A recombinant Escherichia coli strain E. coli Lac for producing L-lactic acid, a construction method thereof, and an application thereof

Abstract

The present invention relates to a recombinant Escherichia coli strain E. coli Lac for producing L-lactic acid, a construction method thereof and an application thereof, belonging to the field of bioengineering. The construction method of the recombinant Escherichia coli strain E. coli Lac: The method uses the Escherichia coli genetic recombinant strain E. coli XZ132 as the starting strain to obtain the BcldhL gene, the nucleotide sequence of the BcldhL gene is as shown in SEQ ID NO.1, and the λ homologous recombination method is used to replace the ldhA gene in the genome of the starting strain with the BcldhL gene to obtain the strain E. coli pldhA::BcldhL; then the BcldhL gene is used to replace the ppsA gene in the starting strain gene to obtain the strain E. coli pldhA::BcldhL-pppsA::BcldhL, the BcldhL gene is inserted into the plasmid pTrc99a to obtain the plasmid pTrc99a-BcldL; the chemical competent state of the strain E. coli pldhA::BcldhL-pppsA::BcldhL is prepared and the plasmid pTrc99a-BcldhL is transformed to obtain the strain E. coli pldhA::BcldhL-pppsA::BcldhL-pTrc::BcldhL, which is renamed E. coli Lac. The present invention also provides an application of using the strain to ferment and prepare L-lactic acid. By overexpressing the L-lactic acid dehydrogenase gene with high enzyme activity, the present invention greatly improves the lactic acid yield.

Description

Technical Field

[0001] The present invention belongs to the field of microbial engineering, and particularly relates to a recombinant Escherichia coli strain E. coli Lac for producing L-lactic acid, a construction method thereof and an application thereof. Background Art

[0002] Lactic acid (Lac; CH3CHCOOH) is one of the three major organic acids. Due to its optical isomerism, it exists in three forms in nature: D-Lac, L-Lac, and DL-Lac. In recent years, various L-lactic acid-derived products have been put on the market and are used in the medical industry, such as surgical sutures, drug controlled-release preparations, internal fixation materials for fractures, etc. L-lactic acid has also made important contributions in the field of environmental protection and can be used to produce green solvents such as methyl L-lactate and ethyl L-lactate, and biodegradable plastics such as polylactic acid (PLA). With the increasingly serious environmental problems, the demand for degradable plastics is growing at a compound annual growth rate of 33%. It is of great significance to improve the production capacity of L-lactic acid and reduce the production cost.

[0003] Production strategies of L-lactic acid include chemical synthesis, enzymatic conversion, and microbial fermentation. In the chemical synthesis method, acetaldehyde and hydrocyanic acid are used as substrates to synthesize L-lactic acid. However, this production pipeline is polluting, costly, and it is difficult to synthesize L-lactic acid with a single configuration. Meanwhile, there are many inevitable by-products and residual harmful intermediates, which will pose serious safety risks if consumed by humans. Enzymatic production of L-lactic acid uses pyruvate (Pyr) or 2-chloropropionic acid as substrates, catalyzed by highly specific lactate dehydrogenase (LDH) or l-2-halogenase. In this way, optically pure L-lactic acid can be obtained. However, due to the complex conditions of enzymatic conversion, there are disadvantages such as low yield and high cost, so it is rarely used industrially. Compared with chemical synthesis and enzymatic synthesis, microbial fermentation for producing L-lactic acid not only meets the needs of large-scale industrial production but also reduces the production cost due to the cheap biomass resources. In addition, the production of L-lactic acid with high optical purity can be achieved using engineered microorganisms. Therefore, microbial fermentation has become the main route for L-lactic acid production. In recent years, many microbial fermentation methods for producing L-lactic acid have emerged. Many microbial cell factories have been developed, and progress has been made in the efficient synthesis of L-lactic acid by fermentation. However, there are still many problems in the microbial production of L-lactic acid, such as the imbalance of metabolic flux between cell growth and L-lactic acid synthesis, the high production cost during fermentation, and the adverse effects of low pH caused by the accumulation of L-lactic acid during fermentation. To improve the synthesis efficiency of L-lactic acid, it is crucial to establish a global L-lactic acid metabolic regulation strategy through genome-scale metabolic models to balance the metabolic flux among cell growth, cofactor supply, and L-lactic acid synthesis. Secondly, the accumulation of L-lactic acid will reduce the pH value of the fermentation medium, further inhibiting the growth of bacteria. However, adding neutralizing agents will increase the fermentation cost and introduce salt ion impurities. High concentrations of ions will consume cell energy and directly inhibit cell activity. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a recombinant Escherichia coli strain for producing L-lactic acid and its application. In the present invention, the Escherichia coli strain E. coli XZ132, which is a genetically recombinant strain derived from the chassis bacterium E. coli W (ATCC 9637), is used as the starting strain. The L-lactate dehydrogenase (FDP-activated L-lactate dehydrogenase) gene of Bacillus coagulans, hereinafter abbreviated as gene BcldhL, is used to replace the endogenous lactate dehydrogenase A (D-lactate dehydrogenase, ldhA) of Escherichia coli. The promoter of the endogenous lactate dehydrogenase A of Escherichia coli is used to overexpress the L-lactate dehydrogenase gene BcldhL of Bacillus coagulans. The phosphoenolpyruvate synthase (ppsA) gene is knocked out and its promoter is used to express BcldhL, and the plasmid ptrc99A-BcldhL that overexpresses L-lactate dehydrogenase BcldhL under the Trc promoter is introduced to construct an Escherichia coli strain capable of efficiently fermenting and producing L-lactic acid, named E. coli Lac.

[0005] The present invention is achieved by the following technical solutions:

[0006] A recombinant Escherichia coli strain E. coli Lac for producing L-lactic acid, wherein the recombinant Escherichia coli strain E. coli Lac is E. coli pldhA::BcldhL-pppsA::BcldhL-pTrc::BcldhL. The E. coli pldhA::BcldhL-pppsA::BcldhL-pTrc::BcldhL uses the genetically recombinant Escherichia coli strain E. coli XZ132 as the starting strain, and replaces the lactate dehydrogenase A (D-lactate dehydrogenase, ldhA) gene in the starting strain with the L-lactate dehydrogenase gene shown in SEQ ID NO.1. The L-lactate dehydrogenase gene is abbreviated as the BcldhL gene. At the same time, the BcldhL gene replaces the phosphoenolpyruvate synthase (ppsA) in the starting strain, and a plasmid ptrc99A-BcldhL containing the Trc promoter for overexpressing L-lactate dehydrogenase is introduced into the starting strain.

[0007] The present invention also provides a method for constructing the recombinant Escherichia coli strain E. coli Lac. The method is to obtain the gene of L-lactate dehydrogenase (BcldhL), and the nucleotide sequence of the BcldhL gene is shown in SEQ ID NO.1. The λ homologous recombination method is used to replace the ldhA gene in the genome of the starting strain with the BcldhL gene to obtain the strain E. coli pldhA::BcldhL; then the ppsA gene in the starting strain gene is replaced with the BcldhL gene to obtain the strain E. coli pldhA::BcldhL-pppsA::BcldhL. The gene BcldhL is inserted into the commercial plasmid pTrc99a (purchased from Addgene) to obtain the plasmid pTrc99a-BcldL; the chemically competent cells of the strain E. coli pldhA::BcldhL-pppsA::BcldhL are prepared and transformed with the plasmid pTrc99a-BcldhL to obtain the strain E. coli pldhA::BcldhL-pppsA::BcldhL-pTrc::BcldhL, which is renamed E. coli Lac.

[0008] The nucleotide sequence of the above-mentioned knocked-out ppsA gene is shown in SEQ ID NO.2.

[0009] As a preferred embodiment, the obtained recombinant Escherichia coli strain E. coli Lac is subjected to acid acclimation and high-salt acclimation, and strains resistant to acid and high-salt osmotic stress are screened, and finally the high-yield L-lactic acid strain E. coli Lac is obtained.

[0010] The present invention also relates to the application of the above-mentioned recombinant Escherichia coli strain E. coli Lac in the preparation of L-lactic acid by microbial fermentation. The application is as follows: inoculate the recombinant Escherichia coli strain E. coli Lac into the fermentation medium, culture at 30 °C and 180 rpm until the cell growth reaches OD600 = 0.5, then add IPTG with a final concentration of 0.2 mM to induce gene expression, and continue to culture for 48 h until the fermentation ends to obtain a fermentation broth containing L-lactic acid. The fermentation broth is separated and purified to obtain L-lactic acid.

[0011] The formula of the fermentation medium is as follows: the mass percentage of glucose is 10-12%, ammonium sulfate is 100 mM, AM1 medium (the components are shown in Table 1) is 100 ml / 500 ML, the rotation speed is 2000 rpm within 0-11 h, 80 rpm after 11 h, seal the mouth, anaerobic fermentation, and dilute ammonia water is used to adjust the pH = 7.0.

[0012] Seed culture medium: 3% glucose by mass percentage, 100 mM ammonium sulfate, 100 ml of NBS medium (composition shown in Table 1), 37 °C, rotation speed 180 rpm, pH 7.0, OD of the fermentation broth without dilution 600 nm value is 1.313, inoculation amount 10%;

[0013] Table 1 a Composition and concentration of NBS + 1 mM betaine and AM1

[0014]

[0015] a NBS + 1 mM betaine.

[0016] b Calculation includes KOH for neutralizing the stock solution of betaine HCl.

[0017] c Stock solution of trace metals (1000x) is prepared with 120 mM hydrochloric acid.

[0018] Before fermentation of the recombinant genetically engineered bacterium, first inoculate the genetically engineered bacterium into LB medium, culture overnight at 37 °C and 180 rpm to prepare a seed solution, and inoculate the seed solution into the fermentation medium at an inoculation amount of 5% by volume concentration.

[0019] The fermentation is carried out in a fermenter: inoculate the recombinant genetically engineered bacterium onto an LB plate containing 50 mg / L ampicillin resistance, culture overnight at 37 °C, pick a single colony into an LB test tube containing 50 mg / L ampicillin resistance, culture overnight at 37 °C and 150 rpm to prepare a seed solution; inoculate the seed solution into LB medium at a volume concentration of 10% and culture overnight at 37 °C and 150 rpm as the secondary seed solution; inoculate the secondary seed solution into a 5-L fermenter containing 2 L of fermentation medium at a volume concentration of 15%, and add IPTG with a final concentration of 0.2 mM, and ferment and culture at 30 °C, 500 rpm, and an aeration rate of 0.5 V / V·min. When the pH value is higher than 6.80 (when the initial sugar in the fermenter is consumed), automatic feeding is started, and the feeding medium is added until the pH is lower than 6.80 and then the feeding stops. Culture for 90 h to obtain a fermentation broth containing L-lactic acid. The feeding medium: 500 g / L glucose, 16 g / L (NH4)2SO4, 4 g / L yeast extract, 14 g / L KH2PO4, 10 g / L NaHCO3, 0.4 mg / L VB1, 0.2 mg / L VB 12 , the solvent is water, and the pH is adjusted to 6.8 with 50% ammonia water.

[0020] The feeding rate is 25 mL / h, and the total addition amount of the feeding medium is 800 mL / 2 L.

[0021] Advantages of the present invention compared with the prior art:

[0022] The technical solution of the present invention can provide a new method for improving the production of L-lactic acid. Through bioinformatics comparison and experimental verification, a highly active L-lactic acid dehydrogenase was found in Bacillus coagulans, which can efficiently oxidize pyruvate to lactic acid. The pathway from glucose to pyruvate was strengthened, the side branches were weakened by gene knockout to reduce unnecessary consumption, and the gene of highly active L-lactic acid dehydrogenase was overexpressed to obtain a strain with significantly increased lactic acid production and conversion rate without affecting cell growth. The engineered bacterium uses glucose as a substrate, and under the given fermentation conditions, the L-lactic acid concentration reaches 177 g / L, the production intensity reaches 25 g / L / h, and the pyruvate yield reaches 0.98 g / g. The Escherichia coli engineered strain constructed by the present invention has high lactic acid production, low cost, and simple method, and has high practical value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Plasmid map of pKD4-BcldhL for gene replacement;

[0024] Figure 2 Plasmid map of pTrc99a-BcldhL for overexpressing BcldhL gene with promoter Trc;

[0025] Figure 3 Bar graph of biomass (by OD 600nm value) and fermentation product L-lactic acid concentration of the starting strains E. coli XZ132 and E. coli Lac;

[0026] Figure 4 Curve graph of biomass (OD 600nm ) and fermentation product L-lactic acid concentration of strain E. coli Lac in fed-batch fermentation in a 6 L fermenter. DETAILED DESCRIPTION OF THE EMBODIMENTS

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

[0028] In the embodiments, the final concentrations of kanamycin in the liquid medium and the solid medium are both 50 mg / L, the final concentration of ampicillin is 50 mg / L, the final concentration of chloramphenicol is 25 mg / L, and the final concentration of IPTG is 0.2 mM.

[0029] In the following embodiments, the materials, reagents, plasmids, special reagent kits, strains, etc. used are all obtained from commercial channels unless otherwise specified.

[0030] Brief description of the strains involved in the embodiments of the present invention and their sources:

[0031] The Bacillus coagulans involved in the present invention was purchased from the American Type Culture Collection (ATCC) standard strain preservation center, and the strain number is 7050 TM .

[0032] The original strain selected in the present invention is Escherichia coli E.coli W (ATCC 9637). On this basis, the G.stearothermophilus alaD gene was used to replace the lactate dehydrogenase D-lactate dehydrogenase ldhA. The acetate kinase ackA acetate kinase, aldehyde dehydrogenase adhE alcohol / aldehyde dehydrogenase, fumarate reductase frd fumarate reductase, methylglyoxal synthase gene mgsA methylglyoxalsynthase and pyruvate formatelyase pflB pyruvate formatelyase were knocked out to increase the yield, and the catabolic alanine racemase gene (dadX) was knocked out to reduce the conversion of L-alanine to D-alanine. Finally, a new engineered strain, named E.coli XZ132, was constructed, which can efficiently produce L-alanine. For the specific construction method, please refer to the paper "Production of L-alanine by metabolically engineered Escherichia coli" by Xueli Zhang&KaemwichJantama&J.C.Moore&K.T.Shanmugam&L.O.Ingram

[0033] Example 1: The recombinant strain overexpresses BcldhL lactate dehydrogenase (the amino acid sequence of which is shown in SEQ ID NO.13) and replaces the gene ldhA

[0034] (1) Preparation of chemically competent E.coli XZ132

[0035] The E.coli XZ132 cells in the culture medium need to be collected into a centrifuge tube, placed on ice for a period of time, and then centrifuged at 4000 rpm for 10 min to remove the supernatant. The cells are gently suspended with pre-cooled CaCl2 solution, placed on ice for a period of time, and then centrifuged again. After discarding the supernatant, pre-cooled CaCl2 solution containing glycerol is added, the cells are gently suspended, and placed on ice for a few minutes to form a suspension of competent cells.

[0036] (2) Transformation of plasmid pKD46

[0037] Take competent E. coli XZ132, add 1 μl of commercial plasmid pKD46 (purchased from Addgene), mix well and place on ice for 30 min. Heat shock at 42 °C for 20 - 30 s, cool on ice for 2 min, add 1 ml of LB medium, resuscitate at 28 °C for 1 - 2 h, spread on a solid Amp (50) plate, invert for 16 h to obtain E. coli XZ132-pKD46. Take multiple clones of E. coli XZ132-pKD46 and shake culture overnight for 16 h at 28 °C in liquid LB containing 50 μg / ml ampicillin.

[0038] (3) Preparation of electrocompetent E. coli XZ132-pKD46

[0039] Take 500 μl of the small shake culture and add it to 50 ml of liquid LB medium, then add ampicillin (abbreviated as Amp) at a concentration of 50 μg / ml, culture at 220 r at 28 °C for 110 - 120 min, OD600 ≈ 0.2. Add arabinose with a final concentration of 100 mM (0.75 g of L-arabinose dissolved in 5 ml of double-distilled water, filter sterilized and then added). Continue to culture at 28 °C for 45 min, OD600 ≈ 0.45 (any value between 0.4 - 0.5 is acceptable). Pour the bacterial solution into a 50 ml centrifuge tube, place on ice for 30 min, centrifuge at 4000 rpm at 4 °C for 10 min, discard the supernatant, add half the volume of 10% glycerol to resuspend, centrifuge for 10 min again, repeat 2 times to completely wash away impurities such as ions in the bacteria. Finally, resuspend the pellet with 500 μl of 10% glycerol and it can be used after placing on ice for 30 min.

[0040] (4) Obtaining the DNA fragment for gene replacement

[0041] Using the Bacillus coagulans genome as a template, the BcldhL gene fragment with the upstream homologous arm of ldhA was obtained using primers LdhA-BcldhL F and BcldhL R. Using the commercial plasmid pKD4 purchased from Addgene as a template, the kana gene fragment with the downstream homologous arm of the ldhA gene was amplified using primers BcldhLkana F and LdhA-kana R. Purify the DNA fragments, use the two fragments together as a template, and perform overlap PCR using primers LdhA-BcldhL F and LdhA-kana R to obtain the DNA fragment for gene replacement.

[0042] LdhA-BcldhL F:

[0043] ttagtagcttaaatgtgattcaacatcactggagaaagtcttatgaaaaaagtcaatcgtattgcag(SEQ ID NO.3);

[0044] LdhA - BcldhL - R:

[0045] cgaagcagctccagcctacacaatcgcttacagaaccggtgccattgtctc(SEQ ID NO.4).

[0046] BcldhL - kana F: cgaagcagctccagcctacacaatcgc ttacaatacagg tgccatcgtt(SEQID NO.5);

[0047] LdhA - kana R:

[0048] gggattatctgaatcagctcccctggaatgcaggggagcggcaagatgggaattagccatggtccatatgaatatc(SEQ ID NO.6).

[0049] Take 100 μl of E. coli XZ132 - pKD46 competent cells, add 2.5 μl of the DNA fragment for gene replacement (too much DNA also affects the transformation efficiency. After adjustment, only 2.5 μl of the DNA fragment for gene replacement is used, with a final concentration of about 1 ng), mix well, and gently add it to the pre - cooled electroporation cuvette. The capacitance is 5 μF, the voltage is 1800 V, and the resistance is 100 Ω. After taking it out, add 1 ml of LB and resuscitate at 28 °C with a rotation speed of 150 rpm for 1 - 3 h. The longer the time, the better the effect. After resuscitation, spread it on a solid medium containing the antibiotic kanamycin (abbreviated as kana), perform PCR amplification verification on single colonies, and sequence the selected positive strains by a sequencing company to obtain the correct strains. Incubate at a high temperature to lose the temperature - sensitive plasmid pKD46, and obtain the strain with kana resistance where the DNA for gene replacement replaces the original gene BcldhL, named E. coli XZ132 - BcldhL - kana.

[0050] (5) Prepare electrocompetent E. coli XZ132-ldhA::BcldhL-kana according to the method for preparing electrocompetent cells, transform the commercial plasmid pCP20 (purchased from Addgene), and screen on chloramphenicol plates. Verify the positive strains by PCR to screen the strains with the kana gene successfully removed, and verify the chromosomal gene sequence by sequencing. Culture at high temperature to lose the temperature-sensitive plasmid pCP20, and obtain the strain with the BcldhL gene replaced, named E. coli pldhA::BcldhL.

[0051] Example 2 Replacement of phosphoenolpyruvate synthase (ppsA) gene with BcldhL lactate dehydrogenase in recombinant strains

[0052] Prepare chemically competent E. coli pldhA::BcldhL according to the method of Example 1 (1)-(3) and transform the plasmid pKD46, then prepare electrocompetent E. coli pldhA::BcldhL-pKD46.

[0053] Using the genome of Bacillus coagulans as a template, obtain the BcldhL gene fragment with NdeI restriction sites at both ends using primers BcldhLNdeI F and BcldhLNdeI R. Treat the DNA fragments of pKD46 and BcldhL with NdeI enzyme, and ligate and transform E. coli competent cells overnight at 4°C with T4 ligase to obtain the plasmid pKD46-BcldhL, Figure 1 as shown.

[0054] Using the plasmid pKD46-BcldhL as a template, amplify the fragment with the upstream and downstream homologous arms of the ppsA gene, the BcldhL gene, and the kana resistance gene using primers ppsA-BcldhL F and ppsA-BcldhL R, and transform the electrocompetent E. coli pldhA::BcldhL-pKD46 to obtain the strain with the ppsA gene knocked out and the BcldhL gene expressed under the promoter of the ppsA gene. Remove the kana resistance gene according to the method of Example 1 (5) to obtain the strain E. coli pldhA::BcldhL-pppsA::BcldhL.

[0055] ppsA-BcldhLF:

[0056] cgcagaaatgtgtttctcaaaccgttcatttatcacaaaaggattgttcga tgaaaaaagtcaatcgtattgcag (SEQ ID NO.7);

[0057] ppsA-BcldhL R:

[0058] gtttcatcttcggggatcacataaacccggcgactgaacgccgccggggatttattgcgattgtgtaggctggag(SEQ ID NO.8).

[0059] BcldhLNdeI F: catatga tgaaaaaagtcaatcgtattgcag(SEQ ID NO.9);

[0060] BcldhLNdeI R: catatgttacaatacaggtgccatcgt(SEQ ID NO.10).

[0061] Construction and Overexpression of Plasmid pTrc99a-BcldhL in Example 3

[0062] The commercial plasmid pTrc99a (purchased from Addgene) was treated with BamHI and EcoRI enzymes. The target gene sequence was amplified using primers BcldhL 99a F and BcldhL 99a R. The PCR product was recovered by gel extraction and then treated with BamHI and EcoRI enzymes. The target gene fragment and the plasmid were ligated with T4 ligase, and the ligation product was chemically transformed into Escherichia coli to obtain the pTrc99a-BcldhL expression vector as Figure 2 shown. According to the method for preparing competent cells in Example 1(1), the chemical competent cells of the strain E.coli pldhA::BcldhL-pppsA::BcldhL were prepared and transformed with the plasmid pTrc99a-BcldhL to obtain the strain E.coli pldhA::BcldhL-pppsA::BcldhL-pTrc::BcldhL, which was renamed E.coli Lac. BcldhL 99a F: ggatccatgaaaaaagtcaatcgtattgcagtg(SEQ ID NO.11); BcldhL 99aR: aagcttttacaatacaggtgccatcgtttc(SEQ ID NO.12).

[0063] Adaptive Evolution of Escherichia coli E.coli Lac in Example 4

[0064] To eliminate the effect of gene knockout on cell growth, single colonies of E.coli Lac were selected for culture, and the strain with the fastest growth rate in the liquid medium was selected. According to the OD 600nm change within 48 h for judgment, the strain with the fastest OD 600nm increase and continuous growth was selected.

[0065] To improve the high tolerance of the strain to the product L-lactic acid, adaptive domestication was carried out using high-concentration L-lactic acid. L-lactic acid with a concentration gradient of 5 - 100 g / L was added to the medium, and the concentration was gradually increased to screen for strains that grew faster in high-concentration L-lactic acid. The high-concentration adaptability to D-lactic acid can cross-influence the adaptability to high-concentration L-lactic acid, so the strain was also subjected to high-concentration adaptability domestication to D-lactic acid, and strains with good growth were screened in the D-lactic acid medium with a concentration gradient of 5 - 100 g / L.

[0066] During the organic acid fermentation process, the production of acid causes the pH value to decrease, which in turn inhibits cell growth and acid accumulation. The soluble salts generated during the pH regulation process can also exert osmotic stress on the cells, affecting cell growth and acid accumulation. Therefore, the strain also needs to be subjected to high-concentration salt adaptability domestication. NaCl with a concentration gradient of 5 - 70 g / L was added to the medium to screen for strains resistant to osmotic stress and gradually improve their adaptability to osmotic stress. Finally, the high-yield L-lactic acid-producing strain E. coli Lac was obtained.

[0067] Medium formulation: Glucose 3% (mass percentage), peptone 0.5% (mass percentage), yeast powder 0.5% (mass percentage), (NH4)2SO4 100 mM, K2HPO4 5 g / L, KH2PO4 3.5 g / L, (NH4)2HPO4 3.5 g / L, MgSO4·7H2O 1 mM, CaCl2·2H2O 0.1 mM, trace element solution (1000×) 1 mL, adjust the pH to 7.0, sterilize at 121 °C for 20 minutes. Among them, the formulation of the trace element solution (1000×) is: ZnCl2 0.2 g / L, FeCl3·2H2O 1.6 g / L, CuCl2·2H2O 0.1 g / L, CoCl2·2H2O 0.2 g / L, H3BO3 0.05 g / L, NaMoO4·2H2O 0.24 g / L, 37 °C, 180 rpm.

[0068] In Example 5, the starting strain E. coli XZ132 and the modified E. coli Lac fermented L-lactic acid using glucose as the substrate

[0069] 1) Plate culture: Streak the recombinant strain E. coli Lac onto an LB medium containing 1.6 - 1.8% (mass / volume) agar and culture at 37 ± 1 °C for 10 ± 1 hours;

[0070] (2) Seed culture: Under aseptic conditions, pick a single colony on the plate in step (1) with a sterile pipette tip, and then inoculate it into 5 mL of LB liquid medium. Incubate it on a shaker at 37 ± 1 °C for 10 ± 1 hours; then inoculate it into 50 mL of LB liquid medium at an inoculation amount of 1% (v / v) and incubate it on a shaker at 37 ± 1 °C for 10 ± 1 hours;

[0071] (3) 5 L fermenter culture: Under aseptic conditions, take the bacterial liquid obtained in step (2) and inoculate it into the fermentation medium at an inoculation amount of 5% (v / v). Among them, the fermentation conditions are: the culture temperature is 37 °C, the culture method is stirring culture, the stirring speed is 450 revolutions per minute, the ventilation volume is 1.5 vvm, and the pH is adjusted to 6.8 with 10 M NaOH. Samples are taken every 3 hours to detect OD 620 nm and the glucose concentration in the fermentation sample. According to the glucose concentration, glucose dry powder is supplemented to maintain the glucose concentration at 20 - 30 g / L; at the same time, high performance liquid chromatography analysis is performed on the fermentation sample to determine the concentration in the fermentation broth. When glucose is no longer consumed, fermentation is stopped, and L-lactic acid is obtained from the fermentation broth.

[0072] In the above method, the detection method of the fermentation product is:

[0073] After diluting the sample by an appropriate multiple, boil it in a metal bath at 105 °C for 15 minutes, then centrifuge it at 14680 rpm for 15 minutes, and take the supernatant for liquid phase detection. The specific liquid phase detection conditions are as follows:

[0074] The model of the liquid chromatograph used is Shimadzu LC20AT, and the chromatographic column is HPX-87H (inner diameter 7.8 mm, length 300 mm); the column temperature during the detection process is set at 55 °C, the flow rate is 0.4 mL per minute, and the injection volume is 5 μL for liquid phase detection.

[0075] The results show that as Figure 3 and Figure 4 shown, when the recombinant strain E. coli llac is cultured for 17 h, it consumes 180 g / L of glucose, the L-lactic acid concentration reaches 177 g / L, the production intensity reaches 25 g / L / h, and the pyruvate yield reaches 0.98 g / g.

[0076] Among them: The formula of the LB medium described in the above steps (1) - (2) is: peptone 10 g / L; yeast extract 5 g / L; NaCl 10 g / L, pH 7.0; sterilize at 121 °C for 20 minutes.

[0077] The fermentation medium formulation described in step (3) above is as follows: glucose 60 - 80 g / L, yeast powder 5 g / L, K2HPO4 10 g / L, NaH2PO4 2 g / L, NH4Cl 3.5 g / L, MgSO4·7H2O 0.1 g / L, trace element solution (1000×) 1 mL, adjust the pH to 6.8, sterilize at 121 °C for 20 minutes. Among them, the trace element solution (1000×) formulation is: CaCl2·2H2O 3.2 g / L, ZnCl2 3.8 g / L, FeCl3·2H2O 30 g / L, MnCl2·2H2O 11.14 g / L, CuCl2·2H2O 0.96 g / L, CoCl2·2H2O 2.64 g / L, H3BO3 0.35 g / L, NaMoO4·2H2O 0.024 g / L.

[0078] From the experimental results of the examples, it can be seen that by knocking out the genes ldhA and ppsA and overexpressing the gene BcldhL with its promoter, and applying the strong promoter Trc to overexpress the gene BcldhL, L-lactic acid can be efficiently produced by the fermentation of Escherichia coli engineering strains. The fermentation time is 17 h, 150 g / L of glucose is consumed, the L-lactic acid concentration reaches 177 g / L, the production intensity reaches 25 g / L / h, and the pyruvate yield reaches 0.98 g / g.

[0079] The nucleotide sequence of the BcldhL gene (SEQ ID NO.1)

[0080] atgaaaaaagtcaatcgtattgcagtggttggaacgggtgcagttggtacaagttactgctacgccatgattaatcagggtgttgcagaagagcttgttttaatcgatattaacgaagcaaaagcagaaggggaagccatggacctgaaccacggcctgccatttgcgcctacgtcgacccgcgtttggaaaggcgattattccgattgcggcactgccgatcttgttgtcattacggcaggttccccgcaaaaaccgggcgaaacaaggcttgatcttgttgccaaaaacgcaaaaatttttaaaggcatgattaagagcatcatggacagcggctttaacgggatttttcttgttgccagcaacccggttgacattttgacatatgtaacttggaaagagtccggcctgccgaaagaacatgttatcggttcgggcacagtgcttgactccgcgcgtctccgcaactctttgagcgcccaatttggaattgacccgcgcaatgtgcatgctgcgattatcggcgaacacggcgatacggaacttccggtatggagccatacaaatatcggttacgatacgattgaaagctatctacaaaaaggaattattgacgaaaagacgttagatgacatttttgtcaatacgagagatgcggcttatcatattattgaacgaaaaggggccacattttacggcatcgggatgtccctgacccggattacaagggcaatcctgaacaatgaaaacagcgtattgacggtctctgcatttcttgaaggccaatacggaaacagcgatgtgtacgttggcgttccggccatcatcaatcgccagggcatccgtgaagtggttgaaatcaaactgaacgaaaaagaacaggaacagttcaatcattctgtaaaagtgctaaaagaaacgatggcacctgtattgtaa

[0081] Amino acid sequence of the protein encoded by the BcldhL gene (SEQ ID NO.13)

[0082] MKKVNRIAVVGTGAVGTSYCYAMINQGVAEELVLIDINEAKAEGEAMDLNH

[0083] GLPFAPTSTRVWKGDYSDCGTADLVVITAGSPQKPGETRLDLVAKNAKIFKG

[0084] MIKSIMDSGFNGIFLVASNPVDILTYVTWKESGLPKEHVIGSGTVLDSARLRNS

[0085] LSAQFGIDPRNVHAAIIGEHGDTELPVWSHTNIGYDTIESYLQKGIIDEKTLDD

[0086] IFVNTRDAAYHIIERKGATFYGIGMSLTRITRAILNNENSVLTVSAFLEGQYGNSDVYVGVPAIINRQGIREVVEIKLNEKEQEQFNHSVKVLKETMAPVL*

[0087] Nucleotide sequence of phosphoenolpyruvate synthase gene (ppsA) (SEQ ID NO.2)

[0088]

Claims

1. A recombinant E. coli strain E. coli Lac for producing L-lactic acid, characterized in that, The recombinant Escherichia coli strain E. coli Lac is E. coli pldhA::BcldhL-pppsA::BcldhL-pTrc::BcldhL. The E. coli pldhA::BcldhL-pppsA::BcldhL-pTrc::BcldhL uses the Escherichia coli gene recombinant strain E. coli XZ132 as the starting strain and the L-lactate dehydrogenase gene shown in SEQ ID NO.1 BcldhL to replace the lactate dehydrogenase A gene in the starting strain ldhA ; meanwhile BcldhL the gene replaces the phosphoenolpyruvate synthase gene in the starting strain ppsA ; meanwhile, the plasmid ptrc99A-BcldhL overexpressing L-lactate dehydrogenase containing the Trc promoter was introduced into the starting strain; the obtained recombinant Escherichia coli strain was acid-acclimated and high-salt-acclimated, and strains resistant to acid and high-salt osmotic stress were screened, and finally the high-yield L-lactic acid strain E. coli Lac was obtained; The acid domestication is carried out by adding L-lactic acid with a concentration gradient of 5-100 g / L and D-lactic acid with a concentration gradient of 5-100 g / L for domestication; The high-salt tolerance domestication is carried out by adding NaCl with a concentration gradient of 5-70 g / L to screen strains resistant to osmotic stress.

2. Use of the recombinant Escherichia coli strain E. coli Lac described in claim 1 in the preparation of L-lactic acid by microbial fermentation method, characterized in that, The application is as follows: inoculate the recombinant strain E. coli Lac into a fermentation medium, and culture the cells at 30 °C and 180 rpm until the cell growth reaches OD 600 = 0.

5. At this time, add IPTG with a final concentration of 0.2 mM, and continue to culture for 48 h until the fermentation ends to obtain a fermentation broth containing L-lactic acid. Separate and purify the fermentation broth to obtain L-lactic acid.

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