Genetically engineered escherichia coli for producing d-lactic acid and application thereof

Abstract

This invention discloses a recombinant strain of *Escherichia coli* for producing D-lactic acid, a method for producing D-lactic acid, and the application of the recombinant strain described above in D-lactic acid production. This invention differs from traditional glycolysis pathways, which require a microaerobic / anaerobic environment, by using the methylglyoxal (MGA) pathway to produce D-lactic acid. Fermentation can proceed without strict oxygen control, significantly simplifying equipment and environmental control requirements. The engineered strain EcoL15 (MGP14) constructed using the heterologous MGA pathway exhibits excellent performance—with 9.809 g / L glycerol + 2.128 g / L acetic acid as the carbon source, the glycerol to D-lactic acid conversion rate reaches 0.956 g / g (approaching the theoretical value of 0.978 g / g), and the enantiomeric purity of the obtained D-lactic acid reaches 99.9%. Acetic acid can significantly improve the conversion rate, and the lactic acid yield can be controlled by adjusting the glycerol concentration in the culture medium.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering and biomanufacturing technology. Specifically, it relates to a genetically engineered Escherichia coli that produces D-lactic acid based on the methylglyoxal pathway and its applications. Background Technology

[0002] Lactic acid, also known as 2-hydroxypropionic acid, with the molecular formula CH3CH(OH)COOH, is a naturally occurring hydroxycarboxylic acid widely used in food, pharmaceuticals, textiles, leather, and chemicals. Because the lactic acid molecule contains both hydroxyl and carboxyl groups, it serves as an important precursor for many high-value-added chemicals, used in the preparation of pyruvic acid, acrylic acid, 1,2-propanediol, lactic acid esters, polylactic acid, and polyhydroxyalkanoates containing lactic acid components. With the increasing demand for renewable chemicals and bio-based materials, the market demand for lactic acid and its derivatives continues to grow.

[0003] Compared with traditional chemical synthesis methods, the production of lactic acid using biotechnology offers advantages such as milder reaction conditions, lower energy consumption, and environmental friendliness. However, current industrial and research-based lactic acid fermentation processes primarily use sugars such as glucose and xylose as carbon sources, resulting in relatively high raw material costs. Therefore, developing bio-manufacturing technologies for lactic acid using low-cost carbon sources is of great significance for reducing production costs and improving process economics.

[0004] Glycerol is a carbon source with high reducing power (κ = 4.67), higher than glucose and xylose (κ = 4). Theoretically, glycerol metabolism can provide more reducing equivalents, which is beneficial for driving reductive biosynthetic reactions. In traditional lactate production pathways, microorganisms typically use NADH-dependent lactate dehydrogenases to reduce pyruvate to lactate; therefore, glycerol has a potential metabolic advantage as a carbon source. Furthermore, the biodiesel and bioethanol industries are important global biomass energy industries, producing large amounts of glycerol as a byproduct during their production; the oleochemical industry also generates significant amounts of glycerol as a byproduct during the processing of animal and vegetable oils. These sources make glycerol widely available, inexpensive, and suitable for large-scale utilization.

[0005] Escherichia coli is a model microorganism widely used in industrial biotechnology and metabolic engineering research, possessing advantages such as rapid growth, a clear genetic background, mature molecular manipulation tools, and the ability to utilize multiple carbon sources. Furthermore, E. coli can grow in relatively simple culture media, making it suitable as an engineering chassis strain for the production of lactic acid and other organic acids. Lactic acid exists in two optical isomers: D-lactic acid and L-lactic acid. E. coli endogenously contains NADH-dependent D-lactic acid dehydrogenase but lacks NADH-dependent L-lactic acid dehydrogenase; therefore, its natural metabolism primarily converts pyruvate to D-lactic acid.

[0006] Currently, several studies have used *E. coli* as the chassis strain to produce D-lactic acid from glycerol. For example, Ramon Gonzalez et al. used engineered *E. coli* MG1655 as the carbon source and obtained 34 g / L D-lactic acid in 72 h (see article "..."). Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol (published in *Applied and Environmental Microbiology*, 2010). Wang Zhengxiang et al. used engineered *Escherichia coli* B0013 to obtain 100.3 g / L D-lactic acid in a fermenter using crude glycerol as a carbon source in 36 h (see article "Efficient bioconversion of crude glycerol from biodiesel to optically pure D-lactate by metabolically engineered"). Escherichia coli The study, published in *Green Chemistry* in 2014, used exogenous thiamine to control final cell quality. In a fermenter using pure glycerol as the carbon source, 119.8 g / L D-lactic acid was obtained within 32 h (see article "Limitation of thiamine pyrophosphate supply to growing cells"). Escherichia coli "Switches metabolism to efficient d-lactate formation," published in *Biotechnology and Bioengineering* in 2016. Zhao Yunpeng and Jiang Zhongren et al. used engineered *Escherichia coli* BL21 to obtain 105 g / L D-lactic acid in a fermenter using crude glycerol as a carbon source in 40 h (see article "Systematic Engineering of..."). Escherichia coliThe paper, "For d-Lactate Production from Crude Glycerol," was published in the *Journal of Agricultural and Food Chemistry* in 2015. Further optimization yielded 115 g / L D-lactic acid in 35 h using crude glycerol as the carbon source (see article "A simple strategy to effectively produce d-lactate in crude glycerol-utilizing"). Escherichia coli The above metabolic engineering strategy mainly relies on the conversion of glycerol to pyruvate via glycolysis, followed by the reduction of pyruvate to D-lactic acid by D-lactic acid dehydrogenase. The fermentation process is mostly carried out under microaerophilic or anaerobic conditions.

[0007] The methylglyoxal pathway also exists naturally in *E. coli*. Compared to the glycolysis-pyruvate-lactic acid pathway, the methylglyoxal pathway has the advantages of shorter reaction steps, a relatively simple metabolic bypass, and the ability to generate D-lactic acid without additional reducing power. This pathway uses dihydroxyacetone phosphate as a precursor, which is converted to methylglyoxal by methylglyoxal synthase, and then further converted to D-lactic acid by the glyoxalase system or related detoxification pathways. However, methylglyoxal is a highly reactive electrophilic intermediate. Its accumulation in cells can modify proteins and DNA, and at low concentrations, it can lead to cell growth inhibition or even cell death. Therefore, in its natural state, cells usually limit the accumulation of methylglyoxal in several ways, such as the allosteric inhibition of methylglyoxal synthase MgsA by phosphate, and the limitation of methylglyoxal production by the low intracellular level of dihydroxyacetone phosphate. Cells are more likely to produce methylglyoxal when the intracellular level of dihydroxyacetone phosphate is increased and the phosphate level is decreased.

[0008] Although the endogenous methylglyoxal pathway in Escherichia coli has been systematically biochemically characterized, its application as the main pathway for lactate synthesis in metabolic engineering remains limited. Current technologies mainly focus on generating pyruvate via glycolysis and further reducing it to D-lactic acid, without fully utilizing the potential advantages of the methylglyoxal pathway in terms of short pathway length, low reducing power requirement, and carbon flux regulation. Summary of the Invention

[0009] The purpose of this invention is to overcome the problems of existing D-lactic acid production technologies, such as the need for a microaerobic / anaerobic environments, reliance on high-priced sugar carbon sources, and the ineffective utilization of the natural methylglyoxal pathway due to the accumulation of toxic intermediates. Therefore, this invention proposes a genetically engineered Escherichia coli based on the methylglyoxal pathway and its application, which uses inexpensive glycerol as the main carbon source and acetic acid as an auxiliary source to produce D-lactic acid under aerobic conditions.

[0010] To achieve the above objectives, a first aspect of the present invention provides a recombinant Escherichia coli strain that produces D-lactic acid, said strain being produced by knocking out a certain gene in the genome of Escherichia coli MG1655. tpiA Gene, gldA Gene, dld Genes and glcDEF Gene clusters were transferred into recombinant plasmids containing engineered expression cassettes; The backbone of the recombinant plasmid is pColADuet-1, and the expression cassette is selected from: J23100-B0029- mgsA (Escherichia coli) -B0029- gloA -B0029- gloB -RrnB T1 / T2terminator; Or J23100-B0029- mgsA ( Clostridium acetobutylicum )-B0029- At1g08110 -B0029- At3g10850 -RrnB T1 / T2 terminator.

[0011] According to a preferred embodiment of the present invention, the nucleotide sequence of the J23100 gene is shown in SEQ ID NO:1; The nucleotide sequence of the B0029 gene is shown in SEQ ID NO:2.

[0012] The nucleotide sequence of the RrnB T1 / T2 terminator gene is shown in SEQ ID NO:3.

[0013] Preferably, the mgsA (Escherichia coli) The nucleotide sequence of the gene is shown in SEQ ID NO:36; The gloA The nucleotide sequence of the gene is shown in SEQ ID NO:37; The gloB The nucleotide sequence of the gene is shown in SEQ ID NO:38.

[0014] Preferably, the mgsA ( Clostridium acetobutylicum The nucleotide sequence of the gene is shown in SEQ ID NO:39; The At1g08110 The nucleotide sequence of the gene is shown in SEQ ID NO:40; The At3g10850 The nucleotide sequence of the gene is shown in SEQ ID NO:41.

[0015] A second aspect of the present invention provides a method for producing D-lactic acid, comprising the step of inoculating the genetically engineered strain as described above into a fermentation medium for cultivation; The fermentation medium contains glycerol and acetic acid.

[0016] According to a preferred embodiment of the present invention, the initial concentration of glycerol in the fermentation medium is from 0 g / L to 9.8 g / L, and the initial concentration of acetic acid is from 0 g / L to 2.2 g / L.

[0017] Preferably, the cultivation process is divided into a seed culture stage and a fermentation culture stage; The temperature during the seed culture stage is 37°C, and the temperature during the fermentation culture stage is 30°C.

[0018] Preferably, the cultivation process is carried out under aerobic conditions, and the pH value of the fermentation broth is controlled within the range of 6.8 to 7.2 by adding acid or alkali.

[0019] A third aspect of the invention provides the use of the genetically engineered strains described above in the production of D-lactic acid.

[0020] The beneficial effects of this invention are as follows: 1. Unlike the traditional glycolysis pathway which requires a microaerobic / anaerobic environment, this invention uses the methylglyoxal pathway to produce D-lactic acid, which can be fermented without strict oxygen control, greatly simplifying equipment and environmental control requirements.

[0021] 2. The engineered strain EcoL15 (MGP14) constructed via the heteromethylglyoxal pathway exhibited excellent performance—when using 9.809 g / L glycerol + 2.128 g / L acetic acid as the carbon source, the conversion rate of glycerol to D-lactic acid reached 0.956 g / g (approaching the theoretical value of 0.978 g / g), and the purity of the D-lactic acid enantiomeric strains obtained reached 99.9%; acetic acid can significantly improve the conversion rate, and the lactic acid yield can be controlled by the glycerol concentration in the culture medium. Attached Figure Description

[0022] Figure 1 This is a metabolic diagram of D-lactic acid production by *E. coli* using glycerol / acetic acid. The English annotations in the diagram are as follows: Glycerol, 3P-glycerol, dihydroxyacetone-P, methylglyoxal, DS-lactoylglutathione, D-lactate, dihydroxyacetone, hydroxyacetone, 1,2-propanediol, L-lactaldehyde, L-lactate, 6P-fructose Fructose), 3P-glyceraldehyde, 3P-glycerate, phosphoenolpyruvate, pyruvate, acetate, acetyl-CoA, mgsA, glyoxal synthase, gloA, glyoxalase I, gloB, tpiA, glyoxal isomerase, gldA, dldA, glucosyl-dependent D-lactate dehydrogenase, glcDEF, extracellular, glycerol utilization, acetate utilization, methylglyoxal module, gene knockout, gene overexpression. Detailed Implementation

[0023] The present invention will be described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are all available through conventional commercial channels.

[0025] 1. The specific strains and plasmids used in this invention are shown in Table 1.

[0026] Table 1. Sources and Functional Descriptions of Strains and Plasmids Among them, plasmid MGP1 was constructed by this invention, its plasmid backbone is pColADuet-1, and its expression cassette structure is as follows: J23100-B0029- mgsA (Escherichia coli) -B0029- gloA -B0029- gloB -RrnB T1 / T2terminator.

[0027] in, mgsA , gloA and gloB All were derived from Escherichia coli MG1655.

[0028] Plasmid MGP14, constructed using this invention, has a plasmid backbone of pColADuet-1 and an expression cassette structure as follows: J23100-B0029- mgsA ( Clostridium acetobutylicum )-B0029-At1g08110-B0029-At3g10850-RrnB T1 / T2 terminator.

[0029] in, mgsA The bacteria were derived from Clostridium acetone-butanol, while At1g08110 and At3g10850 were derived from Arabidopsis thaliana.

[0030] The plasmid MGPB (blank control plasmid) was constructed using this invention. Its plasmid backbone is pColADuet-1, and its expression cassette structure is as follows: J23100-B0029-RrnB T1 / T2 terminator.

[0031] 2. The sequence and function description of the control elements used in this invention are shown in Table 2.

[0032] Table 2. Sequence and Functional Description of Control Elements 3. The primer sequences used in this invention are shown in Table 3.

[0033] Table 3. Primer sequences and functional descriptions 4. The specific gene coding sequences used in this invention are shown in Table 4.

[0034] Table 4. Genes used in this invention Example 1: Construction of engineered strains In this embodiment, CRISPR-Cas9 gene editing technology was used to knock out the gene in Escherichia coli MG1655 to construct a combined deletion engineered strain.

[0035] 1. General Operating Procedures for Gene Editing All gene knockouts follow these steps (in order to) tpiA For example, the other gene manipulations are performed in the same way, only the primers are different. S1. Homologous arm amplification: Using E. coli MG1655 genomic DNA as a template, primers were used... tpiA -upF / tpiA -upR and tpiA -downF / tpiA -downR was used for PCR amplification to obtain tpiA upstream homologous arm of gene tpiA -up and downstream homologous arms tpiA -down, each segment is approximately 500 bp in length.

[0036] S2, Donor DNA Fusion: with tpiA -up and tpiA -down is the template, and primers are used. tpiA -upF / tpiA -downR was used for fusion PCR to obtain [the desired product / product]. tpiA The donor DNA fragments of the upstream and downstream homologous arms.

[0037] S3 and sgRNA vector construction: According to tpiA Gene sequence, design a suitable sgRNA sequence on the Benchling website, and ligate it into the pGRB plasmid using primers. tpiA sgRNA-F / tpiA pGRB-sgRNA plasmid was constructed using sgRNA-R.

[0038] S4. Preparation of Electrocompetent Cells: The glycerol-preserved bacteria stored at -80℃ were removed and thawed, then streaked onto LB solid medium and incubated at 37℃ for 8-12 h. A single colony was picked and inoculated into 3 mL of LB liquid medium and incubated at 37℃ for 8-12 h. Subsequently, 1 mL of the bacterial culture was inoculated into 50 mL of LB liquid medium and incubated at 37℃ until OD... 600 The concentration should be 0.4-0.6. After incubating the bacterial culture on ice for 20 min, centrifuge at 4℃ and 5500 rpm for 5 min, and discard the supernatant. Add 10-15 mL of pre-chilled 10% glycerol to resuspend the bacterial cells, and centrifuge again at 4℃ and 5500 rpm for 5 min, discarding the supernatant. Repeat the above 10% glycerol washing step once. Finally, add 300-500 μL of pre-chilled 10% glycerol to resuspend the bacterial cells, and aliquot 50 μL into 1.5 mL centrifuge tubes, storing at -80℃ for later use.

[0039] S5. Gene Knockout Transformation: Thaw one tube of electroporated competent cells on ice, add 100 ng of pREDCas9 plasmid, transfer to a 1 mm electroporation cuvette, and perform electroporation transformation using a MicroPulser (Bio-Rad Laboratories, USA) according to bacterial model. Immediately after transformation, add 950 μL of LB liquid medium and incubate at 30°C for 2 h. Then centrifuge at 5500 rpm for 5 min, discard approximately 900 μL of supernatant, resuspend the cells in the remaining approximately 100 μL of bacterial culture, and spread them all on LB solid medium containing 100 mg / L zizomycin. Incubate at 30°C for 12-16 h. The resulting single colony is Escherichia coli MG1655 carrying the pREDCas9 plasmid.

[0040] S6. Target Gene Knockout: Following the above method, *E. coli* MG1655 carrying pREDCas9 was further prepared into electrotransformable competent cells. During preparation, 100 mg / L gesmycin was added as needed. One tube of competent cells was thawed on ice, and 100 ng donor DNA and 100 ng pGRB-sgRNA plasmid were added simultaneously for electrotransformation. After transformation, 950 μL of LB liquid medium was added, and the cells were incubated at 30°C for 2-3 h. Subsequently, the cells were centrifuged at 5500 rpm for 5 min, approximately 900 μL of supernatant was discarded, and the cells were resuspended in approximately 100 μL of the remaining bacterial culture. All cells were then plated onto LB solid medium containing 100 mg / L gesmycin and 100 mg / L ampicillin, and incubated at 30°C for 16-20 h. Single colonies were picked and used with primers. tpiA verF / tpiA VerR is used for colony PCR verification; colonies that test positive by PCR are considered valid. tpiA An engineered strain that successfully knocked out its genes.

[0041] S7, Plasmid elimination: Elimination of pGRB-sgRNA: PCR-positive colonies were picked and inoculated into 3 mL of LB liquid medium containing 100 mg / L genomic trimethoprim, with 60 μL of 1 mL-arabinose added. The culture was incubated at 30°C for 12–16 h. The bacterial culture was then streaked onto LB solid medium containing 100 mg / L genomic trimethoprim and incubated at 30°C for 12–16 h. Single colonies were picked and inoculated again into 3 mL of LB liquid medium containing 100 mg / L genomic trimethoprim, with 60 μL of 1 mL-arabinose added. The culture was incubated at 30°C for 12–16 h to complete the elimination of the pGRB-sgRNA plasmid.

[0042] Elimination of pREDCas9: After eliminating the pGRB-sgRNA plasmid, inoculate 1 μL of bacterial culture into 3 mL of LB liquid medium and incubate at 42°C for 8–12 h. Then, streak the bacterial culture onto LB solid medium and incubate at 42°C for 8–12 h. Pick a single colony and inoculate it into 3 mL of LB liquid medium, incubating at 42°C for 8–12 h to complete the elimination of the pREDCas9 plasmid.

[0043] 2. Construction of combined deletion engineered strains 1. Following the general procedure described above, knock out genes consecutively. tpiA Gene, gldA Gene, dld Genes were used to obtain the engineered strain EcoL1: MG1655 Δ tpiA Δ gldA Δ dld .

[0044] 2. Following the general procedure described above, knock out the genes sequentially. tpiA Gene, gldA Gene, dld Gene, glcDEF Gene clusters were used to obtain the engineered strain EcoL15:MG1655 Δ tpiA Δ gldA Δ dld Δ glcDEF .

[0045] Example 2: Production of D-lactic acid by shake-flask fermentation of engineered strains using glycerol / acetic acid as a dual carbon source. In this embodiment, the engineered strain constructed in Example 1 was further transformed into an engineered plasmid related to the methylglyoxal pathway, and shake-flask fermentation was carried out under glycerol / acetic acid dual carbon source conditions to investigate the ability of the engineered strain to produce D-lactic acid.

[0046] 1. Transformation of engineered strains Electrocompetent cells of engineered strains (EcoL1, EcoL15) were prepared according to the method in Example 1. One tube of electrocompetent cells was thawed on ice, and 100 ng of the engineered plasmid was added for electroporation transformation. Immediately after transformation, 950 μL of LB liquid medium was added, and the cells were incubated at 37°C for 2 h. 100 μL of the bacterial culture was directly spread onto LB solid medium containing 50 mg / L kanamycin and incubated at 37°C for 12-16 h. The resulting single colonies are the engineered strains carrying the corresponding engineered plasmids.

[0047] The following four recombinant engineered strains were obtained in this embodiment: 1) Plasmid MGP1 was transformed into EcoL1 to obtain strain EcoL1 (MGP1). 2) Plasmid MGP1 was transformed into EcoL15 to obtain strain EcoL15 (MGP1). 3) Plasmid MGP14 was transformed into EcoL15 to obtain strain EcoL15 (MGP14). 4) Plasmid MGPB was transformed into EcoL15 to obtain strain EcoL15 (MGPB).

[0048] 2. The shake-flask fermentation process is as follows: (1) Primary seed culture: Three fresh single colonies were picked from LB solid medium and inoculated into the same tube of 3 mL LB liquid medium containing 50 mg / L kanamycin. The culture was carried out at 37°C and 220 rpm for 12 h.

[0049] (2) Secondary seed culture: The primary seed culture was inoculated into 50 mL of LB liquid medium containing 50 mg / L kanamycin at an inoculation rate of 2%, and cultured at 37℃ and 220 rpm for 12 h.

[0050] (3) Shake-flask fermentation: The secondary seed culture was inoculated into 50 mL shake-flask fermentation medium at an inoculation rate of 2%, and cultured at 30℃ and 220 rpm for 60 h. During the fermentation process, samples were taken every 12 h, and the contents of lactic acid, glycerol, and acetic acid in the fermentation broth were analyzed by HPLC. The enantiomeric composition of lactic acid was also detected by chiral HPLC. At the same time, the pH of the fermentation broth was adjusted every 12 h using 6 M H₂SO₄ or 6 M NaOH to maintain it within the range of 6.8-7.2.

[0051] The shake-flask fermentation medium consists of the following: 5 g / L NaCl, 1.5 g / L KCl, 1 g / L NH4Cl, 0.2 g / L MgCl2, 0.07 g / L Na2SO4, 0.01% (v / v) trace element stock solution, 2 mg / L thiamine hydrochloride, 10 g / L 3-(N-morpholine)propanesulfonic acid, 5 g / L yeast extract, 50 mg / L kanamycin, and carbon sources of varying concentrations.

[0052] Some components need to be prepared into stock solutions before being added to the shake-flask fermentation medium in appropriate proportions to achieve the desired final concentration. The concentration of thiamine hydrochloride stock solution is 10 g / L, and the concentration of 3-(N-morpholine)propanesulfonic acid stock solution is 100 g / L (prepared with half-sodium salt). The trace element stock solution is prepared with 3 M HCl, and its composition and concentration are shown in Table 5.

[0053] Table 5. Composition and Concentration of Trace Element Stock Solution 3. Metabolite assay: (1) Quantitative analysis of lactic acid, glycerol, and acetic acid: Take 1 mL of fermentation broth sample, centrifuge at 12,000 rpm for 10 min, collect the supernatant, and filter it through a 0.22 μm PES membrane. The resulting filtrate is used as the sample to be tested. Lactic acid, glycerol, and acetic acid in the sample are quantitatively analyzed using HPLC LC-2050 (Shimadzu, Japan). During the analysis, 0.5 mM H2SO4 is used as the mobile phase, and an HPX-87H column (Bio-Rad, USA) is used for separation; the column temperature is 65℃, the differential refractive index detector temperature is 45℃, and the flow rate is 0.5 mL / min.

[0054] (2) Lactate enantiomeric analysis: After shake-flask culture, fermentation broth samples were collected, centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm PES membrane. The filtrate was used for lactate enantiomeric analysis. Separation was performed using a Superchiral D-PE chiral column with a 2 mM CuSO4 solution in water-isopropanol (95:5, v / v) as the mobile phase. Detection conditions were: flow rate 0.8 mL / min, UV detection wavelength 254 nm, and column temperature 25℃. The results after 60 h of shake-flask fermentation are shown in Table 6.

[0055] Table 6. Detection results after 60 hours of shake-flask fermentation. * indicates that no acetic acid or glycerol was added at the beginning of fermentation.

[0056] This invention uses wild-type Escherichia coli MG1655 as the starting strain, and through knockout... tpiA , gldA , dld and glcDEF Genetically engineered strains capable of producing D-lactic acid via the methylglyoxal pathway were constructed using genes (clusters).

[0057] By modifying the metabolic pathway, glycerol is mainly used to generate D-lactic acid via the methylglyoxal pathway, while acetic acid is mainly used to supplement the carbon source and energy supply required for cell growth and metabolism. The two carbon sources have a clear division of labor in metabolic function.

[0058] As shown in Table 6, this method can achieve a near-theoretical conversion of glycerol to D-lactic acid under aerobic conditions, with a conversion rate of 0.956 g / g, close to the theoretical conversion rate of 0.978 g / g. At this point, D-lactic acid accounts for 99.9% of the total conversion. Furthermore, the amount of D-lactic acid produced can be controlled by adjusting the amount of glycerol added to the culture medium; this control method is simple and easy to implement.

[0059] MGP14 exhibited a higher conversion efficiency than MGP1, indicating that the heteromethylglyoxal pathway can significantly enhance the conversion of glycerol to D-lactic acid. Meanwhile, the blank control results showed that the engineered strain without pathway enhancement possessed some glycerol conversion capacity, but its conversion efficiency was lower than that of the enhanced methylglyoxal pathway. These results demonstrate that the strain modification strategy and glycerol / acetic acid dual-carbon source fermentation method described in this invention can be used to produce D-lactic acid and have good potential for fermentation process regulation.

[0060] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A recombinant strain of *Escherichia coli* that produces D-lactic acid, characterized in that, The strain is obtained by knocking out the genes tpiA , gldA , dld and glcDEF gene cluster in the genome of E. coli MG1655 and transforming with a recombinant plasmid containing an engineered expression cassette; The backbone of the recombinant plasmid is pColADuet-1, and the expression cassette is selected from: J23100-B0029- mgsA (Escherichia coli) -B0029- gloA -B0029- gloB -RrnB T1 / T2terminator; OrJ23100-B0029- mgsA ( Clostridium acetobutylicum )-B0029- At1g08110 -B0029- At3g10850 -RrnB T1 / T2 terminator.

2. The recombinant strain according to claim 1, characterized in that, The nucleotide sequence of the J23100 gene is shown in SEQ ID NO:1; The nucleotide sequence of the B0029 gene is shown in SEQ ID NO:

2.

3. The recombinant strain according to claim 1, characterized in that, The nucleotide sequence of the RrnB T1 / T2 terminator gene is shown in SEQ ID NO:

3.

4. The recombinant strain according to claim 1, characterized in that, The mgsA (Escherichia coli) The nucleotide sequence of the gene is shown in SEQ ID NO:36; The gloA The nucleotide sequence of the gene is shown in SEQ ID NO:37; The gloB The nucleotide sequence of the gene is shown in SEQ ID NO:

38.

5. The recombinant strain according to claim 1, characterized in that, The mgsA ( Clostridium acetobutylicum The nucleotide sequence of the gene is shown in SEQ ID NO:39; The At1g08110 The nucleotide sequence of the gene is shown in SEQ ID NO:40; The At3g10850 The nucleotide sequence of the gene is shown in SEQ ID NO:

41.

6. A method for producing D-lactic acid, characterized in that, Includes the step of inoculating the genetically engineered strain according to any one of claims 1 to 5 into a fermentation medium for cultivation; The fermentation medium contains glycerol and acetic acid.

7. The method according to claim 6, characterized in that, The initial concentration of glycerol in the fermentation medium was from 0 g / L to 9.8 g / L, and the initial concentration of acetic acid was from 0 g / L to 2.2 g / L.

8. The method according to claim 6, characterized in that, The cultivation process is divided into a seed culture stage and a fermentation culture stage; The temperature during the seed culture stage is 37°C, and the temperature during the fermentation culture stage is 30°C.

9. The method according to claim 6, characterized in that, The cultivation process is carried out under aerobic conditions, and the pH value of the fermentation broth is controlled within the range of 6.8 to 7.2 by adding acid or alkali.

10. The use of the recombinant strain according to any one of claims 1 to 5 in the production of D-lactic acid.

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