells, the obtained positive clones were subjected to colony PCR and sequencing verification using SEQ ID NO.43 and SEQ ID NO.44. After eliminating the gene editing tool plasmid, another round of colony PCR and sequencing verification was performed to obtain the genetically engineered strain XHX-DPA-05-02 [Δglk::ilvC: p.A71N / A417Q, ilvC: p.A71N / A417Q], which was named XHX-DPA-10.

[0086] pBR322-BspanB / BspanC (a plasmid containing panB and panC genes) was transformed into XHX-DPA-10, and strain XHX-DPA-11 was obtained after resistance screening on LB plates.

[0087] Example 9: Shake flask fermentation of D-pantothenic acid producing strain XHX-DPA-11

[0088] The obtained XHX-DPA-11 pantothenic acid producing strain was subjected to shake flask fermentation and then the D-pantothenic acid production titer was tested to detect the effects of the ilvC mutant and multiple copies on the production of D-pantothenic acid by the chassis strain.

[0089] The control strain XHX-DPA-01 and the experimental strain XHX-DPA-11 were inoculated into 4 ml SOB medium, respectively, and cultured at 37°C and 220 rpm for 12 h; 2.5 ml was inoculated into 50 ml fermentation medium, and cultured at 37°C and 220 rpm for 48 h; after the fermentation, 1 ml of the fermentation liquid was diluted 30 times and the OD was measured 600 , take another 1 ml of fermentation broth, centrifuge at 12000 rpm for 2 min, take the supernatant and dilute it 4 times, perform HPLC detection according to Example 1, OD 600 and D-pantothenic acid titer Figure 3 shown.

[0090] The experimental results showed that the titer of D-pantothenic acid was significantly increased to 3.116 g / L by constructing the ilvC:p.A71N / A417Q mutant (i.e., XHX-DPA-11) in the chassis strain and increasing the mutant copy, which was about 40% higher than that of the control strain. The data are presented as the mean ± SD of three independent biological replicates (n = 3). The significance was tested by the two-tailed t-test method, *P < 0.05.

[0091] SOB medium: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4.

[0092] Fermentation medium: glucose monohydrate 30 g / L, ammonium sulfate 10 g / L, dipotassium hydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 0.3 g / L, calcium carbonate 25 g / L, yeast powder 5 g / L, β-alanine 3 g / L.

[0093] SEQ ID NO.45 is

[0094] ATGGCTAACTACTTCAATACACTGAACCTGCGCCAGCAGCTGGCACAGCTGGGCAAATGTCGCTTTATGGGGCGCGACGAATTCGCCGACGGCGCGAGCTACCTTCAGGGTAAAAAAGTGGTTATCGTCGGCTGTGGCGCACAGGGGCTGAACCAGGGCCTGAACATGCGTGATTCTGGTCTCGATATCTCCTACGCTCTGCGTAAAGAAGCGATTGCCGAGAAGCGCGCGTCCTGGCGTAAAGCGACCGAAAACGGTTTTAAAGTTGGTACTTACGAAGAACTGATCCCGCAGGCGGATCTGGTGGTTAACCTGACGCCGGACAAGCAGCACTCTGATGTAGTGCGCACCGTACAGCCACTGATGAAAGACGGCGCGGCGCTGGGTTACTCGCACGGTTTCAACATCGTCGAAGTGGGCGAGCAGATCCGTAAAGATATCACCGTAGTCATGGTTGCGCCGAAATGCCCTGGCACCGAAGTGCGTGAAGAGTACAAACGTGGATTCGGCGTACCGACGCTGATTGCCGTTCACCCGGAAAACGATCCGAAAGGCGAAGGTATGGCGATCGCTAAAGCATGGGCGGCAGCAACCGGTGGTCACCGTGCGGGCGTGCTGGAATCGTCCTTCGTTGCGGAAGTGAAATCTGACCTGATGGGTGAGCAAACCATCCTGTGCGGTATGTTGCAGGCTGGCTCGCTGCTGTGCTTCGACAAGCTGGTGGAAGAGGGCACCGATCCGGCATACGCAGAAAAACTAATTCAGTTCGGTTGGGAAACCATCACCGAAGCGCTGAAGCAGGGCGGCATCACCCTGATGATGGACCGTCTCTCTAACCCGGCGAAACTGCGTGCTTACGCGCTGTCTGAACAGCTGAAAGAGATCATGGCACCCCTGTTCCAGAAACATATGGACGACATCATCTCCAGCGAATTCTCCTCCGGTATGATGGCGGACTGGGCCAACGATGATAAGAAACTGCTGACCTGGCGTGAAGAGACCGGCAAAACCGCCTTCGAAACCGCGCCGCAGTATGAAGGCAAAATCGGCGAGCAGGAGTACTTCGATAAAGGCGTACTGATGATCGCGATGGTGAAAGCGGGCGTTGAACTGGCGTTCGAAACCATGGTCGATTCCGGCATCATTGAAGAGTCTGCATATTATGAATCACTGCACGAGCTGCCGCTGATTGCCAACACCATCGCCCGTAAGCGTCTGTACGAAATGAACGTGGTTATCTCTGATACCGCTGAGTACGGTAACTATCTGTTCTCTTACGCTTGTGTGCCGTTGCTGAAACCGTTTATGGCAGAGCTGCAACCGGGCGACTTGGGTAAAGCTATTCCGGAAGGCGCGGTAGATAACGCGCAACTGCGTGATGTGAACGAAGCGATTCGCAGCCATGCGATTGAGCAGGTAGGTAAGAAACTGCGCGGCTATATGACAGATATGAAACGTATTGCTGTTGCGGGTTAA。

[0095] SEQ ID NO. 46 is

[0096] ATGGCTAACTACTTCAATACACTGAACCTGCGCCAGCAGCTGGCACAGCTGGGCAAATGTCGCTT

[0097] TATGGGGCGCGACGAATTCGCCGACGGCGCGAGCTACCTTCAGGGTAAAAAAGTGGTTATCGTCGGC

[0098] TGTGGCGCACAGGGGCTGAACCAGGGCCTGAACATGCGTGATTCTGGTCTCGATATCTCCTACGCTCT

[0099] GCGTAAAGAAAACATTGCCGAGAAGCGCGCGTCCTGGCGTAAAGCGACCGAAAACGGTTTTAAAGT

[0100] TGGTACTTACGAAGAACTGATCCCGCAGGCGGATCTGGTGGTTAACCTGACGCCGGACAAGCAGCAC

[0101] TCTGATGTAGTGCGCACCGTACAGCCACTGATGAAAGACGGCGCGGCGCTGGGTTACTCGCACGGTT

[0102] TCAACATCGTCGAAGTGGGCGAGCAGATCCGTAAAGATATCACCGTAGTCATGGTTGCGCCGAAATGC

[0103] CCTGGCACCGAAGTGCGTGAAGAGTACAAACGTGGATTCGGCGTACCGACGCTGATTGCCGTTCACC

[0104] CGGAAAACGATCCGAAAGGCGAAGGTATGGCGATCGCTAAAGCATGGGCGGCAGCAACCGGTGGTC

[0105] ACCGTGCGGGCGTGCTGGAATCGTCCTTCGTTGCGGAAGTGAAATCTGACCTGATGGGTGAGCAAAC

[0106] CATCCTGTGCGGTATGTTGCAGGCTGGCTCGCTGCTGTGCTTCGACAAGCTGGTGGAAGAGGGCACC

[0107] GATCCGGCATACGCAGAAAAACTAATTCAGTTCGGTTGGGAAACCATCACCGAAGCGCTGAAGCAGG

[0108] GCGGCATCACCCTGATGATGGACCGTCTCTCTAACCCGGCGAAACTGCGTGCTTACGCGCTGTCTGAA

[0109] CAGCTGAAAGAGATCATGGCACCCCTGTTCCAGAAACATATGGACGACATCATCTCCAGCGAATTCTC

[0110] CTCCGGTATGATGGCGGACTGGGCCAACGATGATAAGAAACTGCTGACCTGGCGTGAAGAGACCGGC

[0111] AAAACCGCCTTCGAAACCGCGCCGCAGTATGAAGGCAAAATCGGCGAGCAGGAGTACTTCGATAAAG

[0112] GCGTACTGATGATCGCGATGGTGAAAGCGGGCGTTGAACTGGCGTTCGAAACCATGGTCGATTCCGG

[0113] CATCATTGAAGAGTCTGCATATTATGAATCACTGCACGAGCTGCCGCTGATTGCCAACACCATCGCCCG

[0114] TAAGCGTCTGTACGAAATGAACGTGGTTATCTCTGATACCCAGGAGTACGGTAACTATCTGTTCTCTTA

[0115] CGCTTGTGTGCCGTTGCTGAAACCGTTTATGGCAGAGCTGCAACCGGGCGACTTGGGTAAAGCTATTC

[0116] CGGAAGGCGCGGTAGATAACGCGCAACTGCGTGATGTGAACGAAGCGATTCGCAGCCATGCGATTGAGCAGGTAGGTAAGAAACTGCGCGGCTATATGACAGATATGAAACGTATTGCTGTTGCGGGTTAA.

[0117] SEQ ID NO.47 is

[0118] MANYFNTLNLRQQLAQLGKCRFMGRDEFADGASYLQGKKVVIVGCGAQGLNQGLNMRDSGLDISYALRKEAIAEKRASWRKATENGFKVGTYEELIPQADLVVNLTPDKQHSDVVRTVQPLMK DGAALGYSHGFNIVEVGEQIRKDITVVMVAPKCPGTEVREEYKRGFGVPTLIAVHPENDPKGEGMAIAKAWAAATGGHRAGVLESSFVAEVKSDLMGEQTILCGMLQAGSLLCFDKLVEEGTD PAYAEKLIQFGWETITEALKQGGITLMMDRLSNPAKLRAYALSEQLKEIMAPLFQKHMDDIISSEFSSGMMADWANDDKKLLTWREETGKTAFETAPQYEGKIGEQEYFDKGVLMIAMVKAGV ELAFETMVDSGIIEESAYYESLHELPLIANTIARKRLYEMNVVISDTAEYGNYLFSYACVPPLKPFMAELQPGDLGKAIPEGAVDNAQLRDVNEAIRSHAIEQVGKKLRGYMTDMKRIAVAG.

[0119] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0120] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. A ketol-acid reductoisomerase mutant, characterized in that: The ketol-acid reductoisomerase is obtained by subjecting the ketol-acid reductoisomerase to site-directed mutagenesis at positions 71, 110 and / or 417, wherein the site-directed mutagenesis is performed in such a way that position 71 is mutated from A to N, Q or Y, position 110 is mutated from Q to Y or R, and position 417 is mutated from A to Q, Y or R; the sequence of the ketol-acid reductoisomerase is SEQ ID NO.

47.

2. The ketol-acid reductoisomerase mutant according to claim 1, characterized in that: The 71st position mutates from A to N, or the 110th position mutates from Q to R, or the 417th position mutates from A to Q.

3. The ketol-acid reductoisomerase mutant according to claim 2, characterized in that: The 71st position mutated from A to N, and the 417th position mutated from A to Q.

4. A ketol-acid reductoisomerase mutant gene, characterized in that: Encoding the ketol-acid reductoisomerase mutant according to any one of claims 1 to 3.

5. A high-yield D-pantothenic acid bacterium, characterized in that: Contains the ketol-acid reductoisomerase mutant gene as claimed in claim 4.

6. The high-yielding D-pantothenic acid bacteria according to claim 5, characterized in that: The high-yield D-pantothenic acid bacteria use Escherichia coli, which is preserved in China Center for Type Culture Collection and has a preservation number of CCTCC No.M 20231938, as a host cell.

7. The high-yielding D-pantothenic acid bacteria according to claim 6, characterized in that: The host cell knocks out the glk gene, and the ketol-acid reductoisomerase mutant gene is inserted into the knockout site of the glk gene.

8. Use of the high-yield D-pantothenic acid bacteria according to any one of claims 5 to 7 in the preparation of D-pantothenic acid by microbial fermentation.

9. The use according to claim 8, characterized in that: The high-yield D-pantothenic acid bacteria are inoculated into a fermentation medium containing ampicillin resistance, and fermentation culture is carried out at 25-37° C. and 100-220 rpm. After the fermentation is completed, the fermentation supernatant is collected, and D-pantothenic acid is separated and purified.

10. The use according to claim 9, characterized in that: The fermentation is terminated when the OD600 is 10-20 and the pH value is 5.5-8.5; the fermentation medium comprises: 15-30 g / L of glucose monohydrate, 10-15 g / L of ammonium sulfate, 0.5-1.0 g / L of dipotassium hydrogen phosphate, 0.2-0.4 g / L of magnesium sulfate heptahydrate, 10-25 g / L of calcium carbonate, 2-5 g / L of yeast powder, and 2-6 g / L of beta-alanine.

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