Keto-acid reductoisomerase mutant, genetically engineered bacteria with high yield of d-pantothenic acid and application

Abstract

The application provides a ketol-acid reductoisomerase mutant and application thereof in construction of a genetically engineered bacterium with high D-pantothenic acid yield. In order to solve the technical problem of low yield of D-pantothenic acid synthesized by microbial fermentation in the prior art, the application uses semi-rational design of enzymes to obtain a ketol-acid reductoisomerase mutant with substrate specificity, and applies the mutant to construction of a genetically engineered bacterium with high D-pantothenic acid yield. The application also uses the ketol-acid reductoisomerase mutant to construct a genetically engineered bacterium with high D-pantothenic acid yield by CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04 %, and compared with a starting strain, the accumulation amount of valine in the obtained fermentation liquor is almost unchanged compared with a control, but the accumulation of branched-chain amino acid isoleucine is less, achieving modification of the substrate specificity of the ketol-acid reductoisomerase.

Description

[0001] The present application is a divisional application of Chinese Patent Application No. 202411766945.4, filed on December 4, 2024, and the original application has the title "Ketol-acid reductoisomerase mutant, genetically engineered bacteria with high yield of D-pantothenic acid". TECHNICAL FIELD

[0002] The present application belongs to the field of bioengineering, and specifically relates to a ketol-acid reductoisomerase mutant with substrate specificity and its application in constructing genetically engineered bacteria with high yield of D-pantothenic acid, genetically engineered bacteria with high yield of D-pantothenic acid and a method for constructing the same. BACKGROUND

[0003] Pantothenic acid (PA) is an important member of the vitamin B family, also known as vitamin B5 or pantothenic acid, and is an essential vitamin for maintaining normal energy metabolism. Pantothenic acid was first isolated from yeast in 1933, and it is widely present in various foods in nature, especially in animal liver, vegetables and whole grain foods. As a component of coenzyme A (CoA) and acyl carrier protein (ACP), D-pantothenic acid is involved in biochemical reactions such as fatty acid synthesis, protein metabolism and energy metabolism, and has multiple biological functions, which are essential for maintaining normal physiological functions and are widely used in the pharmaceutical industry, food industry, feed processing and cosmetics industry.

[0004] Currently, chemical enzyme method is the mainstream method for producing D-pantothenic acid, which has the advantages of substrate specificity and mild reaction conditions, but has the disadvantages of low production rate, high cost, difficulty in industrialization and generation of toxic and harmful substances in the production process. Compared with traditional chemical production methods, microbial cell factories produce more environmentally friendly products, reducing environmental pollution, meeting the urgent needs of modern society for green production. In addition, this production method can achieve zero or negative carbon emissions during production, thereby reducing the negative impact on the content of carbon dioxide in the atmosphere, which is conducive to addressing the challenge of climate change, and provides a more environmentally friendly and more economically beneficial method for producing D-pantothenic acid.

[0005] However, there are still defects in the production of D-pantothenic acid by chemical enzyme method, such as unstable fermentation process, low yield and other problems. Therefore, it is still a great challenge to construct a strain with higher yield of D-pantothenic acid. SUMMARY

[0006] In order to solve the technical problem that the yield of D-pantothenic acid synthesized by microbial fermentation is not high in the prior art, the present application uses semi-rational design of enzymes to obtain a ketol-acid reductoisomerase mutant with substrate specificity, and applies it to the construction of a genetically engineered bacterium with high yield of D-pantothenic acid. The present application also uses the ketol-acid reductoisomerase mutant to construct a genetically engineered bacterium with high yield of D-pantothenic acid by CRISPR-Cas9 gene editing technology.

[0007] The technical solution adopted by the present application is that a ketol-acid reductoisomerase mutant is obtained by single mutation at positions 47, 69, 75, 115, and 414 of the amino acid sequence shown in SEQ ID NO. 1.

[0008] Ketol-acid reductoisomerase (EC 1.1.1.86, AHAIR), also known as acetolactate reductoisomerase, is involved in the biosynthesis of branched-chain amino acids. It is a bifunctional enzyme that can catalyze two completely different reactions at a common active site; it can catalyze two completely different reactions, acting as an isomerase and a reductase. In the isomerase reaction, 2-acetolactate is converted to 2,3-dihydroxyisovalerate by Mg 2+ The rearrangement-dependent methyl shift generates pantothenate pathway intermediate 2,3-dihydroxyisovalerate, so it is one of the key enzymes in the D-pantothenic acid biosynthesis pathway, which is encoded by the ilvC gene in Escherichia coli.

[0009] In order to construct a D-pantothenic acid-producing strain with better performance, the present application modifies the site near the interaction between the substrate acetolactate molecule and ketol-acid reductoisomerase, and changes the substrate promiscuity. The active pocket of the enzyme molecule is shallow, and the substrate is more easily bound to the enzyme, so ketol-acid reductoisomerase can catalyze reactions of multiple 2-keto acids. In one aspect, the present application selects 6 amino acid positions 68, 75, 110, 132, 155, and 414 near the substrate and enzyme binding site as the object of site-directed mutagenesis. Since alanine has a simple structure and does not significantly change the spatial configuration of the protein, the 7 positions are all mutated to alanine. On the other hand, the present application also selects 4 amino acid positions 47, 69, 43, and 115 that are beneficial to enhancing the affinity of acetolactate as a substrate to ketol-acid reductoisomerase for site-directed mutagenesis. Subsequently, the above-mentioned mutants are overexpressed in the ilvC gene-knocked-out chassis bacterium by plasmid pACYC, and a better ketol-acid reductoisomerase mutant ilvC A47S , ilvC K69L , ilvC K75A , ilvC V115I , ilvC S414A , and a genetically engineered bacterium with high yield of D-pantothenic acid containing the gene encoding the above-mentioned better mutant.

[0010] As preferred, the ketol-acid reductoisomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1 by one of the following mutations:

[0011] (1) the alanine at position 47 is mutated into serine;

[0012] (2) the lysine at position 69 is mutated into leucine;

[0013] (3) the lysine at position 75 is mutated into alanine;

[0014] (4) the valine at position 115 is mutated into isoleucine;

[0015] (5) the serine at position 414 is mutated into alanine.

[0016] The present application also provides a gene encoding the ketol-acid reductoisomerase mutant.

[0017] The present application also provides a recombinant vector containing the gene encoding the ketol-acid reductoisomerase mutant.

[0018] The present application also provides a genetically engineered bacterium containing the gene encoding the ketol-acid reductoisomerase mutant.

[0019] The present application also provides the use of the ketol-acid reductoisomerase mutant in constructing a genetically engineered bacterium for high-yield production of D-pantothenate.

[0020] The present application also provides a method for constructing a genetically engineered bacterium for high-yield production of D-pantothenate, comprising: knocking out the ilvC gene in the genome of a chassis bacterium, introducing the gene encoding the ketol-acid reductoisomerase mutant into the chassis bacterium and overexpressing it, thereby constructing the genetically engineered bacterium for high-yield production of D-pantothenate.

[0021] As preferred, the gene encoding the ketol-acid reductoisomerase mutant is introduced into the chassis bacterium by using a low-copy plasmid pACYC and overexpressed.

[0022] As preferred, the chassis bacterium is Escherichia coli W3110, Trc-panC panE panB ilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔilvA / Trc-lpd / Δglk / ilvA* / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA T (referred to as DPAL6), which has been disclosed in CN113637618A.

[0023] As preferred, the method for constructing the genetically engineered bacterium for high-yield production of D-pantothenate comprises:

[0024] (1) using CRISPR-Cas9 gene editing technology, knocking out ilvC gene in the genome of the chassis DPAL6, to obtain strain DPAL6 / ΔilvC;

[0025] (2) overexpressing the coding gene of the ketol-acid reductoisomerase mutant in strain DPAL6 / ΔilvC through low-copy plasmid pACYC, to obtain the genetically engineered strain for high-yield D-pantothenate.

[0026] The application also provides the genetically engineered strain for high-yield D-pantothenate obtained by the method.

[0027] As preferred, the genetically engineered strain for high-yield D-pantothenate is one of the following:

[0028] (1) Escherichia coli W3110, Trc-panC panE panB ilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔilvA / Trc-lpd / Δglk / ilvA* / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA* T / ΔilvC / pACYC-ilvC A47S ;

[0029] (2) Escherichia coli W3110, Trc-panC panE panB ilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔilvA / Trc-lpd / Δglk / ilvA* / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA* T / ΔilvC / pACYC-ilvC K69L ;

[0030] (3) Escherichia coli W3110, Trc-panC panE panB ilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔilvA / Trc-lpd / Δglk / ilvA* / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA* T / ΔilvC / pACYC-ilvC K75A ;

[0031] (4) Escherichia coli W3110, Trc-panC panE panB ilvC / ilvG / ΔavtA / ilvE / coaA / ΔilvA / Trc-lpd / Δglk / ilvA / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA T / ΔilvC / pACYC-ilvC S414A ;

[0032] (5) Escherichia coli W3110, Trc-panC panE panB ilvC / ilvG / ΔavtA / ilvE / coaA / ΔilvA / Trc-lpd / Δglk / ilvA / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA T / ΔilvC / pACYC-ilvC V115I .

[0033] The application further provides application of the genetically engineered bacterium with high D-pantothenic acid yield in microbial fermentation for preparing D-pantothenic acid.

[0034] Preferably, the application comprises inoculating the genetically engineered bacterium with high D-pantothenic acid yield into a fermentation medium containing chloramphenicol, and carrying out fermentation culture at 37°C and 100-200 rpm until OD 600 = 0.8-1.0, then transferring to 30°C and continuing fermentation at 180 rpm for 48 h, and obtaining the D-pantothenic acid by separating and purifying the supernatant of the fermentation liquid after the fermentation.

[0035] Preferably, the fermentation medium comprises 10-30 g / L glucose, 10-25 g / L ammonium sulfate, 1-5 g / L anhydrous betaine, 1-5 g / L yeast powder, 1-5 g / L potassium dihydrogen phosphate, 0.5-2 g / L anhydrous magnesium sulfate, 1-5 g / L β-alanine, 1-5 ml / L trace element solution, and deionized water as a solvent, and the pH value is natural; the trace element solution comprises 10 g / L CuCl2, 10 g / L FeSO4·7H2O, 10 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, and deionized water as a solvent.

[0036] Specifically, the fermentation culture method comprises the following steps: taking out genetically engineered bacteria strains with high yield of D-pantothenic acid to be fermented, streaking on LB solid plates, culturing at 37 DEG C for 12-16 hours, picking single colonies to inoculate in LB test tubes, culturing at 37 DEG C and 180 rpm for 12 hours to obtain seed liquid. 1.5 mL of the seed liquid is inoculated in a 250 mL shaking flask containing 50 mL of culture medium, and the shaking culture is carried out at 30 DEG C and 180 rpm for 48 hours.

[0037] The present application has the following advantages: the present application uses molecular docking to perform site-directed mutation on ketol-acid reductoisomerase, enhances the binding capacity of substrates and enzymes, improves the affinity of acetyl lactate to substrates, and finally obtains a better ketol-acid reductoisomerase mutant. The ketol-acid reductoisomerase mutant ilvC V115I While the D-pantothenic acid pathway is strengthened, the synthesis of L-valine is not affected; finally, through the shaking flask fermentation, the yield of D-pantothenic acid is increased by about 12.04%, and compared with the starting strain, the accumulation of valine in the obtained fermentation liquid is almost unchanged compared with the control, but the accumulation of branched chain amino acid isoleucine is less, which achieves the modification of the substrate specificity of ketol-acid reductoisomerase. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The OD600 and D-pantothenic acid titer changes of DPAL7 (DPAL6 / ΔilvC) in the present application comparative example 1.

[0039] Figure 2 The OD600 and D-pantothenic acid titer changes of DPAL8 (DPAL7 / pACYC-ilvC) in the present application comparative example 2.

[0040] Figure 3 The OD600 and D-pantothenic acid titer changes of DPAL15 (DPAL7 / pACYC-ilvC A47S ) in the present application example 1.

[0041] Figure 4 The OD600 and D-pantothenic acid titer changes of DPAL16 (DPAL7 / pACYC-ilvC K69L ) in the present application example 2.

[0042] Figure 5 The OD600 and D-pantothenic acid titer changes of DPAL10 (DPAL7 / pACYC-ilvC K75A ) in the present application example 3.

[0043] Figure 6 The amino acid and D-pantothenic acid titer changes of DPAL18 (DPAL7 / pACYC-ilvC V115I ) in the present application example 4.

[0044] Figure 7 In Embodiment 5 of the present invention, DPAL14(DPAL7 / pACYC-ilvC) S414A Changes in OD600 and D-pantothenic acid potency.

[0045] Figure 8 DPAL9 (DPAL7 / pACYC-ilvC) in Comparative Example 3 of this invention R68A Changes in OD600 and D-pantothenic acid potency.

[0046] Figure 9 DPAL11(DPAL7 / pACYC-ilvC) in Comparative Example 4 of this invention Q110A Changes in OD600 and D-pantothenic acid potency.

[0047] Figure 10 DPAL12 (DPAL7 / pACYC-ilvC) in Comparative Example 5 of this invention H132A Changes in OD600 and D-pantothenic acid potency.

[0048] Figure 11 DPAL13 (DPAL7 / pACYC-ilvC) in Comparative Example 6 of this invention K155A Changes in OD600 and D-pantothenic acid potency.

[0049] Figure 12 DPAL17 (DPAL7 / pACYC-ilvC) in Comparative Example 7 of this invention V43I Changes in OD600 and D-pantothenic acid potency. Detailed Implementation

[0050] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0051] In the following examples, the final concentration of spectinomycin and kanamycin in the culture medium was 0.05 mg / L.

[0052] LB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent is deionized water, pH value is natural.

[0053] MS medium: glucose 20 g / L, (NH4)2SO4 16 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, yeast extract 2 g / L, CaCO3 10 g / L, 1 ml / L trace element solution, solvent is deionized water, pH value is natural; 10 g / L calcium carbonate (sterilized separately); the composition of the trace element solution is: 10 g / L CuCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.20 g / L CuSO4, 0.02 g / L NiCl2·7H2O, solvent is deionized water.

[0054] The parent strain E. coli W3110 in the application is from the CGSC (Coli Genetic Stock Center) of Yale University, the preservation date is August 5, 1975, the preservation number is CGSC #4474, and has been disclosed in patents US2009 / 0298135A1 and US2010 / 0248311A1.

[0055] HPLC determination of D-pantothenic acid content: chromatographic conditions: C18 column (250*4.6 mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200 nm, column temperature: 30 °C; sample treatment: dilute the sample with ultrapure water to keep the D-pantothenic acid content between 0.05 g / L and 0.40 g / L; mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1); data collection time: 25 min.

[0056] Determination of amino acid content: analysis conditions: Hitachi custom ion exchange resin (4.6 mm ID*60 mm*), wavelength: 570 nm, 440 nm, separation column temperature: 57 °C: reaction column temperature: 135 °C; sample treatment: dilute the sample with ultrapure water to keep the L-valine content between 0.05 g / L and 0.4 g / L; mobile phase: B1 (water 700 mL / sodium citrate 6.19 g / 1M NaOH / sodium chloride 5.66 g / citric acid 19.8 g / ethanol 135 mL); R3 (ethanol 50 mL / water 950 mL); data collection time: 30 min.

[0057] The primer sequence information used in the examples is shown in Table 1.

[0058] Table 1: Primer sequences

[0059]

[0060] Comparative Example 1: Construction of DPAL7 (DPAL6 / ΔilvC) and shake flask fermentation

[0061] Using DPAL6 as the starting strain, the ilvC gene in the genome was knocked out using CRISPR-Cas9 mediated gene editing technology, in order to determine the effect of the ketol-acid reductoisomerase mutant on the pantothenate pathway in the subsequent step.

[0062] (1) Construction of pTarget-ΔilvC plasmid: pTarget F plasmid (Addgene Plasmid#62226) was used as a template, and pT-ΔilvC-F / pT-ΔilvC-R was used as a primer for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3h. The PCR product of pTarget-ΔilvC was amplified by linear primers pTarget-XF and pTarget-XR, and the purified pTarget-ΔilvC linear vector was used for subsequent ligation of Donor DNA.

[0063] (2) Construction of pTD-ΔilvC plasmid: E. coli W3110 genome was used as a template, and ΔilvC-up-F and ΔilvC-up-R were used as primers to amplify the upstream part of the donor DNA (F1). ΔilvC-down-F and ΔilvC-down-R were used as primers to amplify the downstream part of the donor DNA (F2). F1 and F2 were obtained by gel recovery and purification of PCR fragments. According to the instructions of One step clone kit (Vazyme Biotech, Nanjing, China), pTarget-ΔilvC linear vector, fragments F1 and F2 were ligated together, and pTD-ΔilvC plasmid was obtained by sequencing verification. (One step clone kit, Vazyme Biotech, Nanjing, China) instructions pTarget-ΔilvC linear vector, fragments F1 and F2 were ligated together, and pTD-ΔilvC plasmid was obtained by sequencing verification.

[0064] (3) Introduce pCas plasmid (Addgene Plasmid#62225) into DPAL6, transfer single colony to LB test tube containing 0.05 mg / L kanamycin, and incubate at 30°C overnight. Then inoculate into 250 mL shake flask containing 50 mL LB medium with 1% volume concentration of inoculum, and add 500 μl 1 mol / L L-arabinose, 150 rpm, 30°C culture to OD 6000.4-0.6; 4000 rpm, 4°C centrifugation for 10 min to collect cells, and then prepare electrotransformation competent cells, see detailed process (Molecular Cloning: A Laboratory Manual, 3ed Edition, 99-102).

[0065] (4) Use a pipette to take an appropriate amount of pTD-ΔilvC (about 200 ng) plasmid and mix with the previously prepared 100 μl electroshock competent cells, and then transfer into a pre-cooled 2 mm electroshock cup, and then ice bath for 1-2 min, and then use an electroporator (MicroPluser TM , BIO-RAD) to perform electroshock transformation, and then immediately add 800 μl LB medium and immediately gently suck out, and then transfer into a 2 mL Ep tube, and then recover at 30°C for 3-4 h, and then plate on an LB solid plate containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin, and then incubate at 30°C for 12-16 h, and then perform colony PCR using ΔilvC-VF / R as primers, and then if a fragment of about 1500 bp can be successfully cloned, then it is proved that the ΔilvC positive colony of DPAL7 (DPAL6 derivative, ΔilvC) is obtained.

[0066] (5) Plasmid elimination: use a loop to pick the positive single colony and inoculate into an LB liquid test tube containing 1 mM IPTG and 0.05 mg / L kanamycin, and then incubate at 30°C overnight, and then streak the bacterial liquid on an LB solid plate containing 0.05 mg / L kanamycin the next day, and then incubate at 30°C for 24 h, and then pick some single colonies and streak on an LB plate containing 0.05 mg / L spectinomycin, and then the single colony which cannot grow on the LB plate containing 0.05 mg / L spectinomycin indicates that the pTarget-ΔilvC plasmid is successfully eliminated, and then pick the single colony with successfully eliminated pTarget-ΔilvC plasmid on an LB test tube, and then incubate at 37°C overnight, and then use it to eliminate the pCas plasmid, and then streak the bacterial liquid on an LB plate the next day, and then incubate at 37°C for 12 h, and then pick some single colonies and streak on an LB plate containing 0.05 mg / L kanamycin, and then the single colony which cannot grow on the LB plate containing 0.05 mg / L kanamycin indicates that the pCas plasmid is successfully eliminated, and then finally obtain the plasmid-free strain DPAL7 (DPAL6 derivative, ΔilvC).

[0067] (6) Shake flask fermentation: DPAL7 (DPAL6 derivative, ΔilvC), with DPAL6 as the control group, was inoculated into 10 mL of LB medium, and cultured at 37°C, 200 rpm as a preculture; 8-12 h later, 1 mL of the preculture was inoculated into a 500 mL flask containing 50 mL of MS medium at a 2% inoculation amount, and then cultured in a constant-temperature shaker at 30°C, 180 rpm for 48 h for strain fermentation; after the fermentation was completed, 1 mL of the fermentation broth was taken to measure the OD 600 value, and at the same time, 1 mL of the fermentation broth was taken using a pipette, centrifuged at 12000 rpm at room temperature for 3 min, and the fermentation supernatant was diluted 5 times, then the diluted sample was treated to remove impurities using a water-based filter membrane, and then HPLC detection was performed, OD 600 and the D-pantothenic acid content in the fermentation supernatant were as shown in Figure 1 .

[0068] As can be seen from the figure, the knockout of the ilvC gene in the genome, compared with DPAL6, the strain DPA in which ilvC was knocked out, the yield decreased by about 80%, indicating that ketol-acid reductoisomerase plays a key role in the DPA production pathway.

[0069] Comparative Example 2: Construction and shake flask fermentation of DPAL8 (DPAL7 / pACYC-ilvC)

[0070] DPAL7 was used as the starting strain, and the recombinant plasmid pACYC-ilvC was constructed and transformed into the chassis strain DPAL7 to verify the effect of ilvC on the potency of D-pantothenic acid

[0071] (1) Construction of pACYC plasmid linearization vector: using the original pACYC plasmid as the template, the primers pACYC-F and pACYC-R were used to linearize the open loop. After the PCR product was verified by nucleic acid gel electrophoresis, DPN I was used to digest the residual template at 37°C for 1 h, and after the digestion was completed, the product was purified, the nucleic acid concentration was measured, and finally the pACYC linearization vector fragment was obtained

[0072] (2) Amplification of the original ilvC fragment: using the wild-type E. coli W3110 genome as the template, the primers ilvCEc-F and ilvCEc-R were used to amplify it by PCR, and after the product was verified by nucleic acid gel electrophoresis, the product was purified, the nucleic acid concentration was measured, and finally the amplified ilvC fragment (nucleotide sequence as shown in SEQ ID NO. 2) was obtained.

[0073] (3) Plasmid pACYC-ilvC construction: using pACYC linearized vector and amplified ilvC fragment as template, adding reaction system according to the instruction of ClonExpress Ultra One Step Cloning Kit, placing at 50℃ for 15 min, then immediately placing on ice to cool, then the ilvC gene fragment can be integrated into the multiple cloning site of pACYC plasmid, then the ligation product is transformed into DH5a competent cells, after colony PCR verification, the recombinant plasmid pACYC-ilvC is obtained by picking bacteria into test tubes.

[0074] (4) Transforming the plasmid pACYC-ilvC into the chassis strain DPAL7 to obtain DPAL8. The specific steps are referred to step (6) of Comparative Example 1. The OD600 of the strain DPAL8 after fermentation and the D-pantothenic acid content in the supernatant of the fermentation broth are shown in Table 2. Figure 2

[0075] As can be seen from the figure, by constructing the recombinant plasmid pACYC-ilvC to overexpress ilvC, the isomerase activity of DPAL6 is restored, and the D-pantothenic acid yield is almost the same as that of the control.

[0076] Example 1: Construction of DPAL15 (DPAL7 / pACYC-ilvC A47S ) and shake flask fermentation

[0077] Using DPAL7 as the starting strain, site-directed mutagenesis is used to mutate the 47th codon gca of the original ilvC of E. coli to agc, so that the encoded amino acid is mutated from alanine to serine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0078] (1) Plasmid pACYC-ilvC A47S construction: using plasmid pACYC-ilvC as template, using primers A47S-F and A47S-R for site-directed mutagenesis, after verifying the PCR product of the mutated product by nucleic acid gel electrophoresis, using DPN I to digest the residual template at 37℃ for 1 h, after digestion, purifying the product, measuring the nucleic acid concentration, and finally obtaining the pACYC-ilvC A47S plasmid linearized vector fragment with ilvC mutation.

[0079] (2) Transforming the plasmid pACYC-ilvC A47S into the chassis strain DPAL7 to obtain DPAL15. The specific steps are referred to step (6) of Comparative Example 1. The OD600 of the strain DPAL15 after fermentation and the D-pantothenic acid content in the supernatant of the fermentation broth are shown in Table 2. Figure 3

[0080] ​​As can be seen from the figure, compared with the DPAL6 strain, the OD of the strain DPAL15 has greatly improved, and the pantothenic acid yield has increased, which shows that the mutation of the 47th codon of ilvC may improve the isomerase activity, thereby causing the D-pantothenic acid yield to rise.

[0081] Example 2: Construction and shake flask fermentation of DPAL16 (DPAL7 / pACYC-ilvC K69L )

[0082] Using DPAL7 as the starting strain, site-directed mutation was used to mutate the 69th codon aaa of the original ilvC of E. coli to ctg, so that the encoded amino acid was mutated from lysine to leucine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0083] Using plasmid pACYC-ilvC as the template, site-directed mutation was performed on it using primers K69L-F and K69L-R, and plasmid pACYC-ilvC K69L was constructed. The plasmid pACYC-ilvC K69L was transformed into the chassis strain DPAL7, and DPAL16 was obtained. The specific steps are referred to in Example 1. The OD600 after fermentation of the strain DPAL16 and the D-pantothenic acid content in the supernatant of the fermentation broth are shown in Figure 4 .

[0084] As can be seen from the figure, compared with the DPAL6 strain, the OD of the strain DPAL16 has greatly improved, and the pantothenic acid yield has increased, which shows that the mutation of the 69th codon of ilvC may improve the isomerase activity, thereby causing the D-pantothenic acid yield to rise.

[0085] Example 3: Construction and shake flask fermentation of DPAL10 (DPAL7 / pACYC-ilvC K75A )

[0086] Using DPAL7 as the starting strain, site-directed mutation was used to mutate the 75th codon aag of the original ilvC of E. coli to gcg, so that the encoded amino acid was mutated from lysine to alanine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0087] Using plasmid pACYC-ilvC as the template, site-directed mutation was performed on it using primers K75A-F and K75A-R, and plasmid pACYC-ilvC K75A was constructed. The plasmid pACYC-ilvC K75A was transformed into the chassis strain DPAL7, and DPAL10 was obtained. The specific steps are referred to in Example 1. The OD600 after fermentation of the strain DPAL10 and the D-pantothenic acid content in the supernatant of the fermentation broth are shown in Figure 5 .

[0088] As shown in the figure, compared with DPAL6 strain, the D-pantoic acid titer of strain DPAL10 is slightly increased, which indicates that the mutation of the 75th codon of ilvC may increase the isomerase activity, thereby slightly increasing the yield of D-pantoic acid.

[0089] Example 4: Construction and shake flask fermentation of DPAL18 (DPAL7 / pACYC-ilvC V115I )

[0090] Using DPAL7 as the starting strain, site-directed mutation was used to mutate the 115th codon gtg of the original ilvC of E. coli to att, so that the encoded amino acid was mutated from valine to isoleucine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0091] Using plasmid pACYC-ilvC as the template, site-directed mutation was performed on it using primers V115I-F and V115I-R, and plasmid pACYC-ilvC V115I was constructed. Plasmid pACYC-ilvC V115I was transformed into the chassis strain DPAL7, and DPAL18 was obtained. The specific steps are referred to Example 1. The OD600 after fermentation and the D-pantoic acid content in the supernatant of the fermentation broth of strain DPAL18 are shown in Figure 6 .

[0092] As shown in the figure, by mutating ilvC and constructing recombinant plasmid pACYC-ilvC V115I , compared with DPAL6 strain, the yield of D-pantoic acid is increased by about 12.04%, the accumulation of valine is almost unchanged, but the accumulation of isoleucine is reduced by 54.1%, the specificity of ketol-acid substrate is realized, and the D-panthotenic acid synthesis pathway is strengthened.

[0093] Example 5: Construction and shake flask fermentation of DPAL14 (DPAL7 / pACYC-ilvC S414A )

[0094] Using DPAL7 as the starting strain, site-directed mutation was used to mutate the 414th codon tct of the original ilvC of E. coli to gcg, so that the encoded amino acid was mutated from serine to alanine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0095] Using plasmid pACYC-ilvC as the template, site-directed mutation was performed on it using primers S414A-F and S414A-R, and plasmid pACYC-ilvC S414A was constructed. Plasmid pACYC-ilvC S414AThe plasmid pACYC-ilvC was transformed into the chassis strain DPAL7 to obtain DPAL9. The specific steps refer to Example 1. The OD600 of the fermentation of the strain DPAL9 and the D-pantoic acid content in the supernatant of the fermentation broth are shown in Table 3. Figure 7

[0096] As can be seen from the figure, compared with the strain DPAL6, the OD of the strain DPAL14 has been greatly improved, and the yield has also been improved to a certain extent, which is presumably because the enzyme activity is improved and the pantoic acid synthesis pathway is strengthened.

[0097] Comparative Example 3: Construction and shake flask fermentation of DPAL9 (DPAL7 / pACYC-ilvC R68A )

[0098] DPAL7 as the starting strain, site-directed mutagenesis was used to mutate the 68th codon cgt of the original ilvC of E. coli to gcg, so that the encoded amino acid was mutated from arginine to alanine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0099] The plasmid pACYC-ilvC was used as a template, and primers R68A-F and R68A-R were used for site-directed mutagenesis to construct the plasmid pACYC-ilvC R68A . The plasmid pACYC-ilvC R68A was transformed into the chassis strain DPAL7 to obtain DPAL9. The specific steps refer to Example 1. The OD600 of the fermentation of the strain DPAL9 and the D-pantoic acid content in the supernatant of the fermentation broth are shown in Table 3. Figure 8

[0100] As can be seen from the figure, compared with the strain DPAL6, the D-pantoic acid of the strain DPAL9 has almost no change, indicating that the mutation of the 68th codon of ilvC may have little effect on the enzyme activity.

[0101] Comparative Example 4: Construction and shake flask fermentation of DPAL11 (DPAL7 / pACYC-ilvC Q110A )

[0102] DPAL7 as the starting strain, site-directed mutagenesis was used to mutate the 68th codon cgt of the original ilvC of E. coli to gcg, so that the encoded amino acid was mutated from arginine to alanine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0103] The plasmid pACYC-ilvC was used as a template, and primers R68A-F and R68A-R were used for site-directed mutagenesis to construct the plasmid pACYC-ilvC Q110A . The plasmid pACYC-ilvC Q110A ​​The plasmid pACYC-ilvC was transformed into the chassis strain DPAL7 to obtain DPAL11. The specific steps refer to Example 1. The OD600 of the fermentation of the strain DPAL11 and the content of D-pantothenate in the supernatant of the fermentation broth are shown in Table 2. Figure 9

[0104] As can be seen from the figure, compared with the strain DPAL6, the D-pantothenate yield of the strain DPAL11 is reduced, indicating that the mutation of the 110th codon of ilvC may reduce the isomerase activity, thereby causing the reduction of the D-pantothenate yield.

[0105] Comparative Example 5: Construction and shake flask fermentation of DPAL12 (DPAL7 / pACYC-ilvC H132A )

[0106] DPAL7 as the starting strain, site-directed mutagenesis was used to mutate the 132nd codon cac of the original ilvC of E. coli to gcg, so that the encoded amino acid was mutated from histidine to alanine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0107] The plasmid pACYC-ilvC was used as a template, and primers H132A-F and H132A-R were used for site-directed mutagenesis to construct the plasmid pACYC-ilvC H132A . The plasmid pACYC-ilvC H132A was transformed into the chassis strain DPAL7 to obtain DPAL12. The specific steps refer to Example 1. The OD600 of the fermentation of the strain DPAL12 and the content of D-pantothenate in the supernatant of the fermentation broth are shown in Table 3. Figure 10

[0108] As can be seen from the figure, compared with the strain DPAL6, the OD of the strain DPAL12 has been greatly improved, but the D-pantothenate yield is reduced, and it is speculated that the mutation of the 132nd codon of ilvC may reduce the isomerase activity.

[0109] Comparative Example 6: Construction and shake flask fermentation of DPAL13 (DPAL7 / pACYC-ilvC K155A )

[0110] DPAL7 as the starting strain, site-directed mutagenesis was used to mutate the 155th codon aaa of the original ilvC of E. coli to gcg, so that the encoded amino acid was mutated from lysine to alanine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0111] The plasmid pACYC-ilvC was used as a template, and primers K155A-F and K155A-R were used for site-directed mutagenesis to construct the plasmid pACYC-ilvC K155A . The plasmid pACYC-ilvC K155A ​​The plasmid pACYC-ilvC was transformed into the chassis strain DPAL7 to obtain DPAL13. The specific steps refer to Example 1. The OD600 of the strain DPAL13 after fermentation and the content of D-pantothenate in the supernatant of the fermentation broth are shown in Table 2. Figure 11

[0112] As can be seen from the figure, compared with the strain DPAL6, the OD of the strain DPAL13 has been greatly improved, but the isomerase activity has been reduced, thereby causing the yield of D-pantothenate to be reduced.

[0113] Comparative Example 7: Construction and shake flask fermentation of DPAL17 (DPAL7 / pACYC-ilvC V43I )

[0114] DPAL7 as the starting strain, site-directed mutagenesis was used to mutate the 43rd codon gtc of the original ilvC of E. coli to atc, so that the encoded amino acid was changed from valine to isoleucine, so as to change the substrate preference of ketol-acid reductoisomerase.

[0115] The plasmid pACYC-ilvC was used as a template, and primers V43I-F and V43I-R were used for site-directed mutagenesis to construct the plasmid pACYC-ilvC V43I . The plasmid pACYC-ilvC V43I was transformed into the chassis strain DPAL7 to obtain DPAL17. The specific steps refer to Example 1. The OD600 of the strain DPAL17 after fermentation and the content of D-pantothenate in the supernatant of the fermentation broth are shown in Table 2. Figure 12

[0116] As can be seen from the figure, compared with the strain DPAL6, the OD of the strain DPAL17 has been greatly improved, but it is speculated that the mutation of the 43rd codon of ilvC reduces the isomerase activity, thereby causing the yield of D-pantothenate to be reduced.

[0117] The above examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope of the present application.​​

Claims

1. A ketohydric acid reductase mutant, characterized in that, It was obtained by mutating lysine at position 69 of the amino acid sequence shown in SEQ ID NO.1 to leucine.

2. The gene encoding the ketool acid reductase mutant of claim 1.

3. A recombinant vector containing the gene encoding the ketohydric acid reductase mutant as described in claim 2.

4. Genetically engineered bacteria containing the gene encoding the ketohydric acid reductase mutant as described in claim 2.

5. The application of the ketool acid reductase mutant as described in claim 1 in the construction of genetically engineered bacteria that produce D-pantothenic acid.

6. A method for constructing a genetically engineered bacterium that produces D-pantothenic acid, characterized in that, include: Knockout of the chassis bacteria genome ilvC The gene encoding the ketol acid reductase mutant of claim 1 was introduced into the spores and overexpressed to construct a genetically engineered bacterium that produces D-pantothenic acid.

7. The genetically engineered bacteria producing D-pantothenic acid constructed by the method described in claim 6.

8. The application of the genetically engineered bacteria producing D-pantothenic acid as described in claim 7 in the microbial fermentation preparation of D-pantothenic acid.

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