Succinic acid-producing genetically engineered Saccharomyces cerevisiae, their construction methods and applications

Abstract

This invention discloses a genetically engineered *Saccharomyces cerevisiae* strain that produces succinic acid, its construction method, and its applications. Using *Saccharomyces cerevisiae* as the starting strain, a series of genes involved in byproduct metabolic pathways were knocked out to obtain *Saccharomyces cerevisiae* genetically engineered strain HM1, which can achieve anaerobic fermentation for succinic acid production while reducing byproduct generation. Based on *Saccharomyces cerevisiae* genetically engineered strain HM1, overexpression of the pyruvate carboxylase gene pyc2, the fumarate reductase gene frd, the *Saccharomyces cerevisiae* mitochondrial citrate transporter Yhm2, and the *Schizosaccharomyces cerevisiae* malate transporter Spmae1 resulted in *Saccharomyces cerevisiae* genetically engineered strain HM2 with increased succinic acid production. This strain, through anaerobic domestication and screening, yielded *Saccharomyces cerevisiae* genetically engineered strain HM3. This strain can accumulate succinic acid at 21 g / L under anaerobic conditions with low levels of byproducts, effectively reducing carbon flux loss and achieving high succinic acid production through anaerobic fermentation, demonstrating industrial application value and potential.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a genetically engineered Saccharomyces cerevisiae that produces succinic acid, its construction method, and its application. Background Technology

[0002] Succinic acid, scientifically known as succinic acid, is a tetracarboxylic acid in the tricarboxylic acid cycle. It is primarily used as a surfactant, ion chelating agent, and food additive. It is also used in the production of biodegradable materials such as polyurethane and polybutylene succinate, and has a wide range of applications in the pharmaceutical, food, chemical, and agricultural industries, with substantial market demand. In 2004, the U.S. Department of Energy ranked succinic acid as the most promising bulk bio-based chemical in a report.

[0003] Currently, the main technologies for the industrial production of succinic acid include catalytic hydrogenation, electrolysis, and bio-fermentation. Catalytic hydrogenation and electrolysis are both chemical synthesis methods using maleic anhydride as a raw material. However, due to the depletion of petroleum resources and increasingly severe environmental pollution, the drawbacks of chemical synthesis methods are becoming increasingly apparent. Compared with chemical synthesis, bio-fermentation has advantages such as a wide availability of raw materials, low cost, renewability, mild reaction conditions, and low carbon footprint, thus showing promising development prospects.

[0004] Currently, succinic acid fermentation strains are divided into two main categories. The first category consists of naturally occurring succinic acid-producing strains, such as Actinobacillus, anaerobic spirochetes, and Mannheim bacteria. The second category comprises engineered strains modified through metabolic processes, primarily Escherichia coli and yeast. While naturally occurring succinic acid-producing strains can produce high levels of succinic acid, they have significant drawbacks, such as insensitivity to acid and oxygen, reliance on glucose as a carbon source, and high cultivation costs, limiting their large-scale industrial production. Engineered E. coli strains exhibit lower fermentation efficiency, are intolerant to acid, and produce byproducts such as lactic acid, formic acid, acetic acid, and ethanol during fermentation. Furthermore, they suffer from metabolic imbalances due to factors such as cofactor metabolism imbalances, intolerance to high product concentrations, high substrate glucose concentrations, high osmotic pressure, and excessively rapid glucose absorption and utilization. Currently, engineered lipolytic yeast can achieve high succinic acid production of up to 110 g / L through fed-batch fermentation. However, lipolytic yeast is a strictly aerobic bacterium and utilizes expensive glycerol as a raw material, resulting in high production costs. As a eukaryotic model microorganism, *Saccharomyces cerevisiae* possesses characteristics such as a clear genetic background, ease of operation, tolerance to low pH and high substrate concentrations, simple nutritional requirements, low cost, and facultative anaerobic properties. Currently, through genetic engineering, *Saccharomyces cerevisiae* can achieve a yield of 43 g / L under aerobic fed-batch fermentation conditions, demonstrating that it is a potentially optimal microorganism for the fermentation production of succinic acid.

[0005] However, the production of succinic acid using genetically engineered Saccharomyces cerevisiae faces several challenges. Oxaloacetic acid is synthesized by the pyruvate carboxylase gene pyc2, but during anaerobic fermentation, it is degraded by PCK to release ATP and promote cell growth. Therefore, oxaloacetic acid production needs strict regulation to ensure both succinic acid accumulation during the TCA cycle and the provision of ATP for cell growth during anaerobic fermentation. Consequently, it is necessary to combine molecular genetic techniques with traditional microbial breeding methods to modify the starting Saccharomyces cerevisiae strain and develop an engineered Saccharomyces cerevisiae strain capable of high-yield succinic acid production through anaerobic fermentation while minimizing byproduct accumulation. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention uses *Saccharomyces cerevisiae* as the starting strain. By knocking out genes that produce byproducts ethanol, glycerol, and acetic acid, enhancing the supply of oxaloacetic acid, strengthening the metabolic flux of the succinate reduction pathway, and accelerating the efflux and secretion of succinate, a genetically engineered *Saccharomyces cerevisiae* strain capable of anaerobic fermentation to produce succinate was obtained. Further domestication and screening of this genetically engineered *Saccharomyces cerevisiae* strain yielded a genetically engineered *Saccharomyces cerevisiae* strain capable of anaerobic fermentation with high succinate production, which has significant industrial application value.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for constructing a genetically engineered Saccharomyces cerevisiae strain that produces succinic acid, wherein the Saccharomyces cerevisiae strain is modified by one or more of the following (1)-(8):

[0009] (1) Knock out the alcohol dehydrogenase gene adh1;

[0010] (2) Knock out the glycerol-3-phosphate dehydrogenase gene gpd1;

[0011] (3) Knock out the acetyl-CoA hydrolase gene ach1;

[0012] (4) Knock out the succinate dehydrogenase gene sdh5;

[0013] (5) Overexpression of the pyruvate carboxylase gene pyc2;

[0014] (6) Overexpression of the fumarate reductase gene frd;

[0015] (7) Overexpression of Yhm2, a mitochondrial citrate transporter in Saccharomyces cerevisiae;

[0016] (8) Overexpression of Spmae1 malic acid transporter in Schizosaccharomyces cerevisiae.

[0017] In one embodiment of the present invention, the Saccharomyces cerevisiae strain is modified in the following ways (1)-(4): by knocking out the alcohol dehydrogenase gene adh1 to reduce the production of ethanol under high sugar concentration fermentation, knocking out the glycerol 3-phosphate dehydrogenase gene gpd1 to reduce the production of the byproduct glycerol, knocking out the acetyl-CoA hydrolase gene ach1 to eliminate the effect of acetic acid accumulation leading to slower cell growth and reduce carbon flow loss, and knocking out the succinate dehydrogenase gene sdh5 to block the downstream metabolism of the succinate oxidation pathway.

[0018] In one embodiment of the present invention, the Saccharomyces cerevisiae strain is modified in the following ways (1)-(6): by knocking out the alcohol dehydrogenase gene adh1 to reduce the production of ethanol under high sugar concentration fermentation, knocking out the glycerol 3-phosphate dehydrogenase gene gpd1 to reduce the production of the byproduct glycerol, knocking out the acetyl-CoA hydrolase gene ach1 to eliminate the effect of acetic acid accumulation leading to slower cell growth and reduce carbon flow loss, knocking out the succinate dehydrogenase gene sdh5 to block the downstream metabolism of the succinate oxidation pathway, overexpressing the pyruvate carboxylase gene pyc2 to increase the supply of upstream oxaloacetate, and overexpressing the fumarate reductase gene frd from Trypanosoma brucellis to enhance the metabolic flux of the succinate reduction pathway.

[0019] In one embodiment of the present invention, the *Saccharomyces cerevisiae* strain is modified in the following ways (1)-(8): by knocking out the alcohol dehydrogenase gene adh1 to reduce ethanol production under high sugar concentration fermentation, knocking out the glycerol 3-phosphate dehydrogenase gene gpd1 to reduce the production of the byproduct glycerol, knocking out the acetyl-CoA hydrolase gene ach1 to eliminate the effect of acetic acid accumulation leading to slower cell growth and reduce carbon flow loss, knocking out the succinate dehydrogenase gene sdh5 to block the downstream metabolism of the succinate oxidation pathway, overexpressing the pyruvate carboxylase gene pyc2 to increase the supply of upstream oxaloacetic acid, overexpressing the fumarate reductase frd gene from *Brucella brucella* to enhance the metabolic flux of the succinate reduction pathway, and overexpressing the *Saccharomyces cerevisiae* mitochondrial citrate transporter Yhm2 and the *Schizosaccharomyces cerevisiae* malate transporter Spmae1 to facilitate the efflux and secretion of succinate.

[0020] In one embodiment of the present invention, the nucleotide sequence of the alcohol dehydrogenase gene adh1 has the gene accession number Gene ID: 854068 (SEQ ID NO: 1); the nucleotide sequence of the 3-glycerol phosphate dehydrogenase gene gpd1 has the gene accession number Gene ID: 851539 (SEQ ID NO: 2); the nucleotide sequence of the acetyl-CoA hydrolase gene ach1 has the gene accession number Gene ID: 852266 (SEQ ID NO: 3); the nucleotide sequence of the succinate dehydrogenase gene sdh5 has the gene accession number Gene ID: 854083 (SEQ ID NO: 4); the pyruvate carboxylase gene pyc2 is derived from Saccharomyces cerevisiae, and its nucleotide sequence has the gene accession number Gene ID: 852519 (amino acid sequence shown in SEQ ID NO: 5); the fumarate reductase gene frd is derived from Trypanosoma brucellae, and its accession number is GenBank: ALM30213.1 (amino acid sequence shown in SEQ ID NO: 5). The FRD amino acid sequence (shown in SEQ ID NO: 6) was obtained by codon optimization to suit the preferences of Saccharomyces cerevisiae; the mitochondrial citrate transporter Yhm2 was derived from Saccharomyces cerevisiae, and its nucleotide sequence was obtained by Gene ID: 855282 (amino acid sequence shown in SEQ ID NO: 7); the malate transporter Spmae1 was derived from Schizosaccharomyces cerevisiae, and its nucleotide sequence was obtained by Gene ID: 254334 (amino acid sequence shown in SEQ ID NO: 8).

[0021] Secondly, the present invention provides a succinic acid-producing Saccharomyces cerevisiae genetically engineered strain prepared using the above-described construction method.

[0022] In one embodiment of the present invention, the strain of Saccharomyces cerevisiae BY4741 was modified by the above (1)-(4) and is denoted as Saccharomyces cerevisiae genetically engineered strain HM1.

[0023] In one embodiment of the present invention, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae BY4741, and its genotype is MATa his3Δ1leu2 met15Δura3-52.

[0024] In one embodiment of the present invention, the upper and lower homologous arms of the alcohol dehydrogenase gene adh1, glycerol 3-phosphate dehydrogenase gene gpd1, acetyl-CoA hydrolase gene ach1, and succinate dehydrogenase gene sdh5 knockout elements are obtained by PCR amplification using the Saccharomyces cerevisiae BY4741 genome as a template and the selection marker using the pYES2 plasmid as a template.

[0025] In one embodiment of the present invention, the strain of Saccharomyces cerevisiae BY4741 was modified by the above (1)-(8) and is denoted as Saccharomyces cerevisiae genetically engineered strain HM2.

[0026] In one embodiment of the present invention, the overexpression of the pyruvate carboxylase gene pyc2 is achieved by introducing the plasmid pESC-HIS-PYC2 containing pyc2 into the genetically engineered Saccharomyces cerevisiae HM1.

[0027] In one embodiment of the present invention, the method for introducing the pyruvate carboxylase gene pyc2 includes the following steps: fusion of constitutive promoter PGK1 with pyc2, linearization of pESC-HIS plasmid by double enzyme digestion, transformation of the linearized plasmid and the fused sequence into DH5α by one-step cloning, selection of transformants for PCR verification, inoculation of correctly sequenced transformants into bacteria, extraction of plasmid pESC-HIS-pyc2, and introduction of the plasmid into the Saccharomyces cerevisiae HM1 genetically engineered bacteria.

[0028] In one embodiment of the present invention, the overexpression of the fumarate reductase gene frd is achieved by introducing a plasmid containing the fumarate reductase gene frd into the genetically engineered Saccharomyces cerevisiae HM1, wherein the plasmid is pESC-HIS-PYC2-FRD.

[0029] In one embodiment of the present invention, the method for introducing the fumarate reductase gene frd includes the following steps: codon optimization is performed based on the FRD amino acid sequence of Trypanosoma brucellosa (accession number GenBank: ALM30213.1) to optimize it to the codon preference of Saccharomyces cerevisiae, and the gene TDH3-frd-TPI1 is routinely synthesized, 5' (XmaI) and 3' (SalI) are added, the gene is cloned into the vector pESC-HIS-PYC2 plasmid through 5' (XmaI) and 3' (SalI), mini-scale recombinant plasmid DNA and skeletal bacteria containing the recombinant plasmid are prepared, and the plasmid is extracted, which is pESC-HIS-PYC2-FRD expressing frd under the TDH3 strong promoter.

[0030] In one embodiment of the present invention, the overexpression of the Saccharomyces cerevisiae mitochondrial citrate transporter Yhm2 and the Saccharomyces cerevisiae malate transporter Spmae1 is achieved by introducing a plasmid containing the genes Yhm2 and Spmae1 into the Saccharomyces cerevisiae HM1 genetically engineered strain. The plasmid is pESC-HIS-PYC2-FRD-Yhm2-Spmae1.

[0031] In one embodiment of the present invention, the method for introducing the Yhm2 and Spmae1 genes includes the following steps: constructing a pESC-HIS-PYC2-FRD-Yhm2-Spmae1 plasmid expressing Yhm2 under the FBA1 promoter and Spmae1 under the ENO1 promoter, and introducing the plasmid into the Saccharomyces cerevisiae genetically engineered strain HM1, and the constructed strain is denoted as Saccharomyces cerevisiae genetically engineered strain HM2.

[0032] Thirdly, the present invention provides a succinic acid-producing Saccharomyces cerevisiae genetically engineered strain HM3, which was deposited at the China Center for Type Culture Collection on October 31, 2022, with accession number CCTCC NO:M 20221705, classified as Saccharomyces cerevisiae, and deposited at Wuhan University, Wuhan, Hubei, China.

[0033] Fourthly, the present invention provides the application of the above-mentioned succinic acid-producing Saccharomyces cerevisiae genetically engineered strains or Saccharomyces cerevisiae genetically engineered strains HM1 or HM2 or HM3 in the production of succinic acid.

[0034] In one embodiment of the present invention, the method for producing succinic acid includes: fermenting and culturing the above-mentioned succinic acid-producing Saccharomyces cerevisiae genetically engineered strain or Saccharomyces cerevisiae genetically engineered strain HM1 or Saccharomyces cerevisiae genetically engineered strain HM2 or Saccharomyces cerevisiae genetically engineered strain HM3 on a fermentation medium.

[0035] In one embodiment of the present invention, the fermentation culture method is as follows: the seed culture of the above-mentioned succinic acid-producing Saccharomyces cerevisiae genetically engineered strain or Saccharomyces cerevisiae HM1 or Saccharomyces cerevisiae HM2 or Saccharomyces cerevisiae HM3 is inoculated into the fermentation medium at an inoculation rate of 3-6%, and fermented at 28-32℃ and 180-240rpm for 65-80h, with the pH controlled at 6.0-7.0 during the fermentation process.

[0036] In one embodiment of the present invention, the fermentation culture method includes the following steps: transferring 5% of the seed culture of Saccharomyces cerevisiae genetically engineered bacteria cultured at 30°C and 220 rpm for 20 h into the fermentation medium and culturing it at 30°C and 220 rpm.

[0037] In one embodiment of the invention, the fermentation broth is transferred to a new fermentation medium every 24 hours to bring the initial OD600 to 0.1.

[0038] In one embodiment of the present invention, the fermentation process uses 5M NaHCO3 to control the pH at 7.0.

[0039] In one embodiment of the present invention, the fermentation medium is: glucose 50g / L, YNB 6.7g / L, DOSupplement-HIS 1.29g / L, potassium dihydrogen phosphate 10g / L, magnesium sulfate 5g / L, NaHCO3 8.4g / L, trace element storage solution 8ml / L, and vitamin storage solution 8ml / L.

[0040] The trace element storage solution consists of: EDTA 15 g·L -1 ZnSO4·7H2O 4.5g·L -1 MnCl2·2H2O 0.84 g·L -1 CoCl2·6H2O 0.30g·L -1 CuSO4·5H2O 0.30 g·L -1 Na₂MoO₄·2H₂O 0.40 g·L⁻¹ -1 CaCl2·2H2O 4.50 g·L -1 FeSO4·7H2O 3.00 g·L -1 H3BO3 1.00g·L -1 KI 0.10 g·L -1 .

[0041] The vitamin storage solution contains: Biotin (D-)(C 10 H 16 N2O3S) 0.05 g·L -1 Calcium pantothenate D(+)(C 18 H 32 CaN2O 10 1.00 g·L -1 Nicotinic acid (C6H5NO2) 1.00 g·L -1 Inositol (C6H) 12 O6) 25.00g·L -1 Thiamine chloride hydrochloride (C 12 H 18 C l2 (N4OS×H2O) 1.0 g·L -1 pyridoxine hydrochloride (C8H) 12 ClNO3) 1.00 g·L -1 p-Aminobenzoic acid (C7H7NO2) 0.20 g·L -1 .

[0042] In one embodiment of the present invention, the fermentation medium is: glucose 20g / L, yeast extract 10g / L, and peptone 20g / L.

[0043] In one embodiment of the present invention, the Saccharomyces cerevisiae genetically engineered strain seed liquid is obtained by inoculating a single colony of fresh Saccharomyces cerevisiae genetically engineered strain into a seed culture medium containing 10 mL, and then culturing at 30°C and 220 rpm for 18 h with shaking.

[0044] In one embodiment of the present invention, the seed culture medium is YPD: glucose 20g / L, yeast extract 10g / L, peptone 20g / L, and water as the solvent.

[0045] In one embodiment of the present invention, the seed culture medium is SD-HIS: glucose 20g / L, YNB 6.7g / L, DO Supplement-HIS 1.29g / L.

[0046] Compared with existing technologies, this invention has the following beneficial technical effects: Using *Saccharomyces cerevisiae* as the starting strain, this invention knocks out a series of genes involved in byproduct metabolic pathways, overexpresses genes related to enhanced succinic acid metabolic flux, and identifies transport proteins that facilitate succinic acid efflux and secretion. Furthermore, through metabolic evolution technology, this invention, for the first time, obtains a *Saccharomyces cerevisiae* strain capable of producing succinic acid under anaerobic conditions, exhibiting industrial practicality. This strain can accumulate succinic acid up to 21 g / L under anaerobic conditions, demonstrating a high conversion rate, effectively reducing carbon flux loss, and producing extremely low byproduct yields. This invention has extremely high industrial application value. Attached Figure Description

[0047] Figure 1 The flowchart shows the preparation process of the knockout cassette, where: (a) is overlap PCR amplification; and (b) is a two-step homologous recombination method.

[0048] Figure 2 Diagram showing the construction of the pESC-HIS-PYC2-FRD-Yhm2-Spmae1 overexpression plasmid;

[0049] Figure 3 The images show the cell growth and succinic acid accumulation results of the genetically engineered Saccharomyces cerevisiae HM3, where: (a) is a cell growth diagram of metabolic evolution, and (b) is a succinic acid accumulation diagram.

[0050] Figure 4 The liquid chromatograms show the accumulation of succinic acid by anaerobic fermentation of the genetically engineered Saccharomyces cerevisiae HM3, where: (a) is the succinic acid standard; (b) is the succinic acid produced by the genetically engineered Saccharomyces cerevisiae HM3. Detailed Implementation

[0051] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims, and those skilled in the art will clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical solutions of the present invention, and such modifications and changes are also included within the scope of the present invention.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents and materials used are commercially available unless otherwise specified.

[0053] I. Experimental Materials

[0054] 1. The strain and plasmid information constructed in this invention are as follows:

[0055] Saccharomyces cerevisiae BY4741, with genotype MATa his3Δ1leu2 met15Δura3-52, was purchased from Beina Biotechnology - Henan Provincial Industrial Microbial Strains Engineering Technology Research Center.

[0056] pESC-URA, containing the URA3 screening marker, catalog number P0236, was purchased from Miaoling Biotechnology (Miaoling Plasmid Platform).

[0057] pESC-HIS, containing HIS screening markers, catalog number P1269, purchased from Miaoling Biotechnology (Miaoling Plasmid Platform).

[0058] The culture medium used in this invention is as follows:

[0059] SD-URA liquid medium: glucose 20 g / L, YNB 6.7 g / L, DO Supplement-URA 1.29 g / L.

[0060] SD-URA plates: glucose 20g / L, YNB 6.7g / L, DO Supplement-URA 1.29g / L, agar powder 20g / L.

[0061] YPD plates: glucose 20g / L, peptone 20g / L, yeast extract 10g / L, agar powder 20g / L;

[0062] YPD liquid medium: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L;

[0063] SD-HIS liquid culture medium: glucose 20 g / L, YNB 6.7 g / L, DO Supplement-HIS 1.29 g / L.

[0064] SD-HIS plate: glucose 20g / L, YNB 6.7g / L, DO Supplement-HIS 1.29g / L, agar powder 20g / L.

[0065] LB liquid medium: 10 g / L tryptone -1 5g·L yeast extract -1 10 g·L sodium chloride -1 pH 7.0;

[0066] LB plates: Add 20 g / L agar powder to LB liquid medium. -1 ;

[0067] 3. Information on the primers used in this invention is shown in Table 1.

[0068] Table 1 Primers used in this invention

[0069]

[0070]

[0071]

[0072]

[0073] Example 1: Construction of the genetically engineered Saccharomyces cerevisiae HM1

[0074] Using Saccharomyces cerevisiae BY4741 as the starting strain, a two-step homologous recombination method was employed to knock out the alcohol dehydrogenase gene adh1, thereby reducing ethanol production under high sugar concentration fermentation. The glycerol 3-phosphate dehydrogenase gene gpd1 was knocked out to reduce the production of the byproduct glycerol. The acetyl-CoA hydrolase gene ach1 was knocked out to eliminate the effect of acetic acid accumulation leading to slower cell growth and reduce carbon flux loss. Furthermore, the succinate dehydrogenase gene sdh5 was knocked out to block the downstream metabolism of the succinate oxidation pathway.

[0075] 1.1 Construction of gene knockout cassette

[0076] 1.1.1 Construction of the adh1 knockout box

[0077] 1) Using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair ADH1-UP-F / ADH1-UP-R, and verified by agarose gel electrophoresis, yielding a single target band ADH1-UP; using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair ADH1-DN-F / ADH1-DN-R, and verified by agarose gel electrophoresis, yielding a single target band adh1-DN; using plasmid pESC-URA as a template, PCR amplification was performed using primer pair URA-F1 / URA-R1, and verified by agarose gel electrophoresis, yielding a single target band URA3.

[0078] The amplification system consisted of: 25 μl of 2×Phanta Max Buffer (Vazyme), 1 μl of dNTPs (10 mM each), 20 ng of DNA template, 2 μl of primers (10 μM each), 1 μl of Phanta Max Super-Fidelity DNA polymerase (2.5 U / μl), and 20 μl of distilled water, for a total volume of 50 μl.

[0079] The amplification conditions were: 95℃ pre-denaturation for 3 minutes (1 cycle); 95℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, and 72℃ extension for 1 minute (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0080] 2) After purifying and recovering the above single target bands ADH1-UP, ADH1-DN and URA3, PCR fusion was performed using the mixture of ADH1-UP, ADH1-DN and URA3 as a common template with primers ADH1-UP-F / ADH1-DN-R. The fusion product was then purified and sent for sequencing verification to obtain the adh1 knockout cassette.

[0081] 1.1.2 Construction of the gpd1 knockout box

[0082] 1) Using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair GPD1-UP-F / GPD1-UP-R, and verified by agarose gel electrophoresis, yielding a single target band GPD1-UP; using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair GPD1-DN-F / GPD1-DN-R, and verified by agarose gel electrophoresis, yielding a single target band gpd1-DN; using plasmid pESC-URA as a template, PCR amplification was performed using primer pair URA-F1 / URA-R1, and verified by agarose gel electrophoresis, yielding a single target band URA3.

[0083] The amplification system consisted of: 25 μl of 2×Phanta Max Buffer (Vazyme), 1 μl of dNTPs (10 mM each), 20 ng of DNA template, 2 μl of primers (10 μM each), 1 μl of Phanta Max Super-Fidelity DNA polymerase (2.5 U / μl), and 20 μl of distilled water, for a total volume of 50 μl.

[0084] The amplification conditions were: 95℃ pre-denaturation for 3 minutes (1 cycle); 95℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, and 72℃ extension for 1 minute (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0085] 2) After purifying and recovering the above single target bands GPD1-UP, GPD1-DN and URA3, PCR fusion was performed using the mixture of GPD1-UP, GPD1-DN and URA3 as a common template with primers GPD1-UP-F / GPD1-DN-R. The fusion product was then purified and sent for sequencing verification to obtain the gpd1 knockout cassette.

[0086] 1.1.3 Construction of the ach1 knockout box

[0087] 1) Using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair ACH1-UP-F / ACH1-UP-R, and verified by agarose gel electrophoresis, yielding a single target band ACH1-UP; using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair ACH1-DN-F / ACH1-DN-R, and verified by agarose gel electrophoresis, yielding a single target band ACH1-DN; using plasmid pESC-URA as a template, PCR amplification was performed using primer pair URA-F1 / URA-R1, and verified by agarose gel electrophoresis, yielding a single target band URA3.

[0088] The amplification system consisted of: 25 μl of 2×Phanta Max Buffer (Vazyme), 1 μl of dNTPs (10 mM each), 20 ng of DNA template, 2 μl of primers (10 μM each), 1 μl of Phanta Max Super-Fidelity DNA polymerase (2.5 U / μl), and 20 μl of distilled water, for a total volume of 50 μl.

[0089] The amplification conditions were: 95℃ pre-denaturation for 3 minutes (1 cycle); 95℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, and 72℃ extension for 1 minute (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0090] 2) After purifying and recovering the above single target bands ACH1-UP, ACH1-DN and URA3, PCR fusion was performed using the mixture of ACH1-UP, ACH1-DN and URA3 as a common template with primers ACH1-UP-F / ACH1-DN-R. The fusion product was then purified and sent for sequencing verification to obtain the ach1 knockout cassette.

[0091] 1.1.4 Construction of the sdh5 knockout box

[0092] 1) Using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair SDH5-UP-F / SDH5-UP-R, and verified by agarose gel electrophoresis, yielding a single target band SDH5-UP; using the genome of *Saccharomyces cerevisiae* BY4741 as a template, PCR amplification was performed using primer pair SDH5-DN-F / SDH5-DN-R, and verified by agarose gel electrophoresis, yielding a single target band SDH5-DN; using plasmid pESC-URA as a template, PCR amplification was performed using primer pair URA-F1 / URA-R1, and verified by agarose gel electrophoresis, yielding a single target band URA3.

[0093] The amplification system consisted of: 25 μl of 2×Phanta Max Buffer (Vazyme), 1 μl of dNTPs (10 mM each), 20 ng of DNA template, 2 μl of primers (10 μM each), 1 μl of Phanta Max Super-Fidelity DNA polymerase (2.5 U / μl), and 20 μl of distilled water, for a total volume of 50 μl.

[0094] The amplification conditions were: 95℃ pre-denaturation for 3 minutes (1 cycle); 95℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, and 72℃ extension for 1 minute (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0095] 2) After purifying and recovering the above single target bands SDH5-UP, SDH5-DN and URA3, PCR fusion was performed using the mixture of SDH5-UP, SDH5-DN and URA3 as a common template with primers SDH5-UP-F / SDH5-DN-R. The fusion product was then purified and sent for sequencing verification to obtain the sdh5 knockout cassette.

[0096] 1.2 Transformation

[0097] 1.2.1 Construction of BY4741-Δadh1 strain

[0098] The adh1 knockout box was used to transform Saccharomyces cerevisiae BY4741 using the LiAc transformation method. The resulting transformants were plated on SD-URA plates and incubated upside down at 30°C for 2 days. Transformants were picked and verified by PCR using primers ADH1-YZ-F / ADH1-YZ-R. Successfully verified PCR products were sent for sequencing. Positive transformants with correct sequencing results were cultured in SD-URA liquid medium for preservation. To recover the URA selection marker, the genome of positive transformants was extracted as a template. PCR amplification was performed using primer pairs ADH1-UP-F / URA-UP1-R and URA-DN1-F / ADH1-DN-R, with a 20bp overlap between the primers. The resulting fragments were URA-UP1 and URA-DN1. After purification, the fragments were fused using primers ADH1-UP-F / ADH1-DN-R. The fusion product was purified, and after successful sequencing, it was transferred to the positive transformants. Reverse screening was performed using YPD plates containing 5-FOA (final concentration 1.0 g / L). Transformants were picked and verified by PCR using primers ADH1-YZ-F / ADH1-YZ-R. After successful sequencing, the transformants were inoculated in YPD liquid medium and cultured at 30℃ and 180-200 rpm for 16-18 h. The resulting bacteria were the URA-recovered and adh1-deficient strain, denoted as BY4741-Δadh1.

[0099] 1.2.2 Construction of BY4741-Δadh1-Δgpd1 strain

[0100] Based on the deletion of the adh1 gene, the gpd1 knockout cassette was transformed into BY4741-Δadh1. The resulting transformed bacterial culture was plated on SD-URA plates and incubated upside down in a 30°C incubator for 2 days. Transformants were picked and PCR was performed using primers GPD1-YZ-F / GPD1-YZ-R for verification. Successfully verified PCR products were sent for sequencing. Genomic samples from correctly sequenced positive transformants were used as templates for PCR amplification using primer pairs GPD1-UP-F / URA-UP2-R and URA-DN2-F / GPD1-DN-R, respectively. The resulting fragments were URA-UP2 and URA-DN2. After purification, the fragments were fused using primer pairs GPD1-UP-F / GPD1-DN-R. The fusion product was purified, and after successful sequencing, it was transferred to the aforementioned positive transformants. Transformants were screened using YPD plates containing 5-FOA (final concentration 1.0 g / L). Transformants were picked and verified by PCR using primer pairs GPD1-YZ-F / GPD1-YZ-R. After successful sequencing, the PCR product was inoculated into transformants in YPD liquid medium and cultured at 30℃ and 180-200 rpm for 16-18 h. The resulting bacteria were the URA-recovered and gpd1-deficient strains, denoted as BY4741-Δadh1-Δgpd1.

[0101] 1.2.3 Construction of the BY4741-Δadh1-Δgpd1-Δach1 strain

[0102] The ach1 knockout cassette was transformed into BY4741-Δadh1-Δgpd1. The resulting transformed bacterial culture was plated on SD-URA plates and incubated upside down at 30°C for 2 days. Transformants were picked and verified by PCR using primers ACH1-YZ-F / ACH1-YZ-R. Successfully verified PCR products were sent for sequencing. Genomic samples from correctly sequenced positive transformants were used as templates for PCR amplification using primer pairs ACH1-UP-F / URA-UP3-R and URA-DN3-F / ACH1-DN-R. The resulting fragments were URA-UP3 and URA-DN3. After purification, the fragments were fused using primers ACH1-UP-F / ACH1-DN-R. The fusion product was purified, and after successful sequencing, it was transferred to the aforementioned positive transformants. Transformants were screened using YPD plates containing 5-FOA (final concentration 1.0 g / L). Transformants were picked and verified by PCR using primers ACH1-YZ-F / ACH1-YZ-R. After successful sequencing, the PCR product was inoculated into transformants in YPD liquid medium and cultured at 30℃ and 180-200 rpm for 16-18 h. The resulting bacteria were the URA-recovered strain with ach1 deletion, denoted as BY4741-Δadh1-Δgpd1-Δach1.

[0103] 1.2.4 Construction of strain BY4741-Δadh1-Δgpd1-Δach1-Δsdh5

[0104] The sdh5 knockout cassette was transformed into BY4741-Δadh1-Δgpd1. The resulting transformed bacterial culture was plated on SD-URA plates and incubated upside down at 30°C for 2 days. Transformants were picked and verified by PCR using primers SDH5-YZ-F / SDH5-YZ-R. Successfully verified PCR products were sent for sequencing. Genomic samples from correctly sequenced positive transformants were extracted as templates and amplified by PCR using primer pairs SDH5-UP-F / URA-UP4-R and URA-DN4-F / SDH5-DN-R, respectively. The URA-UP4 and URA-DN4 fragments were amplified, purified, and fused using primers SDH5-UP-F / SDH5-DN-R. The fusion product was purified, and after successful sequencing, it was transferred into the above positive transformants. Y chromosomes containing 5-FOA (final concentration 1.0 g / L) were used. PD plates were used for reverse screening, and transformants were picked and verified by PCR using primers SDH5-YZ-F / SDH5-YZ-R. After the PCR product was correctly sequenced, the transformants were inoculated in YPD liquid medium and cultured at 30℃ and 180-200rpm for 16-18h to obtain the URA-recovered and sdh5-deficient strain BY4741-Δadh1-Δgpd1-Δach1-Δsdh5, which is the Saccharomyces cerevisiae genetically engineered strain HM1 with reduced by-products and succinic acid accumulation.

[0105] Example 2: Construction of the genetically engineered Saccharomyces cerevisiae HM2

[0106] To further increase succinic acid production, using the Saccharomyces cerevisiae genetically engineered strain HM1 as the starting strain (genotype MATahis3Δ1leu2 met15Δura3-52Δadh1Δgpd1Δach1Δsdh5), the Saccharomyces cerevisiae genetically engineered strain HM2 was obtained by overexpressing the pyruvate carboxylase gene pyc2, the fumarate reductase gene frd, the mitochondrial citrate transporter Yhm2, and the malate transporter Spmae1.

[0107] Among them, the pyruvate carboxylase gene pyc2 is derived from *Saccharomyces cerevisiae*, with the gene accession number GeneID:852519 (amino acid sequence shown in SEQ ID NO: 5). The fumarate reductase gene frd is derived from *Trypanosoma brucellosa*, and the codons were optimized to suit *Saccharomyces cerevisiae* preferences based on the FRD amino acid sequence of accession number GenBank:ALM30213.1 (amino acid sequence shown in SEQ ID NO: 6). The mitochondrial citrate transporter Yhm2 is derived from *Saccharomyces cerevisiae*, with the gene accession number GeneID:855282 (amino acid sequence shown in SEQ ID NO: 7). The malate transporter Spmae1 is derived from *Schizosaccharomyces cerevisiae*, with the gene accession number GeneID:254334 (amino acid sequence shown in SEQ ID NO: 8).

[0108] 2.1 Construction of overexpression plasmids

[0109] 2.1.1 Construction of pESC-HIS-PYC2 plasmid

[0110] Using the genome of Saccharomyces cerevisiae BY4741 as a template, PCR amplification was performed using primer pairs PGK1p-F / PGK1p-R and PYC2-F / PYC2-R, with a 20bp inter-primer ratio. The obtained sequences PGK1p and PYC2 were purified and mixed as a common template. PCR fusion was performed using primers PGK1p-F and PYC2-R, and the fusion product was purified and sent for sequencing. The pESC-HIS plasmid was linearized by double digestion with BamHI and EcoRI. The fusion fragment and the linearized pESC-HIS plasmid were ligated by one-step cloning. The resulting recombinant plasmid was transformed into E. coli DH5α. The transformed bacterial culture was plated on LB agar plates containing Amp antibiotics (final concentration 100 μg / mL) and cultured overnight at 37°C. Transformants were picked and verified using primers PYC2-YZ-F / PYC2-YZ-R. The correct PCR product band size was 5250 bp. The PCR product was sent for sequencing. The correctly sequenced transformants were inoculated into LB liquid medium containing Amp antibiotics (final concentration 100 μg / mL), preserved, and the plasmid was extracted to obtain the pESC-HIS-PYC2 plasmid.

[0111] 2.1.2 Construction of pESC-HIS-PYC2-FRD plasmid

[0112] Codon optimization was performed based on the FRD amino acid sequence of Brucella brucella (accession number: GenBank: ALM30213.1) to optimize it to a codon-biased form suitable for Saccharomyces cerevisiae. The gene TDH3-frd-TPI1 was synthesized routinely, and 5' (XmaI) and 3' (SalI) were added. The gene was cloned into the vector pESC-HIS-PYC2 plasmid via 5' (XmaI) and 3' (SalI). Mini-scale recombinant plasmid DNA and skeletal bacteria containing the recombinant plasmid were prepared. The plasmid was extracted, which is pESC-HIS-PYC2-FRD expressing frd under the TDH3 strong promoter.

[0113] 2.1.3 Construction of pESC-HIS-PYC2-FRD-Yhm2 plasmid

[0114] Using the genome of Saccharomyces cerevisiae BY4741 as a template, primer pair FBA1 was used. p -F / FBA1 p -R, Yhm2-F / Yhm2-R were respectively subjected to PCR amplification to obtain the FBA1 sequence. p After purification, it was mixed with Yhm2 and used as a common template, using primer FBA1. p The PCR fusion of -F and Yhm2-R was performed, and the fusion product was purified and sent for sequencing. The pESC-HIS-PYC2-FRD plasmid was linearized by SalI single enzyme digestion. The fusion fragment and the linearized plasmid were ligated by one-step cloning. The resulting recombinant plasmid was transformed into E. coli DH5α. The transformed bacterial solution was plated on LB agar containing Amp resistance (final concentration 100 μg / mL) and cultured overnight at 37°C. Transformants were picked and verified using primers Yhm2-YZ-F / Yhm2-YZ-R. The PCR product with correct verification was sent for sequencing. The correctly sequenced transformants were inoculated into LB liquid medium containing Amp resistance (final concentration 100 μg / mL), preserved, and the plasmid was extracted to obtain the pESC-HIS-PYC2-FRD-Yhm2 plasmid.

[0115] 2.1.4 Construction of pESC-HIS-PYC2-FRD-Yhm2-Spmae1 plasmid

[0116] Using the genome of Saccharomyces cerevisiae BY4741 as a template, primer pair ENO1 was used. p -F / ENO1 p -R, TDH2 t -F / TDH2 t -R were used for PCR amplification to obtain the ENO1 sequence. p and TDH2 tUsing the genome of Schizosaccharomyces pombe (Jiang Zhennan. Engineering modification of carboxylic acid transporter to produce succinic acid efficiently in Yersinia lipolytica [D]. Shandong University, 2021. DOI:10.27272 / d.cnki.gshdu.2021.000822. / Grobler J, Bauer F, Subden RE, Van Vuuren HJ. The maelgene of Schizosaccharomyces pombe encodes a permease for malate and other C4dicarboxylic acids. Yeast 1995; 11(15): 1485·1491.) as a template, PCR amplification was performed using primer pair Spmae1-F / Spmae1-R. The obtained sequence Spmae1 and the obtained sequence ENO1 were obtained. p Spmae1 and TDH2 t After purification, the mixture was used as a common template, and primer ENO1 was used. p -F / TDH2 t PCR fusion was performed using the -R method, and the fusion product was purified and sent for sequencing. The pESC-HIS-PYC2-FRD-Yhm2 plasmid was linearized by SalI single enzyme digestion. The fusion fragment and the linearized plasmid were ligated by one-step cloning. The resulting recombinant plasmid was transformed into E. coli DH5α. The transformed bacterial culture was plated on LB agar containing Amp resistance (final concentration 100 μg / mL) and cultured overnight at 37°C. Transformants were picked and verified using primers Spmae1-YZ-F / Spmae1-YZ-R. The PCR product with correct verification was sent for sequencing. The correctly sequenced transformants were inoculated into LB liquid medium containing Amp resistance (final concentration 100 μg / mL), preserved, and the plasmid was extracted to obtain the pESC-HIS-PYC2-FRD-Yhm2-Spmae1 plasmid.

[0117] 2.2 Transformation

[0118] The constructed plasmid pESC-HIS-PYC2-FRD-Yhm2-Spmae1 was transformed into the engineered Saccharomyces cerevisiae HM1 using the LiAc transformation method. The resulting transformed bacterial solution was plated on SD-HIS plates and incubated upside down at 30°C for 2 days. The obtained positive transformants were verified by colony PCR using the primers Spmae1-YZ-F / Spmae1-YZ-R. After preservation, the engineered Saccharomyces cerevisiae HM2 was obtained.

[0119] Example 3: Production of succinic acid by shake-flask fermentation of HM2 genetically engineered Saccharomyces cerevisiae

[0120] In this embodiment, the seed culture medium is SD-HIS medium, composed of the following components (solvent is water): glucose 20 g / L, YNB 6.7 g / L, DO Supplement-His 1.29 g / L. The fermentation medium consists of: glucose 50 g / L, YNB 6.7 g / L, DO Supplement-His 1.29 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 5 g / L, NaHCO3 8.4 g / L, trace element storage solution 8 ml / L, and vitamin storage solution 8 ml / L; wherein the trace element storage solution contains: EDTA 15 g / L. -1 ZnSO4·7H2O 4.5g·L -1 MnCl2·2H2O 0.84 g·L -1 CoCl2·6H2O 0.30g·L -1 CuSO4·5H2O 0.30 g·L -1 Na₂MoO₄·2H₂O 0.40 g·L⁻¹ -1 CaCl2·2H2O 4.50 g·L -1 FeSO4·7H2O 3.00 g·L -1 H3BO3 1.00 g·L -1 KI 0.10 g·L -1 The vitamin storage solution contains: Biotin (D-)(C 10 H 16 N2O3S) 0.05 g·L -1 Calcium pantothenate D(+)(C 18 H 32 CaN2O 10 1.00 g·L -1 Nicotinic acid (C6H5NO2) 1.00 g·L -1 Inositol (C6H) 12 O6) 25.00g·L -1 Thiamine chloride hydrochloride (C 12 H 18 C l2 (N4OS×H2O) 1.0 g·L -1 pyridoxine hydrochloride (C8H) 12 ClNO3) 1.00 g·L -1 p-Aminobenzoic acid (C7H7NO2) 0.20 g·L -1 .

[0121] Shake-flask fermentation of the genetically engineered Saccharomyces cerevisiae HM2 includes the following steps:

[0122] (1) Seed culture: Fresh single colonies on SD-HIS plates were inoculated into 10 mL of seed culture medium and cultured at 30℃ and 220 rpm for 18 h. The resulting seed culture solution was used for inoculation of fermentation medium.

[0123] (2) Fermentation culture: The seed culture solution was transferred to a 250ml Erlenmeyer flask containing 50ml of fermentation medium at an inoculation rate of 1% (V / V), and cultured at 30℃ and 220rpm for 3 days to obtain the fermentation broth.

[0124] Analytical methods: Components in the fermentation broth after 3 days of fermentation were determined using an Agilent 1260 high-performance liquid chromatograph. The concentrations of glucose and organic acids in the fermentation broth were determined using a Biorad Aminex HPX-87H organic acid analytical column. The mobile phase was 5 mM H₂SO₄, the flow rate was 0.5 mL / min, the sampling time was 20 min, the injection volume was 20 μL, and the column temperature was 60 °C.

[0125] The results showed that the genetically engineered Saccharomyces cerevisiae HM2, fermented under anaerobic conditions for 3 days, produced 1.3 g / L of succinic acid, ethanol, glycerol, acetic acid and other byproducts. Compared with the original strain BY4741, the contents of ethanol, glycerol and acetic acid were reduced by 70%, 24% and 54%, respectively.

[0126] Example 4: Anaerobic domestication to obtain the genetically engineered Saccharomyces cerevisiae HM3

[0127] As can be seen from Example 3, the constructed Saccharomyces cerevisiae genetically engineered strain HM2 exhibits weak cell growth and low OD value under anaerobic fermentation conditions. Therefore, the Saccharomyces cerevisiae genetically engineered strain HM2 can simultaneously improve cell growth and succinic acid production capacity through evolutionary metabolism.

[0128] The metabolic evolution process was performed using a 500 mL fermenter containing 250 mL of fermentation medium. 5M NaHCO3 was used as a neutralizing agent to maintain the pH at 7.0. The seed culture medium used for metabolic evolution was SD-HIS: glucose 20 g / L, YNB 6.7 g / L, DO Supplement-HIS 1.29 g / L; the fermentation medium consisted of: glucose 50 g / L, YNB 6.7 g / L, DO Supplement-HIS 1.29 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 5 g / L, NaHCO3 8.4 g / L, trace element stock solution 8 ml / L, and vitamin stock solution 8 ml / L. The trace element stock solution contained EDTA 15 g·L⁻¹. -1 ZnSO4·7H2O 4.5g·L -1 MnCl2·2H2O 0.84 g·L -1CoCl2·6H2O 0.30g·L -1 CuSO4·5H2O 0.30 g·L -1 Na₂MoO₄·2H₂O 0.40 g·L⁻¹ -1 CaCl2·2H2O 4.50 g·L -1 FeSO4·7H2O 3.00 g·L -1 H3BO3 1.00 g·L -1 KI 0.10 g·L -1 The vitamin storage solution contains: Biotin (D-)(C 10 H 16 N2O3S) 0.05 g·L -1 Calcium pantothenate D(+)(C 18 H 32 CaN2O 10 1.00 g·L -1 Nicotinic acid (C6H5NO2) 1.00 g·L -1 Inositol (C6H) 12 O6) 25.00g·L -1 Thiamine chloride hydrochloride (C 12 H 18 C l2 (N4OS×H2O) 1.0 g·L -1 pyridoxine hydrochloride (C8H) 12 ClNO3) 1.00 g·L -1 p-Aminobenzoic acid (C7H7NO2) 0.20 g·L -1 Every 24 hours, the fermentation broth was transferred to a new fermenter to achieve an initial OD600 of 0.1. After 29 generations of anaerobic metabolic evolution and 3 days of cultivation, the final OD reached 8.8, yielding the genetically engineered Saccharomyces cerevisiae strain HM3. Its cell growth is shown in [the table below]. Figure 3 a. The highest yield of succinic acid is 21 g / L ( Figure 3 b).

[0129] The genetically engineered Saccharomyces cerevisiae strain HM3 was deposited on October 31, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei, China, with accession number CCTCC NO:M 20221705 and classified as Saccharomyces cerevisiae.

[0130] Example 5: Anaerobic fermentation of *Saccharomyces cerevisiae* genetically engineered strain HM3 in a 5L fermenter to produce succinic acid.

[0131] The composition and preparation of the seed culture medium and fermentation culture medium are the same as those described in Example 4 for the domestication and acquisition of the genetically engineered Saccharomyces cerevisiae HM3, and the analytical methods are the same as those described in Example 3.

[0132] Fermentation was carried out anaerobically in a 5L fermenter, and included the following steps:

[0133] (1) Seed culture: Fresh single colonies on SD-HIS plates were inoculated into 10 mL of seed culture medium and cultured at 30°C and 220 rpm for 16-20 h to obtain the first-stage seed culture. Then, the culture was transferred to a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium at an inoculation rate of 1% (V / V) and cultured at 30°C and 220 rpm for 16-20 h to obtain the second-stage seed culture, which was used for inoculation of the third-stage fermentation medium.

[0134] (2) Fermentation culture: 2L of fermentation medium was placed in a 5L fermenter and sterilized at 115℃ for 25min. The seed culture was inoculated into the fermentation medium at a final concentration of OD600 = 0.1. After the OD600 reached 8, the pH was controlled at 7.0 with 5M NaHCO3. During the fermentation process, glucose was added to maintain the sugar concentration not higher than 4g / L. Anaerobic culture was carried out at 30℃ and 250rpm for 3 days.

[0135] The results showed that after 72 hours of fermentation with the genetically engineered Saccharomyces cerevisiae HM3, the succinic acid yield reached 21 g / L, the conversion rate reached 1.29 mol succinic acid / mol glucose (0.85 g succinic acid / g glucose), and the production intensity was 0.29 g / L / h. Byproducts included small amounts of ethanol, glycerol, and acetic acid. Figure 4 ).

[0136] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

[0137] sequence list

[0138] ADH1: SEQ ID NO: 1, Saccharomyces cerevisiae

[0139]

[0140] GPD1:SEQ ID NO:2,Saccharomyces cerevisiae

[0141]

[0142] ACH1:SEQ ID NO:3,Saccharomyces cerevisiae

[0143]

[0144] SDH5:SEQ ID NO:4,Saccharomyces cerevisiae

[0145] atgcacaatatgtttccagcactcacaaagacactgtcgttgcaaggctacaagattatcaactctcaaacagggtccgctgcatggtcgtgcggtcgtaggtggtttagtagtgataaaga tgaccacgacgatgtggtgacgaggattaaaattgcccccataaagagaactaacgagccattggataagaaaagagctcggttgatatatcaatcacgcaaaagagggatcttggagacgg acttgctgctatctgggttcgctgccaaatatttgaagaagatgaacgaagaggaactggaagaatacgattcgctattgaatgagttggactgggacatatactattgggccacaaaaaatttcaaaaccagccccttgcctgacaagtgggccaattctaaattgctgaagcagctacaagaattcagtgaaaataaagagaaggaaattttaagtatgccggacttgtccaagtatcaatga

[0146] PYC2:SEQ ID NO:5,Saccharomyces cerevisiae

[0147]

[0148] FRD:SEQ ID NO:6,Trypanosoma brucei

[0149]

[0150] Yhm2:SEQ ID NO:7,Saccharomyces cerevisiae

[0151] MPSTTNTAAANVIEKKPVSFSNILLGACLNLSEVTTLGQPLEVVKTTMAANRNFTFLESVKHVWSRGGILGYYQGLIPWAWIEASTKGAVLLFVSAEAEYRFKSLGLNNFASGILGGVTGGVTQAYLTMGFCTCMKTVEITRHKSASAGGVPQSSWSVFKNIYKKEGIRGINKGVNAVAIRQMTNWGSRFGLSRLVEDGIRKITGKTNKDDKLNPFEKIGASALGGGLSAWNQPIEVIRVEMQSKKEDPNRPKNLTVGKTFKYIYQSNGLKGLYRGVTPRIGLGIWQTVFMVGFGDMAKEFVARMTGETPVAKH*

[0152] Spmae1:SEQ ID NO:8,Schizosaccharomyces pombe

[0153] MGELKEILKQRYHELLDWNVKAPHVPLSQRLKHFTWSWFACTMATGGVGLIIGSFPFRFYGLNTIGKIVYILQIFLFSLFGSCMLFRFIKYPSTIKDSWNHHLEKLFIATCLLSISTFIDMLAIYAYPDTGEWMVWVIRILYYIYVAVSFIYCVMAFFTIFNNHVYTIETASPAWILPIFPPMICGVIAGAVNSTQPAHQLKNMVIFGILFQGLGFWVYLLLFAVNVLRFFTVGLAKPQDRPGMFMFVGPPAFSGLALINIARGAMGSRPYIFVGANSSEYLGFVSTFMAIFIWGLAAWCYCLAMVSFLAGFFTRAPLKFACGWFAFIFPNVGFVNCTIEIGKMIDSKAFQMFGHIIGVILCIQWILLMYLMVRAFLVNDLCYPGKDEDAHPPPKPNTGVLNPTFPPEKAPASLEKVDTHVTSTGGESDPPSSEHESV*

Claims

1. A genetically engineered Saccharomyces cerevisiae strain HM3 that produces succinic acid, characterized in that, The preservation number of the genetically engineered Saccharomyces cerevisiae HM3 is CCTCC NO:M 20221705.

2. The application of the genetically engineered Saccharomyces cerevisiae HM3 as described in claim 1 in the production of succinic acid.

3. The application according to claim 2, characterized in that, The method for producing succinic acid includes: fermenting the genetically engineered Saccharomyces cerevisiae HM3 of claim 1 on a fermentation medium.

4. The application according to claim 3, characterized in that, The fermentation culture method is as follows: the seed culture of the genetically engineered Saccharomyces cerevisiae HM3 described in claim 1 is inoculated into the fermentation medium at an inoculation rate of 3-6%, and fermented at 28-32℃ and 180-240rpm for 65-80h, with the pH controlled at 6.0-7.0 during the fermentation process.

5. The application according to claim 3, characterized in that, The fermentation medium consisted of: glucose 50 g / L, YNB 6.7 g / L, DO Supplement-HIS 1.29 g / L, potassium dihydrogen phosphate 10 g / L, magnesium sulfate 5 g / L, NaHCO3 8.4 g / L, trace element stock solution 8 ml / L, and vitamin stock solution 8 ml / L. The trace element storage solution consists of: EDTA 15 g·L -1 ZnSO4•7H2O 4.5g·L -1 MnCl2•2H2O 0.84 g·L -1 CoCl2•6H2O 0.30g·L -1 CuSO4•5H2O 0.30g·L -1 Na₂MoO₄•₂H₂O 0.40 g·L⁻¹ -1 CaCl2•2H2O 4.50 g·L -1 FeSO4•7H2O 3.00g·L -1 H3BO3 1.00g·L -1 KI 0.10 g·L -1 ; The vitamin storage solution contains: D-Biotin 0.05 g·L⁻¹ -1 Calcium pantothenate D(+) 1.00 g·L -1 Niacin 1.00 g·L -1 Inositol 25.00 g·L -1 Thiamine chloride hydrochloride 1.0 g·L -1 pyridoxine hydrochloride 1.00 g·L -1 p-Aminobenzoic acid 0.20 g·L -1 .

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