CN114921429A - An acetylation-regulated lactate dehydrogenase mutant and its application - Google Patents

Abstract

The invention discloses an acetylation-regulated lactic dehydrogenase mutant and application thereof in improving synthesis of lactic acid, wherein the mutant is formed by mutating lysine at the 9 th position of a lactic dehydrogenase LdhA protein into arginine, is named as a K9R mutant, and has an amino acid sequence shown as SEQ ID No. 1. The invention also discloses an acetylation-regulated double mutant of lactate dehydrogenase and application thereof in inhibiting synthesis of lactic acid, wherein the mutant is formed by mutating the 154 th lysine and the 248 th lysine of lactate dehydrogenase LdhA protein into glutamine, and is named as a K154Q-K248Q double mutant, and the amino acid sequence of the double mutant is shown as SEQ ID NO. 2. Experiments prove that the K9R mutant improves the enzyme activity of the LdhA by 2.5 times, and the lactic acid yield of the strain overexpressing the K9R mutant is improved by 1.67 times compared with the lactic acid yield of the strain overexpressing the wild type LdhA; the activity of the K154Q-248Q double mutant is reduced to 12.6 percent of that of a wild type, and the double mutant strain in which the ldhA gene on the genome is in situ mutated into K154Q-K248Q can realize the effect similar to that of the currently commonly used ldhA gene knock-out method on the aspect of inhibiting the synthesis of lactic acid.

Description

An acetylation-regulated lactate dehydrogenase mutant and its application

technical field

The present invention relates to a lactate dehydrogenase mutant and its application, in particular to an acetylation-regulated lactate dehydrogenase mutant and its application. It belongs to the field of genetic engineering technology.

Background technique

Lactic acid is an important bulk chemical commonly used in food flavorings and preservatives. Lactic acid is also a natural moisturizing ingredient with little irritation, which is used in various cosmetics. In medicine, lactic acid is widely used as a preservative, a carrier, a cosolvent, a pharmaceutical preparation, a pH adjuster, and the like. At the same time, lactic acid can also be used as a monomer to synthesize polylactic acid, which is a new type of biodegradable plastic. Therefore, lactic acid has a wide range of applications in food, cosmetics, medicine, agriculture, chemical industry, degradable plastics and other fields. At present, the synthesis methods of lactic acid mainly include microbial fermentation and chemical synthesis. Among them, microbial fermentation has become the main method for lactic acid synthesis due to its advantages of environmental protection and sustainable raw materials.

NAD-dependent lactate dehydrogenase (LdhA) is a conserved enzyme widely found in various species that specifically catalyzes the conversion of pyruvate to lactate. In engineering bacteria that synthesize lactic acid by microorganisms, the production of lactic acid is often improved by overexpressing the lactate dehydrogenase LdhA. However, the natural enzymatic activity of LdhA limits the efficiency of lactate synthesis. Optimizing the catalytic activity of LdhA has become an important method to further improve the yield and conversion efficiency of lactic acid.

In addition, lactic acid is also an important by-product in the synthesis of other bio-based chemicals. LdhA catalyzes the conversion of pyruvate to lactate when the rapid depletion of substrates and limited cellular respiration capacity are unbalanced, resulting in massive accumulation of pyruvate and NADH. The synthesis of lactic acid will waste the carbon source of the target product synthesis pathway, affecting the yield and conversion efficiency of the target product. In order to avoid the synthesis of lactic acid by-products during the synthesis of other bio-based chemicals, a common method is to directly knock out the ldhA gene on the genome. Although direct knockout of the ldhA gene inhibited lactate production, it also affected biomass accumulation. Therefore, exploring a new metabolic regulation method to inhibit the synthesis of lactic acid byproducts under the precursors that maintain the normal biomass accumulation of cells is expected to solve the common problem of lactic acid byproducts accumulation in the current microbial synthesis process.

Lysine acetylation is a conservative post-translational modification mechanism that affects various cellular physiological and metabolic processes such as enzyme activity, metabolic flux distribution, replication and transcription. However, the role of lysine acetylation in the regulation of bacterial lactate dehydrogenase LdhA activity and lactate synthesis has not yet been reported. Further, the related methods of regulating the lysine acetylation modification of lactate dehydrogenase, changing the activity of lactate dehydrogenase, and realizing the regulation of lactate synthesis, as well as the related acetylation-regulated lactate dehydrogenase mutants and their applications are also disclosed. Not reported.

SUMMARY OF THE INVENTION

Aiming at the problem of the natural activity limitation of lactate dehydrogenase LdhA in the microbial synthesis process of lactic acid as the target product, and the problem of accumulation of lactic acid as a by-product in the synthesis process of other bio-based chemicals, the object of the present invention is to provide a Acetylation-regulated lactate dehydrogenase mutants and their applications. By regulating the specific lysine acetylation site of lactate dehydrogenase LdhA, the activity of lactate dehydrogenase can be increased or decreased according to the synthetic requirements, so as to realize the regulation of lactate synthesis.

The acetylation-regulated lactate dehydrogenase mutant of the present invention is characterized in that: the mutant is formed by mutating the 9th lysine of the lactate dehydrogenase LdhA protein to arginine, and is named as K9R mutation body, the amino acid sequence of which is shown in SEQ ID NO.1.

Further, the application of the acetylation-regulated lactate dehydrogenase mutant of the present invention in improving the synthesis of lactic acid.

The present invention provides an expression strain of the above acetylation-regulated lactate dehydrogenase K9R mutant, characterized in that: the strain carries the K9R mutant lactate dehydrogenase gene ldhA shown in SEQ ID NO.3.

The expression strain of the above-mentioned acetylation-regulated lactate dehydrogenase K9R mutant is preferably a strain named E. coli BL21(DE3)/pETDuet1-ldhA(K9R). Its build method is:

1) Using the genomic DNA of Escherichia coli as a template, the upstream of K9R mutant lactate dehydrogenase gene ldhA was cloned with primers pETDuet1-ldhA-5'(CCG GGATCC GATGAAACTCGCCGTTTATAGC) and pETDuet1-ldhA(K9R)-3'(AGGTACTTCTTGTCGTACTGACGTGTGCTATAAACGGCGAGTT) The fragment was cloned with primers pETDuet1-ldhA-3'(CCGGAGCTCTTAAACCAGTTCGTTCGGGC) and pETDuet1-ldhA(K9R)-5'(AACTCGCCGTTTATAGCACACG TCAGTA CGACAAG AAGTACCT) to obtain the downstream fragment of K9R mutated lactate dehydrogenase gene ldhA, and used fragment bridging method to bridge K9R The upstream and downstream fragments of the mutated lactate dehydrogenase gene ldhA are obtained to obtain a K9R mutated lactate dehydrogenase gene ldhA, the nucleotide sequence of which is shown in SEQ ID NO.3;

2) connecting the K9R mutant lactate dehydrogenase gene ldhA obtained in step 1) to the expression vector to obtain a recombinant plasmid;

3) The recombinant plasmid obtained in step 2) was introduced into the recipient cell E.coli BL21(DE3) to obtain the K9R mutant expression strain E.coli BL21(DE3)/pETDuet1-ldhA(K9R).

Wherein, the expression vector in step 2) is pETDuet1, which was purchased from Novagen Company.

The application of the expression strain of the K9R mutant of the present invention in improving lactic acid production and transformation efficiency.

Wherein, the preferred technical solution for improving lactic acid production and transformation efficiency is: inoculating the above-mentioned K9R mutant expression strain in a lactic acid fermentation medium, and fermenting for 24 hours at 37° C. and 100 rpm with glucose as the carbon source.

The invention also discloses an acetylation-regulated lactate dehydrogenase double mutant, characterized in that: the double mutant is that the lysines at the 154th and 248th positions of the lactate dehydrogenase LdhA protein are mutated into glutamate amide formed, named as K154Q-K248Q double mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

Further, the application of the acetylation-regulated lactate dehydrogenase double mutant in inhibiting the synthesis of lactate.

Correspondingly, the present invention provides a K154Q-K248Q double mutant strain of lactate dehydrogenase regulated by the above-mentioned acetylation, characterized in that: the lactate dehydrogenase gene ldhA in the genome of the strain is mutated, and the in situ replacement is as shown in SEQ The K154Q-K248Q double-mutated lactate dehydrogenase gene ldhA shown in ID NO.4, the strain was named E. coli BL21(DE3)ldhA::ldhA(K154Q-K248Q). Its build method is:

1) Using the genomic DNA of Escherichia coli as a template, the primers pRE112-ldhA(K154Q-K248Q)-5'(CAACATCACTGGAGAAAGTCTTATGAAACTCGCCGTTTATAGC) and ldhA(K154Q)-3'(CGCAGCATCGCCACACCGATCTGACCGGTACCGATAACGCCTG) were cloned to obtain the K154Q mutant lactate dehydrogenase gene ldhA The upstream fragment was cloned with primers pRE112-ldhA(K154Q-K248Q)-3'(GGTTGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGC) and ldhA(K154Q)-5'(CAGGCGTTATCGG TACCGGTCAGATCGGTGTGGCGATGCTGCG) to obtain the downstream fragment of K154Q-mutated lactate dehydrogenase gene ldhA, using the fragment bridging method By bridging the upstream and downstream fragments of the K154Q-mutated lactate dehydrogenase gene ldhA, the K154Q-mutated lactate dehydrogenase gene ldhA was obtained;

2) Using the K154Q-mutated lactate dehydrogenase gene ldhA as a template, the K154Q-mutated lactate was cloned with primers pRE112-ldhA(K154Q-K248Q)-5'(CAACATCACTGGAGAAAGTCTTATGAAACTCGCCGTTTATAGC) and ldhA(K248Q)-3'(AACGAACCAATTTTCTGATTCTGCAGCGCTTCAATTGCTGCCT) The upstream fragment of dehydrogenase gene ldhA was cloned with primers pRE112-ldhA(K154Q-K248Q)-3'(GGTTGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGC) and ldhA(K248Q)-5'(AACGAACCAATTTTCTGATTCTGCAGCGCTTCAATTGCTGCCT) to obtain K154Q-K248Q mutant lactate dehydrogenase gene ldhA The downstream fragment is used to bridge the upstream and downstream fragments of the K154Q-K248Q mutant lactate dehydrogenase gene ldhA by using the fragment bridging method to obtain the K154Q-K248Q double mutant lactate dehydrogenase gene ldhA, and its nucleotide sequence is as SEQ ID NO.4 shown;

3) Taking the genomic DNA of Escherichia coli as a template, using primers pRE112-up-5' (CCG TCTAGA CAGTTGCTGGATATCAGAGG) and pRE112-up-3' (GCTATAAACGGCGAGTTTCATAAGACTTTCTCCAGTGATGTTG) to clone the upstream homologous fragment Up sequence of the ldhA gene, and use the primers pRE112- dwon-5'(GCCCGAACGAACTGGTTTAATCTTGCCGCTCCCCTGCAACC) and pRE112-dwon-3'(CCGGAGCTCGTCGATGTCCAGTAGTGGAG) were cloned to obtain the Down sequence of the downstream homologous fragment of the ldhA gene, and the Up-ldhA(K154Q-248Q)-Down fragment was obtained by bridging by fragment bridging method. The acid sequence is shown in SEQ ID NO.5.

4) The Up-ldhA(K154Q-248Q)-Down fragment obtained in step 3) was constructed into the suicide plasmid pRE112, and the lactate dehydrogenase gene ldhA of the recipient cell was in situ using the homologous recombination mediated by the suicide plasmid pRE112. The K154Q-K248Q double mutant ldhA gene was replaced to obtain a K154Q-K248Q double mutant strain (E. coli BL21(DE3)ldhA::ldhA(K154Q-K248Q).

The application of the K154Q-K248Q double mutant strain of the present invention in inhibiting the synthesis of lactic acid.

Wherein, the preferred technical solution for inhibiting the synthesis of lactic acid is: in 3-hydroxypropionic acid fermentation medium, inoculate the above-mentioned K154Q-K248Q double mutant strain, use glucose as carbon source, and ferment at 37°C and 180rpm. 48h.

The application of the K154Q-K248Q double mutant strain of the present invention in the biosynthesis of 3-hydroxypropionic acid.

Wherein, the preferred technical scheme of the 3-hydroxypropionic acid biosynthesis is:

1) Prepare the competent cells of the K154Q-K248Q double mutant strain according to the operation instructions of the competent preparation kit;

2) introducing the synthetic plasmids pA-accADBC and pMCR-N-C-N940V/K1106W/S1114R of 3-hydroxypropionic acid into the competent cells prepared in step 1) to prepare a recombinant strain of 3-hydroxypropionic acid;

3) Inoculate the recombinant strain prepared in step 2) into a 3-hydroxypropionic acid fermentation medium, use glucose as a carbon source, and ferment for 48 hours at 37° C. and 180 rpm.

The invention discloses an acetylation-regulated lactate dehydrogenase mutant and its application. For the first time, by regulating the specific lysine acetylation modification site of lactate dehydrogenase LdhA, the activity of lactate dehydrogenase LdhA can be increased or decreased according to the demand to realize the regulation of lactate synthesis. Experiments confirmed that: after the lysine (K) at position 9 of LdhA was mutated to arginine (R), the enzyme activity of the K9R mutant was 2.5 times higher than that of the wild-type LdhA. When the lactate dehydrogenase gene ldhA containing the K9R mutant was overexpressed, the production and transformation efficiency of lactate were increased by 1.63 times compared with the control strain. After the lysine (K) at positions 154 and 248 of LdhA was mutated to glutamine (Q), the enzyme activity of the K154Q-K248Q mutant decreased to 12.6% of the wild type. When the ldhA gene on the genome of E.coli BL21(DE3) strain was in situ replaced with the ldhA gene containing the K154Q-K248Q mutant, the accumulation of lactate was significantly reduced, which was directly related to the currently commonly used method for controlling lactate synthesis, namely ldhA. Compared with the knockout method, the lactate inhibitory effect was similar, but the biomass accumulation was significantly higher than that of the ldhA knockout strain. During the synthesis of the biobased chemical 3-hydroxypropionic acid, the K154Q-K248Q mutant strain was able to reduce lactate by-products by 82.3%, similar to the ldhA knockout strain. The 3HP production of the double mutant strain was significantly higher than that of the control strain and the ldhA knockout strain. This shows that the method of the present invention to reduce ldhA enzyme activity by protein lysine acetylation modification inhibits the synthesis of lactic acid by-products, and can achieve a similar effect to the currently commonly used method (direct knockout of ldhA gene), but the method of the present invention is in cells. It has more advantages in biomass accumulation and target product synthesis. Further, the present invention obtains the lactate dehydrogenase mutant by regulating the lysine acetylation modification site of the protein for the first time, which can increase or decrease the synthesis of lactic acid as needed, so as to realize the flexible regulation of microbial synthesis. Given that protein acetylation modification is a very conservative and extensive post-translational modification mechanism, the technical solution of the present invention has reference value in the regulation of lactic acid synthesis in other species, and can be widely applied to inhibit lactic acid in the synthesis process of other bio-based chemicals accumulation of by-products.

Description of drawings

Figure 1 shows the effect of acetylation modification site mutation of lactate dehydrogenase on enzyme activity.

Figure 2 shows the effect of K9R mutant lactate dehydrogenase on cell growth, lactate synthesis and conversion rate during lactate synthesis.

Figure 3 is a comparison of the double mutant strain of K154Q-K248Q with the ldhA knockout strain in terms of cell growth and lactate accumulation.

Figure 4 is a comparison of cell growth, lactic acid accumulation and 3-hydroxypropionic acid synthesis in the K154Q-K248Q double mutant strain and the ldhA knockout strain during the fermentation of 3-hydroxypropionic acid.

Detailed ways

General notes:

The materials, reagents, instruments and methods used in the following examples, unless otherwise specified, are conventional materials, reagents, instruments and methods in the art, and can be obtained through commercial channels.

The enzyme reagents used were purchased from MBI Fermentas, and the kits used for plasmid extraction and DNA fragment recovery were purchased from OMEGA, the United States. The corresponding operation steps were carried out according to the product instructions; all media were deionized water unless otherwise specified. formulate.

Medium formula:

1) LB medium: 5g/L yeast powder, 10g/L NaCl, 10g/L peptone, the rest is water, sterilized at 121°C for 20min.

2) Lactic acid fermentation medium: 0.42g/L citric acid, 2g/L KH ₂ PO ₄ , 1.6g/L K ₂ HPO ₄ , 5.4g/L NH ₄ Cl, 0.25g/L MgSO ₄ ·7H ₂ O, 1 /1000 trace elements (3.7g/L(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O, 2.9g/L ZnSO ₄ ·7H ₂ O, 24.7g/LH ₃ BO ₃ , 2.5g/L CuSO ₄ ·5H ₂ O, 15.8 g/L MnCl ₂ ·4H ₂ O).

3) 3-Hydroxypropionic acid fermentation medium: 14g/LK ₂ HPO ₄ ·3H2O, 5.2g/L KH ₂ PO, 1g/L NaCl, 1g/LNH ₄ Cl, 0.25g/L MgSO ₄ ·7H ₂ O, 0.2g/L yeast powder, 20g/L glucose.

In the actual culture process, a certain concentration of antibiotics can be added to the above medium to maintain the stability of the plasmid, such as 50 mg/L chloramphenicol, 100 mg/L ampicillin.

The Escherichia coli described in the present invention is E. coli BL21 (DE3), purchased from Invitrogen Company.

In the present invention, the lactate dehydrogenase gene ldhA is derived from Escherichia coli E. coli BL21 (DE3).

The method for introducing the recombinant plasmid of the present invention into a host cell is a heat shock transformation method.

The pRE112-mediated homologous recombination method of the present invention is one of the commonly used techniques for gene replacement in the art, and can be operated according to standard operation methods.

The recombinant plasmids of 3-hydroxypropionic acid in the examples are pA-accADBC and pMCR-N-C-N940V/K1106W/S1114R. For the information and construction method of the recombinant plasmids, please refer to Metabolic Engineering 34 (2016) 104-111.

The definitions and abbreviations involved in the present invention are contrasted as follows:

Lactate dehydrogenase: LdhA.

3-Hydroxypropionic acid: 3HP.

Escherichia coli: E. coli.

In the present invention, "heat shock transformation" or "heat transformation" refers to a kind of transfection technology in molecular biology, which is used to integrate foreign genes into host genes and express them stably. The introduction of foreign genes into host genes or the introduction of foreign plasmids into host protoplasts, and heat shock transformation or thermal transformation, etc.

In the present invention, "overexpression" means that a specific gene is highly expressed in an organism, and the expression level exceeds the normal level (ie, the wild-type expression level), which can be achieved by enhancing endogenous expression or introducing exogenous genes.

The present invention employs techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art can use other conventional techniques, methods and reagents in the art on the basis of the embodiments provided by the present invention, and are not limited to the limitations of the specific embodiments of the present invention.

The technical content of the present invention will be further elaborated below in conjunction with specific embodiments.

Example 1: Construction and enzymatic activity assay of acetylation-regulated lactate dehydrogenase LdhA mutants.

Construct the lactate dehydrogenase LdhA mutant K9R in which the 9th lysine of the lactate dehydrogenase LdhA protein is mutated to arginine; LdhA double mutant K154Q-K248Q of aminoamide, and the enzyme activity of the mutant was determined.

Specific steps are as follows:

1) Amplification of LdhA mutants

Using the genomic DNA of Escherichia coli as a template, the upstream fragment of K9R mutant lactate dehydrogenase gene ldhA was cloned with primers pETDuet1-ldhA-5'(CCG GGATCC GATGAAACTCGCCGTTTATAGC) and pETDuet1-ldhA(K9R)-3'(AGGTACTTCTTGTCGTACTGACGTGTGCTATAAACGGCGAGTT). Using primers pETDuet1-ldhA-3'(CCGGAGCTCTTAAACCAG TTCGTTCGGGC) and pETDuet1-ldhA(K9R)-5'(AACTCGCCGTTTATAGCACACG TCAGTA CGACAAG AAGTACCT), the downstream fragment of K9R mutated lactate dehydrogenase gene ldhA was obtained by PCR cloning, using fragment bridging method By bridging the upstream and downstream fragments of the K9R-mutated lactate dehydrogenase gene ldhA, the K9R-mutated lactate dehydrogenase gene ldhA is obtained, and its nucleotide sequence is shown in SEQ ID NO.3;

Using the genomic DNA of Escherichia coli as a template, using primers pETDuet1-ldhA-5'(CCG GGATCC GATGAAACTCGCCGTTTATAGC) and ldhA(K154Q)-3'(CGCAGCATCGCCACACCGATCTGACCGGTACCGATAACGCCTG), the upstream fragment of K154Q mutant lactate dehydrogenase gene ldhA was obtained by PCR cloning , using primers pETDuet1-ldhA-3'(CCGGAGCTCTTAAACCAG TTCGTTCGGGC) and ldhA(K154Q)-5'(CAGGCGTTATCGGTACCGGTCAGATCGGTGTGGCGATGCTGCG), the downstream fragment of K154Q mutant lactate dehydrogenase gene ldhA was obtained by PCR cloning, and the K154Q mutant was bridged by the fragment bridge method. The upstream and downstream fragments of the lactate dehydrogenase gene ldhA, to obtain the K154Q mutant lactate dehydrogenase gene ldhA;

K154Q-mutated lactate dehydrogenase gene ldhA was used as a template and primers pETDuet1-ldhA-5' (CCG GGATCC GATGAAACTCGCCGTTTATAGC) and ldhA(K248Q)-3' (AACGAACCAATTTTCTGATTCTGCAGCGCTTCAATTGCTGCCT) were cloned to obtain K154Q-K248Q-mutated lactate dehydrogenase by PCR. The upstream fragment of the enzyme gene ldhA was cloned by PCR with primers pETDuet1-ldhA-3'(CCGGAGCTCTTAAAACCAGTTCGTTCGGGC) and ldhA(K248Q)-5'(AACGAACC AATTTTCTGAT TCTGC AGCGC TTCAA TTGCTGCCT) to obtain K154Q-K248Q mutant lactate dehydrogenase gene ldhA The downstream fragment, utilizes the fragment bridging method to bridge the upstream and downstream fragments of the lactate dehydrogenase gene ldhA of K154Q-K248Q mutation, and obtains the lactate dehydrogenase gene ldhA of K154Q-K248Q double mutation, and its nucleotide sequence is such as SEQ ID NO. 4 shown;

The above PCR amplification system is 50 μl, which contains: 25 μL 2×PrimeSTARMAX, 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 23 μL deionized water, and E. coli colony is added as a template.

The above PCR amplification procedure is: 95°C for 3 min (95°C for 30s, 58°C for 30s, 72°C for 1 min, 30 cycles) and 72°C for 3 min.

2) Construction of recombinant plasmid

The K9R mutated lactate dehydrogenase gene ldhA and the K154Q-K248Q double mutated lactate dehydrogenase gene ldhA obtained in step 1) and the expression vector pETDuet1 were digested with BamHI and SacI, respectively, and the digestion system was 50 μL, including: 20 μL gene or vector, 1 μL BamHI, 1 μL SacI, 5 μL 10*buffer, 23 μL deionized water. After recovery of the digested product, T4 ligase was used to ligate the digested mutant ldhA gene fragment and the vector fragment, respectively, to obtain recombinant plasmids, which were named as: pETDuet1-ldhA(K9R) and pETDuet1-ldhA(K154Q-K248Q).

3) Conversion

Prepare competent cells of E.coli BL21 (DE3) according to the operation instructions of the competent preparation kit, add the recombinant plasmids prepared in step 2) to the prepared competent cells respectively, and take an ice bath for 30 minutes; Heat shock in a water bath at 42°C for 90s; take out after heat shock, quickly place on ice for 10min; take out the centrifuge tube after ice bath, add 500 μL of liquid LB medium, and activate at 37°C for about 1h; activate The resulting cells were centrifuged at 4000g for 2 min at 4°C, the supernatant was discarded, the cells were resuspended with 100 μL of liquid LB, and coated on solid LB plates containing 100 mg/L ampicillin, and the coated plates were incubated at 37°C at a constant temperature. Incubate overnight to grow a single clone, correspondingly obtain the expression strain of lactate dehydrogenase mutant K9R, named E. coli BL21(DE3)/pETDuet1-ldhA(K9R) and K154Q-248Q double mutant expression strain, named E.coli BL21(DE3)/pETDuet1-ldhA(K154Q-248Q).

4) Enzyme activity assay

The strains obtained in step 3) were respectively inoculated into Erlenmeyer flasks containing 50 mL of LB medium. When the OD600 was about 0.8 at 180 rpm at 37 °C, 0.1 mM IPTG was added to induce expression, and the cells were collected by centrifugation at 30 °C and 180 rpm for 18 h. , the crude protein product was obtained by high pressure crushing, protein purification was carried out by nickel affinity chromatography column, and the K9R mutant and K154Q-K248Q mutant of lactate dehydrogenase were obtained correspondingly, and the enzyme activity was determined.

Enzyme activity assay reaction system 200 μL contains: 0.2M phosphate buffer (A solution: dissolve 71.64g Na ₂ HPO ₄ .12H ₂ O in 1L pure water; B solution: dissolve 31.21g NaH ₂ PO ₄ .2H ₂ O Dissolve in 1L pure water: mix 84ml A solution and 16ml B solution evenly, pH7.5, that is, 0.2M phosphate buffer), 20mM NADH, 20mM sodium pyruvate, 40nM purified protein sample, using a microplate reader The absorbance value at 340 nm wavelength was measured.

The enzyme activity assay results are shown in Figure 1. The enzyme activity of the K9R mutant was 2.5 times higher than that of the wild-type LdhA, and the enzyme activity of the K154Q-K248Q mutant decreased to 12.6% of the wild-type.

The application of the expression strain of embodiment 2.K9R mutant in improving lactic acid production and transformation efficiency

This example will compare the differences in cell growth, lactate accumulation and substrate conversion rate during lactic acid fermentation between the wild-type lactate dehydrogenase expressing strain and its K9R mutant expressing strain. In this embodiment, two sets of experiments are carried out to illustrate the effects that can be obtained by the present invention.

Control group: E.coli BL21(DE3)/pETDuet1-ldhA

Experimental group: E.coli BL21(DE3)/pETDuet1-ldhA(K9R)

Specific steps are as follows:

1) With reference to the steps of Example 1, using the genomic DNA of Escherichia coli as a template, the wild-type ldhA gene was obtained by PCR cloning with primers pETDuet1-ldhA-5' (CCG GGATCC GATGAAACTCGCCGTTTATAGC) and pETDuet1-ldhA-3' (CCGGAGCTCTTAA ACCAGTTCGTTCGGGC) , and construct it into the pETDuet1 expression plasmid to obtain the recombinant plasmid pETDuet1-ldhA, transform the recombinant plasmid into the competent cells of E.coli BL21(DE3), and obtain the expression strain E.coli BL21(DE3) of wild-type lactate dehydrogenase /pETDuet1-ldhA (control group).

2) The wild-type lactate dehydrogenase expression strain (control group) and the K9R mutant expression strain (experimental group) were respectively inoculated into test tubes containing 3 mL of LB medium, and cultured at 37°C overnight.

2) Inoculate the strains after overnight culture into 250mL shake flasks containing 100mL lactic acid fermentation medium (containing 100mg/L ampicillin) at a volume ratio of 1:50, and cultivate to about OD600 at 37°C and 180rpm. When it was 0.8, 0.1 mM IPTG was added to induce protein expression, and then it was transferred to 30 °C and 100 rpm for lactic acid fermentation for 24 h.

3) After the fermentation, use a spectrophotometer to measure the OD600 absorbance value of the cells respectively. At the same time, 1 mL of fermentation broth was taken, centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was taken, filtered with a 0.22 μm filter membrane, and the lactic acid concentration was detected by HPLC.

The test results are shown in Figure 2. The cell biomass accumulation of the K9R mutant expressing strain is basically the same as that of the control group, and the lactic acid production and transformation efficiency of the K9R mutant are about 1.63 times higher than that of the control strain.

Example 3. Application of K154Q-K248Q double mutant strain in inhibiting the synthesis of lactic acid

This example will compare the wild-type E.coli BL21(DE3) strain, the lactate dehydrogenase gene ldhA knockout strain (E.coli BL21(DE3)ΔldhA) and the K154Q-K248Q double mutant strain of ldhA on the genome (E.coli Differences in cell growth and lactate synthesis of BL21(DE3)ldhA::ldhA(K154Q-K248Q)). In this embodiment, three groups of experiments are carried out to illustrate the effects that the present invention can obtain.

Control 1: E.coli BL21(DE3)

Control 2: E. coli BL21(DE3)ΔldhA

Experimental group: E.coli BL21(DE3)ldhA::ldhA(K154Q-K248Q)

Specific steps are as follows:

1) Construction of a knockout strain of ldhA

According to the standard experimental procedure of P1 phage transduction method, the ldhA gene of E.coli BL21(DE3) was knocked out to obtain the ldhA knockout strain E.coli BL21(DE3)ΔldhA (control group 2).

2) Construction of K154Q-K248Q double mutant strain

Referring to the PCR amplification system and procedure of Example 1, the genomic DNA of Escherichia coli was used as a template, and the primers pRE112-ldhA(K154Q-K248Q)-5'(CAACATCACTGGAGAAAGTCTTATGAAACTCGCCGTTTATAGC) and ldhA(K154Q)-3'(CGCAGCATCGCCACACCGATCTGACCGGTACCGATAACGCCTG) were cloned and obtained The upstream fragment of K154Q-mutated lactate dehydrogenase gene ldhA was cloned with primers pRE112-ldhA(K154Q-K248Q)-3'(GGTTGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGC) and ldhA(K154Q)-5'(CAGGCGTTATCGGTACCGGTCAGATCGGTGTGGCGATGCTGCG) to obtain K154Q-mutated lactate dehydrogenase gene The downstream fragment of ldhA was used to bridge the upstream and downstream fragments of the K154Q-mutated lactate dehydrogenase gene ldhA to obtain the K154Q-mutated lactate dehydrogenase gene ldhA;

K154Q-mutated lactate dehydrogenase gene ldhA was used as a template and primers pRE112-ldhA(K154Q-K248Q)-5'(CAACATCACTGGAGAAAGTCTTATGAAACTCGCCGTTTATAGC) and ldhA(K248Q)-3'(AACGAACCAATTTTCTGATTCTGCAGCGCTTCAATTGCTGCCT) were cloned to obtain K154Q-K248Q-mutated lactate dehydrogenase The upstream fragment of the enzyme gene ldhA was cloned with primers pRE112-ldhA(K154Q-K248Q)-3'(GGTTGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGC) and ldhA(K248Q)-5'(AACGAACCAATTTTCTGATTCTGCAGCGCTTCAATTGCTGCCT) to obtain the downstream fragment of the K154Q-K248Q mutant lactate dehydrogenase gene ldhA , the upstream and downstream fragments of the K154Q-K248Q mutated lactate dehydrogenase gene ldhA were bridged by the fragment bridging method to obtain a K154Q-K248Q double-mutated lactate dehydrogenase gene ldhA, the nucleotide sequence of which is shown in SEQ ID NO.4 ;

Using the genomic DNA of Escherichia coli as a template, the upstream homologous fragment Up sequence of the ldhA gene was cloned with primers pRE112-up-5' (CCG TCTAGA CAGTTGCTGGATATCAGAGG) and pRE112-up-3' (GCTATAAACGGCGAGTTTCATAAGACTTTCTCCAGTGATGTTG), followed by primers pRE112-dwon- 5'(GCCCGAACGAACTGGTTTAATCTTGCCGCTCCCCTGCAACC) and pRE112-dwon-3'(CCGGAGCTCGTCGATGTCCAGTAGTGGAG) were cloned to obtain the Down sequence of the downstream homologous fragment of the ldhA gene, and the Up-ldhA(K154Q-248Q)-Down fragment was obtained by using the fragment bridging method. As shown in SEQ ID NO.5.

The Up-ldhA(K154Q-248Q)-Down fragment obtained in step 3) and the suicide plasmid pRE112 were double digested with XbaI and SacI, and then ligated with T4 ligase to form a homology arm-containing recombinant plasmid pRE112-Up-ldhA(K154Q- 248Q)-Down, this recombinant plasmid is transformed into recipient cell E.colix7213 (method reference embodiment 1 step 3) using heat shock transformation method, E.coliχ7213 is a DAP auxotrophic strain, and every milliliter of culture medium needs to add during culturing 50 μg/mL of DAP. The E.coliχ7213 strain that has been transformed into the above recombinant plasmid and the ldhA knockout strain E.coli BL21(DE3)ΔldhA undergo conjugation and two homologous recombinations, and the lactate dehydrogenase on the genome is mediated by the suicide plasmid pRE112. The gene ldhA was replaced in situ with the K154Q-K248Q double mutant ldhA gene to obtain the K154Q-K248Q double mutant strain (E. coli BL21(DE3)ldhA::ldhA(K154Q-K248Q) (experimental group).

3) The ldhA knockout strain constructed in step 1) (control group 2), the ldhA (K154Q-248Q) double mutant strain constructed in step 2) (experimental group) and the wild-type E. coli BL21 (DE3) strain (control group) 1) Inoculate into test tubes of 3 mL LB medium respectively, and cultivate overnight at 37°C. The strains after overnight culture were inoculated into 250 mL shake flasks containing 50 mL of 3HP fermentation medium at a volume ratio of 1:50, and cultured at 37°C and 180 rpm for 48 hours. During the fermentation process, multiple samples were taken to detect cell growth and lactic acid accumulation.

The test results are shown in Figure 3. Both the ldhA knockout strain and the ldhA(K154Q-248Q) double mutant strain can control the lactate level below 0.5g/L, while the lactate content of the wild strain is 1.04g/L-3.23 between g/L. Although the ldhA knockout strain and the ldhA(K154Q-248Q) double mutant strain had better initial cell growth rate than the control strain, the ldhA knockout strain entered a stable phase after 24 h, and the cell biomass accumulation remained unchanged, while the wild strain and ldhA The cellular biomass accumulation of the (K154Q-248Q) double mutant strain continued to increase and achieved similar levels. This indicated that the ldhA(K154Q-248Q) double mutant reached the lactate inhibitory ability similar to the ldhA knockout strain, but was significantly better than the ldhA knockout strain in terms of cell growth and biomass accumulation.

Example 4. Application of K154Q-K248Q double mutant strain in 3-hydroxypropionic acid biosynthesis

In this example, the wild-type E. coli BL21 (DE3) strain, the lactate dehydrogenase gene ldhA knockout strain and the K154Q-K248Q double mutant strain will be compared in the fermentation process of the biobased chemical 3-hydroxypropionic acid, cell growth, Differences between lactic acid synthesis and 3-hydroxypropionic acid synthesis. In this embodiment, three groups of experiments are carried out to illustrate the effects that the present invention can obtain.

The specific experimental methods are as follows:

1) Competent cells of the control group 1, the control group 2 and the experimental group in Example 3 were prepared by using the competent index kit operation instructions.

2) The synthetic plasmids pA-accADBC and pMCR-N-C-N940V/K1106W/S1114R of 3-hydroxypropionic acid were introduced into the three competent cells prepared in step 1) by the heat shock transformation method in step 3) of reference example 1, respectively. , to obtain recombinant strains for synthesizing 3-hydroxypropionic acid, respectively

Control 1: E.coli BL21(DE3)/pA-accADBC/pMCR-N-C-N940V/K1106W/S1114R

Control 2: E.coli BL21(DE3)ΔldhA/pA-accADBC/pMCR-N-C-N940V/K1106W/S1114R

Experimental group: E.coli BL21(DE3)ldhA::ldhA(K154Q-K248Q)/pA-accADBC/

pMCR-N-C-N940V/K1106W/S1114R

For the information and construction methods of the above-mentioned 3-hydroxypropionic acid recombinant plasmids pA-accADBC and pMCR-N-C-N940V/K1106W/S1114R, please refer to Metabolic Engineering 34 (2016) 104-111.

3) The recombinant strains of 3-hydroxypropionic acid obtained in step 2), namely the control group 1, the control group 2 and the experimental group strains described in this example, were respectively inoculated into a test tube of 3 mL LB medium, and cultured at 37°C overnight. . The strains after overnight culture were inoculated into 250mL shake flasks containing 50mL 3HP fermentation medium at a volume ratio of 1:50, cultured at 37°C at 180rpm to an OD600 of about 0.8, added 0.1M IPTG to induce protein expression, and transferred to 30°C Fermentation was continued at 180rpm for 48h.

4) After the fermentation, the OD600 absorbance values of the cells were measured using a spectrophotometer. At the same time, 1 mL of fermentation broth was taken, centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was taken, filtered with a 0.22 μm filter membrane, and the concentrations of 3-hydroxypropionic acid and lactic acid were detected by HPLC.

The fermentation results of 3-hydroxypropionic acid are shown in Figure 4. The accumulation of lactic acid in the experimental group and control group 2 is basically the same, indicating that the ldhA double mutant strain regulated by acetylation modification can inhibit lactic acid synthesis similar to the ldhA gene knockout. Effect. However, the production of 3-hydroxypropionic acid in the experimental group reached 2.05 g/L, which was higher than the 1.57 g/L of the control group 1 and the 1.79 g/L of the control group 2. It is suggested that the K154Q-K248Q double mutant strain has broad application prospects in the biosynthesis of 3-hydroxypropionic acid.

Those skilled in the art should understand that each of the above steps is performed according to standard molecular cloning techniques.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, , the protection scope of the present invention should be defined by the claims.

sequence list

<110> Shandong University

<120> An acetylation-regulated lactate dehydrogenase mutant and its application

<141> 2022-05-05

<160> 5

<210> 1

<211> 272

<212> PRT

<213> Artificial sequences

<221> Amino acid sequence of an acetylation-regulated lactate dehydrogenase K9R mutant

<222> (1)…(272)

<400> 1

MKLAVYSTRQ YDKKYLQQVN ESFGFELEFF DFLLTEKTAK TANGCEAVCI FVNDDGSRPV 60

LEELKKHGVK YIALRCAGFN NVDLDAAKEL GLKVVRVPAY DPEAVAEHAI GMMMTLNRRI 120

HRAYQRTRDA NFSLEGLTGF TMYGKTAGVI GTGKIGVAML RILKGFGMRL LAFDPYPSAA 180

ALELGVEYVD LPTLFSESDV ISLHCPLTPE NYHLLNEAAF EQMKNGVMIV NTSRGALIDS 240

QAAIEALKNQ KIGSLGMDVY ENERDLFFED KS 272

<210> 2

<211> 272

<212> PRT

<213> Artificial sequences

<221> Amino acid sequence of acetylation-regulated lactate dehydrogenase K154Q-K248Q double mutant

<222> (1)…(272)

<400> 2

MKLAVYSTKQ YDKKYLQQVN ESFGFELEFF DFLLTEKTAK TANGCEAVCI FVNDDGSRPV 60

LEELKKHGVK YIALRCAGFN NVDLDAAKEL GLKVVRVPAY DPEAVAEHAI GMMMTLNRRI 120

HRAYQRTRDA NFSLEGLTGF TMYGKTAGVI GTGRIGVAML RILKGFGMRL LAFDPYPSAA 180

ALELGVEYVD LPTLFSESDV ISLHCPLTPE NYHLLNEAAF EQMKNGVMIV NTSRGALIDS 240

QAAIEALRNQ KIGSLGMDVY ENERDLFFED KS 272

<210> 3

<211> 990

<212> DNA

<213> Artificial sequences

<221> K9R mutant lactate dehydrogenase gene ldhA nucleotide sequence

<222>(1)…(990)

<400> 3

atgaaactcg ccgtttatag cacacgtcag tacgacaaga agtacctgca acaggtgaac 60

gagtcctttg gctttgagct ggaatttttt gactttctgc tgacggaaaa aaccgctaaa 120

actgccaatg gctgcgaagc ggtatgtatt ttcgtaaacg atgacggcag ccgcccggtg 180

ctggaagagc tgaaaaagca cggcgttaaa tatatcgccc tgcgctgtgc cggtttcaat 240

aacgtcgacc ttgacgcggc aaaagaactg gggctgaaag tagtccgtgt tccagcctat 300

gatccagagg ccgttgctga acacgccatc ggtatgatga tgacgctgaa ccgccgtatt 360

caccgcgcgt atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt 420

actatgtatg gcaaaacggc aggcgttatc ggtaccggta aaatcggtgt ggcgatgctg 480

cgcattctga aaggttttgg tatgcgtctg ctggcgttcg atccgtatcc aagtgcagcg 540

gcgctggaac tcggtgtgga gtatgtcgat ctgccaaccc tgttctctga atcagacgtt 600

atctctctgc actgcccgct gacaccggaa aactatcatc tgttgaacga agccgccttc 660

gaacagatga aaaatggcgt gatgatcgtc aataccagtc gcggtgcatt gattgattct 720

caggcagcaa ttgaagcgct gaaaaatcag aaaattggtt cgttgggtat ggacgtgtat 780

gagaacgaac gcgatctatt ctttgaagat aaatccaacg acgtgatcca ggatgacgta 840

ttccgtcgcc tgtctgcctg ccacaacgtg ctgtttaccg ggcaccaggc attcctgaca 900

gcagaagctc tgaccagtat ttctcagact acgctgcaaa acttaagcaa tctggaaaaa 960

ggcgaaacct gcccgaacga actggtttaa 990

<210> 4

<211> 990

<212> DNA

<213> Artificial sequences

<221> K154Q-K248Q double mutant lactate dehydrogenase gene ldhA nucleotide sequence

<222>(1)…(990)

<400> 4

atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca acaggtgaac 60

gagtcctttg gctttgagct ggaatttttt gactttctgc tgacggaaaa aaccgctaaa 120

actgccaatg gctgcgaagc ggtatgtatt ttcgtaaacg atgacggcag ccgcccggtg 180

ctggaagagc tgaaaaagca cggcgttaaa tatatcgccc tgcgctgtgc cggtttcaat 240

aacgtcgacc ttgacgcggc aaaagaactg gggctgaaag tagtccgtgt tccagcctat 300

gatccagagg ccgttgctga acacgccatc ggtatgatga tgacgctgaa ccgccgtatt 360

caccgcgcgt atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt 420

actatgtatg gcaaaacggc aggcgttatc ggtaccggtc atatcggtgt ggcgatgctg 480

cgcattctga aaggttttgg tatgcgtctg ctggcgttcg atccgtatcc aagtgcagcg 540

gcgctggaac tcggtgtgga gtatgtcgat ctgccaaccc tgttctctga atcagacgtt 600

atctctctgc actgcccgct gacaccggaa aactatcatc tgttgaacga agccgccttc 660

gaacagatga aaaatggcgt gatgatcgtc aataccagtc gcggtgcatt gattgattct 720

caggcagcaa ttgaagcgct gcataatcag aaaattggtt cgttgggtat ggacgtgtat 780

gagaacgaac gcgatctatt ctttgaagat aaatccaacg acgtgatcca ggatgacgta 840

ttccgtcgcc tgtctgcctg ccacaacgtg ctgtttaccg ggcaccaggc attcctgaca 900

gcagaagctc tgaccagtat ttctcagact acgctgcaaa acttaagcaa tctggaaaaa 960

ggcgaaacct gcccgaacga actggtttaa 990

<210> 5

<211> 2351

<212> DNA

<213> Artificial sequences

<221> Nucleotide sequence of Up-ldhA(K154Q-248Q)-Down

<222>(1)…(2351)

<400> 5

cagttgctgg atatcagagg ttaatgcgag agagagtttt ccctgccatt cctgccaggg 60

agaaaaaatc agtttatcga tattgatcca ggtgttaggc agcatggact gccactgcgc 120

gagggttttt ggagcagctg gcgattgctc cgtctgcggc aatttcgcca gacaagcaga 180

atcaagttct accatgccga cgttcaataa ccagcggctg ggatgtgaaa ggctggcgtt 240

ggtgatatgc gcaagctgac aatctcccac cagataacgg agatcgggaa tgattaaacc 300

tttacgcgta atgcgtgggc tttcatctaa tgcaatacgt gtcccgagcg gtagccagat 360

gcccgccagc gtgggaaccc acagcccgag cgtcatcagc agcgtcaacg gcacaagaat 420

aatcagtaat aacagcgcga gaacggcttt atatttaccc agcatgggta gttaatatcc 480

tgatttagcg aaaaattaag cattcaatac gggtattgtg gcatgtttaa ccgttcagtt 540

gaaggttgcg cctacactaa gcatagttgt tgatgaattt ttcaatatcg ccatagcttt 600

caattatatt tgaaattttg taaaatattt ttagtagctt aaatgtgatt caacatcact 660

ggagaaagtc ttatgaaact cgccgtttat agcacaaaac agtacgacaa gaagtacctg 720

caacaggtga acgagtcctt tggctttgag ctggaatttt ttgactttct gctgacggaa 780

aaaaccgcta aaactgccaa tggctgcgaa gcggtatgta ttttcgtaaa cgatgacggc 840

agccgcccgg tgctggaaga gctgaaaaag cacggcgtta aatatatcgc cctgcgctgt 900

gccggtttca ataacgtcga ccttgacgcg gcaaaagaac tggggctgaa agtagtccgt 960

gttccagcct atgatccaga ggccgttgct gaacacgcca tcggtatgat gatgacgctg 1020

aaccgccgta ttcaccgcgc gtatcagcgt acccgtgatg ctaacttctc tctggaaggt 1080

ctgaccggct ttactatgta tggcaaaacg gcaggcgtta tcggtaccgg tcatatcggt 1140

gtggcgatgc tgcgcattct gaaaggtttt ggtatgcgtc tgctggcgtt cgatccgtat 1200

ccaagtgcag cggcgctgga actcggtgtg gagtatgtcg atctgccaac cctgttctct 1260

gaatcagacg ttatctctct gcactgcccg ctgacaccgg aaaactatca tctgttgaac 1320

gaagccgcct tcgaacagat gaaaaatggc gtgatgatcg tcaataccag tcgcggtgca 1380

ttgattgatt ctcaggcagc aattgaagcg ctgcataatc agaaaattgg ttcgttgggt 1440

atggacgtgt atgagaacga acgcgatcta ttctttgaag ataaatccaa cgacgtgatc 1500

caggatgacg tattccgtcg cctgtctgcc tgccacaacg tgctgtttac cgggcaccag 1560

gcattcctga cagcagaagc tctgaccagt atttctcaga ctacgctgca aaacttaagc 1620

aatctggaaa aaggcgaaac ctgcccgaac gaactggttt aatcttgccg ctcccctgca 1680

acccagggga gctgattcag ataatcccca atgacctttc attctctatt cttaaaatag 1740

tcctgagtca gaaactgtaa ttgagaacca caatgaagaa agtagccgcg tttgttgcgc 1800

taagcctgct gatggcggga tgtgtaagta atgacaaaat tgctgttacg ccagaacagc 1860

tacagcatca tcgctttgtg ctggaaagcg taaacggtaa gcccgtgacc agcgataaaa 1920

atccgccaga aatcagcttt ggtgaaaaaa tgatgatttc cggcagcatg tgtaaccgct 1980

ttagcggtga aggcaaactg tctaatggtg aactgacagc caaagggctg gcaatgaccc 2040

gtatgatgtg cgctaacccg cagcttaatg aactcgataa caccattagc gaaatgctga 2100

aagaaggtgc acaagtggat ctgaccgcga accagttaac gctggcgacc gcgaaacaga 2160

cattaactta taagctggcg gatttaatga attaatagct gccacagctc ccggcggcaa 2220

gtgactgttc gctacagcgt ttgccgttgg gtaatgcaca catcccaatc gccgtaccat 2280

ccagttgacg ggcaacagaa agcgaaccgc cgatcattgc acaatttgct tctccactac 2340

tggacatcga c 2351

Claims (10)

1. An acetylation-regulated lactate dehydrogenase mutant, comprising: the mutant is formed by mutating lysine at the 9 th site of lactate dehydrogenase LdhA protein into arginine, is named as K9R mutant, and has an amino acid sequence shown as SEQ ID No. 1.

2. Use of the acetylation-regulated lactate dehydrogenase mutant according to claim 1 for improving the synthesis of lactic acid.

3. An expression strain of the acetylation regulated lactate dehydrogenase K9R mutant according to claim 1, wherein: the strain carries the lactate dehydrogenase gene ldhA with the K9R mutation shown in SEQ ID NO. 3.

4. Expression strain of the acetylation regulated lactate dehydrogenase K9R mutant according to claim 3, wherein: the expression strain was designated as E.coli BL21(DE3)/pETDuet1-ldhA (K9R).

5. Use of the K9R mutant expression strain of claim 3 or 4 for increasing the lactic acid production and transformation efficiency.

6. An acetylation-regulated lactate dehydrogenase double mutant, which is characterized in that: the double mutants are formed by mutating the lysine at 154 th site and 248 th site of the lactate dehydrogenase LdhA protein into glutamine, are named as K154Q-K248Q double mutants, and have the amino acid sequence shown as SEQ ID NO. 2.

7. Use of the acetylation-regulated lactate dehydrogenase double mutant according to claim 6 for inhibiting the synthesis of lactic acid.

8. An acetylation regulated lactate dehydrogenase K154Q-K248Q double mutant strain of claim 6, wherein: the genomic lactate dehydrogenase gene ldhA in the strain was mutated and replaced in situ with the K154Q-K248Q double-mutated lactate dehydrogenase gene ldhA as shown in SEQ ID NO.4, and the strain was named E.coli BL21(DE3) ldhA:: ldhA (K154Q-K248Q).

9. Use of the K154Q-K248Q double mutant strain of claim 8 for inhibiting the synthesis of lactic acid.

10. Use of the double mutant strain K154Q-K248Q of claim 8 in the biosynthesis of 3-hydroxypropionic acid.

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