Pichia kudriavzevii with high yield of l-malic acid, construction method and application

Abstract

The application discloses a high-yield L-malic acid Pichia kudriavzevii, a construction method and application, and belongs to the technical field of biology. The Pichia kudriavzevii is named YM-023, classified as Pichia kudriavzevii Pichia kudriavzevii , the preservation number is CGMCC No. 35487, the preservation time is July 31, 2025, and the preservation unit is the China General Microbiological Culture Collection Center. The application starts from the acid-resistant yeast Pichia kudriavzevii strain YL-000 (the preservation number is CGMCC No. 35680), obtains a modified strain through metabolic engineering modification, and ensures that the modified strain can realize high-efficiency production of L-malic acid in a fermentation mode with a small amount of calcium carbonate neutralizer added.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Pichia kudriavzevii with high yield of L-malic acid, a construction method and application. BACKGROUND

[0002] L-malic acid, also known as hydroxybutanedioic acid, is a four-carbon dicarboxylic acid. L-malic acid is an important intermediate of the TCA cycle and widely exists in fruits and vegetables. L-malic acid has the sour taste of natural malic acid and is often used as an acidifier and flavor enhancer in the food and beverage industry; it can also be used as a raw material for the synthesis of poly-malic acid (PMA), alkyl and unsaturated polyester resins, coatings, etc. in the chemical industry; some malic acid derivatives are also used in medicine, such as citrate-malate calcium, which is often used to treat hyperammonemia and liver dysfunction, and citrulline, which can enhance the exercise performance of male resistance training.

[0003] Currently, the main methods for synthesizing L-malic acid are chemical synthesis, enzyme catalysis and microbial fermentation. The chemical synthesis method mainly uses maleic anhydride as a raw material to generate a mixture of D and L malic acid through a series of hydration and isomerization reactions, and then L-malic acid is obtained by separation and extraction with chemical reagents. Although this process is mature, it has the disadvantages of harsh process conditions, difficult separation and extraction, high pollution and high energy consumption. The enzyme catalysis method uses fumaric acid as a raw material to obtain high-purity L-malic acid under mild conditions through the catalysis of immobilized enzymes or whole cells, or to generate L-malic acid by hydrolyzing poly-malic acid. However, this method has the disadvantages of high cost of substrates, high cost of enzyme purification, short half-life of enzymes and increased production cost due to substrate recovery.

[0004] Microbial fermentation method is a method that uses renewable carbon sources such as glucose and glycerol as raw materials to produce high-purity L-malic acid under mild conditions. This method is environmentally friendly, green and low-carbon, and has the advantages of mild reaction conditions, pure product and high yield. Microbial fermentation method is divided into one-step fermentation method and two-step fermentation method. The two-step fermentation method uses two microorganisms to produce L-malic acid in two stages, which has the disadvantages of long fermentation period, many by-products and high cost of separation and purification, which is not conducive to large-scale production. The one-step fermentation method refers to the fermentation of L-malic acid by one microorganism, which has the advantages of simple fermentation conditions and low production cost.

[0005] Currently, the commonly used microorganisms for microbial fermentation are Aspergillus oryzae (A. oryzae) (WO 2018 / 230 1 1 1 ), Aspergillus terreus (A. terreus) (CN 1 1 1 1 1 1 1 1 1 1 1 1 A), and Aspergillus flavus (A. flavus) (CN 1 1 1 1 1 1 1 1 1 1 1 1 1 A). Aspergillus oryzae Aspergillus terreus Aspergillus flavus ​​) etc. The use of the above-mentioned filamentous fungi for the fermentation production of L-malic acid requires the addition of a large amount of neutralizing agent to the culture medium to maintain the pH at neutral, making the downstream separation and purification more complicated, and the production of a large amount of waste salt increases the production cost. Moreover, the fermentation cycle of filamentous fungi is long, and the mycelium is easy to clump, increasing the difficulty of fermentation control. Some yeasts can tolerate lower pH, which can reduce the amount of neutralizing agent added to the culture medium, or can produce L-malic acid under the condition of not adding neutralizing agent, which can simplify the downstream separation and purification process and reduce the production cost. East Issa yeast (Pichia kudriavzevii, now renamed Pichia kudriavzevii (P. kudriavzevii) Issatchenkia orientalis ), has the ability to degrade malic acid and citric acid, has outstanding environmental stress tolerance, and can grow at a pH of less than 3.0, which is a potential host for the fermentation production of L-malic acid. Pichia kudriavzevii SUMMARY

[0006] The present application discloses a Pichia kudriavzevii with high yield of L-malic acid, a construction method and application. Starting from the acid-tolerant yeast Pichia kudriavzevii strain YL-000 (preservation number CGMCC No. 35680), a modified strain is obtained through metabolic engineering modification, and the modified strain can realize efficient production of L-malic acid under the fermentation mode of adding a small amount of calcium carbonate neutralizing agent.

[0007] To solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0008] A Pichia kudriavzevii with high yield of L-malic acid, the Pichia kudriavzevii is named YM-023, and the classification name is Pichia kudriavzevii Pichia kudriavzevii , the preservation number is CGMCC No. 35487, the preservation time is July 31, 2025, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No. 1, Beichen West Road, Beijing City, China Academy of Microbiology Institute.

[0009] ​The method for constructing the high L-malic acid-producing Pichia kudriavzevii comprises the following steps: taking Pichia kudriavzevii as a starting strain, knocking out the orotidine-5-phosphate decarboxylase URA3 gene, overexpressing the malate dehydrogenase ScMDH gene, the transporter SpMAE1 gene, the pyruvate carboxylase AoPYC gene, the phosphoenolpyruvate carboxylase EcPPC gene, the pyruvate carboxylase AoPYC gene, the NADH-dependent malate dehydrogenase AoMDH gene, the NADH-dependent malate dehydrogenase PkMDH2 gene, the phosphoenolpyruvate carboxykinase PkPCK1 gene, the NADPH-dependent malate dehydrogenase SbMDHP1 gene, knocking out the oxaloacetate decarboxylase OAD gene, overexpressing the NADPH-dependent malate dehydrogenase SbMDHP2 gene, the biotin transporter SpVHT1 gene and the phosphoenolpyruvate carboxylase EcPPC gene in sequence, knocking out the mitochondrial succinate / fumarate transporter SFC1 gene, overexpressing the soluble pyridine nucleotide transhydrogenase EcSthA gene, and knocking out the transcriptional regulator ROX1 gene to obtain the Pichia kudriavzevii strain;

[0010] The classification name of the starting strain is Pichia kudriavzevii. Pichia kudriavzevii The preservation number is CGMCC No. 35680, the preservation date is August 20, 2025, the preservation unit is the General Microbiological Center of China, and the preservation address is No. 1, Xibaheyi, Chaoyang District, Beijing, China Institute of Microbiology;

[0011] The amino acid sequence corresponding to the orotidine-5-phosphate decarboxylase URA3 is shown in SEQ ID NO: 13.

[0012] The malate dehydrogenase ScMDH gene is derived from Saccharomyces cerevisiae.

[0013] The nucleotide sequence of the malate dehydrogenase ScMDH gene is shown in SEQ ID NO: 1.

[0014] The transporter SpMAE1 gene is derived from Schizosaccharomyces pombe.

[0015] The nucleotide sequence of the transporter SpMAE1 gene is shown in SEQ ID NO: 2.

[0016] The pyruvate carboxylase AoPYC gene is derived from Aspergillus oryzae.

[0017] The nucleotide sequence of the pyruvate carboxylase AoPYC gene is shown in SEQ ID NO: 3.

[0018] The nucleotide sequence of the phosphoenolpyruvate carboxylase EcPPC gene overexpressed for the first time is shown as SEQ ID NO: 4;

[0019] The NADH-dependent malate dehydrogenase AoMDH gene is derived from Aspergillus oryzae;

[0020] The nucleotide sequence of the NADH-dependent malate dehydrogenase AoMDH gene is shown as SEQ ID NO: 5;

[0021] The NADH-dependent malate dehydrogenase PkMDH2 gene is derived from itself;

[0022] The nucleotide sequence of the NADH-dependent malate dehydrogenase PkMDH2 gene is shown as SEQ ID NO: 6;

[0023] The phosphoenolpyruvate carboxykinase PkPCK1 gene is derived from itself;

[0024] The nucleotide sequence of the phosphoenolpyruvate carboxykinase PkPCK1 gene is shown as SEQ ID NO: 7;

[0025] The NADPH-dependent malate dehydrogenase SbMDHP1 gene is derived from Sorghum bicolor;

[0026] The nucleotide sequence of the NADPH-dependent malate dehydrogenase SbMDHP1 gene is shown as SEQ ID NO: 8;

[0027] The corresponding amino acid sequence of the oxaloacetate decarboxylase OAD is shown as SEQ ID NO: 21;

[0028] The NADPH-dependent malate dehydrogenase SbMDHP2 gene is derived from Sorghum bicolor;

[0029] The nucleotide sequence of the NADPH-dependent malate dehydrogenase SbMDHP2 gene is shown as SEQ ID NO: 9;

[0030] The biotin transporter SpVHT1 gene is derived from Schizosaccharomyces pombe;

[0031] The nucleotide sequence of the biotin transporter SpVHT1 gene is shown as SEQ ID NO: 10;

[0032] The phosphoenolpyruvate carboxylase EcPPC gene is derived from Escherichia coli;

[0033] The nucleotide sequence of the phosphoenolpyruvate carboxylase EcPPC gene overexpressed for the second time is shown as SEQ ID NO: 11;

[0034] The amino acid sequence corresponding to the mitochondrial succinate / fumarate transporter SFC1 is shown as SEQ ID NO: 22;

[0035] The soluble pyridine nucleotide transhydrogenase EcSthA gene is derived from Escherichia coli;

[0036] The nucleotide sequence of the soluble pyridine nucleotide transhydrogenase EcSthA gene is shown as SEQ ID NO: 12;

[0037] The amino acid sequence corresponding to the transcriptional regulator ROX1 is shown as SEQ ID NO: 23.

[0038] The application of the high L-malic acid-producing Pichia kudriavzevii in the fermentation of L-malic acid.

[0039] Compared with the prior art, the application has the following beneficial effects:

[0040] The application starts with the acid-tolerant yeast Pichia kudriavzevii strain YL-000, and through metabolic engineering modification, obtains the high L-malic acid-producing Pichia kudriavzevii, which can realize efficient production of L-malic acid in the fermentation mode of adding a small amount of calcium carbonate neutralizer. DETAILED DESCRIPTION

[0041] The application will be further illustrated by the following non-limiting examples, and those skilled in the art will understand that many modifications can be made to the application without departing from the spirit of the application, and such modifications also fall within the scope of the application.

[0042] The following experimental methods are conventional methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.

[0043] Example 1 Construction of YM-001 strain overexpressing ScMDH gene

[0044] The starting strain YL-000 was obtained from Yuanli Chemical Group Co., Ltd., and was classified and named as Pichia kudriavzevii Pichia kudriavzevii , with the preservation number of CGMCC No. 35680, the preservation date of August 20, 2025, the preservation unit of General Microbial Center of China Microbial Culture Collection Committee, and the preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences;

[0045] Park HJ, Bae JH, Ko HJ, Lee SH, Sung BH, Han JI, Sohn JH. Low-pH production of d-lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii The URA3 gene (the corresponding amino acid sequence is SEQ ID NO: 13) of the starting strain YL-000 was knocked out to obtain strain YL-001 (genotype: YL-000, △URA3), which is a uracil-deficient Pichia kudriavzevii, and the endogenous URA3 gene encoding orotidine-5-phosphate decarboxylase (Uniprot database retrieval number: Q6IUR4, EC 4.1.1.23) is knocked out. Subsequently, pWSYL-Cas9 was optimized to construct CRISPR / Cas9 plasmid pYL-Cas9 containing a URA3 selection marker, and positive transformants can be obtained by auxotrophic selection.

[0046] Then the NADH-dependent malate dehydrogenase ScMDH (Uniprot database retrieval number: P32419, EC 1.1.1.37) from Saccharomyces cerevisiae was overexpressed in strain YL-001. The ScMDH gene (SEQ ID NO: 1) was synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and was optimized according to the codon bias of YL-001. The ScMDH gene was integrated into the ADH3 gene (the corresponding amino acid sequence is SEQ ID NO: 14) site of the genome of strain YL-001, and the promoter and terminator used were the promoter (sequence is SEQ ID NO: 24) and terminator (sequence is SEQ ID NO: 25) of the Alcohol dehydrogenase gene ADH3 of strain YL-001 itself. The specific construction method is as follows:

[0047] 1. Construction of donor DNA fragment for homologous recombination

[0048] The genomic DNA of strain YL-001 was used as a template, and the primer ADH3-F1 and ADH3-ScMDH-R (see Table 1 for sequences) were used to amplify the upstream homologous arm fragment of the ADH3 gene (fragment 1); the amplification system and procedure were referred to the product manual of TAKARA PrimeSTAR® GXL DNA polymerase; the plasmid containing the ScMDH synthetic sequence was used as a template, and the primers ScMDH-ADH3-F and ScMDH-ADH3-R (see Table 1) were used to amplify the ScMDH gene sequence (fragment 2); the genomic DNA of strain YL-001 was used as a template, and the primers ADH3-ScMDH-F and ADH3-R1 (see Table 1 for sequences) were used to amplify the downstream homologous arm fragment of the ADH3 gene (fragment 3). The three fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 1, 2 and 3 were used as templates, and the amplification system and procedure were referred to the product manual of Novagen 2xPhanta Max Master Mix, and then the primerless PCR amplification was performed. The above primerless amplification PCR reaction liquid was used as a template, and the primers ADH3-F2 and ADH3-R2 (see Table 1 for sequences) were used for nested PCR, and then the correct size bands were cut and recovered after agarose gel electrophoresis, and fragment 4 was obtained.

[0049] 2. Construction of pYL-Cas9-ADH3 plasmid for editing YL-001's own ADH3 site

[0050] The pYL-Cas9 plasmid was used as a template, and the primers gRNA-F and ADH3-N20-R (see Table 1 for sequences) were used to amplify a 10035 bp fragment containing the sgRNA sequence of the endogenous ADH3 gene of YL-001 strain (fragment 5). Then Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above product was purified using SanPrep Column PCR Product Purification Kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zixingjin Biotechnology Co., Ltd., product number: CB101-01) according to the method of E. coli electroporation (Nováková J, Izsáková A, Grivalský T. Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids. doi: 10.1007 / s12223-013-0267-1), and the obtained positive plasmid was named pYL-Cas9-ADH3.

[0051] Table 1. Primers required for overexpression of ScMDH gene and construction of CRISPR / Cas9 plasmid

[0052]

[0053] 3. Construction of ScMDH gene overexpression strain

[0054] According to the yeast electroporation method (Fang ZJ, Chen T, Hao HL, et al. New exploration of Saccharomyces cerevisiae transformation method. doi:10.3969 / j.issn.1006-7167.2012.04.002), 1 μg of pYL-Cas9-ADH3 plasmid and 1 μg of fragment 4 were electroporated into YL-001 strain, and positive transformants were screened, followed by multiple passages in YPD medium to lose pYL-Cas9-ADH3 plasmid, and obtain malic acid producing yeast YM-001 (genotype: YL-001, ADH3::ADH3p-ScMDH-ADH3t).

[0055] Example 2 Overexpression of SpMAE1 gene in YM-001 strain

[0056] On the basis of YM-001 strain, the transporter SpMAE1 (Uniprot database accession number: P50537) from Schizosaccharomyces pombe was overexpressed. The SpMAE1 gene (SEQ ID NO: 2) was synthesized by Nanjing Kings River Biotechnology Co., Ltd., and was optimized according to the codon bias of YL-001. The SpMAE1 gene was integrated into the JEN1 gene (the corresponding amino acid sequence is SEQ ID NO: 15) site of the YM-001 genome, and the promoter and terminator used were the promoter of the cell wall glycoprotein gene SED1 of YL-001 (the sequence is SEQ ID NO: 26) and the terminator of the Triosephosphate isomerase gene TPI1 (the sequence is SEQ ID NO: 27), respectively. The specific construction method is as follows:

[0057] 1. Construction of donor DNA fragment for homologous recombination

[0058] YL-001 genomic DNA as a template, the primer SED1p-JEN1-F and SED1p-SpMAE1-R (see Table 2) to amplify the SED1 promoter fragment (fragment 7); YL-001 genomic DNA as a template, the primer SpMAE1-SED1p-F and SpMAE1-TPI1t-R (see Table 2) to amplify SpMAE1 gene sequence (fragment 8); YL-001 genomic DNA as a template, the primer TPI1t-SpMAE1-F and TPI1t-JEN1-R (see Table 2) to amplify the TPI1 gene terminator fragment (fragment 9); YL-001 genomic DNA as a template, the primer JEN1-TPI1t-F and JEN1-R1 (see Table 2) to amplify the JEN1 gene downstream homologous arm fragment (fragment 10). The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 6-10 were used as templates for primer-free PCR amplification. The above-mentioned primer-free amplification PCR reaction liquid was used as a template, and the primer JEN1-F2 and JEN1-R2 (see Table 2) were used for nested PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 11.

[0059] 2. Constructing pYL-Cas9-JEN1 plasmid for editing JEN1 site

[0060] YL-001 genomic DNA as a template, the primer SED1p-JEN1-F and SED1p-SpMAE1-R (see Table 2) to amplify the SED1 promoter fragment (fragment 7); YL-001 genomic DNA as a template, the primer SpMAE1-SED1p-F and SpMAE1-TPI1t-R (see Table 2) to amplify SpMAE1 gene sequence (fragment 8); YL-001 genomic DNA as a template, the primer TPI1t-SpMAE1-F and TPI1t-JEN1-R (see Table 2) to amplify the TPI1 gene terminator fragment (fragment 9); YL-001 genomic DNA as a template, the primer JEN1-TPI1t-F and JEN1-R1 (see Table 2) to amplify the JEN1 gene downstream homologous arm fragment (fragment 10). The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 6-10 were used as templates for primer-free PCR amplification. The above-mentioned primer-free amplification PCR reaction liquid was used as a template, and the primer JEN1-F2 and JEN1-R2 (see Table 2) were used for nested PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 11.

[0061] Table 2. Primers required for overexpression of SpMAE1 gene and construction of CRISPR / Cas9 plasmid

[0062]

[0063] 3. Construction of ScMDH gene overexpressing strain

[0064] 1 μg of pYL-Cas9-JEN1 plasmid and 1 μg of fragment 11 were electroporated into YM-001 strain according to the yeast electroporation method, and positive transformants were screened, followed by multiple passages in YPD medium to lose the pYL-Cas9-JEN1 plasmid, and obtain malate-producing yeast YM-002 (genotype: YM-001, JEN1::SED1p-SpMAE1-TPI1t).

[0065] Example 3 Overexpression of AoPYC gene in YM-002 strain

[0066] AoPYC (Uniprot database accession number: I8TVE3, EC 6.4.1.1) derived from Aspergillus oryzae was overexpressed in the YM-002 strain. The AoPYC gene (SEQ ID NO: 3) was synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. and optimized according to the codon preference of YL-001. The AoPYC gene was integrated into the PDC6 gene (the corresponding amino acid sequence is SEQ ID NO: 16) site of the YM-002 genome, and the promoter and terminator used were the promoter (sequence is SEQ ID NO: 28) and terminator (sequence is SEQ ID NO: 29) of the YL-001 own pyruvate decarboxylase (Pyruvate decarboxylase) gene PDC6. The specific construction method is as follows:

[0067] 1. Construction of donor DNA (Donor DNA) fragment for homologous recombination

[0068] YL-001 genomic DNA as a template, primer PDC6-F1 and PDC6-AoPYC-R (see Table 3 for sequence) were used to amplify the PDC6 gene upstream homologous arm fragment (fragment 13); the plasmid containing the AoPYC synthetic sequence was used as a template, and the primers AoPYC-PDC6-F and AoPYC-PDC6-R (see Table 3 for sequence) were used to amplify the AoPYC gene sequence (fragment 14); YL-001 genomic DNA as a template, primer PDC6-AoPYC-F and PDC6-R1 (see Table 3 for sequence) were used to amplify the PDC6 gene downstream homologous arm fragment (fragment 15). The above three fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragment 13, fragment 14 and fragment 15 were used as templates for primer-free PCR amplification. Using the above primer-free amplification PCR reaction solution as a template, primers PDC6-F2 and PDC6-R2 (see Table 3 for sequence) were used for nested PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 16.

[0069] 2. Construction of pYL-Cas9-PDC6 plasmid for editing PDC6 locus

[0070] A 10035 bp fragment (fragment 17) containing sgRNA sequence of YL-001 strain endogenous PDC6 gene was amplified using primers gRNA-F (sequence: SEQ ID NO: 50) and PDC6-N20-R (sequence in Table 3) with pYL-Cas9 plasmid as template. Subsequently, the plasmid template was digested using Dpn I enzyme (Thermo Scientific), and the product was purified using SanPrep Column PCR Product Purification Kit (Shanghai Biomed Longlife Co., Ltd.). The purified product was transformed into E. coli Transl-Tl competent cells (purchased from Beijing Zison Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-PDC6.

[0071] Table 3. Primers required for overexpression of AoPYC gene and construction of CRISPR / Cas9 plasmid

[0072]

[0073] 3. Construction of AoPYC gene overexpression strain

[0074] 1 μg of pYL-Cas9-PDC6 plasmid and 1 μg of fragment 16 were electroporated into YM-002 strain according to the yeast electroporation method, and positive transformants were selected. Subsequently, the pYL-Cas9-PDC6 plasmid was lost by multiple passages in YPD medium, and the malate-producing yeast YM-003 (genotype: YM-002, PDC6::PDC6p-AoPYC-PDC6t) was obtained.

[0075] Example 4. Overexpression of EcPPC gene in YM-003 strain

[0076] On the basis of YM-003 strain, phosphoenolpyruvate carboxylase EcPPC (Uniprot database retrieval number: P00864, EC 4.1.1.31) from E. coli was overexpressed, and the EcPPC gene (SEQ ID NO: 4) was synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. and optimized according to the YL-001 codon bias. The EcPPC gene was integrated into the MCH4 gene (the corresponding amino acid sequence is SEQ ID NO: 17) site of the YM-003 genome, and the promoter and terminator used were the promoter of the YL-001 cell wall glycoprotein gene SED1 and the terminator of the UDP-galactose transporter homolog gene GAL2 (the sequence is SEQ ID NO: 30), respectively. The specific construction method is as follows:

[0077] 1. Construction of donor DNA fragment for homologous recombination

[0078] YL-001 genomic DNA as a template, primer MCH4-F1 and MCH4-SED1p-R (see Table 4 for sequences) were used to amplify the MCH4 gene upstream homologous arm fragment (fragment 18); YL-001 genomic DNA as a template, primer SED1p-MCH4-F and SED1p-EcPPC-R (see Table 4 for sequences) were used to amplify the SED1 promoter fragment (fragment 19); the plasmid containing the EcPPC synthetic sequence was used as a template, and primers EcPPC-SED1p-F and EcPPC-GAL2t-R (see Table 4) were used to amplify the EcPPC gene sequence (fragment 20); YL-001 genomic DNA as a template, primer GAL2t-EcPPC-F and GAL2t-MCH4-R (see Table 4 for sequences) were used to amplify the GAL2 gene terminator fragment (fragment 21); YL-001 genomic DNA as a template, primer MCH4-GAL2t-F and MCH4-R1 (see Table 4 for sequences) were used to amplify the MCH4 gene downstream homologous arm fragment (fragment 22). The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 18-22 were used as templates for primer-free PCR amplification. Using the above primer-free amplification PCR reaction solution as a template, primers MCH4-F2 and MCH4-R2 (see Table 4 for sequences) were used for nested PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 23.

[0079] 2. Construction of pYL-Cas9-MCH4 plasmid for editing MCH4 site

[0080] The 10035 bp fragment containing sgRNA sequence of endogenous MCH4 gene of YL-001 strain (fragment 24) was amplified using primers gRNA-F (sequence: SEQ ID NO: 50) and MCH4-N20-R (sequence in Table 4) with pYL-Cas9 plasmid as template. Then, the plasmid template was digested using Dpn I enzyme (Thermo Scientific), and the above product was purified using SanPrep Column PCR Product Purification Kit (Shanghai Biomed Longlife Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biotech Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-MCH4.

[0081] Table 4. Primers required for construction of overexpression EcPPC gene and CRISPR / Cas9 plasmid

[0082]

[0083] 3. Construction of EcPPC gene overexpression strain

[0084] 1 μg of pYL-Cas9-MCH4 plasmid and 1 μg of fragment 23 were electroporated into YM-003 strain according to the yeast electroporation method, and positive transformants were screened, followed by multiple passages in YPD medium to lose the pYL-Cas9-MCH4 plasmid, and obtain malate-producing yeast YM-004 (genotype: YM-003, MCH4::SED1p-EcPPC-GAL2t).

[0085] Example 5. Overexpression of AoPYC gene in YM-004 strain

[0086] Aspergillus oryzae-derived pyruvate carboxylase AoPYC (Uniprot database accession number: I8TVE3, EC 6.4.1.1) was overexpressed in YM-004 strain. AoPYC gene was integrated at the GPD1 gene (corresponding amino acid sequence: SEQ ID NO: 18) site of YM-004 genome, and the used promoter and terminator were the promoter of cell wall glycoprotein gene SED1 and the terminator of Glyceraldehyde-3-phosphate dehydrogenase gene TDH3 (sequence: SEQ ID NO: 31) of YL-001 itself, respectively. The specific construction method is as follows:

[0087] 1. Constructing donor DNA fragment for homologous recombination

[0088] The GPD1 gene upstream homology arm fragment (fragment 25) was amplified from YL-001 genomic DNA using primers GPD1-F1 and GPD1-SED1p-R (see Table 5); the SED1 promoter fragment (fragment 26) was amplified from YL-001 genomic DNA using primers SED1p-GPD1-F and SED1p-AoPYC-R (see Table 5); the AoPYC gene sequence (fragment 27) was amplified from a plasmid containing the AoPYC synthetic sequence using primers AoPYC-SED1p-F and AoPYC-TDH3t-R (see Table 5); the TDH3 gene terminator fragment (fragment 28) was amplified from YL-001 genomic DNA using primers TDH3t-AoPYC-F and TDH3t-GPD1-R (see Table 5); and the GPD1 gene downstream homology arm fragment (fragment 29) was amplified from YL-001 genomic DNA using primers GPD1-TDH3t-F and GPD1-R1 (see Table 5). The five fragments were subjected to agarose gel electrophoresis, and the correct size bands were then cut and recovered. The recovered products of fragments 25-29 were used as templates for primer-free PCR amplification. Nested PCR was performed using primers GPD1-F2 and GPD1-R2 (see Table 5) and the above primer-free PCR reaction solution as the template. The correct size band was recovered after agarose gel electrophoresis to obtain fragment 30.

[0089] 2. Constructing pYL-Cas9-GPD1 plasmid for editing GPD1 site

[0090] The 10035 bp fragment containing the sgRNA sequence of the endogenous GPD1 gene of YL-001 strain (fragment 31) was amplified from the pYL-Cas9 plasmid using primers gRNA-F (SEQ ID NO: 50) and GPD1-N20-R (see Table 5). Subsequently, Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above product was purified using a SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biological Technology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-GPD1.

[0091] Table 5. Primers required for overexpression of AoPYC gene and construction of CRISPR / Cas9 plasmid

[0092]

[0093] 3. Construction of AoPYC gene overexpression strain

[0094] 1 μg of pYL-Cas9-GPD1 plasmid and 1 μg of fragment 30 were electrotransformed into YM-004 strain according to the yeast electrotransformation method, and positive transformants were screened, followed by multiple passages in YPD medium to lose the pYL-Cas9-GPD1 plasmid, and obtain malic acid producing yeast YM-005 (genotype: YM-004, GPD1::SED1p-AoPYC-TDH3t).

[0095] Example 6 Overexpression of AoMDH gene in YM-005 strain

[0096] NADH-dependent malate dehydrogenase AoMDH (Uniprot database accession number: I8U0T6, EC 1.1.1.37) derived from Aspergillus oryzae was overexpressed in YM-005 strain. AoMDH gene (SEQ ID NO: 5) was synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. and optimized according to the codon preference of YL-001. AoMDH gene was integrated into the MCH2 gene (the corresponding amino acid sequence is SEQ ID NO: 19) site of YM-005 genome, and the used promoter and terminator were the promoter of YL-001 own glyceraldehyde-3-phosphate dehydrogenase gene TDH3 (the sequence is SEQ ID NO: 32) and the terminator of 3-phosphoglycerate kinase gene PGK1 (the sequence is SEQ ID NO: 33), respectively. The specific construction method is as follows:

[0097] 1. Construction of donor DNA fragment for homologous recombination

[0098] The YL-001 genomic DNA was used as a template, and primers MCH2-F1 and MCH2-TDH3p-R (sequences are shown in Table 6) were used to amplify the upstream homologous arm fragment (fragment 32) of the MCH2 gene; the YL-001 genomic DNA was used as a template, and primers TDH3p-MCH2-F and TDH3p-AoMDH-R (sequences are shown in Table 6) were used to amplify the TDH3 promoter fragment (fragment 33) itself; the plasmid containing the AoMDH synthetic sequence was used as a template, and primers AoMDH-TDH3p-F and AoMDH-PGK1t-R (sequences are shown in Table 6) were used to amplify the AoMDH gene sequence (fragment 34); the YL-001 genomic DNA was used as a template, and primers PGK1t-AoMDH-F and PGK1t-MCH2-R (sequences are shown in Table 6) were used to amplify the PGK1 gene terminator fragment (fragment 35) itself; the YL-001 genomic DNA was used as a template, and primers MCH2-PGK1t-F and MCH2-R1 (sequences are shown in Table 6) were used to amplify the downstream homologous arm fragment (fragment 36) of the MCH2 gene. The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 32-36 were used as templates for primer-free PCR amplification. The above primer-free amplification PCR reaction liquid was used as a template, and primers MCH2-F2 and MCH2-R2 (sequences are shown in Table 6) were used for nested PCR. After agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 37.

[0099] 2. Construction of pYL-Cas9-MCH2 plasmid for editing MCH2 site

[0100] The pYL-Cas9 plasmid was used as a template, and primers gRNA-F (sequence is SEQ ID NO: 50) and MCH2-N20-R (sequence is shown in Table 6) were used to amplify a 10035 bp fragment containing the sgRNA sequence of the endogenous MCH2 gene of the YL-001 strain (fragment 38). Then, Dpn I enzyme (Thermo Scientific) was used for digestion of the plasmid template, and the above product was purified using a SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Gold Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-MCH2.

[0101] Table 6. Primers required for overexpression of AoPYC gene and construction of CRISPR / Cas9 plasmid

[0102]

[0103] 3. Construction of AoMDH overexpressing strain

[0104] 1 μg of pYL-Cas9-MCH2 plasmid and 1 μg of fragment 37 were electrotransformed into YM-005 strain according to the yeast electrotransformation method, and positive transformants were screened, followed by multiple passages in YPD medium to lose the pYL-Cas9-MCH2 plasmid, and obtain malate-producing yeast YM-006 (genotype: YM-005, MCH2::TDH3p-AoMDH-PGK1t).

[0105] Example 7. Overexpression of PkMDH2 gene in YM-006 strain

[0106] On the basis of YM-006 strain, the NADH-dependent malate dehydrogenase PkMDH2 (SEQ ID NO: 6, EC 1.1.1.37) of its own was overexpressed. The PkMDH2 gene was integrated into the MAE1 gene (the corresponding amino acid sequence is SEQ ID NO: 20) site of the YM-006 genome, and the promoter and terminator used were the promoter of the cell wall glycoprotein gene SED1 of YL-001 itself and the terminator of the pyruvate kinase gene CDC19 (the sequence is SEQ ID NO: 34), respectively. The specific construction method is as follows:

[0107] 1. Construction of donor DNA fragment for homologous recombination

[0108] The MAE1 gene upstream homologous arm fragment (fragment 39) was amplified from YL-001 genomic DNA using primers MAE-F1 and MAE-SED1p-R (sequences are shown in Table 7); the SED1 promoter fragment (fragment 40) was amplified from YL-001 genomic DNA using primers SED1p-MAE-F and SED1p-MDH2-R (sequences are shown in Table 7); the PkMDH2 gene sequence (fragment 41) was amplified from YL-001 genomic DNA using primers MDH2-SED1p-F and MDH2-CDC19t-R (sequences are shown in Table 7); the CDC19 terminator fragment (fragment 42) was amplified from YL-001 genomic DNA using primers CDC19t-MDH2-F and CDC19t-MAE-R (sequences are shown in Table 7); and the MAE1 gene downstream homologous arm fragment (fragment 43) was amplified from YL-001 genomic DNA using primers MAE-CDC19t-F and MAE-R1 (sequences are shown in Table 7). The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were recovered by gel cutting. The recovered products of fragments 39-43 were used as templates for primer-free PCR amplification. The above primer-free amplification PCR reaction solution was used as a template for nest PCR using primers MAE-F2 and MAE-R2 (sequences are shown in Table 7), and then subjected to agarose gel electrophoresis, and the correct size band was recovered by gel cutting to obtain fragment 44.

[0109] 2. Construction of pYL-Cas9-MAE1 plasmid for editing MAE1 site

[0110] A 10035 bp fragment containing the sgRNA sequence of the endogenous MAE1 gene of YL-001 strain (fragment 45) was amplified from the pYL-Cas9 plasmid using primers gRNA-F (sequence is SEQ ID NO: 50) and MAE-N20-R (sequence is shown in Table 7). Then, Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above product was purified using a SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-MAE1.

[0111] Table 7. Primers required for overexpression of PkMDH2 gene and construction of CRISPR / Cas9 plasmid

[0112]

[0113] 3. Construction of PkMDH2 gene overexpressing strain

[0114] 1 μg of pYL-Cas9-MAE1 plasmid and 1 μg of fragment 44 were electroporated into YM-006 strain according to the yeast electroporation method, and positive transformants were screened, followed by multiple passages in YPD medium to lose the pYL-Cas9-MAE1 plasmid, to obtain a malate-producing yeast YM-007 (genotype: YM-006, MAE1::SED1p-PkMDH2-CDC19t).

[0115] Example 8 Overexpression of PkPCK1 gene in YM-007 strain

[0116] On the basis of YM-007 strain, the self-derived phosphoenolpyruvate carboxykinase PkPCK1 (SEQ ID NO: 7, EC 4.1.1.49) was overexpressed. The PkPCK1 promoter was replaced with the promoter of glyceraldehyde-3-phosphate dehydrogenase TDH3 to overexpress PkPCK1. The specific construction method is as follows:

[0117] 1. Construction of donor DNA fragment for homologous recombination

[0118] YL-001 genomic DNA as a template, PCK1-F1 and PCK1-UP-R (sequences see Table 8) were used to amplify the PkPCK1 promoter upstream homologous arm fragment (fragment 46); YL-001 genomic DNA as a template, primers TDH3p-PCK1-F and TDH3p-YLPCK1-R (sequences see Table 8) were used to amplify the TDH3 promoter fragment (fragment 47); YL-001 genomic DNA as a template, primers YLPCK1-TDH3p-F and YLPCK1-R1 (sequences see Table 8) were used to amplify the PCK1 promoter downstream homologous arm fragment (fragment 48); the above three fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 46-48 were used as templates for primer-free PCR amplification. Using the above primer-free amplification PCR reaction solution as a template, primers PCK1-F2 and YLPCK1-R2 (sequences see Table 8) were used for nested PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 49.

[0119] 2. Construction of pYL-Cas9-0402 plasmid for editing PkPCK1 promoter site

[0120] The sgRNA sequence fragment containing the endogenous PkPCK1 promoter of YL-001 strain was amplified by using primers gRNA-F (the sequence is SEQ ID NO: 50) and PCK1-N20-R (the sequence is shown in Table 8) and pYL-Cas9 plasmid as a template (fragment 50). Then, the plasmid template was digested by Dpn I enzyme (Thermo Scientific), and the above product was purified by SanPrep Column PCR Product Purification Kit (Shanghai Biomed Longlife Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biotech Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named as pYL-Cas9-PCK1.

[0121] Table 8. Primers required for constructing PkPCK1 gene overexpression and CRISPR / Cas9 plasmid

[0122]

[0123] 3. Construction of PkPCK1 gene overexpression strain

[0124] According to the yeast electroporation method, 1 μg of pYL-Cas9-PCK1 plasmid and 1 μg of fragment 49 were electroporated into YM-007 strain, and positive transformants were screened, and then multiple passages were carried out in YPD medium to lose the pYL-Cas9-PCK1 plasmid, and apple acid producing yeast YM-011 (genotype: YM-007, PkPCK1p::TDH3p) was obtained.

[0125] Example 9. Overexpression of SbMDHP1 gene in YM-011 strain

[0126] On the basis of YM-011 strain, the NADPH-dependent malate dehydrogenase SbMDHP1 (Uniprot database accession number: P17606, EC 1.1.1.82) from Sorghum bicolor was overexpressed. The SbMDHP1 gene (SEQ ID NO: 8) was synthesized by Nanjing Kingsrosy Biotech Co., Ltd., and was optimized according to the codon bias of YL-001. The SbMDHP1 gene was integrated into the genome of YM-011, and the used promoter and terminator were the promoter (the sequence is SEQ ID NO: 35) of the coding enolase (Enolase) gene ENO1 and the terminator (the sequence is SEQ ID NO: 36) of the translation elongation factor (Translation Elongation Factor) TEF1 of YL-001, respectively. The specific construction method is as follows:

[0127] 1. Constructing a donor DNA fragment for homologous recombination

[0128] The YL-001 genomic DNA was used as a template to amplify the upstream homologous arm fragment (fragment 51) at the insertion site using primers YM011-F1 and YM011-ENO1p-R (sequences are shown in Table 9); the YL-001 genomic DNA was used as a template to amplify the ENO1 promoter fragment (fragment 52) itself using primers ENO1p-F and ENO1p-R (sequences are shown in Table 9); the plasmid containing the SbMDHP1 synthetic sequence was used as a template to amplify the SbMDHP1 gene sequence (fragment 53) using primers SbMDHP1-ENO1p-F and SbMDHP1-TEF1t-R (sequences are shown in Table 9); the YL-001 genomic DNA was used as a template to amplify the TEF1 terminator fragment (fragment 54) itself using primers TEF1t-F and TEF1t-R (sequences are shown in Table 9); and the YL-001 genomic DNA was used as a template to amplify the downstream homologous arm fragment (fragment 55) at the insertion site using primers YM011-TEF1t-F and YM011-R1 (sequences are shown in Table 9). The above five fragments were subjected to agarose gel electrophoresis, and then the correct-sized bands were cut and recovered. The recovered products of fragments 51-55 were used as templates for primer-free PCR amplification. The above primer-free amplification PCR reaction solution was used as a template for nested PCR using primers YM011-F2 and YM011-R2 (sequences are shown in Table 9), and then the correct-sized bands were cut and recovered after agarose gel electrophoresis to obtain fragment 56.

[0129] 2. Constructing pYL-Cas9-YM011 plasmid for editing YM-011 strain

[0130] The pYL-Cas9 plasmid was used as a template to amplify a 10035 bp fragment containing the sgRNA sequence of the endogenous insertion site in the YM-013 strain (fragment 57) using primers gRNA-F (sequence is SEQ ID NO: 50) and YM011-N20-R (sequences are shown in Table 9). Then, Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above product was purified using SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biological Technology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-YM011.

[0131] Table 9. Primers required for overexpression of SbMDHP1 gene and construction of CRISPR / Cas9 plasmid

[0132]

[0133] 3. Construction of SbMDHP1 gene overexpression strain

[0134] 1 μg of pYL-Cas9-YM011 plasmid and 1 μg of fragment 56 were electroporated into YM-011 strain according to the yeast electroporation method, and positive transformants were screened and obtained, followed by multiple passages in YPD medium to lose the pYL-Cas9-YM011 plasmid, and obtain malic acid producing yeast YM-012 (genotype: YM-011:: ENO1p-SbMDHP1-TEF1t).

[0135] Example 10 Knockout of OAD gene in YM-012 strain

[0136] The endogenous oxaloacetate decarboxylase OAD (SEQ ID NO: 21, Uniprot Accession No: A0A2U9R3C9, EC 4.1.1.112) was knocked out in the YM-012 strain. The specific construction method is as follows:

[0137] 1. Construction of donor DNA fragment for homologous recombination

[0138] YL-001 genomic DNA was used as a template, and the OAD gene upstream homologous arm fragment (fragment 58) was amplified using primers OAD-f1 and OAD-up-r (sequences are shown in Table 10); YL-001 genomic DNA was used as a template, and the OAD gene downstream homologous arm fragment (fragment 59) was amplified using primers OAD-down-f and OAD-R1 (sequences are shown in Table 10). The above two fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered product of fragment 58-fragment 59 was used as a template, and overlap extension PCR was performed using primers OAD-f1 and OAD-R1 (sequences are shown in Table 10), followed by agarose gel electrophoresis, and then the correct size bands were cut and recovered to obtain fragment 60.

[0139] 2. Construction of pYL-Cas9-OAD plasmid for editing OAD site

[0140] The 10035 bp sgRNA sequence fragment containing the endogenous OAD gene of YL-001 strain (fragment 61) was amplified using primers gRNA-F (sequence: SEQ ID NO: 50) and OAD-N20-R (sequence in Table 10) with the pYL-Cas9 plasmid as the template. Subsequently, the plasmid template was digested using Dpn I enzyme (Thermo Scientific), and the above product was purified using a SanPrep column PCR product purification kit (Shanghai Biomed Longlife Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-OAD.

[0141] Table 10. Primers required for knocking out the OAD gene and constructing the CRISPR / Cas9 plasmid

[0142]

[0143] 3. Construction of OAD gene knockout strain

[0144] According to the yeast electroporation method, 1 μg of pYL-Cas9-OAD plasmid and 1 μg of fragment 60 were electroporated into YM-012 strain, and positive transformants were screened, followed by multiple passages in YPD medium to lose the pYL-Cas9-OAD plasmid, and obtain the malate-producing yeast YM-013 (genotype: YM-012, ΔOAD).

[0145] Example 11 Overexpression of SbMDHP2 gene in YM-013 strain

[0146] On the basis of YM-013 strain, the NADPH-dependent malate dehydrogenase SbMDHP2 (Uniprot database accession number: P37229) from Sorghum bicolor was overexpressed. The SbMDHP2 gene (SEQ ID NO: 9) was synthesized by Nanjing Kingsrosy Biotech Co., Ltd., and was optimized according to the codon bias of YL-001. The SbMDHP2 gene was integrated into the insertion site of YM-015 genome, and the used promoter and terminator were the promoter of the 3-phosphogly cerate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase) gene TDH3 and the terminator of the 3-phosphoinositol synthase (Inositol-3-phosphate synthase) INO1 of YL-001, respectively (sequence: SEQ ID NO: 37). The specific construction method is as follows:

[0147] 1. Constructing a donor DNA fragment for homologous recombination

[0148] The YL-001 genomic DNA was used as a template, and primers YM015-S1-F and YM015-R (see Table 11 for sequences) were used to amplify the upstream homologous arm fragment (fragment 62) of the insertion site; the YL-001 genomic DNA was used as a template, and primers YLTDH3p-YM015-F and YLTDH3p-SbMDHP2-R (see Table 11 for sequences) were used to amplify the TDH3 promoter fragment (fragment 63) of itself; the plasmid containing the SbMDHP2 synthetic sequence was used as a template, and primers SbMDHP2-F and SbMDHP2-R (see Table 11 for sequences) were used to amplify the SbMDHP2 gene sequence (fragment 64); the YL-001 genomic DNA was used as a template, and primers YLINO1t-SbMDHP2-F and YLINO1t-YM015-R (see Table 11 for sequences) were used to amplify the INO1 gene terminator fragment (fragment 65) of itself; the YL-001 genomic DNA was used as a template, and primers YM015-F1 and YM015-S1-R (see Table 11 for sequences) were used to amplify the downstream homologous arm fragment (fragment 66) of the insertion site. The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 62-66 were used as templates for primer-free PCR amplification. The above primer-free amplification PCR reaction solution was used as a template, and primers YM015-S2-F and YM015-S2-R (see Table 11 for sequences) were used for nested PCR. After agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 67.

[0149] 2. Constructing pYL-Cas9-YM015 plasmid for editing YM-015 strain

[0150] The pYL-Cas9 plasmid was used as a template, and primers gRNA-F (sequence is SEQ ID NO: 50) and gRNA-YM015-N20-R (see Table 11 for sequences) were used to amplify a 10035 bp fragment containing the sgRNA sequence of the endogenous insertion site of the YM-015 strain (fragment 68). Then Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above product was purified using SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-YM015.

[0151] Table 11. Primers required for overexpression of SbMDHP2 gene and CRISPR / Cas9 plasmid construction

[0152]

[0153] 3. Construction of SbMDHP2 gene overexpression strain

[0154] 1 μg of pYL-Cas9-YM015 plasmid and 1 μg of fragment 67 were electroporated into YM-015 strain according to the yeast electroporation method, and positive transformants were screened, followed by multiple passages in YPD medium to lose the pYL-Cas9-YM015 plasmid, and obtain malic acid producing yeast YM-016 (genotype: YM-015::TDH3p-SbMDHP2-INO1t).

[0155] Example 12 Overexpression of SpVHT1 gene in YM-016 strain

[0156] The SpVHT1 (Uniprot database accession number: 013880) gene from S. pombe was overexpressed in the YM-016 strain. The SpVHT1 gene (SEQ ID NO: 10) was synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. and optimized according to the YL-001 codon bias. The SpVHT1 gene was integrated into the YM-016 genome, and the promoter and terminator used were the promoter (sequence is SEQ ID NO: 38) of the YL-001 own phosphoglycerate kinase (Phosphoglycerate kinase) gene PGK1 and the terminator of pyruvate kinase (Pyruvate kinase) CDC19, respectively. The specific construction method is as follows:

[0157] 1. Construction of donor DNA fragment for homologous recombination

[0158] The YL-001 genomic DNA was used as a template, and the primer YM016-S1-F and YM016-R (see Table 12 for sequences) were used to amplify the upstream homologous arm fragment (fragment 69) of the insertion site; the YL-001 genomic DNA was used as a template, and the primer YLPGK1p-YM016-F and YLPGK1p-SpVHT1-R (see Table 12 for sequences) were used to amplify the PGK1 promoter fragment (fragment 70) of itself; the plasmid containing the SpVHT1 synthetic sequence was used as a template, and the primer SpVHT1-F and SpVHT1-R (see Table 12 for sequences) were used to amplify the SpVHT1 gene sequence (fragment 71); the YL-001 genomic DNA was used as a template, and the primer YLCDC19t-SpVHT1-F and YLCDC19t-YM016-R (see Table 12 for sequences) were used to amplify the CDC19 gene terminator fragment (fragment 72) of itself; the YL-001 genomic DNA was used as a template, and the primer YM016-F and YM016-S1-R (see Table 12 for sequences) were used to amplify the downstream homologous arm fragment (fragment 73) of the insertion site. The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 69-73 were used as templates for primer-free PCR amplification. The above primer-free amplification PCR reaction solution was used as a template, and the primer YM016-S2-F and YM016-S2-R (see Table 12 for sequences) were used for nested PCR, which was subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered to obtain fragment 74.

[0159] 2. Construction of pYL-Cas9-YM016 plasmid for editing YM-016 strain

[0160] The pYL-Cas9 plasmid was used as a template, and the primer gRNA-F (sequence is SEQ ID NO: 50) and gRNA-YM016-N20-R (see Table 12 for sequences) were used to amplify a 10035 bp fragment containing the sgRNA sequence of the endogenous insertion site of the YM-016 strain (fragment 75). Then Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above product was purified using SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-YM016.

[0161] Table 12. Primers required for overexpression of SpVHT1 gene and construction of CRISPR / Cas9 plasmid

[0162]

[0163] 3. Construction of SpVHT1 gene overexpressing strain

[0164] 1 μg of pYL-Cas9-YM016 plasmid and 1 μg of fragment 74 were electrotransformed into YM-016 strain according to the method of yeast electroporation, and positive transformants were screened and obtained, followed by multiple passages in YPD medium to lose the pYL-Cas9-YM016 plasmid, and obtain malic acid producing yeast YM-017 (genotype: YM-016::PGK1p-SpVHT1-CDC19t).

[0165] Example 13 Overexpression of EcPPC(K620S) mutant gene in YM-017 strain

[0166] A mutant EcPPC(K620S) (SEQ ID NO: 11) of Escherichia coli-derived phosphoenolpyruvate carboxylase EcPPC (Uniprot database accession number: P00864, EC 4.1.1.31) was overexpressed in the YM-017 strain. The EcPPC(K620S) gene was integrated on the YM-017 genome, and the promoter and terminator used were the promoter (sequence SEQ ID NO: 39) of the YL-001 own coding 2-phosphofructokinase gene FBA1 and the terminator (sequence SEQ ID NO: 40) of the phosphoglycerate mutase GPM1, respectively. The specific construction method is as follows:

[0167] 1. Construction of pUC57-EcPPC(K620S) plasmid containing EcPPC(K620S) mutant gene

[0168] The plasmid pUC57-EcPPC containing EcPPC synthetic sequence was used as a template, and primers K620S-F and K620S-R (sequences see Table 13) were used to amplify a short sequence containing a mutation site (fragment 76), and the fragment was subjected to agarose gel electrophoresis. The pUC57-EcPPC plasmid was used as a template, and the above-mentioned recovered fragment was used as a primer to amplify a 5362bp fragment containing the EcPPC(K620S) mutant gene (fragment 77). Subsequently, Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above-mentioned product was purified using a SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The above-mentioned purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Gold Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pUC57-EcPPC(K620S).

[0169] 2. Construction of donor DNA fragment for homologous recombination

[0170] The YL-001 genomic DNA was used as a template, and primers YM017-F1 and YM017-FBA1p-R (sequences see Table 13) were used to amplify the upstream homologous arm fragment of the insertion site (fragment 78); the YL-001 genomic DNA was used as a template, and primers YLFBA1p-F and YLFBA1p-R (sequences see Table 13) were used to amplify the FBA1 promoter fragment (fragment 79); the pUC57-EcPPC(K620S) plasmid was used as a template, and primers EcPPC-FBA1p-F and EcPPC-GPM1t-R (sequences see Table 13) were used to amplify the EcPPC(K620S) gene sequence (fragment 80); the YL-001 genomic DNA was used as a template, and primers YLGPM1t-F and YLGPM1t-R (sequences see Table 13) were used to amplify the GPM1 gene terminator fragment (fragment 81); the YL-001 genomic DNA was used as a template, and primers YM017-YLGPM1t-F and YM017-R1 (sequences see Table 13) were used to amplify the downstream homologous arm fragment of the insertion site (fragment 82). The above-mentioned five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 78-82 were used as templates for primer-free PCR amplification. The above-mentioned primer-free amplification PCR reaction solution was used as a template, and primers YM017-F2 and YM017-R2 (sequences see Table 13) were used for nested PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 83.

[0171] 3. Construction of pYL-Cas9-YM017 plasmid for editing YM-017 strain

[0172] A 10035 bp fragment containing the sgRNA sequence of the endogenous insertion site of YM-017 strain (fragment 84) was amplified using primers gRNA-F (sequence of SEQ ID NO: 50) and YM017-N20-R (sequence in Table 13) with the pYL-Cas9 plasmid as template. Subsequently, the plasmid template was digested using Dpn I enzyme (Thermo Scientific), and the product was purified using SanPrep Column PCR Product Purification Kit (Shanghai Biogiga Biotech Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biotech Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-YM017.

[0173] Table 13. Primers required for overexpression of EcPPC(K620S) mutant gene and construction of CRISPR / Cas9 plasmid

[0174]

[0175] 3. Construction of EcPPC(K620S) mutant gene overexpression strain

[0176] According to the yeast electroporation method, 1 μg of pYL-Cas9-YM017 plasmid and 1 μg of fragment 83 were electroporated into YM-017 strain, and positive transformants were selected, followed by multiple passages in YPD medium to lose the pYL-Cas9-YM017 plasmid, and obtain the malate-producing yeast YM-018 (genotype: YM-017: FBA1p-EcPPC(K620S)-GPM1t).

[0177] Example 14. Knockout of SFC1 gene in YM-018 strain

[0178] The endogenous mitochondrial succinate / fumarate transporter SFC1 (SEQ ID NO: 22, Uniprot Accession No: A0A099P425) was knocked out in the YM-018 strain. The specific construction method is as follows:

[0179] 1. Construction of donor DNA fragment for homologous recombination

[0180] YL-001 genomic DNA as a template, the SFC1 gene upstream homologous arm fragment (fragment 85) was amplified using primers SFC1-F1 and SFC1-up-r2 (sequences are shown in Table 14); YL-001 genomic DNA as a template, the SFC1 gene downstream homologous arm fragment (fragment 86) was amplified using primers SFC1-dw-f2 and SFC1-R1 (sequences are shown in Table 14). The two fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered product of fragment 85-fragment 86 was used as a template, and primers SFC1-f1 and SFC1-R1 (sequences are shown in Table 14) were used for overlap extension PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 87.

[0181] 2. Construction of pYL-Cas9-SFC1 plasmid for editing SFC1 site

[0182] YL-001 genomic DNA as a template, the SFC1 gene upstream homologous arm fragment (fragment 85) was amplified using primers SFC1-F1 and SFC1-up-r2 (sequences are shown in Table 14); YL-001 genomic DNA as a template, the SFC1 gene downstream homologous arm fragment (fragment 86) was amplified using primers SFC1-dw-f2 and SFC1-R1 (sequences are shown in Table 14). The two fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered product of fragment 85-fragment 86 was used as a template, and primers SFC1-f1 and SFC1-R1 (sequences are shown in Table 14) were used for overlap extension PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 87.

[0183] Table 14. Primers required for knocking out SFC1 gene and constructing CRISPR / Cas9 plasmid

[0184]

[0185] 3. Construction of SFC1 gene knockout strain

[0186] According to the yeast electroporation method, 1 μg of pYL-Cas9-SFC1 plasmid and 1 μg of fragment 87 were electroporated into YM-018 strain, and positive transformants were selected, and then multiple passages were carried out in YPD medium to lose the pYL-Cas9-SFC1 plasmid, and apple acid-producing yeast YM-019 (genotype: YM-018, △SFC1) was obtained.

[0187] Example 15 Overexpression of EcSthA gene in YM-019 strain

[0188] Overexpressing soluble pyridine nucleotide transhydrogenase EcSthA (Uniprot database accession number: P27306, EC 1.6.1.1) from Escherichia coli on the basis of YM-019 strain. EcSthA gene (SEQ ID NO: 12) was synthesized by Nanjing Kingsrosy Biotech Co., Ltd., and optimized according to the codon preference of YL-001. EcSthA gene was integrated on the genome of YM-019, and the promoter and terminator used were the promoter of the cell wall glycoprotein gene SED1 and the terminator of enolase ENO1 (sequence is SEQ ID NO: 41) of YL-001, respectively. The specific construction method is as follows:

[0189] 1. Constructing donor DNA fragment for homologous recombination

[0190] YL-001 genomic DNA as a template, primer YM019-F1 and YM019-SED1p-r (see Table 15 for sequence) were used to amplify the upstream homologous arm fragment (fragment 89) of the insertion site; YL-001 genomic DNA as a template, primer YLSED1p-f and YLSED1p-r (see Table 15 for sequence) were used to amplify the SED1 promoter fragment (fragment 90) of itself; the plasmid containing the EcSthA synthetic sequence was used as a template, and primers EcSthA-SED1p-f and EcSthA-ENO1t-R (see Table 15 for sequence) were used to amplify the EcSthA gene sequence (fragment 91); YL-001 genomic DNA as a template, primer YLENO1t-f and YLENO1t-R (see Table 15) were used to amplify the ENO1 gene terminator fragment (fragment 92) of itself; YL-001 genomic DNA as a template, primer YM019-YLENO1t-f and YM019-R1 (see Table 15 for sequence) were used to amplify the downstream homologous arm fragment (fragment 93) of the insertion site. The above five fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered products of fragments 89-93 were used as templates for primer-free PCR amplification. Using the above primer-free amplification PCR reaction solution as a template, primers YM019-F2 and YM019-R2 (see Table 15 for sequence) were used for nested PCR, and after agarose gel electrophoresis, the correct size band was cut and recovered to obtain fragment 94.

[0191] 2. Constructing pYL-Cas9-YM019 plasmid for editing YM-019 strain

[0192] The sgRNA sequence fragment (fragment 95) containing the endogenous insertion site of YM-019 strain with a length of 10035 bp was amplified using the primer gRNA-F (the sequence is SEQ ID NO: 50) and YM019-N20-R-2 (the sequence is shown in Table 15) as templates, and then digested with Dpn I enzyme (Thermo Scientific). The above product was purified using SanPrep Column PCR Product Purification Kit (Shanghai Biomed Longlife Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Zison Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method. The obtained positive plasmid was named pYL-Cas9-YM019-2.

[0193] Table 15. Primers required for construction of EcSthA gene overexpression and CRISPR / Cas9 plasmid

[0194]

[0195] 3. Construction of EcSthA gene overexpression strain

[0196] According to the yeast electroporation method, 1 μg of pYL-Cas9-YM019-2 plasmid and 1 μg of fragment 94 were electroporated into YM-019 strain, and positive transformants were screened. Then, the pYL-Cas9-YM019-2 plasmid was lost by multiple passages in YPD medium, and the malic acid-producing yeast YM-020-2 (genotype: YM-019::SED1p-EcSthA-ENO1t) was obtained.

[0197] Example 16. Knockout of ROX1 gene in YM-020-2 strain

[0198] The endogenous transcriptional regulator ROX1 (SEQ ID NO: 23) was knocked out based on the YM-020-2 strain. The specific construction method is as follows:

[0199] 1. Construction of donor DNA fragment for homologous recombination

[0200] The YL-001 genomic DNA was used as a template, and the primer ROX1-F1 and ROX1-up-r2 (see Table 16 for sequences) were used to amplify the upstream homologous arm fragment (fragment 96) of the ROX1 gene; the YL-001 genomic DNA was used as a template, and the primer ROX1-dw-f2 and ROX1-R1 (see Table 16 for sequences) were used to amplify the downstream homologous arm fragment (fragment 97) of the ROX1 gene. The two fragments were subjected to agarose gel electrophoresis, and then the correct size bands were cut and recovered. The recovered product of fragment 96-fragment 97 was used as a template, and the primer ROX1-f1 and ROX1-R1 (see Table 16 for sequences) were used for overlap extension PCR, and then the correct size bands were cut and recovered after agarose gel electrophoresis to obtain fragment 98.

[0201] 2. Construction of pYL-Cas9-ROX1 plasmid for editing ROX1 site

[0202] The pYL-Cas9 plasmid was used as a template, and the primer gRNA-F (sequence is SEQ ID NO: 50) and ROX1-N20-R (see Table 16 for sequences) were used to amplify a 10035 bp fragment containing the sgRNA sequence of the endogenous ROX1 gene of the YL-001 strain (fragment 99). Then, Dpn I enzyme (Thermo Scientific) was used to digest the plasmid template, and the above product was purified using a SanPrep column PCR product purification kit (Shanghai Sangon Biological Engineering Co., Ltd.). The purified product was transformed into E. coli Trans1-T1 competent cells (purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., product number: CB101-01) according to the E. coli electroporation method, and the obtained positive plasmid was named pYL-Cas9-ROX1.

[0203] Table 16. Primers required for knockout of ROX1 gene and construction of CRISPR / Cas9 plasmid

[0204]

[0205] 3. Construction of ROX1 gene knockout strain

[0206] According to the yeast electroporation method, 1 μg of pYL-Cas9-ROX1 plasmid and 1 μg of fragment 98 were electroporated into the YM-020-2 strain, and positive transformants were selected, and then the pYL-Cas9-ROX1 plasmid was lost by multiple passages in YPD medium to obtain a malate-producing yeast YM-023 (genotype: YM-020-2, ΔROX1).

[0207] Example 17 Evaluation of L-malic acid production capacity of YL-001 and YM-001-YM-023 strains

[0208] 1. Shake flask fermentation

[0209] OD 600 Strains YL-001 and YM-001-YM-023 (1.5%) were inoculated into 50 mL of yeast inorganic salt medium at an inoculation rate of 10%. Strains YL-001 and YM-001-YM-020-2 were fermented in shake flasks at 30 °C and 70 rpm for 96 h, while strains YM-020-2 and YM-023 were fermented in shake flasks at 30 °C and 110 rpm for 96 h. The yield of L-malic acid was measured by HPLC.

[0210] The yeast inorganic salt culture medium consists of: 10% w / v glucose, 0.04% w / v yeast extract, 0.04% w / v urea, 3 g / L KH2PO4, 0.5 g / L MgSO4∙7H2O, 0.4 mg / L biotin, 76 mg / L uracil, 15 mg / L Na2EDTA, 4.5 mg / L ZnSO4, 0.3 mg / L CoCl2, 1 mg / L MnCl2, 0.3 mg / L CuSO4, 3 mg / L Fe2(SO4)3, 0.4 mg / L NaMoO4·2H2O, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 25 mg / L inositol, and 40 g / L CaCO3.

[0211] Table 17. L-malic acid shake-flask fermentation yields of strains YL-001 and YM-001—YM-020-2 (30℃, 70rpm)

[0212]

[0213] Table 18. L-malic acid shake-flask fermentation yields of strains YM-020-2 and YM023 (30℃, 110 rpm)

[0214]

[0215] The results of L-malic acid shake-flask fermentation above show that YM-023 has the strongest L-malic acid production capacity.

[0216] 2.5 L fermenter

[0217] The YM-023 strain was inoculated into a 5L fermenter containing 3.5L of yeast inorganic salt medium. Fermentation was carried out at 30℃, with DO controlled at 5% and an aeration rate of 1 vvm for 88 hours. The yield of L-malic acid was determined to be 106 g / L by HPLC.

[0218] The components of the yeast inorganic salt culture medium are: 20% w / v glucose, 0.08% w / v yeast powder, 0.08% w / v urea, 3 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.4 mg / L biotin, 76 mg / L uracil, 3 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.4 mg / L biotin, 76 mg / L uracil, 15 mg / L Na2EDTA, 4.5 mg / L ZnSO4, 0.3 mg / L CoCl2, 1 mg / L MnCl2, 0.3 mg / L CuSO4, 3 mg / L Fe2(SO4)3, 0.4 mg / L NaMoO4·2H2O, 1 g / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 25 mg / L myo-inositol, 60 g / L CaCO3.

[0219] The strain YM-023 is preserved, and the classification name is Pichia kudriavzevii Pichia kudriavzevii , the preservation number is CGMCC No. 35487, the preservation time is July 31, 2025, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No. 1, Xiliujie, Beichen West Road, Beijing, China Institute of Microbiology, Chinese Academy of Sciences.

[0220] The above has described the present application in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In short, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application. According to the scope of the following attached claims, some basic features can be applied.

Claims

1. A Pichia ciferrii producing L-malic acid at a high yield, characterized in that, The taxonomic name of the Pichia kudriavzevii is Pichia kudriavzevii Pichia kudriavzevii , the accession number is CGMCC No. 35487, the preservation time is July 31, 2025, and the preservation unit is China General Microbiological Culture Collection Center.

2. Use of a high L-malic acid producing Pichia kudriavzevii according to claim 1 for the fermentation production of L-malic acid.

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