Construction method and application of recombinant yarrowia lipolytica with high succinic acid yield

Abstract

The invention relates to the field of bioengineering, in particular to a construction method and application of recombinant yarrowia lipolytica with high succinic acid yield. The recombinant yarrowia lipolytica disclosed by the invention simultaneously expresses a reduced tricarboxylic acid pathway related gene, a glyoxylic acid circulation pathway related group, a heterologous dicarboxylic acid transport related gene and a mitochondrial transport related gene, and an endogenous dicarboxylic acid transport protein coding gene is also knocked out; and a natural promoter of an isocitrate dehydrogenase coding gene IDH1 is replaced. The recombinant yarrowia lipolytica constructed by the invention can produce 112.85 g / L of succinic acid after fed-batch fermentation under the condition of not controlling pH, and efficient synthesis of succinic acid in the yarrowia lipolytica is realized.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a construction method of a recombinant Yarrowia lipolytica for high succinic acid production and application thereof. Background Art

[0002] Succinic acid, also known as succinic acid, is an important dicarboxylic acid that can be used as a synthetic precursor for a variety of chemicals, such as 2-pyrrolidone, maleimide, γ-butyrolactone and 1,4-butanediol. These succinic acid derivatives are widely used in materials, pharmaceuticals, food and other fields. Succinic acid is also a synthetic precursor for biodegradable plastics, such as polybutylene succinate PBS, polybutylene succinate adipate PBSA, etc. Therefore, succinic acid is listed as one of the twelve major bio-based platform chemicals by the U.S. Department of Energy. In industry, succinic acid is mainly obtained by chemical synthesis, which can be divided into oxidation synthesis based on paraffin materials, catalytic hydrogenation synthesis, and oxidation using electrolysis. These chemical synthesis methods consume large amounts of petrochemical raw materials and cause serious pollution. In comparison, the microbial fermentation method has regenerative raw materials, mild reactions, and a green process. Therefore, the production of succinic acid using microbial fermentation is an economical and efficient way. The microbial synthesis of succinic acid mainly relies on bacteria or yeast. Most bacteria produce succinic acid by anaerobic fermentation and have a high yield, but bacterial growth often requires a near-neutral culture environment. In the production process of succinic acid synthesized by bacteria, alkali needs to be continuously added to maintain a neutral pH, which will make the downstream purification process of bacterial succinic acid synthesis more complicated and more expensive. Compared with bacteria, yeast is more resistant to harsh environments such as acidity, which makes the downstream purification process of yeast succinic acid synthesis under low pH conditions simpler and less expensive. In general, if the yeast-based succinic acid production yield can be increased to the level of bacterial fermentation, yeast may be more suitable for the biomanufacturing of succinic acid than bacteria. There are three main microbial synthesis pathways of succinic acid, including the oxidation tricarboxylic acid (TCA) pathway, the reduction tricarboxylic acid (TCA) pathway, and the glyoxylate cycle pathway. The synthesis of succinic acid based on the oxidation tricarboxylic acid (TCA) pathway requires the reduction of succinate dehydrogenase (Sdh) activity. This process is accompanied by the release of CO2 and the production of NADH, which has a kinetic advantage. The reductive tricarboxylic acid (TCA) pathway mainly exists in bacteria. Although some fungi have some enzymes of this pathway, these enzymes are subject to strict metabolic regulation. This pathway fixes one molecule of CO2 for each molecule of succinate synthesized. Compared with the oxidative tricarboxylic acid (TCA) pathway, it has a higher theoretical yield, but this process requires the conversion of two molecules of NADH into NAD + The glyoxylate cycle is a complementary pathway to the above two pathways, such as supplementing the intermediate metabolite succinate from the oxidative tricarboxylic acid (TCA) pathway. The optimal pathway for succinate synthesis may be a combination of the reductive tricarboxylic acid (TCA) pathway and the glyoxylate cycle pathway.

[0003] Yarrowia lipolytica is an unconventional yeast with a sequenced genome and relatively mature metabolic engineering tools, and it has high tolerance to acidic environments. In the prior art, Yarrowia lipolytica has achieved the production of succinic acid using the reduced tricarboxylic acid (TCA) pathway, and the reduced tricarboxylic acid (TCA) pathway is located in the mitochondrial matrix to couple the oxidative tricarboxylic acid (TCA) pathway to promote the recycling of NADH (see Chinese patents CN107916275A and CN114806913A for details). However, Yarrowia lipolytica has high oxidative tricarboxylic acid (TCA) pathway activity, and will continuously release CO2 during the fermentation process, reducing the conversion rate of the substrate.

[0004] In view of this, if the reductive tricarboxylic acid (TCA) pathway and the glyoxylate cycle pathway are combined through synthetic biology means, and the carbon flux of the oxidative tricarboxylic acid (TCA) pathway is redirected to the glyoxylate cycle pathway, the carbon loss of the substrate can be reduced, and the production of succinic acid is expected to be further increased. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a construction method and application of a recombinant Yarrowia lipolytica with high succinic acid production in view of the deficiencies in the prior art.

[0006] In order to solve the above technical problems, the present invention discloses a method for constructing a recombinant Yarrowia lipolytica with high succinic acid production and its application. The following technical solutions are used:

[0007] A recombinant Yarrowia lipolytica strain with high succinate production, wherein the recombinant Yarrowia lipolytica strain simultaneously expresses genes related to the reduction tricarboxylic acid pathway, groups related to the glyoxylate cycle pathway, and genes related to heterologous dicarboxylic acid transport; wherein the genes related to the reduction tricarboxylic acid pathway include any one or more combinations of pyruvate carboxylase gene PYC, malate dehydrogenase gene MDH, fumarase gene FUMR, and fumarate reductase gene FRD; preferably, the genes related to the reduction tricarboxylic acid pathway are PYC, MDH, FUMR, and FRD. The groups related to the glyoxylate cycle pathway include any one or a combination of two of the isocitrate lyase gene ICL or the malate synthase gene MLS; preferably, ICL and MLS. The heterologous dicarboxylic acid transport-related genes include the heterologous tetracarboxylic acid transporter gene MAE1.

[0008] Wherein, the PYC is derived from Issatchenkia orientalis or Yarrowia lipolytica; preferably, it is derived from Yarrowia lipolytica.

[0009] The MDH is derived from Issatchenkia orientalis or Yarrowia lipolytica, preferably from Issatchenkia orientalis. The FUMR is derived from Issatchenkia orientalis or Escherichia coli, preferably from Issatchenkia orientalis. The FRD is derived from Trypanosoma Brucei.

[0010] Among them, the nucleotide sequence of PYC derived from Issatchenkia orientalis is shown as SEQ ID No.6 (obtained by codon optimization from GenBank No.CAA71993.1), and the GenBank accession number of the nucleotide sequence of PYC derived from Yarrowia lipolytica is YALI0C24101g; the nucleotide sequence of MDH derived from Issatchenkia orientalis is shown as SEQ ID No.1 (obtained by codon optimization from GenBank No.KGK36641.1), and the GenBank accession number of the nucleotide sequence of MDH derived from Yarrowia lipolytica is YALI0E14190g; the nucleotide sequence of FUMR derived from Issatchenkiaorientalis is shown as SEQ ID The nucleotide sequence of FUMR derived from Escherichia coli is shown in SEQ ID No.2 (obtained by codon optimization from GenBank No.KGK38814.1), the nucleotide sequence of FUMR derived from Escherichia coli is shown in SEQ ID No.3 (obtained by codon optimization from GenBank No.b1611); the nucleotide sequence of FRD derived from Trypanosoma Brucei is shown in SEQ ID No.4 (obtained by codon optimization from GenBank No.AAN40014.1).

[0011] Wherein, the ICL and MLS are both derived from Yarrowia lipolytica, and the MAE1 is derived from Schizosaccharomyces pombe; the GenBank accession number of the nucleotide sequence of the ICL is YALI0C16885g; the GenBank accession number of the nucleotide sequence of the MLS is YALI0D19140g; the nucleotide sequence of the MAE1 is shown in SEQ ID No.5 (obtained from GenBank No.SPAPB8E5.03 by codon optimization).

[0012] Wherein, the recombinant Yarrowia lipolytica expresses mitochondrial transport-related genes at the same time, and the mitochondrial transport-related genes include any one or a combination of the mitochondrial citrate transporter encoding gene YHM2 or the mitochondrial pyruvate carrier encoding gene MPC. Preferably, YHM2 and MPC. Wherein, the YHM2 and MPC are both derived from Yarrowia lipolytica, and the GenBank accession number of the YHM2 is YALI0B10736g; the GenBank accession number of the MPC is YALI0F00264g and / or YALI0C03223g, the GenBank accession number of MPC1 is YALI0F00264g, and the GenBank accession number of MPC3 is YALI0C03223g. Preferably, MPC1. Wherein, the recombinant Yarrowia lipolytica simultaneously knocks out the dicarboxylic acid transporter encoding gene JEN1, and the GenBank accession number of the JEN1 is YALI0C15488g.

[0013] In the recombinant Yarrowia lipolytica, the natural promoter of the isocitrate dehydrogenase encoding gene IDH1 is replaced by promoter P1 or P2, the nucleotide sequence of P1 is shown in SEQ ID No.7, and the nucleotide sequence of P2 is shown in SEQ ID No.8, preferably P2. The natural promoter (P1) of the endogenous isocitrate dehydrogenase encoding gene IDH1 is replaced by the constitutive promoter P1 with low endogenous expression in Yarrowia lipolytica or the promoter P2 with reduced expression under high carbon-nitrogen ratio culture conditions. IDH1 , 800 bp before ATG). IDH1 The nucleotide sequence is shown in SEQ ID No.9.

[0014] Preferably, the recombinant Yarrowia lipolytica further expresses a marker gene; the marker gene is selected from an orotidine-5'-phosphate decarboxylase encoding gene expression cassette.

[0015] The recombinant Yarrowia lipolytica is derived from Po1fΔku70-SA0, which is obtained by knocking out the KU70 gene, the succinate dehydrogenase gene SDH5, and the succinyl-CoA transferase gene ACH1 from the Po1f strain. Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection, numbered ATCC MYA-2613, and the construction method of Po1fΔku70-SA0 is disclosed in Cui et al., Metabolic Engineering, 2017, 42, 126-133.

[0016] Preferably, the present invention provides a recombinant Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica expresses the malate dehydrogenase 3 encoding gene IoMDH3 from Issatchenia orientalis, the fumarase encoding gene IoFUMR, the fumarate reductase encoding gene TbFRD from Trypanosoma brucei, and the four-carbon dicarboxylate transporter encoding gene SpMae1 from Schizosaccharomyces pombe, and the endogenous dicarboxylate transporter encoding gene JEN1 is knocked out, and the endogenous pyruvate carboxylase encoding gene PYC, the isocitrate lyase encoding gene ICL, the malate synthase encoding gene MLS, the mitochondrial citrate transporter encoding gene YHM2, and the mitochondrial pyruvate carrier encoding gene MPC1 are expressed, and the endogenous promoter P of the isocitrate dehydrogenase encoding gene IDH1 is knocked out. IDH1 Replace with P2;

[0017] Preferably, the present invention provides a method for constructing the recombinant Yarrowia lipolytica, wherein genes related to the reduced tricarboxylic acid pathway, glyoxylate cycle pathway and heterologous dicarboxylic acid transport related genes are integrated into the yeast genome in the form of a gene expression cassette. Specifically, the method comprises the following steps:

[0018] (1) Construction of a target gene expression cassette: amplify the promoter, target gene and terminator to obtain the promoter, target gene fragment and terminator respectively, and connect them to a plasmid vector to obtain a recombinant plasmid vector containing the gene expression cassette;

[0019] (2) introducing the recombinant plasmid vector containing the gene expression cassette into Yarrowia lipolytica, so that the gene expression cassette is integrated into the Yarrowia lipolytica genome;

[0020] Preferably, the method further comprises step (3), which further comprises constructing any one or more combinations of a mitochondrial transport-related gene expression cassette, an endogenous dicarboxylate transporter encoding gene JEN1 knockout expression cassette, and a promoter replacement expression cassette, and then integrating them into the yeast genome in the form of a gene expression cassette.

[0021] Preferably, the integration is in the Yarrowia lipolytica genome, and the integration site is any one or more of the IntA3 site, IntB site, IntC site, IntC1 site, IntD site, IntE site, IntE2 site, IntE5 site, IntF site, lip1 site, SCP2 site, 7h site, JEN1 site, or YISCD site of Yarrowia lipolytica. The promoter is the promoter P TEF , P hp4d , P TEFin , P FBA , P FBAin , P POX2 , P EXP , P POT1 or P GPD , P TDH Any one of; the terminator is the terminator T of Yarrowia lipolytica xpr2t , T mig1t , T lip2t , T cyc1t , T pex3t , T pex10t or T pex20t Further preferably, the integration site is the IntE5 site, IntE2 site, IntC1 site, IntF site, IntA3 site and 7h site of Yarrowia lipolytica, and the promoter is the promoter P TEFin , P TDH , P EXP and P TEF , the terminator is the terminator T of Yarrowia lipolytica cyc1t , T mig1t and T xpr2t .

[0022] Wherein, in step (3), the promoter replacement expression cassette comprises an upstream homology arm of the isocitrate dehydrogenase encoding gene IDH1 promoter, promoter P1 or P2, and a downstream homology arm of the isocitrate dehydrogenase encoding gene IDH1 start codon. Preferably, the isocitrate dehydrogenase encoding gene IDH1 promoter is 800 bp before the start codon of the IDH1 gene.

[0023] In a second aspect, the present invention provides use of the recombinant Yarrowia lipolytica described in the first aspect in fermentation production of succinic acid.

[0024] Preferably, the fermentation comprises the following steps: culturing the recombinant Yarrowia lipolytica strain described in the first aspect using YPD fermentation medium to obtain a fermentation product, and collecting the upper liquid by centrifugation to obtain a fermentation broth rich in succinic acid.

[0025] Beneficial effects:

[0026] The recombinant Yarrowia lipolytica XJ-RGS1 strain constructed by the present invention is based on the Yarrowia lipolytica in which the non-homologous recombination gene KU70 encoding is knocked out, so that its homologous recombination ability is enhanced, and the integration of genes is achieved through the homologous recombination function of the Yarrowia lipolytica itself, which can greatly improve the genetic stability of the introduced genes. The recombinant Yarrowia lipolytica construction method is efficient and simple to operate. Compared with the prior art, the recombinant Yarrowia lipolytica XJ-RGS1 strain of the present invention overexpresses the malate dehydrogenase 3 encoding gene IoMDH3, the fumarase encoding gene IoFUMR, the fumarate reductase encoding gene TbFRD and the four-carbon dicarboxylic acid transporter encoding gene SpMae1, and knocks out the dicarboxylic acid transporter encoding gene JEN1, and overexpresses the pyruvate carboxylase encoding gene PYC, the isocitrate lyase encoding gene ICL, the malate synthase encoding gene MLS, the mitochondrial citrate transporter encoding gene YHM2, and the mitochondrial pyruvate carrier 1 encoding gene MPC1, and simultaneously replaces the natural promoter of the endogenous isocitrate dehydrogenase 1 encoding gene IDH1 with the promoter P2 whose expression is reduced under high carbon-nitrogen ratio culture conditions. Experiments show that the recombinant Yarrowia lipolytica can produce 112.85 g / L of succinic acid after fed-batch fermentation without controlling pH, thereby realizing the efficient synthesis of succinic acid in Yarrowia lipolytica. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0028] Figure 1 This is a diagram of succinate anabolism provided for an embodiment of the present invention. Glu, glucose; Pyr, pyruvate; OAA, oxaloacetate; Acetyl-CoA, acetyl-CoA; α-KG, α-ketoglutarate; Icit, isocitrate; Cit, citric acid; Mal, malate; Fum, fumarate; SA, succinate; Pyc, pyruvate carboxylase; Mdh, malate dehydrogenase; Fumr, fumarase; Frd, fumarate reductase; Icl, isocitrate lyase; Mpc, mitochondrial pyruvate carrier; Idh1, isocitrate dehydrogenase; Mls, malate synthase; Yhm2, citrate transporter; Jen1, dicarboxylate transporter; Mae1, tetracarboxylate transporter.

[0029] Figure 2 This is the plasmid map of the recombinant plasmid pUC-HUH-IntE5-EcFUMR.

[0030] Figure 3 This is the plasmid map of the recombinant plasmid pUC-HUH-PIDH1-P1.

[0031] Figure 4 It is a comparison of the succinic acid (SA) content produced by fermentation of the initial strain and recombinant strains 1 to 13 (SA-1 to SA-13). DETAILED DESCRIPTION

[0032] The present invention is further described below by specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0033] In the following examples, the YPD fermentation medium has a formula of 20 g / L peptone, 10 g / L yeast extract and 60-100 g / L glucose. The formula of the YPD plate and SD-URA containing 5-fluoroorotic acid is shown in Chinese patent CN118421490A.

[0034] Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection and is numbered ATCCMYA-2613. Yarrowia lipolytica Po1fΔku70 (MatA, Δku70::HisG, leu2-270, URA33-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1fΔku70. Yarrowialipolytica Po1fΔku70 was constructed by knocking out the KU70 coding gene responsible for non-homologous recombination in Yarrowia lipolytica Po1f (published in Kretzschmar et al., Current Genetics, 2013, 59(1-2): 63-72). Po1fΔku70-SA0 was obtained by knocking out the succinate dehydrogenase encoding gene SDH5 and the succinyl-CoA transferase encoding gene ACH1 from Po1fΔku70 (the specific knockout method was disclosed in Gao et al., Biotechnology for Biofuels, 2016, 9(1), 179 and Cui et al., Metabolic Engineering, 2017, 42, 126-133).

[0035] The IntE5 site integration plasmid is obtained by inserting a 1500 bp sequence upstream of the start codon (upstream homology arm) and a 1500 bp sequence downstream of the stop codon (downstream homology arm) of the IntE5 site on chromosome E in the Po1fΔku70-SA0 genome (GenBank accession number: YALI0E31130g) into the pUC57-HUH vector, and two HisG tag encoding genes are located between the upstream and downstream homology arms of the IntE5 site.

[0036] In the following examples, the promoter P TEFin , P TEF , the terminator T cyc1t , T mig1t and T xpr2t The nucleotide sequence of the promoter is shown in Chinese patent CN118421490A. EXP The nucleotide sequence of the promoter is shown in SEQ ID No.10. TDH The nucleotide sequence is shown in SEQ ID No.11.

[0037] The invention discloses the introduction of the malate dehydrogenase 3 encoding gene IoMDH3 from Issatchenkia orientalis, the fumarase encoding gene IoFUMR and the fumarate reductase encoding gene TbFRD from Trypanosoma Brucei to construct a reduced tricarboxylic acid (TCA) succinate synthesis pathway; the overexpression of the endogenous pyruvate carboxylase encoding gene PYC is used to strengthen the supply of precursors in the reduced tricarboxylic acid (TCA) pathway; the overexpression of the Schizosaccharomyces pombe encoding gene PYC is used to strengthen the supply of precursors in the reduced tricarboxylic acid (TCA) pathway; the overexpression of the endogenous pyruvate carboxylase ... pombe-derived four-carbon dicarboxylic acid transporter encoding gene SpMae1 and knockout of endogenous dicarboxylic acid transporter encoding gene JEN1 are used to promote succinate efflux and prevent succinate uptake; the overexpression of isocitrate lyase encoding gene ICL and malate synthase encoding gene MLS is used to construct the glyoxylate cycle pathway to synthesize succinate and provide intermediate malate for the reductive tricarboxylic acid (TCA) pathway; overexpression of mitochondrial citrate transporter encoding gene YHM2 and endogenous mitochondrial pyruvate carrier encoding gene MPC1 strengthens the glyoxylate cycle flux; the promoter P2 with reduced expression under high carbon-nitrogen ratio culture conditions is used to replace the natural promoter of endogenous isocitrate dehydrogenase 1 encoding gene IDH1 to weaken the oxidative tricarboxylic acid (TCA) pathway flux and redirect it to the glyoxylate cycle pathway, thereby reducing carbon loss and providing the precursor citrate for the glyoxylate pathway. Figure 1 shown.

[0038] Example 1 Amplification of genetic elements and preparation of target plasmids

[0039] (I) Preparation of target gene

[0040] According to the nucleotide sequence of the malate dehydrogenase 3 encoding gene (IoMDH3, GenBank No. KGK36641.1) from Issatchenkia orientalis provided by NCBI, after codon optimization, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized malate dehydrogenase 3 encoding gene IoMDH3 (SEQ ID No. 1) and inserted into the plasmid pUC57 (purchased from GenScript) to obtain the plasmid pUC57-IoMDH3.

[0041] According to the nucleotide sequence of the fumarase encoding gene (IoFUMR, GenBank No. KGK38814.1) from Issatchenkia orientalis provided by NCBI, after codon optimization, Anhui Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized fumarase encoding gene IoFUMR (SEQ ID No. 2) and insert it into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-IoFUMR.

[0042] According to the nucleotide sequence of the fumarase encoding gene (EcFUMR, GenBank No.b1611) from Escherichia coli provided by NCBI, after codon optimization, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized fumarase encoding gene EcFUMR (SEQ ID No.3) and insert it into the plasmid pUC57 (purchased from GenScript) to obtain the plasmid pUC57-EcFUMR.

[0043] According to the nucleotide sequence of the fumarate reductase encoding gene (TbFRD, GenBank No. AAN40014.1) from Trypanosoma Brucei provided by NCBI, after codon optimization, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized fumarate reductase encoding gene TbFRD (SEQ ID No. 4) and inserted into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-TbFRD.

[0044] According to the nucleotide sequence of the tetracarboxylic acid transporter encoding gene from Schizosaccharomyces pombe (SpMae1, GenBank No. SPAPB8E5.03) provided by NCBI, after codon optimization, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized tetracarboxylic acid transporter encoding gene SpMae1 (SEQ ID No. 5) and insert it into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-SpMae1.

[0045] According to the nucleotide sequence of the pyruvate carboxylase encoding gene (IoPYC, GenBank No. CAA71993.1) from Issatchenkia orientalis provided by NCBI, after codon optimization, Anhui Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the optimized pyruvate carboxylase encoding gene IoPYC (SEQ ID No. 6) and insert it into plasmid pUC57 (purchased from GenScript) to obtain plasmid pUC57-IoPYC.

[0046] According to the nucleotide sequence of the orotidine-5'-phosphate decarboxylase encoding gene URA3 of Yarrowia lipolytica provided by NCBI (GenBank accession number: AJ306421.1) and the HisG tag (GenBank accession number: AF324729.1), Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the two HisG tag encoding gene sequences into the plasmid pUC57, and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette (driven by the endogenous promoter P of Yarrowia lipolytica) was inserted between the two HisG tag encoding gene sequences. TEFin , orotidine-5'-phosphate decarboxylase encoding gene URA3 and terminator T xpr2t composition) to achieve URA marker recovery, resulting in plasmid pUC57-HisG-URA3-HisG (pUC57-HUH).

[0047] In addition, the target genes malate dehydrogenase encoding gene YlMDH2, pyruvate carboxylase encoding gene PYC, isocitrate lyase encoding gene ICL, malate synthase encoding gene MLS, mitochondrial citrate transporter encoding gene YHM2, mitochondrial pyruvate carrier encoding genes MPC1 and MPC3 are all endogenous genes of Yarrowia lipolytica, the GenBank accession number of the YlMDH2 is YALI0E14190g, the GenBank accession number of the PYC is YALI0C24101g, the GenBank accession number of the ICL is YALI0C16885g, the GenBank accession number of the MLS is YALI0D19140g, the GenBank accession number of the YHM2 is YALI0B10736g, the GenBank accession number of the MPC1 is YALI0F00264g, and the GenBank accession number of the MPC3 is YALI0C03223g.

[0048] (II) Construction of recombinant plasmid

[0049] The structure of the recombinant plasmid is shown in Table 1; the primers used to construct the recombinant plasmid are shown in Table 2.

[0050] 1. Construction of recombinant plasmid pUC-HUH-IntE5-EcFUMR

[0051] The recombinant plasmid pUC-HUH-IntE5-EcFUMR is based on pUC57-HisG-URA3-HisG, and is inserted with the homology arm IntE5-up 1500 bp upstream of the start codon and the homology arm IntE5-dw 1500 bp downstream of the stop codon in the IntE5 locus of Yarrowia lipolytica Po1fΔku70-SA0 (GenBank accession number: YALI0E31130g). The EcFUMR gene expression cassette (P EXP -EcFUMR-T cyc1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. For the specific structure, see Figure 2 .

[0052] Using Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as template, the EcFUMR expression cassette promoter P EXP and terminator T cyc1t .

[0053] The EcFUMR gene was amplified using the primers in Table 2 using plasmid pUC57-EcFUMR as a template.

[0054] The above PCR amplification system is: Primer STAR Max Premix 25μL, template 1μL, primer F 2μL, primer R 2μL, distilled water 20μL. Among them, Primer STAR Max Premix was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd. The above PCR program is as follows: 98℃ denaturation for 10s, 55℃ annealing for 5s, 72℃ extension (extension time = target fragment length / 1kb, unit min), repeated 30 cycles. Each fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Baisai Biotechnology Co., Ltd.). After the IntE5 site integration plasmid was digested with the restriction endonuclease PacⅠ from NEB, the linearized IntE5 site integration plasmid was recovered by agarose gel electrophoresis.

[0055] The linearized IntE5 site was integrated into the plasmid and the elements (promoter P EXP , gene EcFUMR and terminator T cyc1t ) One-step cloning was achieved using ClonExpressMultiS One Step Cloning Kit from Nanjing Novozyme Biotechnology Co., Ltd. The EcFUMR gene expression cassette was inserted between the upstream and downstream homology arms of the IntE5 site integration plasmid to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened on an LB plate with ampicillin resistance, and verified by colony PCR and sequencing to obtain a positive recombinant plasmid pUC-HUH-IntE5-EcFUMR.

[0056] The plasmid pUC-HUH-IntE5-EcFUMR was digested with restriction endonuclease SwaⅠ from NEB Company, and the linearized pUC-HUH-IntE5-EcFUMR plasmid was recovered by agarose gel electrophoresis and used for yeast transformation.

[0057] 2. Construction of recombinant plasmid pUC-HUH-IntE5-EcFUMR-YlMDH2

[0058] The recombinant plasmid pUC-HUH-IntE5-EcFUMR-Y1MDH2 is based on the pUC-HUH-IntE5-EcFUMR constructed in this example, and the Y1MDH2 gene expression cassette is inserted downstream of the EcFUMR gene expression cassette (PTEF -YlMDH2-T mig1t The construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the YlMDH2 expression cassette fragment using the Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as a template. The pUC-HUH-IntE5-EcFUMR plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized pUC-HUH-IntE5-EcFUMR plasmid was recovered by agarose gel electrophoresis. The various elements in the YlMDH2 gene expression cassette (promoter P TEF , gene Y1MDH2 and terminator T mig1t ) and linearized pUC-HUH-IntE5-EcFUMR plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntE5-EcFUMR-YlMDH2.

[0059] 3. Construction of recombinant plasmid pUC-HUH-IntE5-IoFUMR

[0060] The recombinant plasmid pUC-HUH-IntE5-IoFUMR was based on pUC57-HisG-URA3-HisG, into which the homology arm IntE5-up upstream of the start codon and the homology arm IntE5-dw downstream of the stop codon of the IntE5 locus in Yarrowia lipolytica Po1fΔku70-SA0 were inserted, and the IoFUMR gene expression cassette (P EXP -IoFUMR-T cyc1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the IoFUMR expression cassette fragment using Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA and the recombinant plasmid pUC57-IoFUMR as templates. The IntE5 site integration plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized IntE5 site integration plasmid was recovered by agarose gel electrophoresis. The various elements in the IoFUMR gene expression cassette (promoter P EXP , gene IoFUMR and terminator T cyc1t) and the linearized IntE5 site integration plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntE5-IoFUMR.

[0061] 4. Construction of recombinant plasmid pUC-HUH-IntE5-IoFUMR-IoMDH3

[0062] The recombinant plasmid pUC-HUH-IntE5-IoFUMR-IoMDH3 is based on pUC-HUH-IntE5-IoFUMR, and the IoMDH3 gene expression cassette is inserted downstream of the IoFUMR gene expression cassette (P TEF -IoMDH3-T mig1t The construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the IoMDH3 expression cassette fragment using the Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA and the recombinant plasmid pUC57-IoMDH3 as templates. The pUC-HUH-IntE5-IoFUMR plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized pUC-HUH-IntE5-IoFUMR plasmid was recovered by agarose gel electrophoresis. The various elements in the IoMDH3 gene expression cassette (promoter P TEF , gene IoMDH3 and terminator T mig1t ) and linearized pUC-HUH-IntE5-IoFUMR plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntE5-IoFUMR-IoMDH3.

[0063] 5. Construction of recombinant plasmid pUC-HUH-7h-TbFRD

[0064] The recombinant plasmid pUC-HUH-7h-TbFRD is based on pUC57-HisG-URA3-HisG, and inserted into the 1500bp homology arm 7h-up upstream of the start codon and the 1500bp homology arm 7h-dw downstream of the stop codon in the 7h site of Yarrowia polytica Po1fΔku70-SA0 (GenBank accession number: YALI0E26719g), and the TbFRD gene expression cassette (P TEFin -TbFRD-T mig1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowialipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the TbFRD expression cassette fragment using Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA and the recombinant plasmid pUC57-TbFRD as templates. The 7h integration site plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized 7h site integration plasmid was recovered by agarose gel electrophoresis. The various elements in the TbFRD gene expression cassette (promoter P TEFin , gene TbFRD and terminator T mig1t ) and the linearized 7h site integration plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-7h-TbFRD.

[0065] 6. Construction of recombinant plasmid pUC-HUH-IntE2-SpMAE1

[0066] The recombinant plasmid pUC-HUH-IntE2-SpMAE1 is based on pUC57-HisG-URA3-HisG, and is inserted with the homology arm IntE2-up 1500 bp upstream of the start codon and the homology arm IntE2-dw 1500 bp downstream of the stop codon in the IntE2 locus of Yarrowia lipolytica Po1fΔku70-SA0 (GenBank accession number: YALI0E26719g), and the SpMAE1 gene expression cassette (P EXP -SpMAE1-T mig1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. The construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the SpMAE1 expression cassette fragment using Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA and the recombinant plasmid pUC57-SpMAE1 as templates. The IntE2 integration site plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized IntE2 site integration plasmid was recovered by agarose gel electrophoresis. The various elements in the SpMAE1 gene expression cassette (promoter P EXP , gene SpMAE1 and terminator T mig1t) and the linearized IntE2 site integration plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntE2-SpMAE1.

[0067] 7. Construction of recombinant plasmid pUC-HUH-ΔJEN1

[0068] The recombinant plasmid pUC-HUH-ΔJEN1 is based on pUC57-HisG-URA3-HisG, and inserted with the homology arm JEN1-up 1500 bp upstream of the start codon and the homology arm JEN1-dw 1500 bp downstream of the stop codon of the gene JEN1 in Yarrowia lipolytica Po1fΔku70-SA0 (GenBank accession number: YALI0E30965g), and the URA3 expression cassette containing HisG fragments at both ends (containing the endogenous promoter P of Yarrowia lipolytica). TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. Using the primers described in Table 2 and the genomic DNA of Yarrowia lipolytica Po1fΔku70-SA0 as a template, the ΔJEN1 knockout expression cassette fragments (JEN1-up, JEN1-dw) were amplified. The pUC57-HisG-URA3-HisG plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized pUC57-HisG-URA3-HisG plasmid was recovered by agarose gel electrophoresis. The elements (JEN1-up, JEN1-dw) in the ΔJEN1 knockout gene expression cassette and the linearized pUC57-HisG-URA3-HisG plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-ΔJEN1.

[0069] 8. Construction of recombinant plasmid pUC-HUH-IntF-PYC

[0070] The recombinant plasmid pUC-HUH-IntF-PYC is based on pUC57-HisG-URA3-HisG, and is inserted with the homology arm IntF-up 1500 bp upstream of the start codon and the homology arm IntF-dw 1500 bp downstream of the stop codon of the IntF site in Yarrowia polytica Po1fΔku70-SA0 (GenBank accession number: YALI1_F31639g). The PYC gene expression cassette (P EXP -PYC-T cyc1t), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowialipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the PYC expression cassette fragment with Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as a template. The IntF integration site plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized IntF site integration plasmid was recovered by agarose gel electrophoresis. The elements in the PYC gene expression cassette (promoter P EXP , gene PYC and terminator T cyc1t ) and the linearized IntF site integration plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntF-PYC.

[0071] 9. Construction of recombinant plasmid pUC-HUH-IntF-IoPYC

[0072] The recombinant plasmid pUC-HUH-IntF-IoPYC was based on pUC57-HisG-URA3-HisG, into which the homology arm IntF-up upstream of the start codon and the homology arm IntF-dw downstream of the stop codon of the IntF locus in Yarrowia lipolytica Po1fΔku70-SA0 were inserted, and the IoPYC gene expression cassette (P EXP -IoPYC-T cyc1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. Using the primers described in Table 2, the Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA and the recombinant plasmid pUC57-IoPYC were used as templates to amplify the IoPYC expression cassette fragment. The IntF integration site plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized IntF site integration plasmid was recovered by agarose gel electrophoresis. The various elements in the IoPYC gene expression cassette (promoter P EXP , gene IoPYC and terminator T cyc1t) and the linearized IntF site integration plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntF-IoPYC.

[0073] 10. Construction of recombinant plasmid pUC-HUH-IntC1-ICL

[0074] The recombinant plasmid pUC-HUH-IntC1-ICL is based on pUC57-HisG-URA3-HisG, and is inserted with the homology arm IntC1-up 1500 bp upstream of the start codon and the homology arm IntC1-dw 1500 bp downstream of the stop codon in the IntC1 site of Yarrowia lipolytica Po1fΔku70-SA0 (GenBank accession number: YALI0C13002). The ICL gene expression cassette (P TEFin -ICL-T mig1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the ICL expression cassette fragment using the Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as a template. The IntC1 integration site plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized IntC1 site integration plasmid was recovered by agarose gel electrophoresis. The various elements in the ICL gene expression cassette (promoter P TEFin , gene ICL and terminator T mig1t ) and the linearized IntC1 site integration plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntC1-ICL.

[0075] 11. Construction of recombinant plasmid pUC-HUH-IntC1-ICL-MLS

[0076] The recombinant plasmid pUC-HUH-IntC1-ICL-MLS is based on pUC-HUH-IntC1-ICL, with the MLS gene expression cassette inserted downstream of the ICL gene expression cassette (P TDH -MLS-T xpr2tThe construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the MLS expression cassette fragment using the Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as a template. The pUC-HUH-IntC1-ICL plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized pUC-HUH-IntC1-ICL plasmid was recovered by agarose gel electrophoresis. The various elements in the MLS gene expression cassette (promoter P TDH , gene MLS and terminator T xpr2t ) and linearized pUC-HUH-IntC1-ICL plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntC1-ICL-MLS.

[0077] 12. Recombinant plasmid pUC-HUH-ΔJEN1-MPC1

[0078] The recombinant plasmid pUC-HUH-ΔJEN1-MPC1 was based on pUC57-HisG-URA3-HisG, into which the homology arm JEN1-up upstream of the start codon and the homology arm JEN1-dw downstream of the stop codon of the JEN1 gene in Yarrowia lipolytica Po1fΔku70-SA0 were inserted, and the MPC1 gene expression cassette (P TEF -MPC1-T cyc1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the MPC1 expression cassette fragment with Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as a template. The JEN1 knockout plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized JEN1 knockout plasmid was recovered by agarose gel electrophoresis. The various elements in the MPC1 gene expression cassette (promoter P TEF , gene MPC1 and terminator T cyc1t ) and linearized JEN1 knockout plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-ΔJEN1-MPC1.

[0079] 13. Recombinant plasmid pUC-HUH-ΔJEN1-MPC3

[0080] The recombinant plasmid pUC-HUH-ΔJEN1-MPC3 was based on pUC57-HisG-URA3-HisG, into which the homology arm JEN1-up upstream of the start codon and the homology arm JEN1-dw downstream of the stop codon of the JEN1 gene in Yarrowia lipolytica Po1fΔku70-SA0 were inserted, and the MPC3 gene expression cassette (P EXP -MPC3-T mig1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the MPC3 expression cassette fragment with Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as a template. The JEN1 knockout plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized JEN1 knockout plasmid was recovered by agarose gel electrophoresis. The various elements in the MPC1 gene expression cassette (promoter P EXP , gene MPC3 and terminator T mig1t ) and linearized JEN1 knockout plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-ΔJEN1-MPC3.

[0081] 14. Construction of recombinant plasmid pUC-HUH-IntA3-YHM2

[0082] The recombinant plasmid pUC-HUH-IntA3-YHM2 is based on pUC57-HisG-URA3-HisG, and is inserted with the homology arm IntA3-up 1500 bp upstream of the start codon and the homology arm IntA3-dw 1500 bp downstream of the stop codon in the IntA3 locus of Yarrowia lipolytica Po1fΔku70-SA0 (GenBank accession number: YALI0A04785g). The YHM2 gene expression cassette (P TEFin -YHM2-T cyc1t ), a URA3 expression cassette containing HisG fragments at both ends (containing the Yarrowia lipolytica endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t) is also between the upstream and downstream homology arms. Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the YHM2 expression cassette fragment with Yarrowia lipolytica Po1fΔku70-SA0 genomic DNA as a template. The IntA3 integration site plasmid was digested with the restriction endonuclease PacⅠ from NEB, and the linearized IntA3 site integration plasmid was recovered by agarose gel electrophoresis. The various elements in the YHM2 gene expression cassette (promoter P TEFin , gene YHM2 and terminator T cyc1t ) and the linearized IntA3 site integration plasmid were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-IntA3-YHM2.

[0083] 15. Construction of recombinant plasmid pUC-HUH-PIDH1-P1

[0084] The recombinant plasmid pUC-HUH-PIDH1-P1 is based on pUC57-HisG-URA3-HisG, and is inserted with the upstream 1500bp homology arm IDH1-up and the downstream 1500bp homology arm IDH1-dw of the IDH1 start codon 800bp before the start codon of IDH1 (GenBank accession number: YALI0D06303g) in Yarrowia lipolytica Po1fΔku70-SA0 (i.e., the promoter position, whose nucleotide sequence is shown in SEQ ID No.9), and the promoter P1 (the nucleotide sequence of P1 is shown in SEQ ID No.7) and the URA3 expression cassette containing HisG fragments at both ends (containing the endogenous promoter P of Yarrowia lipolytica) are also inserted between the upstream and downstream homology arms. TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ). Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used with the Yarrowialipolytica Po1fΔku70-SA0 genome as a template to amplify the upstream and downstream homology arms and the endogenous promoter P1. The pUC57-HisG-URA3-HisG plasmid was double-digested with restriction endonucleases EcoRⅠ and HindⅢ from NEB, and the linearized pUC57-HisG-URA3-HisG plasmid was recovered by agarose gel electrophoresis. The IDH1-up, IDH1-dw, P1 and linearized pUC57-HisG-URA3-HisG plasmids were cloned and verified in one step to obtain the positive recombinant plasmid pUC-HUH-PIDH1-P1, as shown in FIG. Figure 3 shown.

[0085] 16. Construction of recombinant plasmid pUC-HUH-PIDH1-P2

[0086] The recombinant plasmid pUC-HUH-PIDH1-P2 is based on pUC57-HisG-URA3-HisG, into which the upstream 1500bp homology arm IDH1-up and the downstream 1500bp homology arm IDH1-dw of the IDH1 start codon are inserted 800bp before the start codon of IDH1 (GenBank accession number: YALI0D06303g) in Yarrowia lipolytica Po1fΔku70-SA0, respectively. The promoter P2 (the nucleotide sequence of P2 is shown in SEQ ID No. 8) and the URA3 expression cassette containing HisG fragments at both ends (containing the endogenous promoter P of Yarrowia lipolytica) are also inserted between the upstream and downstream homology arms. TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ). Its construction method is similar to that of the recombinant plasmid pUC-HUH-IntE5-EcFUMR. The primers described in Table 2 were used to amplify the upstream and downstream homology arms and the endogenous promoter P2 using the Yarrowia lipolytica Po1fΔku70-SA0 genome as a template. The pUC57-HisG-URA3-HisG plasmid was double-digested with restriction endonucleases EcoRⅠ and HindⅢ from NEB, and the linearized pUC57-HisG-URA3-HisG plasmid was recovered by agarose gel electrophoresis. The positive recombinant plasmid pUC-HUH-PIDH1-P2 was obtained by one-step cloning and verification of IDH1-up, IDH1-dw, P2 and linearized pUC57-HisG-URA3-HisG plasmids.

[0087] Table 1 Insertion sequence information of each recombinant plasmid

[0088]

[0089]

[0090] Table 2 Primer sequences

[0091]

[0092]

[0093]

[0094]

[0095] Example 2 Construction of recombinant Yarrowia lipolytica (I) Construction of recombinant bacteria 1-3 (expressing TbFRD, EcFUMR-YlMDH2 or IoFUMR-IoMDH3)

[0096] The recombinant plasmid pUC-HUH-7h-TbFRD was introduced into Yarrowia lipolytica Po1fΔku70-SA0, and the TbFRD expression cassette was integrated into the genomic 7h site to obtain recombinant bacteria 1, which then lost a HisG tag and URA3 screening marker under the 5-fluoroorotic acid selection pressure for the next round of URA screening. The recombinant plasmid pUC-HUH-IntE5-EcFUMR-YlMDH2 was introduced into the recombinant bacteria 1 that had lost URA, and the EcFUMR expression cassette and YlMDH2 expression cassette were integrated into the genomic IntE5 site to obtain recombinant bacteria 2. Similarly, the recombinant plasmid pUC-HUH-IntE5-IoFUMR-IoMDH3 was introduced into the recombinant bacteria 1 that had lost URA, and the IoFUMR expression cassette and the IoMDH3 expression cassette were integrated into the genomic IntE5 site to obtain the recombinant bacteria 3, which then lost a HisG tag and URA3 selection marker under 5-fluoroorotic acid selection pressure for use in the next round of URA selection.

[0097] The specific method for constructing the recombinant bacteria 1 is as follows:

[0098] Yarrowia lipolytica Po1fΔku70-SA0 was cultured overnight in YPD liquid medium. Competent cells were prepared using Zymogen Frozen EZ Yeast Transformation Kit II from ZymoResearch Corporation, and the linearized recombinant plasmid pUC-HUH-7h-TbFRD was transformed into Yarrowia lipolytica Po1fΔku70-SA0 competent cells for homologous recombination. Positive clones were screened using screening medium SD-URA, and then identified by PCR. The strain identified correctly by PCR was the recombinant strain 1.

[0099] The recombinant bacteria 1 was coated on a YPD plate containing 5-fluoroorotic acid, and then a single colony was streaked on a YPD plate containing 5-fluoroorotic acid and a SD-URA plate at the same time. A single colony that could grow on a YPD plate containing 5-fluoroorotic acid but failed to grow on a SD-URA plate was selected to obtain a URA-deficient strain of the recombinant bacteria 1. The recombinant plasmid pUC-HUH-IntE5-EcFUMR-YlMDH2 was introduced into the recombinant bacteria 1 that had lost URA, and the EcFUMR expression cassette and the YlMDH2 expression cassette were integrated into the IntE5 site of the genome to obtain recombinant bacteria 2. The specific construction method was the same as that of recombinant bacteria 1. The construction method of recombinant bacteria 3 was the same as that of recombinant bacteria 1, except that the plasmid used for homologous recombination and the host used for transformation needed to be changed accordingly.

[0100] (II) Construction of recombinant bacteria 4-5 (expressing SpMAE1 and knocking out JEN1)

[0101] The recombinant plasmid pUC-HUH-IntE2-SpMAE1 was introduced into the recombinant bacteria 3 that had lost URA, and the SpMAE1 expression cassette was integrated into the genomic IntE2 site to obtain recombinant bacteria 4, which then lost a HisG tag and URA3 screening marker under the 5-fluoroorotic acid selection pressure for the next round of URA screening. The recombinant plasmid pUC-HUH-ΔJEN1 was introduced into the recombinant bacteria 4 that had lost URA, and the ΔJEN1 knockout expression cassette was integrated into the genomic JEN1 gene to obtain recombinant bacteria 5, which then lost a HisG tag and URA3 screening marker under the 5-fluoroorotic acid selection pressure for the next round of URA screening. The construction method of recombinant bacteria 4 and 5 is the same as that of recombinant bacteria 1, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0102] (III) Construction of recombinant bacteria 6-7 (expressing PYC or IoPYC)

[0103] The recombinant plasmid pUC-HUH-IntF-PYC was introduced into the recombinant bacteria 5 that had lost URA, and the PYC expression cassette was integrated into the IntF site of the genome to obtain recombinant bacteria 6, and then a HisG tag and URA3 screening marker were lost under the 5-fluoroorotic acid screening pressure for use in the next round of URA screening. The recombinant plasmid pUC-HUH-IntF-IoPYC was introduced into the recombinant bacteria 5 that had lost URA, and the PYC expression cassette was integrated into the IntF site of the genome to obtain recombinant bacteria 7, and then a HisG tag and URA3 screening marker were lost under the 5-fluoroorotic acid screening pressure for use in the next round of URA screening. The construction method of recombinant bacteria 6-7 is the same as that of recombinant bacteria 1, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0104] (IV) Construction of recombinant bacteria 8-11 (expressing ICL-MLS, YHM2 and MPC1 or MPC3)

[0105] The recombinant plasmid pUC-HUH-IntC1-ICL-MLS was introduced into the recombinant bacteria 6 that had lost URA, and the ICL gene expression cassette and the MLS gene expression cassette were integrated into the genomic IntC1 site to obtain the recombinant bacteria 8, which then lost a HisG tag and URA3 screening marker under the 5-fluoroorotic acid selection pressure for use in the next round of URA screening. The recombinant plasmid pUC-HUH-IntA3-YHM2 was introduced into the recombinant bacteria 8 that had lost URA, and the YHM2 expression cassette was integrated into the genomic IntA3 site to obtain the recombinant bacteria 9, which then lost a HisG tag and URA3 screening marker under the 5-fluoroorotic acid selection pressure for use in the next round of URA screening. The recombinant plasmid pUC-HUH-ΔJEN1-MPC1 was introduced into the recombinant bacteria 9 that had lost URA, and the MPC1 expression cassette was integrated into the genomic JEN1 gene to obtain the recombinant bacteria 10, and then a HisG tag and URA3 screening marker were lost under the 5-fluoroorotic acid screening pressure for the next round of URA screening. The recombinant plasmid pUC-HUH-ΔJEN1-MPC3 was introduced into the recombinant bacteria 9 that had lost URA, and the MPC3 expression cassette was integrated into the genomic JEN1 gene to obtain the recombinant bacteria 11, and then a HisG tag and URA3 screening marker were lost under the 5-fluoroorotic acid screening pressure for the next round of URA screening. The construction method of recombinant bacteria 8-11 is the same as that of recombinant bacteria 1, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0106] (V) Construction of recombinant bacteria 12-13 (replacing the IDH1 promoter with P1 or P2)

[0107] The recombinant plasmid pUC-HUH-PIDH1-P1 was introduced into the recombinant bacteria 10 that had lost URA, and the endogenous promoter P1 replaced the natural promoter P of the isocitrate dehydrogenase encoding gene IDH1. IDH1 , and obtained recombinant bacteria 12. Similarly, the recombinant plasmid pUC-HUH-PIDH1-P2 was introduced into the recombinant bacteria 9 that had lost URA, and the endogenous promoter P2 replaced the natural promoter P of the isocitrate dehydrogenase encoding gene IDH1. IDH1 , and recombinant bacteria 13 was obtained. The construction method of recombinant bacteria 12-13 is the same as that of recombinant bacteria 1, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0108] Example 3 Application of recombinant Yarrowia lipolytica in the production of succinic acid (I) Cultivation of engineered bacteria and product extraction

[0109] The initial bacteria Yarrowia lipolytica Po1f-Δku70-SA0 and the recombinant bacteria 1 to 13 (SA-1 to SA-13) in Example 2 were used to produce succinic acid. The specific method is as follows: the initial bacteria and the recombinant bacteria were activated and cultured in YPD liquid medium at 28°C and 200rpm for 16 hours to obtain seed liquid. The seed liquid was inoculated in 50mL fermentation medium YPD 60-90 at an inoculum of 1% v / v, and cultured at 28°C and 200rpm for 5 days. After the fermentation was completed, the fermentation broth was transferred to a 50mL centrifuge tube, centrifuged at 5000rpm for 15min, and 1mL was taken for liquid phase detection of succinic acid and glucose concentrations. The remaining sample was then removed from the supernatant and placed in an oven (75°C) to dry to constant weight for measuring biomass. The formula of the fermentation medium YPD60-90 is: 20g / L peptone, 10g / L yeast extract and 60-90g / L glucose, and the solvent is water.

[0110] (II) Qualitative and quantitative analysis of succinic acid

[0111] Succinic acid detection detection conditions: HPLC using Aminex HPX-87H column and differential detector was used to determine the concentration of succinic acid and glucose. All samples were filtered with a 0.22 μm filter before loading. 5 mM sulfuric acid was used as the mobile phase, the flow rate was 0.6 mL / min, and the column temperature was 65 °C.

[0112] After 5 days of fermentation, Figure 4 As shown, the succinic acid (SA) concentrations of Yarrowia lipolytica Po1fΔku70-SA0 and recombinant bacteria 1 to 13 (SA-1 to SA-13) were 5.65 g / L and 6.98, 9.98, 10.48, 19.29, 21.44, 24.98, 22.17, 25.27, 27.76, 29.58, 28.98, 32.33 and 35.19 g / L, respectively. Among them, the succinic acid production of recombinant bacteria 13 was the highest, reaching 35.19 g / L, that is, 35.19 g of succinic acid was produced per liter of fermentation liquid, which was significantly higher than that of the initial bacteria. The recombinant bacteria 13 was named Yarrowia lipolytica XJ-RGS1 strain. In the production process of succinic acid, the reduction of tricarboxylic acid (TCA) pathway requires the conversion of 2 molecules of NADH into NAD for each molecule of succinic acid produced. +. The traditional method is to use a substrate with higher reducing power than glucose as a carbon source or to convert NADPH into NADH through a transhydrogenation system. This study couples the glyoxylate pathway and the reductive tricarboxylic acid (TCA) pathway, using the glyoxylate pathway to replace part of the reductive tricarboxylic acid (TCA) pathway, namely the step from oxaloacetate to malate, to reduce the consumption of the cofactor NADH. At the same time, the promoter P2, whose expression is reduced under high carbon-nitrogen ratio culture conditions, is used to replace the natural promoter of the endogenous isocitrate dehydrogenase encoding gene IDH1, redirecting the carbon flux of the oxidative tricarboxylic acid (TCA) pathway to the glyoxylate cycle pathway, thereby reducing carbon loss and providing the precursor citric acid for the glyoxylate cycle pathway. The fermentation results showed that this strategy significantly increased the yield of succinic acid.

[0113] Table 3 Succinic acid production of initial bacteria and recombinant bacteria

[0114] Fermentation method strains Biomass (g / L) Succinic acid yield (g / L) Shake bottle Initial bacteria 4.02 5.65 Shake bottle Recombinant bacteria 3 5.03 10.48 Shake bottle Recombinant bacteria 5 4.81 21.44 Shake bottle Recombinant bacteria 10 5.22 29.58 Shake bottle Recombinant bacteria 13 5.86 35.19 Fermentation tank Recombinant bacteria 13 15.23 112.85

[0115] The recombinant bacteria 13 was inoculated into 50 mL of seed culture medium (YPD medium) and cultured at 28°C and 200 rpm for 24 h to obtain seed solution 1, and then the seed solution 1 was transferred to 100 mL of seed culture medium (YPD medium) at an inoculation amount of 5% v / v and cultured at 28°C and 200 rpm for 24 h to obtain seed solution 2, and then the seed solution 2 was inoculated into a 5L fermenter at an inoculation amount of 5% v / v. 2.5L of fermentation medium was contained in the 5L fermenter, and the fermentation dissolved oxygen was controlled to 20%. The pH value was not controlled during the fermentation process until the fermentation was completed. When the fermentation time was about 48 h, an aqueous solution containing 800 g of glucose per liter was added at a flow rate of 7-15 mL / h. The fermentation temperature was 28°C and the culture time was 7 days. Among them, the components of the fermentation medium were 150 g / L of glucose, 10 g / L of yeast extract and 20 g / L of peptone. After 7 days of fermentation, the maximum succinic acid production of recombinant bacteria 13 reached 112.85 g / L, that is, 112.85 g of succinic acid was produced per liter of fermentation liquid (Table 3), which was significantly higher than that of the initial bacteria and recombinant bacteria 3.

[0116] The present invention provides a method for constructing a recombinant Yarrowia lipolytica with high succinic acid production and a method and idea for its application. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be considered as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A recombinant Yarrowia lipolytica strain with high succinic acid production, characterized in that: The recombinant Yarrowia lipolytica simultaneously expresses genes related to the reduction tricarboxylic acid pathway, genes related to the glyoxylate cycle pathway, and genes related to the transport of heterologous dicarboxylic acids; Wherein, the reductive tricarboxylic acid pathway-related genes include any one or more combinations of pyruvate carboxylase gene PYC, malate dehydrogenase gene MDH, fumarase gene FUMR and fumarate reductase gene FRD; The glyoxylate cycle pathway related groups include any one of the isocitrate lyase gene ICL and the malate synthase gene MLS or a combination of both; The heterologous dicarboxylic acid transport-related genes include the heterologous tetracarboxylic acid transporter gene MAE1.

2. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The PYC is derived from Issatchenkia orientalis or Yarrowia lipolytica; The MDH is derived from Issatchenkia orientalis or Yarrowia lipolytica; The FUMR is derived from Issatchenkia orientalis or Escherichia coli; The FRD is derived from Trypanosoma Brucei.

3. The recombinant Yarrowia lipolytica according to claim 2, characterized in that The nucleotide sequence of PYC from Issatchenkia orientalis is shown in SEQ ID No. 6, and the GenBank accession number of the nucleotide sequence of PYC from Yarrowia lipolytica is YALI0C24101g; The nucleotide sequence of MDH from Issatchenkia orientalis is shown in SEQ ID No. 1, and the GenBank accession number of the nucleotide sequence of MDH from Yarrowia lipolytica is YALI0E14190g; The nucleotide sequence of FUMR derived from Issatchenkia orientalis is shown in SEQ ID No. 2, and the nucleotide sequence of FUMR derived from Escherichia coli is shown in SEQ ID No. 3; The nucleotide sequence of FRD derived from Trypanosoma Brucei is shown in SEQ ID No.

4.

4. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The ICL and MLS are both derived from Yarrowia lipolytica, and the MAE1 is derived from Schizosaccharomyces pombe; The GenBank accession number of the nucleotide sequence of the ICL is YALI0C16885g; The GenBank accession number of the nucleotide sequence of the MLS is YALI0D19140g; The nucleotide sequence of MAE1 is shown in SEQ ID No.

5.

5. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The recombinant Yarrowia lipolytica simultaneously expresses mitochondrial transport-related genes, and the mitochondrial transport-related genes include any one of the mitochondrial citrate transporter encoding gene YHM2 and the mitochondrial pyruvate carrier encoding gene MPC, or a combination of both.

6. The recombinant Yarrowia lipolytica according to claim 5, characterized in that The YHM2 and MPC are both derived from Yarrowia lipolytica, the GenBank accession number of the YHM2 is YALI0B10736g; the GenBank accession number of the MPC is YALI0F00264g and / or YALI0C03223g.

7. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The recombinant Yarrowia lipolytica is also knocked out of the dicarboxylic acid transporter encoding gene JEN1, and the GenBank accession number of the JEN1 is YALI0C15488g.

8. The recombinant Yarrowia lipolytica according to claim 1, characterized in that In the recombinant Yarrowia lipolytica, the natural promoter of the isocitrate dehydrogenase encoding gene IDH1 is replaced by promoter P1 or P2. The nucleotide sequence of P1 is shown in SEQ ID No.7, and the nucleotide sequence of P2 is shown in SEQ ID No.

8.

9. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The recombinant Yarrowia lipolytica has a starting strain of Po1fΔku70-SA0; the Po1fΔku70-SA0 is obtained by knocking out the KU70 gene responsible for non-homologous recombination, the succinate dehydrogenase gene SDH5 and the succinyl-CoA transferase gene ACH1 from the Po1f strain.

10. Use of the recombinant Yarrowia lipolytica according to any one of claims 1 to 9 in the fermentation production of succinic acid.

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