Genetic engineering strain for synthesizing polylactic acid by utilizing dicarbon compound as well as construction method and application of genetic engineering strain

Abstract

The invention discloses a genetic engineering strain for synthesizing polylactic acid by utilizing a dicarbon compound as well as a construction method and application of the genetic engineering strain, and belongs to the technical field of genetic engineering. The genetic engineering strain provided by the invention comprises an original strain, an initial expression vector, a promoter and an exogenous gene, the starting strain comprises escherichia coli; the exogenous genes comprise an ethanol dehydrogenase gene, an acetaldehyde dehydrogenase gene, a lactic dehydrogenase gene, a propionyl coenzyme A transferase gene and a polyhydroxyalkanoate synthetase gene. According to the genetic engineering strain provided by the invention, polylactic acid can be obtained by directly fermenting a dicarbon compound, and the defects of low fermentation rate and low yield of a two-step method in the prior art are overcome. The genetic engineering strain constructed by the method disclosed by the invention can effectively improve the yield of polylactic acid in combination with a mixed culture mode of multiple dicarbonic compounds.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, and in particular relates to a genetically engineered strain that synthesizes polylactic acid using a two-carbon compound, its construction method, and its application. Background Technology

[0002] Polylactic acid (PLA) is a commercially available biodegradable plastic. However, because chiral lactic acid is typically prepared using bio-fermentation methods based on sugars, current PLA production is heavily reliant on food crops. PLA preparation methods include chemical and biological methods. Chemical methods suffer from stringent reaction conditions and complex production processes. With the development of synthetic biology, biological methods based on microbial cell factories are gradually emerging as a promising preparation method.

[0003] In our previous patent applications, we constructed an engineered strain, BW-el, capable of efficiently synthesizing chiral lactic acid from two-carbon compounds, as detailed in patent application number 202311047737.4. The construction of this strain lays the foundation for the biomanufacturing of polylactic acid from two-carbon compounds.

[0004] To upgrade chiral lactic acid to polylactic acid (PLA) via biological methods, one strategy is a two-step fermentation process. First, the BW-el strain produces lactic acid from a two-carbon compound. After centrifugation to obtain the supernatant, another strain directly polymerizes it into PLA. However, this two-step process has three inherent problems that limit its efficiency and application potential. First, the biosynthetic pathway from a two-carbon compound to chiral lactic acid is long, resulting in a slow fermentation rate. To accumulate a sufficient concentration of lactic acid, the first fermentation step is forced to be prolonged. Second, lactic acid undergoes a complex process of "intracellular synthesis → secretion to the extracellular space → transport to the intracellular space → intracellular polymerization," involving intercellular transfer. This can easily lead to carbon source loss or incomplete utilization due to intercellular diffusion, thus affecting PLA yield and conversion rate. Third, the entire process includes two independent fermentation stages, a cell isolation step, and necessary culture medium adjustments. This significantly prolongs the overall production cycle and increases operational risks. In contrast, integrating the lactic acid synthesis module and the PLA synthesis module into the same host strain has the potential to solve these problems.

[0005] Currently, there is limited research on polylactic acid (PLA) fermentation using non-sugar substrates, and the yields are relatively low. Tan et al. [Chunlin Tan, Fei Tao, Ping Xu. Direct carbon capture for the production of high-performance biodegradable plastics by cyanobacterial cell factories[J]. Green Chemistry, 2022, 24(11): 4470-4483] constructed a direct conversion from CO2 to PLA using cyanobacteria as substrate cells, but the yield was only 108 mg / L, and the reaction cycle was as long as 10 days under high-density fermentation. Therefore, although integrating the lactic acid synthesis module and the PLA synthesis module into the same substrate cell to achieve one-step production is conceptually ideal, how to achieve high PLA yield remains a key problem that needs to be solved.

[0006] The basic strain BW-el already stably carries two essential plasmids responsible for carbon source assimilation and lactate synthesis, respectively, and its genetic load is already high. Introducing a third plasmid carrying genes for the polymerization pathway would create a complex overloaded system with three plasmids coexisting. This overloaded system is highly susceptible to plasmid loss or expression imbalance due to plasmid incompatibility, origin of replication conflict, or uneven distribution of selection pressure, leading to the collapse of the entire multi-step catalytic pathway. This is a key obstacle affecting the stability of one-step synthesis. Therefore, we selected pJET, a plasmid backbone with a unique replication mechanism and compatibility set, as the vector for the polymerization pathway. The system design fully considers the compatibility and stability of multiple plasmids coexisting, ensuring the long-term, stable coexistence and synergistic operation of the three plasmids within the engineered strain. Furthermore, to promote the efficient expression of two key exogenous genes, we selected two potent constitutive promoters (PlacUV5 and Ptrc-tho) to regulate their transcription, and globally optimized the gene sequences based on host codon preferences, thereby significantly improving the expression efficiency of heterologous proteins and the yield of soluble proteins. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a genetically engineered strain for synthesizing polylactic acid (PLA) from two-carbon compounds, its construction method, and its applications. This genetically engineered strain, by introducing exogenous genes for alcohol dehydrogenase, acetaldehyde dehydrogenase, lactate dehydrogenase, propionyl-CoA transferase, and polyhydroxyalkanoate synthase, enables the direct synthesis of PLA from two-carbon compounds within the same strain. Compared to the two-step method, this method offers higher yields and a faster production rate.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] On the one hand, the present invention provides a genetically engineered strain for synthesizing polylactic acid using a two-carbon compound, the genetically engineered strain comprising a starting strain, an initial expression vector, a promoter, and a foreign gene;

[0010] The starting strain includes Escherichia coli;

[0011] The exogenous genes include alcohol dehydrogenase gene, acetaldehyde dehydrogenase gene, lactate dehydrogenase gene, propionyl-CoA transferase gene, and polyhydroxy fatty acid ester synthase gene.

[0012] Optionally, the initial expression vector includes pRSFduet-1, pACYC184, and pJET.

[0013] Optionally, when the initial expression vector is pRSFduet-1, the promoter includes Ptrc-tho; the nucleotide sequence of Ptrc-tho is shown in SEQ ID No. 1;

[0014] When the initial expression vector is pACYC184, the promoter includes ParaBAD; the nucleotide sequence of ParaBAD is shown in SEQ ID No. 2.

[0015] When the initial expression vector is pJET, the promoter includes PlacUV5 and Ptrc-tho; the nucleotide sequence of PlacUV5 is shown in SEQ ID No. 3. The functional elements of pJET are derived from pJET1.2 and pRB322. Specifically, the ampicillin resistance selection marker and the origin of replication are derived from pJET1.2; the transcription terminator and the low copy number control element are derived from pBR322.

[0016] Optionally, the NCBI accession number for the alcohol dehydrogenase gene is GenBank:WP_012885841;

[0017] The NCBI accession number for the acetaldehyde dehydrogenase gene is GenBank:NP_014032;

[0018] The nucleotide sequence of the lactate dehydrogenase gene is shown in SEQ ID No. 4.

[0019] The nucleotide sequence of the propionyl-CoA transferase gene is shown in SEQ ID No. 5.

[0020] The nucleotide sequence of the polyhydroxy fatty acid ester synthase gene is shown in SEQ ID No. 6.

[0021] Secondly, the present invention provides a method for constructing the above-mentioned genetically engineered strain, comprising the following steps:

[0022] Step 1) The exogenous gene is linked to the initial expression vector and promoter to obtain a recombinant overexpression vector;

[0023] Step 2) The recombinant overexpression vector is transferred into the starting strain to obtain the genetically engineered strain.

[0024] Optionally, in step 2), the recombinant overexpression vector is transferred into competent cells of the starting strain.

[0025] Optionally, after step 2) transforms the recombinant overexpression vector into the starting strain, the step further includes screening with antibiotics.

[0026] Thirdly, the present invention provides the application of the above-mentioned genetically engineered strains in the synthesis of polylactic acid and its derivatives using two-carbon compounds.

[0027] Fourthly, the present invention provides a method for synthesizing polylactic acid using a two-carbon compound, comprising the following steps:

[0028] The above-mentioned genetically engineered strain was inoculated into a culture medium for biological fermentation to obtain the chiral lactic acid.

[0029] Optionally, the inoculum size of the genetically engineered strain is 0.2~30 OD. 600 .

[0030] Preferably, the inoculum size of the genetically engineered strain is 2-30 OD. 600 .

[0031] Optionally, the inoculum size of the genetically engineered strain is independently selected from 0.2 OD. 600 1 OD 600 2 OD 600 5OD 600 10 OD 600 15 OD 600 20 OD 600 25 OD 600 30OD 600 Any value in the range or any value between the two.

[0032] Optionally, the culture medium includes a two-carbon compound;

[0033] The initial addition amount of the two-carbon compound is 5~20 g / L.

[0034] Preferably, the initial addition amount of the two-carbon compound is 8~12 g / L.

[0035] Optionally, the initial addition amount of the two-carbon compound is independently selected from any value of 5 g / L, 8 g / L, 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, or a range between any two.

[0036] Optionally, the two-carbon compound is selected from one or more of ethanol, acetic acid, ethanol salts, and acetate salts.

[0037] Optionally, the two-carbon compound is a mixture of ethanol and acetic acid, wherein the concentration ratio of ethanol to acetic acid in the mixture is 1 to 2:1.

[0038] Optionally, the culture medium may further include one or more of the following: phosphates, sodium salts, inorganic nitrogen compounds, magnesium salts, chelating agents, and trace elements.

[0039] Optionally, the phosphate is selected from one or more of disodium hydrogen phosphate, potassium dihydrogen phosphate, and diammonium hydrogen phosphate.

[0040] Optionally, the sodium salt includes sodium chloride.

[0041] Optionally, the inorganic nitrogen-containing compound is selected from one or more of ammonium chloride, ammonium sulfate, ammonium phosphate, and ammonium bicarbonate.

[0042] Optionally, the magnesium salt includes magnesium sulfate.

[0043] Optionally, the chelating agent includes EDTA.

[0044] Optionally, the trace element is selected from one or more of soluble cobalt salts, manganese salts, copper salts, molybdenum salts, zinc salts, iron salts, and boric acid.

[0045] Optionally, the bio-fermentation is carried out under aerobic conditions.

[0046] Optionally, the temperature of the bio-fermentation is 27~45℃.

[0047] Preferably, the temperature of the bio-fermentation is 30~37℃.

[0048] Optionally, the bio-fermentation time is not less than 24 hours.

[0049] Optionally, the pH of the culture medium is controlled to be 6-7.5 during the bio-fermentation process.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) Compared with the two-step method of two strains in series, the present invention integrates the lactic acid synthesis and polymerization module into a single engineered strain, realizing the direct intracellular conversion from two-carbon compounds to polylactic acid. This eliminates the transmembrane transfer and diffusion loss of intermediate product lactic acid between cells, shortens the metabolic pathway, and enables carbon flow to be directed to the final product polylactic acid more quickly and efficiently, thereby significantly shortening the production cycle and effectively improving the yield and production rate of polylactic acid.

[0052] (2) This invention successfully constructed an engineered strain that stably and efficiently expresses the entire synthetic pathway by overcoming the compatibility issues of the three-plasmid system, employing a strong constitutive promoter, and optimizing the exogenous polymerase gene with host-preferred codons. Compared with existing one-step methods that use non-sugar substrates, have low yields, and long cycles, this invention achieves increased polylactic acid yield and a shorter fermentation cycle. Attached Figure Description

[0053] Figure 1 This provides plasmid information for the initial expression vector and recombinant overexpression vector used in the production of polylactic acid according to this invention.

[0054] Figure 2 Transmission electron microscopy characterization of intracellular polylactic acid in the strain of this invention;

[0055] Figure 3 The 1H NMR spectrum of polylactic acid synthesized in this invention;

[0056] Figure 4 This is the carbon NMR spectrum of polylactic acid synthesized in this invention;

[0057] Figure 5 Time curves for the two-step production of lactic acid and polylactic acid from two-carbon compounds;

[0058] Figure 6 The time curves for the consumption of two-carbon compounds and the yield of lactic acid during one-step polylactic acid fermentation are shown. Detailed Implementation

[0059] The present application is further illustrated below with reference to specific embodiments. The following descriptions are merely a few embodiments of the present application and are not intended to limit the present application in any way. Although the present application discloses preferred embodiments as follows, they are not intended to limit the present application. Any modifications or variations made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

[0060] Unless otherwise specified, the raw materials used in the embodiments of this application are all purchased commercially and used directly without any special treatment.

[0061] Unless otherwise specified, the analytical methods in the embodiments all adopt conventional instrument or equipment settings and conventional analytical methods.

[0062] The strain of Escherichia coli BW25113 used in the following examples was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (https: / / www.mingzhoubio.com / goods-109218.html), with product number BMZ066594.

[0063] Example 1

[0064] Construction of a recombinant bacterial polylactic acid production module:

[0065] A polylactic acid (PLA) production module was constructed in *Escherichia coli* BW25113 to express heterologous propionyl-CoA transferase and polyhydroxyalkanoate synthase. The propionyl-CoA transferase and polyhydroxyalkanoate synthase genes were synthesized via whole-genome synthesis. The propionyl-CoA transferase gene from *Clostridium propionitum* and the polyhydroxyalkanoate synthase gene from *Pseudomonas* were codon-optimized and then cloned into the pJET expression vector, controlled by the PlacUV5 promoter (nucleotide sequence shown in SEQ ID No. 3) and the Ptrc-tho promoter (nucleotide sequence shown in SEQ ID No. 1), respectively, to obtain the recombinant overexpression vector. The plasmid information for the initial expression vector and the recombinant overexpression vector is as follows: Figure 1 As shown. The recombinant overexpression vector was transformed into competent Escherichia coli BW25113 cells, and the correct genetically engineered bacteria were screened with antibiotics to obtain the pla-w strain. Lactic acid, as a substrate, was catalyzed and converted to polylactic acid after entering E. coli, but this strain did not possess the ability to utilize two-carbon compounds and produce lactic acid. The *E. coli* seed culture was inoculated into a fermentation medium with 200 mM lactic acid as the sole carbon source (fermentation medium: 200 mM lactic acid, 6.8 g / L Na₂HPO₄, 3 g / L KH₂PO₄, 0.5 g / L NaCl, 0.25 g / L NH₄Cl, 1.2 g / L MgSO₄; trace elements: 0.0084 g / L EDTA, 0.0025 g / L CoCl₂, 0.015 g / L MnCl₂, 0.0015 g / L CuCl₂, 0.003 g / L H₃BO₃, 0.0025 g / L Na₂MoO₄, 0.008 g / L Zn(CH₃COO)₂, 0.06 g / L Fe(III)citrate), ensuring an initial inoculum size of 2.0 OD₂. 600 Ampicillin and the inducer IPTG were added, and the mixture was cultured aerobically at 200 rpm and 30°C. The transmission electron microscopy characterization of intracellular polylactic acid in strain pla-w is as follows: Figure 2 As shown, the NMR spectrum of biosynthesized polylactic acid is as follows: Figure 3 and Figure 4 As shown. By Figure 2 It is known that polylactic acid accumulates intracellularly in the form of solid particles, a morphological characteristic consistent with observations of intracellular polyester particles in microorganisms in the literature. Analysis of the composition of these solid particles reveals… 1 H NMR spectrum as shown Figure 3 As shown, 1.6 ppm is the resonance peak of -CH3, and 5.2 ppm is the resonance peak of CH in -OCH-; its 13 The C NMR spectrum is as follows Figure 4 As shown, 169.60 ppm is the characteristic peak of -OCO- in polylactic acid, 69.01 ppm is the characteristic peak of -OCH-, and 16.64 ppm is the characteristic peak of -CH3. Therefore, it can be determined that the solid particles inside the strain cells are polylactic acid.

[0066] Comparative Example 1

[0067] Two-step synthesis of polylactic acid from two-carbon compounds

[0068] The strains BW-el (the strain obtained in Example 2 of the patent application with application number 202311047737.4) and pla-w were selected for a two-step fermentation process using two strains in series. The strain BW-el used ethanol and acetic acid as substrates to produce lactic acid, while the strain pla-w used the obtained lactic acid to produce polylactic acid. First, the seed culture of strain BW-el was inoculated into 100 mL of fermentation medium (fermentation medium: 100 mM ethanol, 100 mM acetic acid, 6.8 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 0.25 g / L NH4Cl, 1.2 g / L MgSO4, trace elements: 0.0084 g / L EDTA, 0.0025 g / L CoCl2, 0.015 g / L MnCl2, 0.0015 g / L CuCl2, 0.003 g / L H3BO3, 0.0025 g / L Na2MoO4, 0.008 g / L Zn(CH3COO)2, 0.06 g / L Fe(III)citrate), with an initial OD... 600 The initial OD was set to 2.2, and ampicillin, arabinose (an inducer), and IPTG were added. During chiral lactic acid production, ethanol and acetic acid were added every 48 hours. After chiral lactic acid fermentation was complete, the bacterial culture containing BW-el was centrifuged, the bacterial cells were discarded, and the supernatant was used for subsequent polylactic acid fermentation. Finally, the seed culture of strain pla-w was inoculated into the lactic acid-containing fermentation supernatant, and the initial OD was adjusted. 600 The pH value was 5.0, and polylactic acid (PLA) production began. Among them, strains BW-el and pla-w produced lactic acid and PLA using two-carbon compounds, as shown in the following results. Figure 5As shown. Strain BW-e1 first produced lactic acid via co-substrate fermentation of ethanol and acetic acid, utilizing 327 mM of dicarbon compounds and accumulating 71 mM of lactic acid within 204 h. Then, after centrifugation to remove BW-el cells, strain pla-w utilized a lactic acid-containing medium for polylactic acid (PLA) production, reaching a peak PLA yield of 127 mg / L within 36 h, while consuming 58 mM of lactic acid. The entire fermentation process lasted 240 h, with a production rate from dicarbon chemicals to PLA of 0.1 mg / L / h / OD. 600 .

[0069] Example 2

[0070] Construction of a pathway for recombinant bacteria to produce polylactic acid from two-carbon compounds:

[0071] A polylactic acid (PLA) production module was constructed in strain BW-el (the strain obtained in Example 2 of patent application No. 202311047737.4) to express heterologous propionyl-CoA transferase and polyhydroxyalkanoate synthase. The propionyl-CoA transferase gene and the polyhydroxyalkanoate synthase gene were synthesized via whole-genome synthesis. The propionyl-CoA transferase gene from *Clostridium propionitum* and the polyhydroxyalkanoate synthase gene from *Pseudomonas* were codon-optimized and then cloned into the pJET expression vector, controlled by the PlacUV5 promoter (nucleotide sequence shown in SEQ ID No. 3) and the Ptrc-tho promoter (nucleotide sequence shown in SEQ ID No. 1), respectively, to obtain the recombinant overexpression vector. The plasmid information of the initial expression vector and the recombinant overexpression vector is as follows: Figure 1 As shown. The recombinant overexpression vector was transformed into competent cells of strain BW-el, and the correct genetically engineered bacteria were screened with antibiotics to obtain strain BW-elP. The two-carbon compound, as a substrate, was catalyzed and converted into polylactic acid after entering *E. coli*. The *E. coli* seed culture was inoculated into a fermentation medium with ethanol and acetic acid as carbon sources (fermentation medium: 100 mM ethanol and 100 mM acetic acid, 6.8 g / L Na₂HPO₄, 3 g / L KH₂PO₄, 0.5 g / L NaCl, 0.25 g / L NH₄Cl, 1.2 g / L MgSO₄, trace elements: 0.0084 g / L EDTA, 0.0025 g / L CoCl₂, 0.015 g / L MnCl₂, 0.0015 g / L CuCl₂, 0.003 g / L H₃BO₃, 0.0025 g / L Na₂MoO₄, 0.008 g / L Zn(CH₃COO)₂, 0.06 g / L Fe(III)citrate), ensuring an initial inoculum size of 2.0 OD₂. 600The appropriate antibiotics and inducers were added, and the mixture was cultured aerobically at 200 rpm and 30°C. The fermentation results of strain BW-elP, which utilizes two-carbon compounds to produce polylactic acid, are as follows: Figure 6 As shown, strain BW-elP consumed 76 mM ethanol and 52 mM sodium acetate within 72 h, ultimately achieving a polylactic acid (PLA) yield of 212 mg / L. Notably, this single-strain system achieved a PLA production rate of 1.5 mg / L / h / OD. 600 Table 1 shows a comparison between the one-step and two-step methods. Compared with the two-step method, the yield of the single-strain fermentation system of strain BW-elP increased by 1.7 times and the production rate increased by 15 times.

[0072] Table 1 Comparison of one-step and two-step methods for the synthesis of polylactic acid from two-carbon compounds.

[0073]

[0074] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A genetically engineered bacterial strain for synthesizing polylactic acid using a two-carbon compound, characterized by comprising a polylactic acid synthase gene and a polylactic acid synthase gene. The genetic engineering strain comprises a starting strain, an initial expression vector, a promoter and an exogenous gene; The starting strain comprises Escherichia coli; The exogenous gene comprises an alcohol dehydrogenase gene, an acetaldehyde dehydrogenase gene, a lactate dehydrogenase gene, a propionyl-CoA transferase gene and a polyhydroxyalkanoate synthase gene; The initial expression vector comprises pRSFduet-1, pACYC184 and pJET.

2. The genetically engineered strain for synthesizing polylactic acid using a two-carbon compound according to claim 1, wherein the polylactic acid is produced by the genetically engineered strain. The functional elements of the pJET are derived from pJET1.2 and pBR322; wherein the ampicillin resistance selection marker and the replication origin are taken from pJET1.2; the transcription terminator and the low copy number control element are taken from pBR322.

3. The genetically engineered strain for synthesizing polylactic acid using a two-carbon compound according to claim 1, wherein the polylactic acid is produced by the genetically engineered strain. When the initial expression vector is pRSFduet-1, the promoter comprises Ptrc-tho; the nucleotide sequence of the Ptrc-tho is shown as SEQ ID No. 1; When the initial expression vector is pACYC184, the promoter comprises ParaBAD; the nucleotide sequence of the ParaBAD is shown as SEQ ID No. 2; When the initial expression vector is pJET, the promoter comprises PlacUV5 and Ptrc-tho; The nucleotide sequence of the PlacUV5 is shown as SEQ ID No.

3.

4. The genetically engineered strain for synthesizing polylactic acid using a two-carbon compound according to claim 1, wherein the polylactic acid is produced by the genetically engineered strain. The NCBI accession number of the alcohol dehydrogenase gene is GenBank: WP_012885841; The NCBI accession number of the acetaldehyde dehydrogenase gene is GenBank: NP_014032; The nucleotide sequence of the lactate dehydrogenase gene is shown as SEQ ID No. 4; The nucleotide sequence of the propionyl-CoA transferase gene is shown as SEQ ID No. 5; The nucleotide sequence of the polyhydroxyalkanoate synthase gene is shown as SEQ ID No.

6.

5. The method for constructing a genetically engineered strain for synthesizing polylactic acid using dicarbon compound according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: Step 1) connecting the exogenous gene with the initial expression vector and the promoter to obtain a recombinant overexpression vector; Step 2) transforming the recombinant overexpression vector into the starting strain to obtain the genetic engineering strain.

6. The construction method of claim 5, wherein, Step 2) transforming the recombinant overexpression vector into the competent cells of the starting strain; after step 2) transforming the recombinant overexpression vector into the starting strain, a step of screening with an antibiotic is further included.

7. The genetic engineering strain for synthesizing polylactic acid from two-carbon compounds according to any one of claims 1-4, is used for synthesizing polylactic acid and its derivatives from two-carbon compounds.

8. Use according to claim 7, characterized in that, The method comprises the following steps: inoculating the genetic engineering strain into a culture medium for biological fermentation to obtain chiral lactic acid.

9. Use according to claim 8, characterized in that, The inoculation amount of the genetically engineered strain is 0.2-30 OD 600 The initial addition amount of the two-carbon compound in the culture medium is 5-20 g / L; and the two-carbon compound is selected from one or more than two kinds of mixture of ethanol, acetic acid, ethanol salt and acetic acid salt.

10. Use according to claim 9, characterized in that, The two-carbon compound is a mixture of ethanol and acetic acid, and the concentration ratio of ethanol to acetic acid in the mixture is 1-2:1; The culture medium further comprises one or more than two kinds of mixtures of phosphate, sodium salt, inorganic nitrogen-containing compound, magnesium salt, chelating agent and trace element; The biological fermentation is carried out under aerobic conditions; The temperature of the biological fermentation is 27-45℃; The time of the biological fermentation is not less than 24h; The pH of the culture medium is controlled to be 6-7.5 during the biological fermentation.

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