CN117305201A

CN117305201A - Genetic engineering strain for synthesizing chiral lactic acid by using two-carbon compound, construction method and application thereof

Info

Publication number: CN117305201A
Application number: CN202311047737.4A
Authority: CN
Inventors: 李�灿; 王旺银; 王迎晨
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2023-08-18
Filing date: 2023-08-18
Publication date: 2023-12-29

Abstract

The application discloses a genetic engineering strain for synthesizing chiral lactic acid by using a two-carbon compound, and a construction method and application thereof, and belongs to the technical field of genetic engineering. The genetic engineering strain provided by the invention comprises a starting strain, an initial expression vector, a promoter and an exogenous gene; the starting strain comprises escherichia coli; the exogenous genes include alcohol dehydrogenase genes, acetaldehyde dehydrogenase genes and lactate dehydrogenase genes. The genetic engineering strain provided by the invention can grow and ferment by utilizing the two-carbon compound to obtain chiral lactic acid, and overcomes the defect that microorganisms cannot ferment by utilizing the two-carbon compound in the prior art. The genetic engineering strain constructed by the method disclosed by the invention can be combined with a mode of mixed culture of various two-carbon compounds, so that the yield and the accumulation time of chiral lactic acid can be effectively improved.

Description

Genetic engineering strain for synthesizing chiral lactic acid by using two-carbon compound, construction method and application thereof

Technical Field

The application belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering strain for synthesizing chiral lactic acid by utilizing a two-carbon compound, and a construction method and application thereof.

Background

The current fermentation industry relies heavily on carbohydrates such as glucose, sucrose. With the progress of society, the development of carbon sources other than sugar is one of the important means for solving the food crisis and energy crisis. Carbon dioxide-based low-carbon compounds, e.g. C1 compounds (CO, CH ₄ Formic acid, methanol) and C2 compounds (ethanol, acetic acid), which are green in source, low in production cost and abundant in natural resources, and do "no contentions with humans" as compared with carbohydrates. For low carbon compounds as carbon sources, a part of autotrophic microorganisms naturally have corresponding metabolic pathways capable of assimilating and converting them into biomass, e.g. with immobilized CO ₂ The growth rate of the competent cyanobacteria, however, tends to be slow, which is disadvantageous for the improvement of the productivity per unit time in fermentation engineering. The production of heterologous microorganisms, in contrast to autotrophic microorganisms, generally relies on carbohydrates such as glucose, which divide rapidly, however they generally do not naturally have the ability to utilize low carbon compounds. The development of synthetic biology gives us the ability to engineer microorganisms to better adapt them to the growth conditions in which the low carbon compounds are the carbon source.

Lactic acid is an important chemical and is widely used in the fields of foods, medicines, cosmetics and the like. It is also the basic monomer of polylactic acid. Polylactic acid is a novel biodegradable material and is currently applied to the disposable industry and the medical industry. However, the properties of polylactic acid are greatly affected by the optical purity of lactic acid monomers, and polylactic acid materials with very good crystallinity can be obtained only by using optically pure lactic acid (pure L-lactic acid or pure D-lactic acid), and the degree of refinement of polylactic acid affects the stability of the material. Currently, optically pure lactic acid is mainly obtained by means of biological fermentation. Because the biocatalytically derived product is naturally pure D-lactic acid or pure L-lactic acid based on the properties of the enzyme itself compared to chemical means, complex and expensive chiral separation processes are avoided. However, currently biocatalytic production of lactic acid is still a carbohydrate that ferments glucose predominantly. Based on the current situation of global food shortage and the rapidly increasing food demand, it is necessary to find and develop more green sustainable compounds as carbon sources for the production of lactic acid.

From the perspective of bioengineering, the solubility of ethanol and acetic acid in aqueous solution is good, the mass transfer is fast, and the development of fermentation engineering is facilitated. Meanwhile, with the development of catalytic chemistry and chemical engineering, ethanol and acetic acid have been able to be synthesized from CO ₂ The method is converted by means of green chemistry such as electrocatalysis, and the like, so that the low pollution and the renewable characteristics of fermentation raw materials are ensured. The energy of C2 compounds such as ethanol and acetic acid is high, and during the assimilation process, an equal concentration of C2 compounds provides sufficient reducing power and energy for microorganisms to reduce the amount of energy used to convert carbon sources to CO for survival ₂ Unnecessary consumption of the respiratory effort is performed. The assimilated acetic acid or ethanol will directly participate in the metabolism of the microorganism in the form of acetyl-coa, an important intermediate metabolite which itself belongs to fine chemicals and is also an important precursor for bio-based macromolecular compounds such as Polyhydroxybutyrate (PHB) and fatty acids. At present, how to use ethanol and acetic acid channels for synthesizing chiral lactic acid has been recently reported.

Disclosure of Invention

In order to solve the technical problems, the invention provides a genetic engineering strain for synthesizing chiral lactic acid by using a two-carbon compound, and a construction method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

in one aspect, the invention provides a genetically engineered strain for synthesizing chiral lactic acid by using a two-carbon compound, wherein the genetically engineered strain comprises a starting strain, an initial expression vector, a promoter and an exogenous gene;

the starting strain comprises escherichia coli;

the exogenous genes include alcohol dehydrogenase genes, acetaldehyde dehydrogenase genes and lactate dehydrogenase genes.

Optionally, the initial expression vector comprises pRSFduet-1 and/or pACYC184.

Alternatively, when the initial expression vector is pRSFduet-1, the promoter comprises Ptrc-tho, and the nucleotide sequence of Ptrc-tho is shown as SEQ ID No. 1;

when the initial expression vector is pACYC184, the promoter comprises ParaBAD, and the nucleotide sequence of the ParaBAD is shown in SEQ ID No. 2.

Optionally, the NCBI of the alcohol dehydrogenase gene has the accession number GenBank:WP_012885841;

the accession number of NCBI of the acetaldehyde dehydrogenase gene is GenBank: NP-014032;

the nucleotide sequence of the lactate dehydrogenase gene is shown as SEQ ID No. 3.

In a second aspect, the invention provides a construction method of the genetic engineering strain, comprising the following steps:

step 1), connecting the exogenous gene with an initial expression vector and a promoter to obtain a recombinant over-expression vector;

and 2) transferring the recombinant over-expression vector into an original strain to obtain the genetic engineering strain.

Alternatively, step 2) transferring the recombinant over-expression vector into competent cells of the starting strain.

Optionally, step 2) after transferring the recombinant overexpression vector into the starting strain, the method further comprises a step of screening with antibiotics.

In a third aspect, the invention provides an application of the genetically engineered strain in synthesizing chiral lactic acid and derivatives thereof by using a two-carbon compound.

In a fourth aspect, the present invention provides a method for synthesizing chiral lactic acid using a two-carbon compound, comprising the steps of:

inoculating the genetic engineering strain into a culture medium for biological fermentation to obtain the chiral lactic acid.

Optionally, the inoculation amount of the genetically engineered strain is 0.2-30 OD ₆₀₀ 。

Preferably, the inoculation amount of the genetically engineered strain is 2-30 OD ₆₀₀ 。

Alternatively, the inoculum size of the genetically engineered strain is independently selected from 0.2OD ₆₀₀ 、1OD ₆₀₀ 、2OD ₆₀₀ 、5OD ₆₀₀ 、10OD ₆₀₀ 、15OD ₆₀₀ 、20OD ₆₀₀ 、25OD ₆₀₀ 、30OD ₆₀₀ Any value therein or any range therebetween.

Optionally, the medium includes a two-carbon compound therein;

the initial addition amount of the two-carbon compound is 5-20 g/L.

Preferably, the initial addition amount of the two-carbon compound is 8 to 12g/L.

Alternatively, the initial addition amount of the two-carbon compound is independently selected from any value or a range of values between any two of 5g/L, 8g/L, 10g/L, 12g/L, 14g/L, 16g/L, 18g/L, 20g/L.

Optionally, the two-carbon compound is selected from one or more of ethanol, acetic acid, ethoxide, and acetate.

Optionally, the two-carbon compound is a mixture of ethanol and acetic acid, and the concentration ratio of the ethanol to the acetic acid in the mixture is 1-2:1.

Optionally, the culture medium further comprises one or more of phosphate, sodium salt, inorganic nitrogen-containing compound, magnesium salt, chelating agent and trace elements.

Optionally, the phosphate is selected from one or more of disodium hydrogen phosphate, potassium dihydrogen phosphate and diammonium hydrogen phosphate.

Optionally, the sodium salt comprises sodium chloride.

Optionally, the inorganic nitrogen-containing compound is selected from one or more of ammonium chloride, ammonium sulfate, ammonium phosphate, and ammonium bicarbonate.

Optionally, the magnesium salt comprises magnesium sulfate.

Optionally, the chelating agent comprises EDTA.

Optionally, the trace elements are selected from one or more of soluble cobalt salts, manganese salts, copper salts, molybdenum salts, zinc salts, iron salts and boric acid.

Optionally, the biological fermentation is performed under aerobic conditions.

Optionally, the temperature of the biological fermentation is 27-45 ℃.

Preferably, the temperature of the biological fermentation is 30-37 ℃.

Optionally, the time of the biological fermentation is not less than 24 hours.

Optionally, the pH of the culture medium is controlled to be 6-7.5 in the biological fermentation process.

Optionally, the chiral lactic acid comprises D-lactic acid and/or L-lactic acid.

Compared with the prior art, the invention has the following beneficial effects:

(1) The genetic engineering strain provided by the invention takes escherichia coli as an original strain, introduces exogenous ethanol dehydrogenase genes, acetaldehyde dehydrogenase genes and lactic acid dehydrogenase genes, can grow and ferment by using the two-carbon compound to obtain chiral lactic acid, and overcomes the defect that microorganisms cannot ferment by using the two-carbon compound in the prior art.

(2) The genetic engineering strain constructed by the method disclosed by the invention can be combined with a mode of mixed culture of various two-carbon compounds, so that the yield and the accumulation time of chiral lactic acid can be effectively improved. In particular, chiral lactic acid yields are highest when fermentation is performed with equal concentrations of ethanol and acetic acid as co-substrates.

Drawings

FIG. 1 shows plasmid information of an initial expression vector and a recombinant overexpression vector when the ethanol pathway is utilized in the present invention;

FIG. 2 is a schematic diagram showing the growth of strains when ethanol is used as the sole carbon source;

FIG. 3 is a schematic diagram showing ethanol consumption when ethanol is used as the sole carbon source in the present invention;

FIG. 4 shows plasmid information of the initial expression vector and the recombinant overexpression vector in the production of chiral lactic acid according to the present invention;

FIG. 5 is a schematic diagram showing the fermentation of lactic acid produced by different strains when ethanol is the sole carbon source;

FIG. 6 is a graph showing ethanol consumption by different strains when ethanol is the only carbon source;

FIG. 7 is a graph showing the effect of adjusting the C/N ratio of the medium on the yield of lactic acid when ethanol is the sole carbon source in the present invention;

FIG. 8 is a schematic diagram showing the effect of adjusting the C/N ratio of the culture medium on the growth of the strain when ethanol is used as the sole carbon source;

FIG. 9 is a graph showing the effect of adjusting the ratio of acetic acid to ethanol on lactic acid production when acetic acid and ethanol are mixedly cultured according to the present invention.

Detailed Description

The present application is further illustrated below in conjunction with specific examples. The following description is given of several embodiments of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art may make various changes or modifications using the technical contents disclosed above without departing from the scope of the technical solutions of the present application, which are equivalent to the equivalent embodiments.

Unless otherwise indicated, all starting materials in the examples of the present application were purchased commercially and used without any particular treatment.

Unless otherwise indicated, the analytical methods in the examples all employed conventional arrangements of instruments or equipment and conventional analytical methods.

The sources of the strain E.coli BW25113 used in the examples below were: https:// www.mingzhoubio.com/good-109218. Html, purchased from Ningbo biosome under the designation BMZ066594.

Example 1

Construction of a pathway for recombinant bacteria to use ethanol:

an ethanol utilization module was constructed in E.coli BW25113, expressing heterologous ethanol dehydrogenase and acetaldehyde dehydrogenase. Synthesis of exogenous alcohol dehydrogenase and B by means of total gene synthesisAldehyde dehydrogenases. The ada gene (NCBI accession number is GenBank: WP_ 012885841) encoding alcohol dehydrogenase from Saccharomyces cerevisiae genome and the adh gene (NCBI accession number is GenBank: NP_ 014032) encoding acetaldehyde dehydrogenase from Rhizoctonia cerealis genome are cloned on pRSFduret-1 expression vector and controlled by Ptrc-tho promoter (nucleotide sequence is shown as SEQ ID No. 1) to obtain recombinant over-expression vector. Plasmid information for the initial expression vector and the recombinant overexpression vector is shown in FIG. 1. Transferring the recombinant over-expression vector into the competence of wild escherichia coli BW25113, and screening correct genetic engineering bacteria by using antibiotics to obtain a BW-e strain. After entering the escherichia coli, the ethanol is catalytically converted into acetyl-CoA, and the acetyl-CoA participates in the metabolic process of the microorganism. Inoculating E.coli seed solution into seed culture medium (seed culture medium: 200mM ethanol, 13.3g/L KH) using 200mM ethanol as sole carbon source ₂ PO ₄ ，4g/L(NH ₄ ) ₂ HPO ₄ ，1.2g/L MgSO ₄ ，0.0084g/L EDTA，0.0025g/L CoCl ₂ ，0.015g/L MnCl ₂ ，0.0015g/L CuCl ₂ ，0.003g/L H ₃ BO ₃ ，0.0025g/L Na ₂ MoO ₄ ，0.008g/L Zn(CH ₃ COO) ₂ 0.06g/L Fe (III) citrate, ensuring an initial inoculum size of 0.05OD ₆₀₀ Aerobic culture was performed at 200rpm and 30 ℃. Wherein the growth of BW-e strain is shown in figure 2, and the consumption of ethanol is shown in figure 3. As can be seen from FIG. 2, the BW-e strain can grow by using ethanol as the only carbon source, and the bacterial load is realized from 0.05OD ₆₀₀ To 16OD ₆₀₀ Is a variation of (c). Also, as can be seen from FIG. 3, BW-e was also very capable of utilizing ethanol, consuming 250mM substrate ethanol within 108 hours.

Example 2

Construction of a pathway for producing chiral lactic acid by recombinant bacteria:

exogenous lactate dehydrogenase is synthesized by total gene synthesis. The ldh gene (nucleotide sequence shown in SEQ ID No. 3) encoding lactate dehydrogenase from the Lactobacillus genome was cloned into pACYC184 plasmid and the L-arabinose inducible promoter ParaBAD, i.e., araBAD promoter, was selectedThe expression of the gene is controlled by the nucleotide sequence shown as SEQ ID No.2, and the recombinant over-expression vector is obtained. Plasmid information for the initial expression vector and the recombinant overexpression vector is shown in FIG. 4. The recombinant over-expression vector was transferred into the BW-e strain of example 1 to give BW-el strain. Based on E.coli introduced with the ethanol utilization module, D-lactate dehydrogenase is heterologously expressed, and pyruvic acid can be converted into D-lactic acid. While neither the strain BW-L, into which lactate dehydrogenase is introduced, nor the wild-type E.coli WT can produce lactate from ethanol. At an initial OD ₆₀₀ 0.3, the bacterial solutions of the different strains were inoculated into a seed medium containing 100mL of kanamycin at a content of 10. Mu.g/mL (seed medium: 10. Mu.g/mL kanamycin, 13.3g/L KH) ₂ PO ₄ ，4g/L(NH ₄ ) ₂ HPO ₄ ，1.2g/L MgSO ₄ ，0.0084g/L EDTA，0.0025g/L CoCl ₂ ，0.015g/L MnCl ₂ ，0.0015g/L CuCl ₂ ，0.003g/L H ₃ BO ₃ ，0.0025g/L Na ₂ MoO ₄ ，0.008g/L Zn(CH ₃ COO) ₂ 0.06g/L Fe (III) citrate), 200rpm,30℃overnight. The cultured cells were collected by centrifugation, and then the cells were redispersed in 100mL of fermentation medium (fermentation medium: 6.8g/L Na) ₂ HPO ₄ ，3g/L KH ₂ PO ₄ ，0.5g/L NaCl，0.25g/L NH ₄ Cl，1.2g/L MgSO ₄ ，0.0084g/L EDTA，0.0025g/L CoCl ₂ ，0.015g/L MnCl ₂ ，0.0015g/L CuCl ₂ ，0.003g/L H ₃ BO ₃ ，0.0025g/L Na ₂ MoO ₄ ，0.008g/L Zn(CH ₃ COO) ₂ 0.06g/L Fe (III) citrate, ensure OD ₆₀₀ 2.2. Kanamycin and the inducer arabinose were added, while 200mM ethanol was added as the sole carbon source, and aerobic fermentation was performed at 30 ℃. Wherein, the condition of D-lactic acid production by fermentation of different strains is shown in figure 5, and the consumption condition of ethanol by different strains is shown in figure 6. As can be seen from FIG. 5, the wild type E.coli BW-25113 (BW) did not have the ability to produce lactic acid, and FIG. 6 also shows that BW could not effectively utilize ethanol in the medium. The BW-L strain is obtained by introducing chiral milk into BW as compared with BW-el strainAcid pathway (exogenous lactate dehydrogenase), but BW-L does not construct a pathway utilizing ethanol. As can be seen from FIG. 5, although BW-L incorporates lactate dehydrogenase, chiral lactic acid still cannot be produced; meanwhile, BW-L cannot effectively utilize ethanol in the medium as BW because no ethanol utilization pathway is constructed. Compared with BW-L, the BW-el introduces an ethanol utilization channel, so that ethanol can be efficiently utilized, and the result of FIG. 5 also shows that the BW-el can produce chiral lactic acid by taking ethanol as the only carbon source. Thus, in order to obtain the target strain for producing lactic acid by fermentation using ethanol as a substrate, the construction of both the ethanol utilization pathway and the lactic acid production pathway is critical.

Example 3

Producing D-lactic acid by using escherichia coli genetic engineering bacteria BW-el fermentation:

(1) The effect of nitrogen content of inorganic salt medium on lactic acid production was studied:

seed culture medium: 13.3g/L KH ₂ PO ₄ ，4g/L(NH ₄ ) ₂ HPO ₄ ，1.2g/L MgSO ₄ Trace elements: 0.0084g/L EDTA,0.0025g/L CoCl ₂ ，0.015g/L MnCl ₂ ，0.0015g/L CuCl ₂ ，0.003g/L H ₃ BO ₃ ，0.0025g/L Na ₂ MoO ₄ ，0.008g/L Zn(CH ₃ COO) ₂ ，0.06g/L Fe(III)citrate；

Fermentation medium: 6.8g/L Na ₂ HPO ₄ ，3g/L KH ₂ PO ₄ ，0.5g/L NaCl，NH ₄ Cl concentration is shown in Table 1,1.2g/L MgSO ₄ Trace elements: 0.0084g/L EDTA,0.0025g/L CoCl ₂ ，0.015g/L MnCl ₂ ，0.0015g/L CuCl ₂ ，0.003g/L H ₃ BO ₃ ，0.0025g/L Na ₂ MoO ₄ ，0.008g/L Zn(CH ₃ COO) ₂ ，0.06g/L Fe(III)citrate；

The culture method comprises the following steps: at an initial OD ₆₀₀ The bacterial solution was inoculated into a seed medium at 0.3 and cultured overnight at 30℃at 200 rpm. The cultured cells were collected by centrifugation. The nitrogen content, i.e., the ammonium content, in the fermentation medium was adjusted to correspond to the C/N ratio of the medium shown in Table 1。

TABLE 1 Nitrogen content and corresponding C/N ratio in fermentation Medium

C/N ratio	NH ₄ Cl concentration (g/L)
		20:1	1
40:1	0.5
		80:1	0.25
400:0	0

Redispersing the bacteria in 100mL fermentation medium with different nitrogen content to ensure OD ₆₀₀ 2.2. 200mM ethanol was added as the sole carbon source and aerobic fermentation was performed at 30 ℃. Under the culture conditions with different nitrogen contents, the fermentation condition of D-lactic acid is shown in FIG. 7, and the growth condition of microorganisms is shown in FIG. 8.

As can be seen from FIG. 7, after fermentation, NH ₄ When the content of Cl is 0g/L or 0.25g/L, the yield of the D-lactic acid synthesized by the genetically engineered bacterium BW-el is highest. With 1g/L NH ₄ Cl, when the culture medium contains 0.25g/L NH ₄ When Cl, the yield of lactic acid was increased from 8mM to 15mM.

(2) Study of the influence of the type and the ratio of the two-carbon compound on the yield of lactic acid:

Fermentation medium: 6.8g/L Na ₂ HPO ₄ ，3g/L KH ₂ PO ₄ ，0.5g/L NaCl，0.25g/L NH ₄ Cl，1.2g/L MgSO ₄ Trace elements: 0.0084g/L EDTA,0.0025g/L CoCl ₂ ，0.015g/L MnCl ₂ ，0.0015g/L CuCl ₂ ，0.003g/L H ₃ BO ₃ ，0.0025g/L Na ₂ MoO ₄ ，0.008g/L Zn(CH ₃ COO) ₂ ，0.06g/L Fe(III)citrate；

The culture method comprises the following steps: at an initial OD ₆₀₀ The bacterial solution was inoculated into a seed medium at 0.3 and cultured overnight at 30℃at 200 rpm. The cultured cells were collected by centrifugation. Then re-dispersing the bacterial precipitate in 100mL fermentation medium to ensure OD ₆₀₀ 2.2. Simultaneously adding ethanol and acetic acid as substrates for fermentation, wherein the ratio of the ethanol and acetic acid mixture was adjusted as shown in Table 2.

TABLE 2 ratio adjustment of ethanol and acetic acid mixtures in fermentation Medium

Ethanol/acetic acid	Ethanol addition (g/L)	Acetic acid addition (g/L)
			1:0	10	0
1:1	5	5
			0:1	0	10

The fermentation of D-lactic acid under the above-mentioned culture conditions in which a mixture of ethanol and acetic acid in different proportions is used as a carbon source is shown in FIG. 9.

As is clear from FIG. 9, the yield of D-lactic acid synthesized by the genetically engineered bacterium BW-el was highest when the ratio of the two carbon compounds was ethanol to acetic acid=1:1 after the fermentation was completed. When fermentation is performed using ethanol and acetic acid at equal concentrations as co-substrates, the yield of lactic acid is increased from 15mM to 53mM, as compared to fermentation using ethanol alone; whereas when fermentation was carried out with ethanol and acetic acid at equal concentrations as co-substrates, the yield of lactic acid was increased from 4mM to 53mM, as compared to fermentation with acetic acid alone.

The foregoing description is only a few examples of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred examples, it is not intended to limit the present application, and any person skilled in the art may make some changes or modifications to the disclosed technology without departing from the scope of the technical solution of the present application, and the technical solution is equivalent to the equivalent embodiments.

Claims

1. The genetically engineered strain is characterized by comprising a starting strain, an initial expression vector, a promoter and an exogenous gene;

the starting strain comprises escherichia coli;

2. Genetically engineered strain for the synthesis of chiral lactic acid using two carbon compounds according to claim 1, in which the initial expression vector comprises prssfduet-1 and/or pACYC184.

3. The genetically engineered strain for the synthesis of chiral lactic acid using two carbon compounds according to claim 2, wherein when the initial expression vector is prsfpuet-1, the promoter comprises Ptrc-tho, the nucleotide sequence of Ptrc-tho is shown in SEQ ID No. 1;

4. The genetically engineered strain for the synthesis of chiral lactic acid using two carbon compounds according to claim 1, wherein the NCBI of the alcohol dehydrogenase gene has accession number GenBank: wp_012885841;

5. The method for constructing a genetically engineered strain according to any one of claims 1 to 4, comprising the steps of:

6. The method of constructing a genetically engineered strain according to claim 5, wherein step 2) the recombinant overexpression vector is transferred into competent cells of the starting strain;

preferably, step 2) further comprises the step of screening with antibiotics after transferring the recombinant overexpression vector into the starting strain.

7. The use of the genetically engineered strain according to any one of claims 1 to 4 for synthesizing chiral lactic acid and its derivatives using a two-carbon compound.

8. A method for synthesizing chiral lactic acid by using a two-carbon compound, which is characterized by comprising the following steps:

inoculating the genetically engineered strain of any one of claims 1-4 into a culture medium for biological fermentation to obtain the chiral lactic acid.

9. The method for synthesizing chiral lactic acid using a two-carbon compound according to claim 8, characterized in that the inoculation amount of the genetically engineered strain is 0.2 to 30OD ₆₀₀ ；

10. The method for synthesizing chiral lactic acid using a two-carbon compound according to claim 8, wherein the two-carbon compound is included in the medium;

the initial addition amount of the two-carbon compound is 5-20 g/L;

preferably, the initial addition amount of the two-carbon compound is 8-12 g/L;

preferably, the two-carbon compound is selected from one or more of ethanol, acetic acid, ethoxide and acetate;

preferably, the two-carbon compound is a mixture of ethanol and acetic acid, and the concentration ratio of the ethanol to the acetic acid in the mixture is 1-2:1;

preferably, the biological fermentation is performed under aerobic conditions;

preferably, the temperature of the biological fermentation is 27-45 ℃;

preferably, the temperature of the biological fermentation is 30-37 ℃;

preferably, the time of the biological fermentation is not less than 24 hours;

preferably, the pH of the culture medium is controlled to be 6-7.5 in the biological fermentation process;

preferably, the chiral lactic acid comprises D-lactic acid and/or L-lactic acid.