Method for producing malonic acid by Saccharomyces cerevisiae through alanine pathway

Abstract

The present invention relates to a method for producing malonic acid by Saccharomyces cerevisiae through the alanine pathway, belonging to the field of bioengineering. In the present invention, Saccharomyces cerevisiae is used as the starting strain, and the aspartate decarboxylase gene (PAND) from Tribolium castaneum and the β-alanine pyruvate aminotransferase gene (BAPAT) from Bacillus cereus are integrated into the delta site of the Saccharomyces cerevisiae BY4741 genome to construct a malonic acid synthesis pathway in Saccharomyces cerevisiae, and the promoters of cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase upstream and downstream of this pathway are replaced with strong constitutive promoters to further increase the malonic acid yield. This application provides a new route for the synthesis of malonic acid in Saccharomyces cerevisiae host. After shake flask fermentation of the recombinant Saccharomyces cerevisiae, the malonic acid yield is 4.4 mg / L, and in a 5-L fermenter, the highest malonic acid yield is 44.31 mg / L.

Description

Technical Field

[0001] The present invention relates to a method for producing malonic acid by Saccharomyces cerevisiae through the alanine pathway, belonging to the field of bioengineering. Background Art

[0002] Malonic acid, also known as carrot acid, malic acid or beet acid, has two functional groups, active methylene and carboxyl, in its molecular structure, so it can participate in various chemical reactions and is a very important organic synthesis intermediate. Malonic acid is one of the top 30 chemicals that can be produced from biomass announced by the US Department of Energy.

[0003] With the rapid development of the domestic and international chemical industries, the output and quality of malonic acid are increasing day by day, and the uses and downstream products of malonic acid have been vigorously developed.

[0004] Currently, the industrial production of malonic acid usually uses the method of hydrolyzing cyanoacetic acid or malonic acid ester. These methods involve cyanide ions in the preparation process. Cyanide ions are highly toxic and cause great harm to the environment. The reaction process is complex and requires complex and cumbersome purification procedures, resulting in low product yield, difficult control of raw material purity, and difficult treatment of three wastes. These problems greatly limit the production capacity of industrial production.

[0005] Based on the above problems, more and more researchers choose to biosynthesize malonic acid through cell factories. Commonly used cells include Escherichia coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae. Sang et al. successfully constructed a malonic acid production pathway with alanine semialdehyde as a precursor in Escherichia coli by heterologous expression of β-alanine pyruvate transaminase, and the yield reached 3.6 g / L. However, using Escherichia coli as a host has problems such as poor cell tolerance and Escherichia coli toxicity, which in turn affect the stability of industrial production and the scope of application of products. Compared with Escherichia coli, Saccharomyces cerevisiae is the simplest eukaryote, with a clear genetic background, easy gene manipulation, strong genetic stability, strong vitality, strong acid resistance and stress resistance. Moreover, Saccharomyces cerevisiae can produce various types of organic acids and there are many endogenous metabolic pathways that support the synthesis of organic acids, making it one of the most commonly used strains for large-scale industrial fermentation production. In previous studies, Dietrich et al. found in Saccharomyces cerevisiae that by site-directed mutation of the EHD3 gene, it can convert malonyl-CoA as a substrate into malonic acid, making it possible to accumulate malonic acid in Saccharomyces cerevisiae, but the yield is low and cannot meet industrial production requirements. Summary of the Invention

[0006] Based on the above problems, the present invention constructs a complete metabolic pathway from aspartic acid to malonic acid by introducing exogenous β-alanine pyruvate transaminase and aspartic acid decarboxylase into Saccharomyces cerevisiae, integrates it into the delta site of Saccharomyces cerevisiae, and replaces the promoters that initiate the expression of cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase on the Saccharomyces cerevisiae genome with strong promoters by homologous recombination.

[0007] The present invention first provides a recombinant Saccharomyces cerevisiae for producing malonic acid, which overexpresses the β-alanine pyruvate transaminase gene (BAPAT) and the aspartic acid decarboxylase gene (PAND), and strongly expresses cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase.

[0008] In one embodiment, the promoter GPD initiates the expression of the BAPAT gene, and the promoter TEF1 initiates the expression of the PAND gene.

[0009] In one embodiment, the strong expression is achieved by initiating the expression of the gene encoding cytoplasmic aspartate aminotransferase by the promoter TEF1, and initiating the expression of the gene encoding succinic semialdehyde dehydrogenase by the promoter GPD.

[0010] In one embodiment, the aspartic acid decarboxylase is from Tribolium castaneum, and the β-alanine pyruvate transaminase is from Bacillus cereus.

[0011] In one embodiment, the nucleotide sequence of the PAND gene is as shown in SEQ ID NO.1, the nucleotide sequence of the BAPAT gene is as shown in SEQ ID NO.2, the nucleotide sequence of the promoter TEF1 is as shown in SEQ ID NO.3, and the nucleotide sequence of the promoter GPD is as shown in SEQ ID NO.4.

[0012] In one embodiment, the recombinant Saccharomyces cerevisiae uses Saccharomyces cerevisiae BY4741 as the starting strain.

[0013] The second object of the present invention is to provide a method for constructing the recombinant Saccharomyces cerevisiae, comprising the following steps:

[0014] (1) Integrate the overexpression cassette HTA-1 into the genome of Saccharomyces cerevisiae BY4741 to construct a recombinant strain BA-1;

[0015] (2) Integrate the overexpression cassette LGU-1 into the genome of the recombinant strain BA-1 in step (1) to construct a recombinant strain BA-2;

[0016] (3) Integrate this fragment, the DBU-1 fragment and the DPU-1 fragment, into the genome of the recombinant bacterium BA-2 in step (2) to construct the recombinant bacterium BA-3.

[0017] In one embodiment, the DBU-1 fragment consists of Delta1, GPD promoter, gene BAPAT, CYC1 terminator and Ura1; the DPU-1 fragment consists of Ura2, TEF1 promoter, PAND gene, ADH terminator and Delta2.

[0018] The third object of the present invention is to provide a method for producing malonic acid, which method is to use the above recombinant Saccharomyces cerevisiae for fermentation with glucose as a carbon source.

[0019] In one embodiment, inoculate the seed liquid of the above recombinant Saccharomyces cerevisiae into the medium at an inoculation amount of 1-3% by volume and culture at 28-30 °C for 72-168 h.

[0020] In one embodiment, the medium comprises 15-25 g / L of glucose, 5-15 g / L of yeast extract and 15-25 g / L of peptone.

[0021] The fourth object of the present invention is to provide a method for improving the extracellular secretion of malonic acid by Saccharomyces cerevisiae, which method is to overexpress the β-alanine pyruvate transaminase gene (BAPAT) and the aspartate decarboxylase gene (PAND), and to enhance the expression of cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase.

[0022] In one embodiment, the GPD promoter initiates the expression of the BAPAT gene, and the TEF1 promoter initiates the expression of the PAND gene.

[0023] In one embodiment, the enhanced expression is to initiate the expression of the gene encoding cytoplasmic aspartate aminotransferase by the TEF1 promoter and initiate the expression of the gene encoding succinic semialdehyde dehydrogenase by the GPD promoter.

[0024] In one embodiment, the aspartate decarboxylase is from Tribolium castaneum, and the β-alanine pyruvate transaminase is from Bacillus cereus.

[0025] In one embodiment, the nucleotide sequence of the PAND gene is as shown in SEQ ID NO.1, the nucleotide sequence of the BAPAT gene is as shown in SEQ ID NO.2, the nucleotide sequence of the TEF1 promoter is as shown in SEQ ID NO.3, and the nucleotide sequence of the GPD promoter is as shown in SEQ ID NO.4.

[0026] The present invention also provides the use of the recombinant Saccharomyces cerevisiae or the method for producing malonic acid in the preparation of malonic acid or its derivative products.

[0027] Beneficial effects

[0028] 1. The present invention uses Saccharomyces cerevisiae as the starting strain, integrates the aspartate decarboxylase gene (PAND) from Tribolium castaneum and the β-alanine pyruvate aminotransferase gene (BAPAT) from Bacillus cereus into the delta site of the Saccharomyces cerevisiae BY4741 genome to construct the malonic acid synthesis pathway. At the same time, by replacing the promoters of the upstream and downstream cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase in this pathway with strong constitutive promoters, a recombinant Saccharomyces cerevisiae with high malonic acid production is constructed, providing a new route for the synthesis of malonic acid in the Saccharomyces cerevisiae host.

[0029] 2. After shake-flask fermentation of the recombinant Saccharomyces cerevisiae constructed in this application, the malonic acid yield is 4.4 mg / L. In a 5-L fermenter, the highest malonic acid yield is 44.31 mg / L. Description of the drawings

[0030] Figure 1 Pathway for alanine to synthesize malonic acid and method for integrating into the Delta site;

[0031] Figure 2 Method for integrating the AAT2 and UGA2 overexpression cassettes;

[0032] Figure 3 Fermentation production of malonic acid in a 5-L fermenter. Detailed implementation manners

[0033] The following media are involved in the following examples:

[0034] (1) SD-His-deficient medium: Glucose 20 g / L, amino acid-free yeast nitrogen source 1.7 g / L, ammonium sulfate 5.0 g / L, 10× essential amino acid mixture 10 mL, 10× Ura mixture 10 mL, 10× Leu mixture 10 mL. Add 2.0% agar powder to prepare a solid medium, and sterilize at 115 °C for 30 min.

[0035] (2) SD-His-Leu-deficient medium: Glucose 20 g / L, amino acid-free yeast nitrogen source 1.7 g / L, ammonium sulfate 5.0 g / L, 10× essential amino acid mixture 10 mL, 10× Ura mixture 10 mL. Add 2.0% agar powder to prepare a solid medium, and sterilize at 115 °C for 30 min.

[0036] (3) SD-His-Leu-Ura Deficient Medium: Glucose 20 g / L, amino acid-free yeast nitrogen source 1.7 g / L, ammonium sulfate 5.0 g / L, 10× essential amino acid mixture 10 mL. Add 2.0% agar powder to prepare solid medium, and sterilize at 115°C for 30 min.

[0037] (4) YPD Liquid Medium: Glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L. Add 2.0% agar powder to prepare solid medium, and sterilize at 115°C for 30 min.

[0038] The experimental methods involved in the following examples:

[0039] (1) Touchdown PCR:

[0040] Reaction system:

[0041] The 50 μL PCR reaction system includes the following components:

[0042]

[0043] Reaction procedure: Pre-denaturation at 98°C for 10 min; denaturation at 98°C for 30 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, for 15 cycles; denaturation at 98°C for 30 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, for 35 cycles; final extension at 72°C for 10 min; keep at 4°C until taken out.

[0044] (2) Malonic Acid Liquid Chromatography-Mass Spectrometry Detection:

[0045] Pretreatment: Centrifuge the fermentation sample at 12,000 rpm for 2 min to separate the fermentation broth from the cells, and treat the fermentation broth with a 0.22 μm filter membrane for liquid chromatography-mass spectrometry detection.

[0046] Liquid chromatography-mass spectrometry conditions: Detection wavelength: 200 - 400 nm, analytical column: BEH C18 (2.1 x 150 mm 1.7 μm), column temperature 45°C, flow rate: 0.3 ml / min, injection volume: 5 μL, detector: Waters Acquity PDA (200 - 400 nm); mobile phase A is 0.1% (v / v) formic acid, and mobile phase B is acetonitrile.

[0047] Table 1 Primer sequence list involved in the following examples

[0048]

[0049]

[0050] The plasmids involved in the following examples:

[0051] pY26-TEF-GPD: It has been published in the article, and the plasmid in this article is named pY26-GPD-TEF; see the article: Na Chen, Jingya Wang, Yunying Zhao* and Yu Deng*. Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer. Microbial Cell Factories, 2018, 17:67.

[0052] pRS313: It has been published in the article, see the article: Sikorski, R.S., Hieter, P., 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122(1), 19–27.

[0053] pHAC181: It has been published in the article, see the article: Yunying Zhao, Huihui Xu, Yan Zhang, Linghuo Jiang. Vcx1-D1(M383I), the Vcx1 mutant with a calcineurin-independent vacuolar Ca(2+) / H(+) exchanger activity, confers calcineurin-independent Mn(2+) tolerance in Saccharomyces cerevisiae. Can J Microbiol, 2016, 62(6):475-484.

[0054] Example 1 Construction of Recombinant Plasmids and Recombinant Saccharomyces cerevisiae

[0055] (1) Construction of plasmid pY26-TEF-GPD-PAND-BAPAT

[0056] The gene PAND fragment is from Tribolium castaneum. After codon optimization, it was fully gene synthesized by GenScript Biotech Corporation into the pUC57-PAND plasmid. The gene BAPAT fragment is from Bacillus cereus. After codon optimization, it was fully gene synthesized by GenScript Biotech Corporation into the pUC57-BAPAT plasmid.

[0057] Using pUC57-PAND as a template and pY26-PANDF / pY26-PANDR as primers, a PAND gene fragment with the nucleotide sequence shown in SEQ ID NO.1 was obtained by PCR amplification. The PAND gene fragment and the plasmid pY26-TEF-GPD were respectively double digested with BglII / NotI. After purifying and recovering the digested products, the plasmid pY26-TEF-GPD-PAND was constructed by overnight ligation with T4 ligase.

[0058] Using pUC57-BAPAT as a template and pY26-BAPATF / pY26-BAPATR as primers, a BAPAT gene fragment with the nucleotide sequence shown in SEQ ID NO.2 was obtained by PCR amplification. The BAPAT gene fragment and the plasmid pY26-TEF-GPD-PAND were respectively double digested with SalI / BamHI. After purifying and recovering the digested products, the plasmid pY26-TEF-GPD-PAND-BAPAT was constructed by overnight ligation with T4 ligase.

[0059] (2) Obtaining of the integrated fragments DBU-1 and DPU-1

[0060] Using the Saccharomyces cerevisiae BY4741 genome as a template and Delta1F / Delta1R and Delta2F / Delta2R as primers respectively, the Delta1 and Delta2 fragments were obtained by PCR amplification;

[0061] Using the plasmid pY26-TEF-GPD-PAND-BAPAT obtained in step (1) as a template and B-GPDF / B-GPDR and P-TEFF / P-TEFR as primers respectively, the fragments B-GPD (including the promoter GPD, the gene BAPAT, and the terminator CYC1) and P-TEF (including the promoter TEF, the gene PAND, and the terminator ADH1) were amplified by PCR;

[0062] Using the plasmid pY26-TEF-GPD-PAND-BAPAT obtained in step (1) as a template, the fragments Ura1 and Ura2 were amplified using the primers Ura1F / Ura1R and Ura2F / Ura2R respectively.

[0063] Perform touchdown PCR on the three fragments of Delta1, B-GPD, and Ura1. Using the PCR product as a template and Delta1F / Ura1R as primers, amplify the integration fragment DBU-1.

[0064] Perform touchdown PCR on Ura2, P-TEF, and Delta2. Using the PCR product as a template and Ura2F / Delta2R as primers, amplify the integration fragment DPU-1, as Figure 1 .

[0065] (3) Obtaining overexpression cassettes HTA-1 and LGU-1

[0066] Using the genome of Saccharomyces cerevisiae BY4741 as a template, and TEF1F / TEF1R and GPDF / GPDR as primers respectively, perform PCR amplification on the TEF1 and GPD promoter fragments;

[0067] Using the plasmid pRS313 as a template and HisF / HisR as primers, perform PCR amplification on the His fragment;

[0068] Using the plasmid pHAC181 as a template and LeuF / LeuR as primers, perform PCR amplification on the Leu fragment;

[0069] Using the genome of Saccharomyces cerevisiae BY4741 as a template, and AAT2UF / AAT2UR, AAT2DF / AAT2DR, UGA2UF / UGA2UR, and UGA2DF / UGA2DR as primers respectively, perform PCR amplification on the fragment AAT2U containing the upstream homologous arm of the AAT2 gene and the fragment AAT2D of the downstream homologous arm, the fragment UGA2U containing the upstream homologous arm of UGA2 and the fragment UGA2D of the downstream homologous arm.

[0070] Perform touchdown PCR on the four fragments of His, AAT2U, AAT2D, and TEF1 prepared above. Using the PCR product as a template and AAT2UF / AAT2DR as primers, amplify the HTA-1 fragment.

[0071] Perform touchdown PCR on the four fragments of Leu, UGA2U, UGA2D, and GPD. Using the PCR product as a template and UGA2UF / UGA2DR as primers, amplify the LGU-1 fragment, as Figure 2 .

[0072] (4) Constructing recombinant Saccharomyces cerevisiae strains

[0073] The overexpression cassette fragment HTA-1 constructed in Example 1 was transformed into Saccharomyces cerevisiae BY4741 by the lithium acetate transformation method, and spread on SD-His-deficient medium. It was cultured at 30 °C for 2-3 days. The transformants picked were the successfully integrated recombinant bacteria, named BA-1. Then, the overexpression cassette fragment LGU-1 constructed in Example 3 was transformed into the recombinant bacterium BA-1 by the lithium acetate transformation method, and spread on SD-His-Leu-deficient medium. It was cultured at 30 °C for 2-3 days. The transformants picked were the successfully integrated recombinant bacteria, named BA-2. The integrated fragments DBU-1 and DPU-1 obtained in Example 1 were transformed into the recombinant bacterium BA-2 by the lithium acetate transformation method, and spread on SD-His-Leu-Ura-deficient medium. It was cultured at 30 °C for 2-3 days. The transformants picked were the successfully integrated recombinant bacteria.

[0074] Example 2 Screening of recombinant bacteria by shake-flask fermentation

[0075] The recombinant bacteria obtained in Example 1 were respectively inoculated into 10 ml of YPD liquid medium and cultured overnight at 30 °C and 220 rpm to obtain seed liquid. The seed liquid was transferred to 50 ml of YPD liquid medium at an inoculation amount of 2% by volume and cultured on a shaker at 30 °C and 220 rpm for 7 d to prepare fermentation broth.

[0076] During the fermentation process, samples were taken every 12 h, centrifuged at 13000 rpm for 5 min to obtain the supernatant, and filtered through a 0.22 μm filter membrane. The filtered supernatant was used for HPLC detection. A differential detector was used, the column temperature was 50 °C, and the injection volume was 20 μl. According to the results of liquid chromatography, a strain with high malonic acid production was screened out. The yield after 7 d of culture was 4.4 mg / L, named BA-3.

[0077] Example 3 Fermentation production of malonic acid in a 5 L fermenter

[0078] The screened malonic acid engineering bacteria were inoculated into 60 mL of YPD medium and cultured overnight at 220 rpm and 30 °C until the OD600 was about 5.0. After transfer at a volume ratio of 2%, the initial OD600 value was 0.1, the initial medium volume of YPD was 3 L, the temperature was 30 °C, the pH was controlled above 4, the rotation speed was 700-800 rpm, and the ventilation volume was 3 L / min. Fermentation was carried out for 216 h, and the final yield was 44.31 mg / L ( Figure 3 ).

[0079] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims. SEQUENCE LISTING <110> Jiangnan University <120> Method for producing malonic acid by Saccharomyces cerevisiae through alanine pathway <130> BAA211101A <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 1620 <212> DNA <213> Artificial sequence <400> 1 atgccggcca ccggtgaaga ccaagacctg gttcaggacc taatagaaga accagcaacc 60 tttagcgacg ctgtactgtc aagcgatgag gagttgtttc accaaaaatg cccgaagcca 120 gccccgattt acagccccgt aagtaagcca gtgtcctttg agagtttgcc aaacagaaga 180 ttgcacgagg aatttttgag aagctcagtt gacgtacttt tgcaggaagc ggtttttgag 240 ggtaccaata gaaaaaatcg tgtcttacaa tggcgtgagc ctgaagaact gcgtagactg 300 atggactttg gagtcagaag tgctccgtca actcatgagg aattgctaga agtgttgaaa 360 aaagtggtaa cttatagcgt gaaaactggc cacccatact ttgtcaatca actgttcagc 420 gcagtagatc catatgggtt agtagctcag tgggcgacgg atgcacttaa tccttccgta 480 tatacatatg aagtaagccc agtcttcgtg ttaatggaag aagtggtgtt aagggagatg 540 agggctattg taggcttcga aggggggaag ggtgacggca tcttttgccc cggtggatct 600 atcgcgaacg gatatgcgat atcctgtgcc aggtacaggt ttatgccaga tataaagaag 660 aagggactac acagtttacc caggcttgtg ttatttacta gtgaagacgc tcactatagt 720 attaagaagt tagcatcctt ccaagggata ggcactgaca acgtatactt aattcgtacg 780 gatgctcgtg gaagaatgga tgtatctcat ctggtagaag agatcgagag gtcactaaga 840 gagggagcag caccatttat ggtatcagcc actgctggta caacagtgat tggtgcgttt 900 gatccaattg aaaaaattgc tgacgtgtgc cagaaataca agttatggct tcatgtcgat 960 gcggcctggg gcggtggggc cctagtaagc gcgaagcata ggcacctttt aaagggtatt 1020 gagcgtgcgg acagtgttac atggaatcct cataagctgc tgactgcccc gcagcaatgc 1080 tccaccttgc tgctgaggca cgaaggcgta ctggcggaag ctcatagcac caacgcggcg 1140 tatttgtttc aaaaggataa gttctatgat acgaagtacg ataccggaga caaacacata 1200 caatgtggga ggagggctga tgtgcttaaa ttctggttca tgtggaaagc taagggtacc 1260 agtggtctgg aaaagcatgt tgacaaagtg tttgagaacg caaggttttt tacggactgc 1320 atcaagaaca gagaaggctt cgaaatggtc attgcggaac cagagtatac aaacatctgt 1380 ttttggtatg tgccgaaatc attacgtggt aggaaagacg aagcggacta taaggacaaa 1440 ttacacaagg ttgcccccag gatcaaggaa aggatgatga aggaaggttc catgatggtt 1500 acataccagg ctcagaaggg gcacccaaac tttttccgta tagtgtttca gaattctgga 1560 cttgataagg ccgacatggt tcatctggta gaagaaatag agaggcttgg ttccgatctg 1620 <210> 2 <211> 1353 <212> DNA <213> Artificial sequence <400> 2 atggagttaa tgattgtgca ggtaaccgaa cagacgcagt cattaaagaa aaccgatgag 60 aagtacctgt ggcacgctat gagaggtgca gccccatccc caaccaactt aataatcact 120 aaggcagaag gtgcgtgggt tactgatatt gatggtaaca ggtatctgga cggaatgtct 180 ggactttggt gtgtaaacgt tggctacggc agaaaggagt tggctagagc cgcgtttgaa 240 caattggagg agatgcccta tttcccgctt acgcaatcac acgtaccagc gatcaaattg 300 gccgagaagc tgaacgaatg gctggatgat gaatatgtta ttttctttag caacagtggt 360 tccgaagcga atgagactgc attcaaaatt gctaggcaat atcaccagca gaagggtgac 420 catggaaggt ataaattcat cagcagatat agagcatatc atggaaatag tatgggagcc 480 ttggccgcca caggtcaggc ccagcgtaaa tacaaatatg agccattagg ccaagggttc 540 ttacatgtag cgccgccgga tacatacaga aacccagagg acgtgcatac gttggcatcc 600 gctgaggaaa tcgacagagt tatgacttgg gagctaagtc agactgtggc tggcgtcata 660 atggaaccta taataacagg cggcgggata ctaatgccac cagacggata tatggaaaag 720 gtgaaggaaa tatgtgaaaa acacggcgct ttattgatct gcgatgaggt aatctgtgga 780 tttggacgta cagggaaacc tttcgggttc atgaattatg gcgtaaagcc agacattatt 840 acgatggcta agggaattac gtctgcttac ctgcctctaa gtgcaacagc cgtgaggagg 900 gaagtgtatg aggcatttgt aggttcagat gactacgaca ggttccgtca tgttaataca 960 tttggaggaa atccggcagc ctgcgcattg gcattaaaaa acttggaaat aatggaaaac 1020 gagaagctga tcgaaagaag taaagaacta ggtgagcgtt tgttgtacga gctggaggac 1080 gtaaaggagc atccgaacgt cggggatgta agagggaagg gtctgctact aggaatcgag 1140 cttgtcgagg acaaacagac caaagagcct gcaagtatag agaaaatgaa caaggttata 1200 aacgcgtgca aggaaaaagg gctaattata ggaaaaaacg gtgatacagt cgcgggttat 1260 aacaatattt tgcaacttgc accaccgtta tccatcacag aagaggactt cacgttcatc 1320 gtgaaaacga tgaaagaatg cctagcacaa tta 1353 <210> 3 <211> 1000 <212> DNA <213> Artificial Sequence <400> 3 cttcatcggt atcttcgcta tattcttttt agtcgaattt gcggggagaa gatggatcta 60 tgctaaacta aataggcatt tgaaaaacga cgacgagtta cacgacatat cgccatcttt 120 aaatgagcaa ccacactggg acctcataga ggacgggtct cgctggagta aatttttcaa 180 cgggataatt aagacgacaa gaaggttcac gaaatcttta atgaggtctt tagtcagagg 240 caggaacagc cgtcaagggg gcataagact acggtcatcc ccatctgcct cttcgtccag 300 ccttgccaac agggagttct tcagagacat ggaggctcaa aacgaaatta ttgacagcct 360 agacatcaat agtcatacaa cagaaagcga ccacccaact ttggctgata atagcgtata 420 aacaatgcat actttgtacg ttcaaaatac aatgcagtag atatatttat gcatattaca 480 tataatacat atcacatagg aagcaacagg cgcgttggac ttttaatttt cgaggaccgc 540 gaatccttac atcacaccca atcccccaca agtgatcccc cacacaccat agcttcaaaa 600 tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac 660 ttcaaaacac ccaagcacag catactaaat ttcccctctt tcttcctcta gggtgtcgtt 720 aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg 780 tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaattt ttttttttga 840 tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc 900 tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag 960 aaagaaagca tagcaatcta atctaagttt taattacaaa 1000 <210> 4 <211> 1000 <212> DNA <213> Artificial Sequence <400> 4 agcccgaaag agttatcgtt actccgatta ttttgtacag ctgatgggac cttgccgtct 60 tcattttttt tttttttcac ctatagagcc gggcagagct gcccggctta actaagggcc 120 ggaaaaaaaa cggaaaaaag aaagccaagc gtgtagacgt agtataacag tatatctgac 180 acgcacgtga tgaccacgta atcgcatcgc ccctcacctc tcacctctca ccgctgactc 240 agcttcacta aaaaggaaaa tatatactct ttcccaggca aggtgacagc ggtccccgtc 300 tcctccacaa aggcctctcc tggggtttga gcaagtctaa gtttacgtag cataaaaatt 360 ctcggattgc gtcaaataat aaaaaaagta accccacttc tacttctaca tcggaaaaac 420 attccattca catatcgtct ttggcctatc ttgttttgtc ctcggtagat caggtcagta 480 caaacgcaac acgaaagaac aaaaaaagaa gaaaacagaa ggccaagaca gggtcaatga 540 gactgttgtc ctcctactgt ccctatgtct ctggccgatc acgcgccatt gtccctcaga 600 aacaaatcaa acacccacac cccgggcacc caaagtcccc acccacacca ccaatacgta 660 aacggggcgc cccctgcagg ccctcctgcg cgcggcctcc cgccttgctt ctctcccctt 720 ccttttcttt ttccagtttt ccctattttg tccctttttc cgcacaacaa gtatcagaat 780 gggttcatca aatctatcca acctaattcg cacgtagact ggcttggtat tggcagtttc 840 gtagttatat atatactacc atgagtgaaa ctgttacgtt accttaaatt ctttctccct 900 ttaattttct tttatcttac tctcctacat aagacatcaa gaaacaattg tatattgtac 960 accccccccc tccacaaaca caaatattga taatataaag 1000

Claims

1. A recombinant Saccharomyces cerevisiae for producing malonic acid, characterized in that, Overexpress the β-alanine pyruvate transaminase gene BAPAT and the aspartate decarboxylase gene PAND , and enhance the expression of cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase; the promoter GPD initiates BAPAT the expression of the gene, and the promoter TEF1 initiates PAND the expression of the gene; The overexpression is to integrate the β-alanine pyruvate transaminase gene BAPAT and the aspartate decarboxylase gene PAND into the delta site; the enhanced expression is achieved by initiating the expression of the gene encoding cytoplasmic aspartate aminotransferase through the promoter TEF1, and initiating the expression of the gene encoding succinic semialdehyde dehydrogenase through the promoter GPD; The said PAND The nucleotide sequence of the BAPAT gene is shown in SEQ ID NO.1, the nucleotide sequence of the gene is shown in SEQ ID NO.2, the nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO.3, and the nucleotide sequence of the promoter GPD is shown in SEQ ID NO.

4.

2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that, using Saccharomyces cerevisiae BY4741 as the starting strain.

3. A method for producing malonic acid, characterized in that, using glucose as the carbon source and fermenting with the recombinant Saccharomyces cerevisiae according to claim 1.

4. The method according to claim 3, characterized in that, inoculating the seed liquid of the recombinant Saccharomyces cerevisiae according to claim 1 into the medium at an inoculation amount of 1 - 3% by volume, and culturing at 28 - 30 °C for 72 - 168 h.

5. A method for enhancing the extracellular secretion of malonic acid by Saccharomyces cerevisiae, characterized in that, The method is to overexpress the β-alanine pyruvate transaminase gene BAPAT and the aspartate decarboxylase gene PAND , and enhance the expression of cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase; the promoter GPD initiates BAPAT the expression of the gene, and the promoter TEF1 initiates PAND the expression of the gene; The overexpression is achieved by integrating the β-alanine pyruvate transaminase gene BAPAT and the aspartate decarboxylase gene PAND into the delta site; the enhanced expression is achieved by initiating the expression of the gene encoding cytoplasmic aspartate aminotransferase through the promoter TEF1, and initiating the expression of the gene encoding succinic semialdehyde dehydrogenase through the promoter GPD; The PAND nucleotide sequence of the BAPAT gene is shown in SEQ ID NO.1, the nucleotide sequence of the gene is shown in SEQ ID NO.2, the nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO.3, and the nucleotide sequence of the promoter GPD is shown in SEQ ID NO.

4.

6. Use of the recombinant Saccharomyces cerevisiae according to claim 1 or the method according to claim 5 in the preparation of malonic acid.

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