Construction method and application of engineering bacteria for improving carbon flow supply in L-malic acid production

Abstract

The invention discloses a construction method and application of engineering bacteria for improving carbon flow supply in L-malic acid production, and belongs to the technical field of biological engineering. A construction method of the engineering bacteria comprises the following steps: amplifying malate dehydrogenase genes mdh1, mdh2 and mdh3 from a genome of saccharomyces cerevisiae, constructing double-gene combined expression recombinant plasmids, and respectively introducing the recombinant plasmids into host bacteria to obtain the engineering bacteria mdh1, 2, mdh1, 3 and mdh2, 3. The yield of accumulated L-malic acid is increased by 13-16 times compared with the yield of the L-malic acid accumulated by the engineering bacteria containing double-gene combined expression, which is constructed by the method disclosed by the invention, compared with the yield of the L-malic acid accumulated by an original strain. According to the engineering bacterium construction method for improving carbon flow supply in L-malic acid production, the capacity of converting oxaloacetic acid into malic acid is improved, carbon flow supply is increased, then the yield of L-malic acid is increased, and good industrial application value and prospects are achieved.

Description

Technical Field

[0001] The present invention relates to a method for constructing an engineering bacterium for improving carbon flow supply in L-malic acid production and its application, belonging to the technical field of bioengineering. Background Art

[0002] L-malic acid, as an important four-carbon dicarboxylic acid, is widely used in the food, industrial, agricultural and pharmaceutical industries. Improving strains for microbial fermentation through random mutagenesis or metabolic engineering is a more sustainable method for producing malic acid. The most competitive pathway is the reduced tricarboxylic acid (rTCA) pathway in the cytoplasm, which has a maximum theoretical yield of 200% glucose. In this pathway, pyruvate is carboxylated to oxaloacetate and further reduced to malic acid. Malate dehydrogenase is an enzyme that promotes the conversion of oxaloacetate to malic acid. By introducing malate dehydrogenase, the metabolic flux can be concentrated on malic acid production. Therefore, the malate dehydrogenase encoding gene and its alleles are key modification sites for producing malic acid using the rTCA pathway, and malic acid production can be effectively increased by combined overexpression methods.

[0003] Currently, bacteria, yeast, and filamentous fungi can all be used as platforms for the development of L-malic acid production via microbial fermentation. Saccharomyces cerevisiae was modified by introducing the pyruvate carboxylase-encoding gene pyc, alleles of the malate dehydrogenase-encoding gene mdh3, and the fission yeast malate transporter gene SpMAE1, either individually or in combination. Results showed that the combined introduction of these three genes significantly increased malic acid production in S. cerevisiae, demonstrating the effectiveness of combined gene introduction. Integrating pyc, mdh, and fum, either individually or in combination, into Pichia pastoris GS115 focused metabolic flux and increased malic acid production. Escherichia coli, which normally cannot accumulate malic acid but produces high levels of acetic acid, lactic acid, ethanol, and formic acid during glucose fermentation, generated a potential L-malic acid-producing strain by inactivating the fumB, frdABCD, ldhA, and pflB genes in E. coli. Overexpressing enzymes involved in the production pathway in E. coli, while reducing target product consumption, can further enhance malic acid production, leading to the construction of a high-yield malic acid-producing strain. Based on Aspergillus oryzae NRRL 3488, overexpressing the native cytoplasmic alleles of pyruvate carboxylase and malate dehydrogenase that constitute the rTCA pathway, and overexpressing the C4-dicarboxylic acid transporter, can increase malic acid production by 27%. Therefore, by overexpressing rate-limiting enzymes and knocking out genes related to byproduct generation and target product consumption pathways, carrying out work related to the modification of the L-malic acid anabolic pathway is crucial for enhancing the carbon flow supply in L-malic acid production and can effectively achieve an increase in L-malic acid production. In addition, the main differences between the relevant reference documents and the invention patent of this application are listed, as shown in Table 1.

[0004] Table 1 Main differences between the relevant comparative documents and the invention patent of this application

[0005]

[0006] The present invention uses Escherichia coli K-12::maeB::fumB::frdBC as a starting strain. By expressing the mitochondrial malate dehydrogenase gene mdh1, the cytoplasmic malate dehydrogenase gene mdh2, and the peroxisome malate dehydrogenase gene mdh3 in the E. coli K-12::maeB::fumB::frdBC using a dual-gene combination, an engineered bacterium is constructed that can improve the carbon flux supply in L-malic acid production. This addresses the issue of insufficient carbon flux in the malate conversion pathway from oxaloacetate in the rTCA pathway. This method provides a method for constructing an engineered bacterium that effectively increases the carbon flux during the conversion of oxaloacetate to L-malic acid in the L-malic acid production pathway, thereby increasing the yield of L-malic acid produced by microbial fermentation. Summary of the Invention

[0007] The present invention provides a method for constructing an engineered bacterium for improving carbon flow supply in L-malic acid production and its application. The carbon flow supply is increased by overexpressing malate dehydrogenase, thereby improving the L-malic acid yield.

[0008] The first purpose of the present invention is to provide a method for constructing an engineered bacterium that improves the carbon flow supply in L-malic acid production, and to construct the engineered bacteria mdh1,2, mdh1,3, and mdh2,3; the second purpose is to provide the use of the engineered bacteria in the fermentation production of L-malic acid.

[0009] Furthermore, the engineered bacteria mdh1, 2 are constructed by introducing a double gene combination of the mitochondrial malate dehydrogenase gene mdh1 and the cytoplasmic malate dehydrogenase gene mdh2 into Escherichia coli K-12::maeB::fumB::frdBC; the engineered bacteria mdh1, 3 are constructed by introducing a double gene combination of the mitochondrial malate dehydrogenase gene mdh1 and the peroxisomal malate dehydrogenase gene mdh3 into Escherichia coli K-12::maeB::fumB::frdBC; the engineered bacteria mdh2, 3 are constructed by introducing a double gene combination of the cytoplasmic malate dehydrogenase gene mdh2 and the peroxisomal malate dehydrogenase gene mdh3 into Escherichia coli K-12::maeB::fumB::frdBC.

[0010] Furthermore, the mitochondrial malate dehydrogenase gene mdh1, the cytoplasmic malate dehydrogenase gene mdh2 and the peroxisomal malate dehydrogenase gene mdh3 are derived from Saccharomyces cerevisiae S288C.

[0011] Furthermore, the nucleotide sequence of the mitochondrial malate dehydrogenase gene mdh1 is shown as SEQ ID NO.1; the nucleotide sequence of the cytoplasmic malate dehydrogenase gene mdh2 is shown as SEQ ID NO.2; and the nucleotide sequence of the peroxisomal malate dehydrogenase gene mdh3 is shown as SEQ ID NO.3.

[0012] Furthermore, the two genes in the double gene combination transformation also include a T7 promoter and an SD fragment with a nucleotide sequence as shown in SEQ ID NO. 4.

[0013] Furthermore, the PCR amplification system involved in the genes is 25-50 μL: ① 2×Rapid Taq MasterMix: 12.5-25 μL; ② Genomic template (10-100 ng / μL): 0.5-1 μL; ③ Upstream primer F (10 μmol / L): 1-2 μL; ④ Downstream primer R (10 μmol / L): 1-2 μL; ⑤ Add Sterilized ddH2O to make up to 25-50 μL.

[0014] Furthermore, the PCR amplification conditions are: ①95℃: 3min; ②95℃: 15s; ③annealing temperature (58-69℃): 15s; ④72℃: 15s / kb; ⑤72℃: 5min, (②-④ are cycled 30-35 times).

[0015] Furthermore, the engineered bacteria mdh1,2, mdh1,3, and mdh2,3 use pET-28a as an expression vector.

[0016] Furthermore, fermenting the recombinant strain to produce L-malic acid includes the following steps: inoculating the L-malic acid-producing engineered bacteria screened from the solid culture medium into the seed culture medium, culturing for 12-18 hours to obtain a seed culture solution; then inoculating the seed culture solution into the fermentation medium in proportion, culturing to the logarithmic growth phase, and performing the second stage fermentation culture.

[0017] Furthermore, the solid culture medium formula is: NaCl 9-12 g / L, tryptone 7-13 g / L, yeast extract 4-9 g / L, agar 15-25 g / L.

[0018] Furthermore, the seed culture medium formula is: NaCl 8-10g / L, tryptone 6-13g / L, yeast extract 4-8g / L.

[0019] Furthermore, the fermentation medium formula is: glucose 15-30g / L, NaHCO3 30-50g / L, Na2HPO4·12H2O 10-20g / L, KH2PO4 2-4g / L, NH4Cl 1-2g / L, NaCl 1-3g / L, MgSO4·7H2O 0.5-2g / L.

[0020] Furthermore, the aerobic culture time of the seed culture solution is 12-18 hours; the aerobic culture temperature of the seed culture solution is 30-42° C.; and the aerobic culture oscillation speed of the seed culture solution is 110-200 r / min.

[0021] Furthermore, the pH value of the fermentation medium is adjusted to 5-8 using sodium carbonate solution or sodium bicarbonate solution.

[0022] Furthermore, the second stage culture time in the fermentation medium is 12-24 hours; the second stage culture temperature in the fermentation medium is 30-42° C.; and the second stage culture shaking speed in the fermentation medium is 110-180 rpm.

[0023] Furthermore, the second stage culture in the fermentation medium includes but is not limited to microaerobic, non-strict anaerobic and anaerobic conditions.

[0024] Furthermore, metabolite analysis was performed using high-pressure liquid chromatography, the method of which was as follows: the obtained sample was placed in a high-speed centrifuge for centrifugation, and then the supernatant was filtered using a filter membrane with a pore size of 0.45 μm, and a UV detector was used; before testing the sample, the L-malic acid standard sample was first subjected to gradient testing and a standard curve was drawn, and then the sample was tested.

[0025] The present invention has the following beneficial effects:

[0026] (1) The present invention overexpresses the mitochondrial malate dehydrogenase gene mdh1, the cytoplasmic malate dehydrogenase gene mdh2, and the peroxisomal malate dehydrogenase gene mdh3 in Escherichia coli K-12::maeB::fumB::frdBC, respectively. The screened engineered bacteria can effectively increase the carbon flux from oxaloacetate to L-malic acid, and the L-malic acid production is significantly improved.

[0027] (2) The L-malic acid production of the genetically engineered bacteria mdh1,2, mdh1,3 and mdh2,3 constructed by the present invention was increased by 13-16 times compared with the starting bacteria, indicating that the strains constructed by the present invention can effectively improve the supply of carbon flow, and the fermentation process is simple and has low equipment requirements. They can be widely used in increasing the production and production of L-malic acid, and are in line with the concepts of green biomanufacturing and sustainable development.

[0028] Description of the drawings

[0029] Figure 1 The following are the construction diagrams of expression plasmids: (a) pET-28a-mdh1-mdh2 plasmid construction diagram, (b) pET-28a-mdh1-mdh3 plasmid construction diagram, (c) pET-28a-mdh2-mdh3 plasmid construction diagram;

[0030] Figure 2 Agarose gel electrophoresis of the constructed recombinant plasmids, including: (a) pET-28a-mdh1-mdh2 agarose gel electrophoresis, (b) pET-28a-mdh1-mdh3, pET-28a-mdh2-mdh3 agarose gel electrophoresis;

[0031] Figure 3 This is a bar graph of L-malic acid production by the original Escherichia coli K-12::maeB::fumB::frdBC strain and different engineered bacteria after 12 hours of fermentation. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with specific embodiments so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art may modify or change the technical solution of the present invention, and such modifications and changes are also within the scope of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents and materials used are all commercially available unless otherwise specified.

[0034] Example 1: Construction of genetically engineered bacteria mdh1,2

[0035] (1) The sequence of mitochondrial malate dehydrogenase mdh1 is shown in SEQ ID NO.1, and the sequence of cytoplasmic malate dehydrogenase mdh2 is shown in SEQ ID NO.2. A multi-fragment homologous recombination primer pair was designed and synthesized. The upstream primer rmdh1-F for mdh1 homologous recombination is shown in SEQ ID NO.5, and the downstream primer 2-rmdh1-R is shown in SEQ ID NO.6; the upstream primer 1-rmdh2-F for mdh2 homologous recombination is shown in SEQ ID NO.7, and the downstream primer 1-rmdh2-R is shown in SEQ ID NO.8.

[0036] (2) The pET-28a plasmid was digested with BamH I to obtain a linearized vector;

[0037] (3) Using the mdh1 gene as a template, PCR amplification was performed using the synthesized primers rmdh1-F and 2-rmdh1-R to obtain a 2-mdh1 DNA fragment with a linearized vector end homologous sequence and another recombinant fragment homologous sequence. The PCR amplification system was as follows: ①2×Rapid Taq Master Mix: 12.5-25 μL; ②Genomic template (10-100 ng / μL): 0.5-1 μL; ③Upstream primer F (10 μmol / L): 1-2 μL; ④Downstream primer R (10 μmol / L): 1-2 μL; ⑤Add Sterilized ddH2O to make up to 25-50 μL. PCR amplification conditions: ① 95°C: 3 min; ② 95°C: 15 s; ③ annealing temperature (59-65°C): 15 s; ④ 72°C: 15 s / kb; ⑤ 72°C: 5 min, (②-④ cycles 30-35 times); using the mdh2 gene obtained in Example 2 as a template, PCR amplification was performed using the synthesized primers 1-rmdh2-F and 1-rmdh2-R to obtain a 1-mdh2 DNA fragment with a linearized vector terminal homologous sequence and another recombinant fragment homologous sequence. The PCR amplification system and PCR amplification conditions were the same as above;

[0038] (4) The linearized vector and the 2-mdh1 and 1-mdh2 inserts were mixed in proportion, and the recombination reaction was completed at 45-50°C for 15-30 minutes under the catalysis of Exnase to obtain the recombinant plasmid pET-28a-mdh1-mdh2;

[0039] (5) The obtained recombinant plasmid pET-28a-mdh1-mdh2 was transformed into Escherichia coli DH5α competent cells, and the correct positive transformants were screened by Kan resistance plates and sequenced for verification;

[0040] (6) The correctly sequenced recombinant plasmid pET-28a-mdh1-mdh2 was transformed into the competent Escherichia coli K-12::maeB::fumB::frdBC by chemical transformation, and positive single clones were screened on a Kan-resistant plate and verified by PCR to obtain the corresponding engineered strain.

[0041] Primer sequences (SEQ ID NO. 5-8)

[0042] rmdh1-F:5'-GTGGACAGCAAATGGGTCGCGGATCCATGTTGTCAAGAGTAGCTAAAC-3'

[0043] 2-rmdh1-R:5'-TCCTATAGTGAGTCGTATTACTATTTACTAGCAACAAAGTTGACAC-3'

[0044] 1-rmdh2-F:5'-TAATACGACTCACTATAGGAAGAAGGAGATATACCATGCCTCACTCAGTTACACC-3'

[0045] 1-rmdh2-R:5'-TGTCGACGGAGCTCGAATTCGGATCCTTAAGATGATGCAGATCTCG-3' Example 2: Construction of genetically engineered bacteria mdh1,3

[0046] (1) The sequence of peroxisomal malate dehydrogenase mdh3 is shown in SEQ ID NO. 3. A primer pair capable of amplifying a kmdh3 gene fragment containing a T7 promoter and an SD sequence was designed and synthesized. The sequence of the upstream primer kmdh3-F is shown in SEQ ID NO. 9, and the sequence of the downstream primer kmdh3-R is shown in SEQ ID NO. 10.

[0047] (2) Using the mdh3 gene as a template, PCR amplification was performed using the synthesized primers kmdh3-F and kmdh3-R to obtain the kmdh3 gene fragment containing the T7 promoter and SD sequence. The PCR amplification system was as follows: ①2×Rapid Taq Master Mix: 12.5-25 μL; ②Genomic template (10-100 ng / μL): 0.5-1 μL; ③Upstream primer F (10 μmol / L): 1-2 μL; ④Downstream primer R (10 μmol / L): 1-2 μL; ⑤Add Sterilized ddH2O to make up to 25-50 μL. PCR amplification conditions: ①95℃: 3 min; ②95℃: 15 s; ③Annealing temperature (58-64℃): 15 s; ④72℃: 15 s / kb; ⑤72℃: 5 min, (②-④ cycles 30-35 times);

[0048] (3) The sequence of mitochondrial malate dehydrogenase mdh1 is shown in SEQ ID NO.1, and the sequence of the kmdh3 gene fragment containing the T7 promoter and the SD sequence is shown in SEQ ID NO.11. A multi-fragment homologous recombination primer pair was designed and synthesized. The upstream primer rmdh1-F sequence of mdh1 homologous recombination is shown in SEQ ID NO.12, and the downstream primer 3-rmdh1-R sequence is shown in SEQ ID NO.13; the upstream primer 1-rmdh3-F sequence of kmdh3 homologous recombination is shown in SEQ ID NO.14, and the downstream primer rmdh3-R sequence is shown in SEQ ID NO.15;

[0049] (4) The pET-28a plasmid was digested with BamH I to obtain a linearized vector;

[0050] (5) Using the mdh1 gene obtained in Example 1 as a template, PCR amplification was performed using the synthesized primers rmdh1-F and 3-rmdh1-R to obtain a 3-mdh1 DNA fragment with a linearized vector terminal homologous sequence and another recombinant fragment homologous sequence. The PCR amplification system and PCR amplification conditions were the same as above. Using the kmdh3 gene obtained as a template, PCR amplification was performed using the synthesized primers 1-rmdh3-F and rmdh3-R to obtain a 1-mdh3 DNA fragment with a linearized vector terminal homologous sequence and another recombinant fragment homologous sequence. The PCR amplification system and PCR amplification conditions were the same as above.

[0051] (7) The linearized vector and the 3-mdh1 and 1-mdh3 inserts were mixed in proportion, and the recombination reaction was completed at 45-50°C for 15-30 minutes under the catalysis of Exnase to obtain the recombinant plasmid pET-28a-mdh1-mdh3;

[0052] (8) The obtained recombinant plasmid pET-28a-mdh1-mdh3 was transformed into Escherichia coli DH5α competent cells, and the correct positive transformants were screened by Kan resistance plates and sequenced for verification;

[0053] (9) The correctly sequenced recombinant plasmid pET-28a-mdh1-mdh3 was transformed into the competent Escherichia coli K-12::maeB::fumB::frdBC by chemical transformation, and positive single clones were screened on a Kan-resistant plate and verified by PCR to obtain the corresponding engineered strain.

[0054] Primer sequences (SEQ ID NO.9-10)

[0055] kmdh3-F:5'-TAATACGACTCACTATAGGAAGAAGGAGATATACCATGGTCAAAGTCGCAATTCTTGGCG-3'

[0056] kmdh3-R:5'-TCATAGCTTGGAAGAGTCTAGGATGAAAACTC-3'

[0057] Primer sequences (SEQ ID NO. 12-15)

[0058] rmdh1-F:5'-GTGGACAGCAAATGGGTCGCGGATCCATGTTGTCAAGAGTAGCTAAAC-3'

[0059] 3-rmdh1-R:5'-AGTCGTATTACTATTTACTAGCAACAAAGTTGACACCCTT-3'

[0060] 1-rmdh3-F:5'-TAGTAAATAGTAATACGACTCACTATAGGAAGAAGGAGATATACCAT GGT-3'

[0061] rmdh3-R:5'-TGTCGACGGAGCTCGAATTCGGATCCTCATAGCTTGGAAGAGTCTAGG ATGAA-3'

[0062] Example 3: Construction of genetically engineered bacteria mdh2,3

[0063] (1) The sequence of cytoplasmic malate dehydrogenase mdh2 is shown in SEQ ID NO.2, and the sequence of the kmdh3 gene fragment containing the T7 promoter and SD sequence is shown in SEQ ID NO.11. A multi-fragment homologous recombination primer pair was designed and synthesized. The upstream primer 3-rmdh2-F for mdh2 homologous recombination is shown in SEQ ID NO.16, and the downstream primer 3-rmdh2-R is shown in SEQ ID NO.17; the upstream primer 2-rmdh3-F for kmdh3 homologous recombination is shown in SEQ ID NO.18, and the downstream primer rmdh3-R is shown in SEQ ID NO.15;

[0064] (2) The pET-28a plasmid was digested with BamH I to obtain a linearized vector;

[0065] (3) Using the mdh2 gene obtained in Example 2 as a template, PCR amplification was performed using the synthesized primers 3-rmdh2-F and 3-rmdh2-R to obtain a 3-mdh2 DNA fragment with a linearized vector terminal homologous sequence and another recombinant fragment homologous sequence. The PCR amplification system was as follows: ① 2× Rapid Taq Master Mix: 12.5-25 μL; ② Genomic template (10-100 ng / μL): 0.5-1 μL; ③ Upstream primer F (10 μmol / L): 1-2 μL; ④ Downstream primer R (10 μmol / L): 1-2 μL; ⑤ Add Sterilized ddH2O to make up to 25-50 μL. PCR amplification conditions: ① 95°C: 3 min; ② 95°C: 15 s; ③ annealing temperature (63-69°C): 15 s; ④ 72°C: 15 s / kb; ⑤ 72°C: 5 min (②-④ cycles 30-35 times); PCR amplification was performed using the obtained kmdh3 gene as a template with the synthesized primers 2-rmdh3-F and rmdh3-R to obtain a 2-mdh3 DNA fragment containing homologous sequences at the ends of the linearized vector and homologous sequences of another recombinant fragment. The PCR amplification system and PCR amplification conditions were the same as above;

[0066] (4) The linearized vector and the 3-mdh2 and 2-mdh3 inserts were mixed in proportion, and the recombination reaction was completed at 45-50°C for 15-30 minutes under the catalysis of Exnase to obtain the recombinant plasmid pET-28a-mdh2-mdh3;

[0067] (5) The obtained recombinant plasmid pET-28a-mdh2-mdh3 was transformed into Escherichia coli DH5α competent cells, and the correct positive transformants were screened by Kan resistance plates and sequenced for verification;

[0068] (6) The correctly sequenced recombinant plasmid pET-28a-mdh2-mdh3 was transformed into the competent Escherichia coli K-12::maeB::fumB::frdBC by chemical transformation, and positive single clones were screened on a Kan-resistant plate and verified by PCR to obtain the corresponding engineered strain.

[0069] Primer sequences (SEQ ID NO. 16-19)

[0070] rmdh3-R:5'-TGTCGACGGAGCTCGAATTCGGATCCTCATAGCTTGGAAGAGTCTAGG ATGAA-3'

[0071] 3-rmdh2-F:5'-GTGGACAGCAAATGGGTCGCGGATCCATGCCTCACTCAGTTACACCA TCC-3'

[0072] 3-rmdh2-R:5'-AGTCGTATTATTAAGATGATGCAGATCTCGATGCAACGAA-3'

[0073] 2-rmdh3-F:5'-ATCATCTTAATAATACGACTCACTATAGGAAGAAGGAGATATACCAT GG-3'

[0074] Example 4: Fermentation of L-malic acid by genetically engineered bacteria mdh1,2, mdh1,3 and mdh2,3

[0075] Solid culture medium: 9-12 g / L NaCl, 7-13 g / L tryptone, 4-9 g / L yeast extract, 15-25 g / L agar. Seed culture medium: 8-10 g / L NaCl, 6-13 g / L tryptone, 4-8 g / L yeast extract. Fermentation medium: 15-30 g / L glucose, 30-50 g / L NaHCO₃, 10-20 g / L Na₂HPO₄·12H₂O, 2-4 g / L KH₂PO₄, 1-2 g / L NH₄Cl, 1-3 g / L NaCl, 0.5-2 g / L MgSO₄·7H₂O. A fresh single colony from a solid culture medium is inoculated into a container containing 15-30% seed culture medium, with mdh1,2, mdh1,3, and mdh2,3 as the constructed genetically engineered bacteria and BBBC as a control bacteria, and cultured with shaking at 30-42°C and 130-180 rpm for 12-18 hours. The seed culture solution is transferred to a container containing 30-55% fermentation medium at an inoculum volume of 5-10% (v / v), cultured to the end of the logarithmic growth phase, and then switched to non-strict anaerobic fermentation. No gas is introduced during the fermentation process, and the container is sealed. The pH is adjusted to 6-8 using sodium bicarbonate solution, and cultured with shaking at 30-42°C and 130-180 rpm for 12-24 hours to obtain a fermentation broth.

[0076] Take the fermentation broth and centrifuge it at 5000-10000r / min for 5-10min, collect the supernatant, and prepare the standard of L-malic acid into standard solutions of different concentrations according to the proportion. After filtering the fermentation broth and the standard solution through a 0.45μm microporous filter membrane, the content of L-malic acid is determined by high pressure liquid chromatography. Aminex HPX-87H (300×7.8mm) chromatographic separation column and UV detector are used, the mobile phase is 20mmol / L potassium dihydrogen phosphate (pH value is adjusted to 2.5-3.5), the column temperature is 25℃, the detection wavelength is 210nm, the injection volume is 10μL, and the flow rate is 0.5mL / min. The production of L-malic acid in the fermentation broth after 12 hours of fermentation is detected, and the results are as follows: Figure 3 As shown in the results, the engineered strain significantly increased the production of L-malic acid.

Claims

1. A method for constructing an engineered bacterium to improve carbon flow supply in L-malic acid production, characterized in that: Using pET-28a as the expression vector in Escherichia coli K-12::maeB::fumB::frdBC, a dual-gene combination expression method was used to express the mitochondrial malate dehydrogenase gene. mdh1 , cytoplasmic malate dehydrogenase gene mdh2 and peroxisomal malate dehydrogenase gene mdh3 , thereby constructing three engineered bacteria mdh1,2, mdh1,3, and mdh2,3.

2. The method for constructing an engineered bacterium for improving carbon flow supply in L-malic acid production according to claim 1, characterized in that: Three engineered bacteria mdh1,2, mdh1,3, and mdh2,3, among which the engineered bacteria mdh1,2 combined to express mdh1 Genes and mdh2 Gene, engineered bacteria mdh1,3 combination expressed mdh1 Genes and mdh3 Gene, engineered bacteria mdh2,3 combination expressed mdh2 Genes and mdh3 Gene.

3. The method for constructing an engineered bacterium for improving carbon flow supply in L-malic acid production according to claim 1, characterized in that: The mdh1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. mdh2 The nucleotide sequence of the gene is shown in SEQ ID NO.

2. mdh3 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

4. The method for constructing an engineered bacterium for improving carbon flow supply in L-malic acid production according to claim 1, characterized in that: The double gene combination expression recombinant plasmid further includes a T7 promoter and an SD sequence between the two genes as shown in the nucleotide sequence of SEQ ID NO.

4.

5. The method for constructing an engineered bacterium for improving carbon flow supply in L-malic acid production according to claim 1, characterized in that: The T7 promoter and SD sequence connecting the two genes were amplified from plasmid pET-28a.

6. Use of the method for constructing an engineered bacterium for improving carbon flow supply in L-malic acid production according to any one of claims 1 to 5 in producing L-malic acid.

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