WO2023197692A1 - 具有线粒体定位还原tca途径的高产琥珀酸酵母工程菌株及其构建方法和应用 - Google Patents

具有线粒体定位还原tca途径的高产琥珀酸酵母工程菌株及其构建方法和应用 Download PDF

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WO2023197692A1
WO2023197692A1 PCT/CN2022/143484 CN2022143484W WO2023197692A1 WO 2023197692 A1 WO2023197692 A1 WO 2023197692A1 CN 2022143484 W CN2022143484 W CN 2022143484W WO 2023197692 A1 WO2023197692 A1 WO 2023197692A1
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succinic acid
gene
yeast
yarrowia lipolytica
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缪汉根
唐金奎
祁庆生
崔志勇
侯进
钟驭涛
邓敬宇
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盛虹控股集团有限公司
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Definitions

  • the invention belongs to the technical field of microorganisms and fermentation engineering, and specifically relates to a high-yield succinic acid yeast engineering strain with a mitochondrial localized reduction TCA pathway and its construction method and application.
  • Succinic acid also known as succinic acid (SA)
  • SA succinic acid
  • SA succinic acid
  • SA succinic acid
  • PBS polybutylene succinate PBS
  • PBSA polybutylene succinate adipate
  • the existing succinic acid production capacity can no longer meet the market supply.
  • the synthesis of succinic acid mainly uses chemical process routes. However, compared with biological fermentation methods, chemical production of succinic acid has disadvantages such as high energy consumption, serious pollution, and greenhouse gas emissions. Therefore, it is of great practical significance to develop green, sustainable and efficient succinic acid fermentation technology.
  • yeast Compared with bacterial hosts, yeast has stronger tolerance to acid, alkali and osmotic pressure, and can perform low pH succinic acid fermentation, which simplifies the fermentation process and reduces downstream product processing costs.
  • many researchers have tried to modify the metabolic pathways of various yeast cells, including Saccharomyces cerevisiae, Pichia kudriavzevii, Issatchenkia orientali and Yarrowia lipolytica, to achieve the fermentative production of succinic acid.
  • Yarrowia lipolytica is an unconventional yeast that has the advantages of high safety, strong acid resistance, secretion of a variety of metabolites, and the ability to utilize a variety of carbohydrates. It is regarded as a potential succinic acid-producing strain.
  • Yarrowia lipolytica can utilize the oxidative TCA pathway for succinic acid production by reducing succinate dehydrogenase activity.
  • the theoretical carbon conversion rate of oxidative TCA pathway to synthesize succinic acid is only 0.65g/g glucose.
  • the actual succinic acid conversion rate of the current Yarrowia lipolytica engineered strain is consistent with other bacterial production strains such as Escherichia coli, Corynebacterium glutamicum and Actinobacillus succinogenes. There is still a big gap in comparison.
  • the theoretical conversion efficiency of the reducing TCA branch is higher (about 1.1 g/g glucose), which can significantly reduce the emission of greenhouse gas CO2 and has obvious advantages over other pathways.
  • Some bacteria isolated from the natural environment such as the rumen bacteria Actinobacterium succinogenes and Succinobacterium Mannheim, can efficiently synthesize succinic acid through their own reduced TCA pathway.
  • the vast majority of yeasts lack the key enzyme that catalyzes fumarate into succinate, namely fumarate reductase, and the construction and expression of exogenous reducing TCA pathways are difficult. Therefore, how to construct a functional reducing TCA pathway in yeast cells and realize the adaptation of the efficient synthesis pathway of exogenous succinic acid to the host is the key to improving the production efficiency of succinic acid.
  • the purpose of the present invention is to provide a high-yield succinic acid yeast engineering strain with a mitochondrial localized reduction TCA pathway and its construction method and application.
  • the present invention uses the strictly aerobic Yarrowia lipolytica strain as the starting strain to locate different types of fumarate reductase to the mitochondrial matrix, proving that the subcellular relocation of the enzyme helps to improve the succinic acid production capacity. .
  • a first aspect of the present invention provides a high-yield succinic acid yeast engineering strain with a mitochondrial localized reducing TCA pathway, the engineered strain expresses fumarate reductase, and the fumarate reductase is overexpressed and relocated to the mitochondrial matrix, while , this engineered strain overexpresses succinate synthesis-related enzymes and dicarboxylate transporters.
  • the above-mentioned yeast engineered strain possesses a functional reductive succinic acid biosynthetic pathway and can efficiently produce succinic acid using culture media containing glucose.
  • a second aspect of the present invention provides a method for constructing the above-mentioned high-yield succinic acid yeast engineering strain with a mitochondrial-localized reducing TCA pathway.
  • the construction method includes:
  • a yeast strain was used as the starting strain to overexpress the mitochondrial localized fumarase and endogenous malate dehydrogenase, and then overexpress the dicarboxylic acid transporter.
  • a third aspect of the present invention provides an industrial production method of succinic acid.
  • the industrial production method includes culturing and fermenting the above-mentioned yeast engineering strain.
  • the fourth aspect of the present invention provides the application of the above-mentioned yeast engineering strain or the above-mentioned industrial production method in the fields of chemical industry, degradable materials, food and pharmaceutical industries.
  • the above technical solution discloses a new Yarrowia lipolytica engineered strain that can be used to produce high-yield succinic acid.
  • succinic acid As an engineered strain of succinic acid, it can bring about ideal yield and conversion rate; the strain can utilize common carbon sources and nitrogen. Source, after fed-batch fermentation and culture, the succinic acid production reached 50.1g/L, and the conversion rate reached 0.75g/g glucose.
  • research has proven that the above-mentioned engineered bacteria can perform low-pH fermentation during the culture process. This feature can effectively reduce the steps of adjusting the pH of the culture solution in the industrial production process and further save costs.
  • the above technical solution uses a new technical method when constructing the engineering strain Mbmr-SA4, that is, the application of the mitochondrial localized reduction TCA pathway in high succinic acid production, which significantly increases the succinic acid production. Therefore, the above engineered bacteria have considerable application value and prospects.
  • Figure 1 is a schematic diagram of the mitochondrial localization of the reduced TCA synthesis pathway used in Examples 2 and 3.
  • Figure 2 is a diagram showing the functional verification results of yeast mitochondrial localization signals in Example 2.
  • hrGFP represents the overexpression of the green fluorescent protein hrGFP using the Po1f strain as the host
  • MLS-mCherry represents the overexpression of the red fluorescent protein mCherry containing a 23-amino acid mitochondrial localization signal sequence using the Po1f strain as the host.
  • the intracellular fluorescence distribution of the two strains was observed under a fluorescence microscope.
  • Figure 3 is a graph showing the effect of mitochondrial localization of fumarase on succinic acid production in Example 2.
  • Mbmr-SA0 is the control strain
  • TbFrd represents the trypanosoma brucei-derived fumarate reductase that uses Mbmr-SA0 as the host to overexpress the cytoplasmic localization
  • mTbFrd represents the trypanosoma brucei-derived fumarate reductase that uses Mbmr-SA0 as the host to overexpress the mitochondrial matrix localization.
  • mScOsm1 is a fumarate reductase derived from Saccharomyces cerevisiae that uses Mbmr-SA0 as a host to overexpress and localize in the mitochondrial matrix
  • mSfFrd represents Shewanella that uses Mbmr-SA0 as a host to overexpress and localize in the mitochondrial matrix.
  • Frigidimarina sources fumarate reductase.
  • Figure 4 is a graph showing the effects of overexpression of genes related to the reducing TCA pathway and dicarboxylic acid transporter on succinic acid production in Example 3.
  • YlMdh1 and YlMdh2 are endogenous malate dehydrogenase coding genes
  • CgMdh is the malate dehydrogenase coding gene derived from Corynebacterium glutamicum
  • mEcFum is the fumarase coding gene derived from Escherichia coli, which is located in the mitochondria.
  • the gene, SpMae1 is a gene encoding a dicarboxylic acid transporter derived from Schizosaccharomyces pombe.
  • Figure 5 is a graph showing the production of succinic acid produced by the engineering strain Mbmr-SA4 in shake flask fermentation in Example 3.
  • a high-yield succinic acid yeast engineering strain with a mitochondrial-localized reducing TCA pathway expresses fumarate reductase, and the fumarate reductase is overexpressed and relocated to the mitochondrial matrix. , at the same time, the engineered strain overexpressed succinate synthesis-related enzymes and dicarboxylate transporters.
  • the above-mentioned yeast engineered strain possesses a functional reductive succinic acid biosynthetic pathway and can efficiently produce succinic acid using culture media containing glucose.
  • the starting strains of the yeast engineering strains include but are not limited to Candida sonorensis, Kluyveromyces marxianus, Kluyveromyces thermotolerana, Issatchenkia orientalis, Candida methanesobosa, Candida lambica, Candida sorboxylosa, Saccharomyces bayanus, Kluyveromyces lactis, Pichia jadinii, Pichia anomala, Zygosacchar omyces lentus, Candida zemplinina, Candida geochares, Pichia membranifaciens, Saccharomyces cerevisiae, Pichia pastoris; further, the starting strain is preferably Yarrowia lipolytica; further, the starting strain is Yarrowia lipolytica (Yarrowia lipolytica) strain Mbmr-SA0, its genotype is MatA, xpr2-322, axp-2, leu2-270, ura3-302, ⁇ Sdh5
  • the fumarate reductase is an exogenous enzyme, and its sources include but are not limited to Trypanosoma brucei, Saccharomyces cerevisiae and Shewanella frigidimarina, preferably Trypanosoma brucei; more specifically, the encoding is described
  • the gene sequence of fumarate reductase is shown in SEQ ID NO.1 (see sequence list seq_1).
  • the succinate synthesis-related enzymes include but are not limited to malate dehydrogenase.
  • the malate dehydrogenase encoding gene is the malate dehydrogenase gene YlMdh2, and its gene sequence number is: YALIOE14190p (Gene ID: 2911503).
  • the dicarboxylic acid transporter encoding gene SpMae1 comes from Schizosaccharomyces pombe, and its nucleotide sequence is as shown in SEQ ID NO. 6 (see sequence list seq_6).
  • a method for constructing the above-mentioned high-yield succinic acid yeast engineering strain with a mitochondrial localized reduction TCA pathway includes:
  • a yeast strain was used as the starting strain to overexpress the mitochondrial localized fumarase and endogenous malate dehydrogenase, and then overexpress the dicarboxylic acid transporter.
  • the yeast strains include but are not limited to Candida sonorensis, Kluyveromyces marxianus, Kluyveromyces thermomotolerana, Issatchenkia orientalis, Candida methanesobosa, Candida lambica, Candida sorboxylosa, Saccharomyces bayanus, Kluyveromyces lactis, Pichia jadinii, Pichia anomala, Zy gosaccharomyces lentus ⁇ Candida zemplinina ⁇ Candida geochares, Pichia membranefaciens, Saccharomyces cerevisiae, Pichia pastoris; further, the starting strain is preferably Yarrowia lipolytica.
  • the construction method includes:
  • the exogenous fumarase TbFrd encoding gene mTbFrd was introduced into the starting strain, and then the malate dehydrogenase encoding gene YlMdh2 and dicarboxylic acid transporter were introduced in sequence
  • the protein gene SpMae1, namely Yarrowia lipolytica Mbmr-SA4, can achieve the effect of high production of succinic acid.
  • Yarrowia lipolytica strain Mbmr-SA0 its genotype is MatA, xpr2-322, axp-2, leu2-270, ura3-302, ⁇ Sdh5::loxP, ⁇ Ach1::loxP, YlPyc, TbFrd, EcFum, YlMdh1 and Pgl G75S ; they are obtained by constructing Yarrowia lipolytica strain (Yarrowia lipolytica) PGC91 through genetic engineering technology.
  • the Yarrowia lipolytica strain (Yarrowia lipolytica) PGC91 is shown in "Cui, Z., Gao,C.,Li,J.,Hou,J.,Lin,C.,&Qi,Q.(2017).Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH.Metabolic engineering,42,126– 133.” and "A method for aerobically synthesizing succinic acid using a Yarrowia lipolytica strain with a reducing TCA pathway", more specifically, the construction method includes: combining TbFrd (SEQ ID NO.
  • YlMdh1 Sequence number YALIOD16753p, Gene ID: 2910208
  • EcFum SEQ ID NO.5, see sequence list seq_5
  • NHEJ non-homology-dependent recombination
  • mTbFrd is a combination of the mitochondrial localization sequence (MLS, SEQ ID NO.9, see sequence listing seq_9) of the Yarrowia lipolytica mitochondrial matrix protein cytochrome c oxidase 5b subunit and the soluble fumarate reductase TbFrd (T. brucei source NAD-dependent, SEQ ID NO.1) was obtained by fusion, so that in the finally obtained engineering bacteria, fumarate reductase was overexpressed and relocated to the mitochondrial matrix; among them, the mitochondrial localization signal peptide (SEQ ID NO.10, see the sequence list seq_10) is located at the N-terminus of the fusion protein.
  • MLS mitochondrial localization sequence
  • SEQ ID NO.9 see sequence listing seq_9
  • the malate dehydrogenase encoding gene is the malate dehydrogenase gene YlMdh2, and its gene sequence number is YALIOE14190p (Gene ID: 2911503).
  • the dicarboxylic acid transporter encoding gene SpMae1 comes from Schizosaccharomyces pombe, and its nucleotide sequence is as shown in SEQ ID NO. 6 (see sequence list seq_6).
  • an industrial production method of succinic acid includes culturing and fermenting the above-mentioned yeast engineering strain.
  • the culture and fermentation method includes: placing the above-mentioned yeast engineering strain in YPD medium for fermentation culture;
  • the fermentation culture method includes: a fermentation culture stage, the temperature is controlled at 25-35°C (preferably 30°C), the ventilation volume is 0.5-5vvm (preferably 1.0vvm), and the stirring speed is controlled at 100-800rpm ( Preferably 500rpm);
  • the fermentation culture method also includes detecting the glucose concentration in the fermentation broth.
  • glucose concentration is lower than 10g/L, sugar is supplemented to 50-60g/L; , there is no need to adjust the pH, making the culture operation more convenient and cost-saving.
  • the YPD medium includes: 2% tryptone, 1% yeast extract, and 6% glucose.
  • the application of the above-mentioned yeast engineering strain or the above-mentioned industrial production method in the fields of chemical industry, degradable materials, food and pharmaceutical industries is provided.
  • the gene synthesis in the present invention is completed by General Biotechnology (Anhui) Co., Ltd.; the primer synthesis and sequencing in the present invention are completed by Beijing Qingke Xinye Biotechnology Co., Ltd.
  • Yarrowia lipolytica strain Mbmr-SA0 (genotype MatA, xpr2-322, axp-2, leu2-270, ura3-302, ⁇ Sdh5::loxP, ⁇ Ach1::loxP, YlPyc, TbFrd) involved in the present invention .
  • the construction steps can be found in the literature (Cui, Z., Jiang, X. ,Zheng,H.,Qi,Q.,& Hou,J.(2019).Homology-independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica.Biotechnology and bioengineering,116(2),354– 363.).
  • the construction of JMP113 plasmid and site-specific recombinase overexpression vector pUB4-CRE can be found in the literature (Fickers, P., Le Dall, MT, Gaillardin, C., Thonart, P., & Nicaud, JM (2003). New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. Journal of microbiological methods, 55(3),727–737.).
  • Genes involved in the present invention including TbFrd from Trypanosoma brucei T. brucei, SfFrd from Shewanella frigidimarina, ScOsm1 from S. cerevisiae, and CgMdh from Corynebacterium glutamicum. , fumC from E. coli, and SpMae from Schizosaccharomyces pombe, S. pombe, were codon-optimized and synthesized (General Biotech, Chuzhou, China).
  • YlMdh1 and YlMdh2 genes were cloned from the Yarrowia lipolytica genome.
  • LB solid medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L agar powder.
  • LB liquid medium 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride.
  • YPD medium 10g/L yeast extract, 20g/L peptone, 20g/L glucose.
  • YPD solid medium 10g/L yeast extract, 20g/L peptone, 20g/L glucose, 2wt% agar powder.
  • SD liquid selection medium 26.7g/L SD basic medium, add appropriate concentration of amino acid deletion mixture, and adjust pH to 6.0.
  • SD solid medium Add 2wt% agar powder to SD liquid selection medium.
  • TbFrd (serial number KT026107.1) from T. brucei provided on NCBI, after codon optimization, it was synthesized and optimized by General Bio (Anhui) Co., Ltd.
  • the NADH-dependent fumarate reductase gene TbFrdY was synthesized and optimized by General Bio (Anhui) Co., Ltd.
  • the nucleotide sequence of TbFrdY is shown in SEQ ID NO.1.
  • EcFum sequence number b1611
  • NCBI General Biotech (Anhui) Co., Ltd.
  • SEQ ID NO.5 The nucleotide sequence of EcFumY is shown in SEQ ID NO.5.
  • Yarrowia lipolytica malate dehydrogenase genes YlMdh1 (serial number YALI0D16753p, Gene ID: 2910208) and YlMdh2 (serial number YALI0E14190p) and the 6-phosphogluconolactonase gene Pgl (serial number YALI0C19085p, Gene) provided on NCBI ID: 2909945) nucleotide sequence, obtained by PCR from the Yarrowia lipolytica genome.
  • PCR polymerase chain reaction
  • TbFrd-F/TbFrd-R primers PCR reaction conditions: pre-denaturation at 97°C for 5 minutes, denaturation at 94°C for 60 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 3 minutes, extension at 72°C for 10 minutes after 30 cycles, and storage at 4°C.
  • pKi-1 was digested with Bsp119I endonuclease and then recovered and purified. Gibson assembly cloning kit (New England Biolabs (NEB), England) was used to connect the above restriction fragments and PCR products to construct the pKi-TbFrd plasmid.
  • YlMdh1-F/YlMdh1-R and EcFum-F/EcFum-R primers were used to PCR amplify the assembly carrying the corresponding terminal homologous sequence. fragment.
  • 113-GPD-TEF was recovered and purified after digestion with SmiI and SalI endonucleases. Gibson assembly was used to connect the above restriction fragments and PCR products to construct the 113-YlMdh1-EcFum plasmid.
  • the above primers were used to PCR amplify the fusion fragment carrying the corresponding terminal overlapping sequence, and then expanded by overlap extension PCR to obtain the Pgl G75S fragment in which glycine at position 75 was mutated to serine.
  • PCR amplification was carried out using UT8-MLS-F/MLS-R and mTbFrd-F/TbFrd-R primers to obtain corresponding terminal overlapping sequences.
  • the fused fragments were then expanded by overlap extension PCR to obtain the assembled fragments.
  • pKi-1 was digested with Bsp119I endonuclease and then recovered and purified. Gibson assembly was used to connect the above restriction fragments and PCR products to construct the pKi-mTbFrd plasmid.
  • UT8-MLS-F/MLS-R and mScOsm1-F/mScOsm1-R primers were used to PCR amplify the genes carrying the corresponding terminal overlapping sequences.
  • the fused fragments were then expanded by overlap extension PCR to obtain the assembled fragments.
  • pKi-1 was digested with Bsp119I endonuclease and then recovered and purified. Gibson assembly was used to connect the above restriction fragments and PCR products to construct the pKi-mScOsm1 plasmid.
  • PCR amplification was carried out using UT8-MLS-F/MLS-R and mSfFrd-F/mSfFrd-R primers to obtain the corresponding terminal overlapping sequence.
  • the fused fragments were then expanded by overlap extension PCR to obtain the assembled fragments.
  • pKi-1 was digested with Bsp119I endonuclease and then recovered and purified. Gibson assembly was used to connect the above restriction fragments and PCR products to construct the pKi-mSfFrd plasmid.
  • YlMdh1-F/YlMdh1-R primers were used to PCR amplify the assembly fragment carrying the corresponding terminal homologous sequence.
  • 113-GPD-TEF is recovered and purified after digestion with SalI endonuclease. Gibson assembly cloning kit (New England Biolabs (NEB), England) was used to connect the above restriction fragments and PCR products to construct the 113-GPD-YlMdh1 plasmid.
  • PCR amplification using YlMdh2-F/YlMdh2-R primers was used to obtain an assembly fragment carrying the corresponding terminal homologous sequence.
  • 113-GPD-TEF is recovered and purified after digestion with SalI endonuclease. Gibson assembly cloning kit (New England Biolabs (NEB), England) was used to connect the above restriction fragments and PCR products to construct the 113-GPD-YlMdh2 plasmid.
  • PCR amplification using CgMdh-F/CgMdh-R primers was used to obtain assembly fragments carrying corresponding terminal homologous sequences.
  • 113-GPD-TEF is recovered and purified after digestion with SalI endonuclease. Gibson assembly cloning kit (New England Biolabs (NEB), England) was used to connect the above restriction fragments and PCR products to construct the 113-GPD-CgMdh plasmid.
  • PCR amplification was carried out using UT8-MLS-F/MLS-R and mEcFum-F/mEcFum-R primers to obtain the corresponding terminal overlapping sequence.
  • the fused fragments were then expanded by overlap extension PCR to obtain the assembled fragments.
  • 113-GPD-YlMdh2 was digested with SmiI endonuclease and then recovered and purified. Gibson assembly cloning kit (New England Biolabs (NEB), England) was used to connect the above restriction fragments and PCR products to construct the 113-YlMdh2-mEcFum plasmid.
  • the SpMae-F/SpMae-R primers were used to PCR amplify the assembled fragment carrying the corresponding terminal homologous sequence.
  • pKi-hyg is digested with Bsp119I endonuclease and then recovered and purified. Gibson assembly was used to connect the above restriction fragments and PCR products to construct the pKi-hyg-SpMae plasmid.
  • the hrGFP-F/hrGFP-R primers were used to PCR amplify the assembled fragment carrying the corresponding terminal homologous sequence.
  • YLEP-leu is recovered and purified after digestion with Bsp119I endonuclease. Gibson assembly was used to connect the above restriction fragments and PCR products to construct the YLEP-hrGFP plasmid.
  • PCR amplification was carried out using UT8-MLS-F/MLS-R and mCherry-F/mCherry-R primers to obtain the corresponding terminal overlapping sequence.
  • the fused fragments were then expanded by overlap extension PCR to obtain the assembled fragments.
  • YLEP-leu is recovered and purified after digestion with Bsp119I endonuclease. Gibson assembly was used to connect the above restriction fragments and PCR products to construct the YLEP-MLS-mCherry plasmid.
  • This example adopts non-homology-dependent recombination (NHEJ) transformation method to introduce linearized plasmids pKi-TbFrd and 113-YlMdh1-EcFum containing TbFrd, YlMdh1 and EcFum gene expression cassettes into the engineering strain Yarrowia lipolytica PGC91, and through the same Source recombination mutated the glycine at position 75 of 6-phosphogluconolactonase Pgl to serine to obtain the starting recombinant strain Mbmr-SA0 with high succinic acid production.
  • NHEJ non-homology-dependent recombination
  • the specific method is as follows: (1) After Yarrowia lipolytica PGC91 was cultured overnight in YPG liquid medium (containing 2% peptone, 1% yeast extract and 2% glycerol), competent cells were prepared using conventional yeast lithium acetate competent preparation methods. (2) Add 2 ⁇ L of pKi-TbFrd and 113-YlMdh1-EcFum plasmid fragments to 40 ⁇ L of competent cells, then add 2 ⁇ L of salmon sperm DNA, and incubate in a water bath at 30°C for 15 minutes.
  • the fermentation and detection method of succinic acid in short: insert the single colony grown on the above plate into a 50mL Erlenmeyer flask containing 10mL of YPG liquid culture medium (2% peptone, 1% yeast powder, 2% glycerol) , 30°C, 220rpm shaking culture activation. Take 1 mL of the activated culture medium and transfer it to a 250 mL Erlenmeyer flask containing 50 ml of fermentation medium (2% peptone, 1% yeast powder, 6% glucose). Cultivate for 96 hours at 30°C and 120 rpm. No external additions are made during the fermentation process. Acid and alkali agents maintain a neutral pH value in the fermentation broth.
  • the specific method is to take 1 mL of fermentation broth and centrifuge it at 12,000 rpm for 2 minutes at room temperature, take the supernatant, filter it with a microporous filter membrane with a pore size of 0.22 ⁇ m, and use high-performance liquid chromatography to detect the concentration of organic acids and glucose.
  • the detection conditions are: the chromatographic column is HPX-87H (BioRad Labs, 300mm ⁇ 7.8mm), the detector is the differential refractive index detector RID-10A, the column temperature is 65°C, the mobile phase is 5mM H 2 SO 4 solution, and the flow rate is 0.6 ml/min.
  • the obtained positive recombinants were continuously passaged in YPG medium for 2-3 days, and the engineering strains that removed the pUB4-CRE plasmid were screened out.
  • (8) Transform the Pgl G75S mutant fragment into the above-mentioned succinic acid high-producing strain that removes the selection marker, and spread it on an SD screening plate. Select positive transformants and verify using primers F: 5'-ACGACTCAACAAGACACAAAG-3' and R: 5'-ACATGATGGACTCTTCATTG-3'.
  • the specific method is as follows: (1) After culturing Yarrowia lipolytica Po1f in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) overnight, the competent cells were prepared using the conventional yeast lithium acetate competent preparation method. (2) Transform YLEP-hrGFP and YLEP-MLS-mCherry plasmids into Po1f competent cells, spread on SD screening plates, and culture at 30°C for 2-3 days. (3) Randomly select transformants, culture them in a 24-well deep well plate containing the corresponding SD screening liquid medium for 24 hours, and detect the fluorescence expression intensity. (4) Select cultures with higher fluorescence intensity for fluorescence microscopy examination.
  • mCherry which carries the mitochondrial localization signal, is expressed in good condition, and the fluorescence distribution is relatively clustered in line with the characteristics of mitochondrial localization, while the green fluorescent protein hrGFP, which is used as a control, shows a uniform distribution after expression. It shows that the N-terminal MLS signal sequence can target the target protein to the mitochondrial matrix.
  • the mitochondrial localization of TbFrd can significantly improve the succinic acid synthesis ability of Yarrowia lipolytica strain Mbmr-SA1, and the succinic acid production and yield are increased to 30.4g/L and 0.84g/g glucose respectively.
  • the succinic acid conversion rate of the mScOsm1 overexpression strain increased to 0.75g/g glucose, but the succinic acid production decreased by 18.3% compared to the control.
  • overexpression of mSfFrd can increase succinic acid production to 22.8g/L, the succinic acid conversion rate has decreased.
  • TbFrd derived from T. brucei can utilize NADH produced by the oxidized TCA cycle in mitochondria for succinic acid synthesis.
  • the linearized plasmids 113-GPD-YlMdh1, 113-GPD-YlMdh2 and 113-GPD-CgMdh were respectively integrated into the mTbFRD overexpression strain Mbmr-SA1.
  • the YPD shake flask fermentation results are as follows As shown in Figure 4. The results show that the individual overexpression of the three malate dehydrogenases all improves the succinic acid production of Mbmr-SA1. Among them, the succinic acid production of the Mbmr-SA2 strain overexpressing the YlMdh2 gene increased by 32.9%, approximately 40.4g/L. .
  • the present invention further overexpresses the mitochondrial-localized fumarase encoding gene mEcFum in the engineering strain Mbmr-SA2 to obtain strain Mbmr-SA3.
  • transformants were selected for shake flask fermentation. The results are shown in Figure 4.
  • the succinic acid production and conversion rate of Mbmr-SA3 strain decreased compared with the control, which were 39.8g/L and 0.84g/g glucose respectively.
  • the present invention further overexpressed the dicarboxylic acid transporter SpMae1 based on the Mbmr-SA3 strain.
  • the succinic acid production, yield and OD 600 of the SpMae1 overexpression strain Mbmr-SA4 reached the highest levels, respectively, which were approximately 46.7g/L, 0.86g/g glucose and 11.3.
  • the succinic acid yield and conversion rate have reached the highest shake flask level reported so far, and it has industrial application prospects.
  • the Yarrowia lipolytica Mbmr-SA4 which was genetically engineered to produce high succinic acid as described in Example 2, was subjected to a continuous fed-batch fermentation experiment in a 1L fermentation tank.
  • the medium selected in this example was YPD medium (2% pancreatic acid).
  • the set temperature of the fermentation tank is 30°C
  • the ventilation volume is 1.0vvm
  • the stirring speed is 500rpm
  • no pH adjustment is performed.
  • the glucose concentration is lower than 10g/L
  • add sugar to 60g/L.
  • the succinic acid and bacterial biomass of the sample were detected.
  • Figure 5 shows the fermentation results. Compared with shake flask fermentation, the succinic acid production rate is faster in the first 54 hours, and the succinic acid productivity can reach 0.8g/h/L.
  • the OD 600 of the bacteria was 12.5, and the production and conversion rate of succinic acid were 50.1g/L and 0.75g/g glucose respectively.

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Abstract

本发明提供一种具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株及其构建方法和应用，属于微生物及发酵工程技术领域。本发明是在酵母细胞中将琥珀酸生物合成途径的相关酶类超表达并定位至线粒体基质，有助于还原TCA途径在真核微生物中的功能表达。所述菌株能够利用普通碳源和氮源，经分批补料发酵培养后，琥珀酸产量达到50.1g/L，转化率达到0.75g/g葡萄糖。作为一种高效生产琥珀酸的优良菌株，在食品、化工及可降解材料等领域具有良好的应用前景。

Description

具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株及其构建方法和应用
本发明要求于2022年4月15日提交中国专利局、申请号为202210395975.3、发明名称为“具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株及其构建方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明属于微生物及发酵工程技术领域，具体涉及一种具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株及其构建方法和应用。
背景技术
本发明背景技术中公开的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
琥珀酸又称丁二酸(Succinic acid，SA)是一种重要的C4化合物，广泛应用于清洁剂、表面活性剂、食品添加剂、抗菌剂以及制药行业，被美国能源部选为十二种最具商业价值的平台化合物之首。琥珀酸也是生产聚丁二酸丁二醇酯PBS、聚丁二酸己二酸丁二醇酯PBSA等生物可降解塑料的重要原料，现有的琥珀酸产能已无法满足市场供给。琥珀酸的合成主要采用化学工艺路线，但是相比生物发酵法而言化学法生产琥珀酸存在能耗高、污染严重、温室气体排放等缺点。因此，开发绿色、可持续的琥珀酸高效发酵技术具有重要的实际意义。
相较于细菌宿主而言，酵母对酸碱和渗透压力具有较强的的耐受性，可以进行低pH的琥珀酸发酵，这简化了发酵工艺流程并且降低下游产物处理成本。近年来许多研究人员尝试对包括Saccharomyces cerevisiae、Pichia kudriavzevii、Issatchenkia orientali和Yarrowia lipolytica在内的多种酵母细胞进行代谢途径改造，以实现琥珀酸的发酵生产。解脂耶氏酵母(Yarrowia lipolytica)是一种非常规酵母，具有安全性高、耐酸能力强、分泌多种代谢产物和能够利用多种碳水化合物等优点，它被视为潜在的琥珀酸生产菌株受到越来越多的关注。解脂耶氏酵母可以通过降低琥珀酸脱氢酶活性，利用氧化TCA途径进行琥珀酸生产。然而，氧化TCA途径合成琥珀酸的理论碳转化率仅为0.65g/g葡萄糖，当前解脂耶氏酵母工程菌株的琥珀酸实际转化率与其他细菌生产菌株如Escherichia coli、Corynebacterium glutamicum和Actinobacillus succinogenes等相比仍然存在很大差距。
在已知的琥珀酸生物合成相关代谢途径中，还原TCA支路的理论转化效率更高(约为1.1 g/g葡萄糖)，能够显著减少温室气体CO 2的排放，相比其他途径具有明显优势。一些自然环境中分离的细菌，例如瘤胃细菌产琥珀酸放线杆菌和曼海姆产琥珀酸菌等，可以通过自身携带的还原TCA途径进行琥珀酸的高效合成。然而，绝大多数的酵母缺乏将延胡索酸催化为琥珀酸的关键酶，即延胡索酸还原酶，并且外源还原TCA途径的构建和表达较为困难。因此，如何在酵母细胞中构建功能性的还原TCA途径，实现外源琥珀酸高效合成途径与宿主的适配，是提高琥珀酸生产效率的关键。
发明内容
针对现有技术中存在的问题，本发明的目的在于提供一种具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株及其构建方法和应用。本发明以严格好氧的解脂耶氏酵母菌株(Yarrowia lipolytica)作为出发菌株，将不同种类的延胡索酸还原酶定位至线粒体基质，证明了该酶的亚细胞重定位有助于提高琥珀酸生产能力。以线粒体定位的NADH依赖延胡索酸还原酶过表达解脂耶氏酵母菌株Mbmr-SA1为宿主，将琥珀酸生物合成相关基因进行过表达，最终得到一种高效生产琥珀酸的解脂耶氏酵母(Yarrowia lipolytica)Mbmr-SA4，其有望成为一种琥珀酸工业化生产的工程菌进行应用。基于上述研究成果，从而完成本发明。
本发明的第一个方面，提供一种具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株，所述工程菌株其表达延胡索酸还原酶，且所述延胡索酸还原酶过表达并重定位至线粒体基质，同时，该工程菌株过表达琥珀酸合成相关酶和二羧酸转运蛋白。上述酵母工程菌株拥有功能的还原性琥珀酸生物合成途径，能够利用含有葡萄糖的培养基高效生产琥珀酸。
本发明的第二个方面，提供上述具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株的构建方法，所述构建方法包括：
以酵母菌株作为出发菌株，过表达线粒体定位的延胡索酸酶和内源的苹果酸脱氢酶，然后过表达二羧酸转运蛋白。
本发明的第三个方面，提供一种琥珀酸的工业化生产方法，所述工业化生产方法包括：对上述酵母工程菌株进行培养发酵。
本发明的第四个方面，提供上述酵母工程菌株或上述工业化生产方法在化工、可降解材料、食品及医药工业领域中的应用。
上述一个或多个技术方案的有益技术效果：
上述技术方案公开了一种新的解脂耶氏酵母工程菌，可用于高产琥珀酸，其作为琥珀酸的工程菌能够带来理想的产量和转化率；所述菌株能够利用普通碳源和氮源，经分批补料发酵培养后，琥珀酸产量达到50.1g/L，转化率达到0.75g/g葡萄糖。同时研究证明，上述工程菌在培 养过程中可以进行低pH发酵，该特点有效的减少工业生产过程中对培养液pH进行调节的步骤，进一步节约成本。
上述技术方案在构建工程菌株Mbmr-SA4时采用新的技术方法，即线粒体定位还原TCA途径在琥珀酸高产中的应用，显著提高了琥珀酸产量，因此上述工程菌具有可观的应用价值和前景。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为实施例2和3中所用到的还原TCA合成途径线粒体定位示意图。
图2为实施例2中酵母线粒体定位信号的功能验证结果图。
其中hrGFP代表以Po1f菌株为宿主过表达了绿色荧光蛋白hrGFP；MLS-mCherry为以Po1f菌株为宿主过表达了含有23个氨基酸的线粒体定位信号序列的红色荧光蛋白mCherry。两种菌株分别在荧光显微镜视野下观察细胞内荧光分布情况。
图3为实施例2中延胡索酸酶线粒体定位对琥珀酸生产的影响结果图。
其中Mbmr-SA0为对照菌株，TbFrd代表了以Mbmr-SA0为宿主过表达细胞质定位的布氏锥虫Trypanosoma brucei来源延胡索酸还原酶；mTbFrd代表了以Mbmr-SA0为宿主过表达线粒体基质定位的布氏锥虫来源延胡索酸还原酶；mScOsm1为以Mbmr-SA0为宿主过表达线粒体基质定位的酿酒酵母Saccharomyces cerevisiae来源延胡索酸还原酶；mSfFrd代表了以Mbmr-SA0为宿主过表达线粒体基质定位的希瓦氏菌Shewanella frigidimarina来源延胡索酸还原酶。
图4为实施例3中还原TCA途径相关基因和二羧酸转运蛋白过表达对琥珀酸生产的影响结果图。
其中，YlMdh1和YlMdh2为内源性苹果酸脱氢酶编码基因，CgMdh为谷氨酸棒杆菌Corynebacterium glutamicum来源的苹果酸脱氢酶编码基因，mEcFum为线粒体定位的大肠杆菌Escherichia coli来源的延胡索酸酶编码基因，SpMae1为粟酒裂殖酵母Schizosaccharomyces pombe来源的二羧酸转运蛋白编码基因。
图5为实施例3中工程菌Mbmr-SA4摇瓶发酵琥珀酸产量图。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
本发明的一个典型具体实施方式中，提供一种具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株，所述工程菌株其表达延胡索酸还原酶，且所述延胡索酸还原酶过表达并重定位至线粒体基质，同时，该工程菌株过表达琥珀酸合成相关酶和二羧酸转运蛋白。上述酵母工程菌株拥有功能的还原性琥珀酸生物合成途径，能够利用含有葡萄糖的培养基高效生产琥珀酸。
其中，所述酵母工程菌株其出发菌株包括但不限于Candida sonorensis、Kluyveromyces marxianus、Kluyveromyces thermotolerana、Issatchenkia orientalis、Candida methanesobosa、Candida lambica、Candida sorboxylosa、Saccharomyces bayanus、Kluyveromyces lactis、Pichia jadinii、Pichia anomala、Zygosaccharomyces lentus、Candida zemplinina、Candida geochares、Pichia membranifaciens、Saccharomyces cerevisiae、Pichia pastoris；进一步的，所述出发菌株优选解脂耶氏酵母Yarrowia lipolytica；更进一步的，所述出发菌株为解脂耶氏酵母(Yarrowia lipolytica)菌株Mbmr-SA0，其基因型为MatA,xpr2-322,axp-2,leu2-270,ura3-302,ΔSdh5::loxP,ΔAch1::loxP,YlPyc,TbFrd,EcFum,YlMdh1和Pgl G75S。
所述延胡索酸还原酶为外源酶，其来源包括但不限于布氏锥虫Trypanosoma brucei、酿酒酵母Saccharomyces cerevisiae和希瓦氏菌Shewanella frigidimarina，优选为布氏锥虫Trypanosoma brucei；更具体的，编码所述延胡索酸还原酶的基因序列如SEQ ID NO.1(见序列表seq_1)所示。
所述琥珀酸合成相关酶包括但不限于苹果酸脱氢酶，所述苹果酸脱氢酶编码基因为苹果酸脱氢酶基因YlMdh2，其基因序列号为:YALI0E14190p(Gene ID:2911503)。
所述二羧酸转运蛋白编码基因SpMae1来自粟酒裂殖酵母，其核苷酸序列如SEQ ID NO.6(见序列表seq_6)所示。
本发明又一具体实施方式中，提供上述具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株的构建方法，所述构建方法包括：
以酵母菌株作为出发菌株，过表达线粒体定位的延胡索酸酶和内源的苹果酸脱氢酶，然后过表达二羧酸转运蛋白。
其中，所述酵母菌株包括但不限于Candida sonorensis、Kluyveromyces marxianus、Kluyveromyces thermotolerana、Issatchenkia orientalis、Candida methanesobosa、Candida  lambica、Candida sorboxylosa、Saccharomyces bayanus、Kluyveromyces lactis、Pichia jadinii、Pichia anomala、Zygosaccharomyces lentus、Candida zemplinina、Candida geochares、Pichia membranifaciens、Saccharomyces cerevisiae、Pichia pastoris；进一步的，所述出发菌株优选解脂耶氏酵母Yarrowia lipolytica。
本发明又一具体实施方式中，所述构建方法包括：
以解脂耶氏酵母菌株(Yarrowia lipolytica)Mbmr-SA0作为出发菌株，将外源延胡索酸酶TbFrd编码基因mTbFrd导入所述出发菌株中，然后依次导入苹果酸脱氢酶编码基因YlMdh2和二羧酸转运蛋白基因SpMae1，即得解脂耶氏酵母(Yarrowia lipolytica)Mbmr-SA4，其能够实现高产琥珀酸的效果。
其中，解脂耶氏酵母菌株(Yarrowia lipolytica)Mbmr-SA0，其基因型为MatA,xpr2-322,axp-2,leu2-270,ura3-302,ΔSdh5::loxP,ΔAch1::loxP,YlPyc,TbFrd,EcFum,YlMdh1和Pgl G75S；其是以解脂耶氏酵母菌株(Yarrowia lipolytica)PGC91通过基因工程技术构建获得，所述解脂耶氏酵母菌株(Yarrowia lipolytica)PGC91见“Cui,Z.,Gao,C.,Li,J.,Hou,J.,Lin,C.,&Qi,Q.(2017).Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH.Metabolic engineering,42,126–133.”和“一种利用具有还原TCA途径的解脂耶氏酵母菌株好氧合成琥珀酸的方法”，更具体的，所述构建方法包括：将TbFrd(SEQ ID NO.1)、YlMdh1(序列号YALI0D16753p，Gene ID:2910208)和EcFum(SEQ ID NO.5，见序列表seq_5)基因导入解脂耶氏酵母菌株(Yarrowia lipolytica)PGC91中，同时将6-磷酸葡萄糖酸内酯酶(序列号YALI0C19085p，Gene ID:2909945)Pgl第75位甘氨酸突变为丝氨酸即得。
更具体的，采用非同源依赖的重组(NHEJ)转化方式，将含有TbFrd、YlMdh1和EcFum基因表达盒的线性化质粒pKi-TbFrd和113-YlMdh1-EcFum导入工程菌株Yarrowia lipolytica PGC91中，并通过同源重组将6-磷酸葡萄糖酸内酯酶Pgl第75位甘氨酸突变为丝氨酸，得到高产琥珀酸的出发重组菌Mbmr-SA0。
mTbFrd是将解脂耶氏酵母线粒体基质蛋白细胞色素c氧化酶5b亚基的线粒体定位序列(Mitochondrial localization sequence，MLS，SEQ ID NO.9，见序列表seq_9)与可溶性延胡索酸还原酶TbFrd(T.brucei来源NAD依赖，SEQ ID NO.1)融合获得，从而使得最终获得的工程菌中，延胡索酸还原酶过表达并重定位至线粒体基质；其中，线粒体定位信号肽(SEQ ID NO.10，见序列表seq_10)位于融合蛋白的N端。
所述苹果酸脱氢酶编码基因为苹果酸脱氢酶基因YlMdh2，其基因序列号为YALI0E14190p(Gene ID:2911503)。
所述二羧酸转运蛋白编码基因SpMae1来自粟酒裂殖酵母，其核苷酸序列如SEQ ID NO.6(见序列表seq_6)所示。
本发明又一具体实施方式中，提供一种琥珀酸的工业化生产方法，所述工业化生产方法包括：对上述酵母工程菌株进行培养发酵。
具体的，所述培养发酵方法包括：将上述酵母工程菌株置于YPD培养基进行发酵培养；
更具体的，所述发酵培养方法包括：发酵培养阶段，温度控制为25-35℃(优选为30℃)，通气量为0.5-5vvm(优选为1.0vvm)，搅拌速度控制为100-800rpm(优选为500rpm)；
更具体的，所述发酵培养方法中，还包括对发酵液中的葡萄糖浓度进行检测，当葡萄糖浓度低于10g/L时，进行补糖，补至50～60g/L；所述培养方法中，无需对pH进行调节，从而使得培养操作更为便捷并节约成本。
其中，所述YPD培养基其包括：2％胰蛋白胨，1％酵母提取物，6％葡萄糖。
本发明又一具体实施方式中，提供上述酵母工程菌株或上述工业化生产方法在化工、可降解材料、食品及医药工业领域中的应用。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
一、材料和方法
1、本发明中的基因合成由通用生物(安徽)股份有限公司完成；本发明中的引物合成及测序由北京擎科新业生物技术有限公司完成。
2、下面实施例中所使用的实验方法包括质粒构建、酶切、感受态细胞的制备、转化等如无特殊说明，均为常规方法。必要时可以通过简单实验确定具体实验条件。
3、下面实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
4、本发明所涉及的解脂耶氏酵母菌株Mbmr-SA0(基因型MatA,xpr2-322,axp-2,leu2-270,ura3-302,ΔSdh5::loxP,ΔAch1::loxP,YlPyc,TbFrd,EcFum,YlMdh1和Pgl G75S)，其以工程菌株PGC91(MatA,xpr2-322,axp-2,leu2-270,ura3-302,ΔSdh5::loxP,ΔAch1::loxP,YlPyc，见“Cui,Z.,Gao,C.,Li,J.,Hou,J.,Lin,C.,& Qi,Q.(2017).Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH.Metabolic engineering,42,126–133.”和“CN107916275A一种利用具有还原TCA途径的解脂耶氏酵母菌株好氧合成琥珀酸的方法”)为基础构建完成。还涉及携带可回收筛选标记的过表达载体pKi-1，pKi-hyg，pKi-ura，113- GPD-TEF和JMP-hyg-GPD，其构建步骤见文献(Cui,Z.,Jiang,X.,Zheng,H.,Qi,Q.,& Hou,J.(2019).Homology-independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica.Biotechnology and bioengineering,116(2),354–363.)。JMP113质粒和位点特异性重组酶过表达载体pUB4-CRE的构建见文献(Fickers,P.,Le Dall,M.T.,Gaillardin,C.,Thonart,P.,&Nicaud,J.M.(2003).New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica.Journal of microbiological methods,55(3),727–737.)。
5、本发明所涉及的基因，其中来自来自布氏锥虫T.brucei的TbFrd，希瓦氏菌Shewanella frigidimarina的SfFrd，来自酿酒酵母S.cerevisiae的ScOsm1，来自谷氨酸棒状杆菌Corynebacterium glutamicum的CgMdh，来自大肠杆菌E.coli的fumC，来自粟酒裂殖酵母S.pombe的SpMae都经密码子优化后并合成(通用生物，滁州，中国)。其中YlMdh1和YlMdh2基因克隆于解脂耶氏酵母基因组。
6、LB固体培养基：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，20g/L琼脂粉。
LB液体培养基：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠。
YPD培养基：10g/L酵母提取物，20g/L蛋白胨，20g/L葡萄糖。
YPD固体培养基：10g/L酵母提取物，20g/L蛋白胨，20g/L葡萄糖，2wt％琼脂粉。
SD液体选择培养基：26.7g/L SD基本培养基，添加适当浓度氨基酸缺失混合物，调pH 6.0。
SD固体培养基：SD液体选择培养基中添加2wt％琼脂粉。
二、基因元件的扩增与目标质粒的制备
(一)目标基因的制备
1、根据NCBI上提供的来自T.brucei的NADH依赖延胡索酸还原酶基因TbFrd(序列号KT026107.1)的核苷酸序列，经过密码子优化后，委托通用生物(安徽)股份有限公司合成优化后的NADH依赖延胡索酸还原酶基因TbFrdY。TbFrdY的核苷酸序列如SEQ ID NO.1所示。
2、根据NCBI上提供的来自S.cerevisiae的FADH 2依赖延胡索酸还原酶基因ScOsm1(序列号YJR051W)的核苷酸序列经过密码子优化后，委托通用生物(安徽)股份有限公司合成优化后的FADH 2依赖延胡索酸还原酶基因ScOsm1Y。ScOsm1Y的核苷酸序列如SEQ ID NO.2(见序列表seq_2)所示。
3、根据NCBI上提供的来自Shewanella frigidimarina的电子传递依赖延胡索酸还原酶基因SfFrd(GenBank:L04283.1)的核苷酸序列经过密码子优化后，委托通用生物(安徽)股份有限公司合成优化后的血红蛋白SfFrdY。SfFrdY的核苷酸序列如SEQ ID NO.3(见序列表seq_3)所 示。
4、根据NCBI上提供的来自C.glutamicum的苹果酸脱氢酶编码基因CgMdh(序列号Cgl2380)的核苷酸序列经过密码子优化后，委托通用生物(安徽)股份有限公司合成优化后的苹果酸脱氢酶编码基因CgMdhY的核苷酸序列如SEQ ID NO.4(见序列表seq_4)所示。
5、根据NCBI上提供的来自E.coli的延胡索酸酶编码基因EcFum(序列号b1611)的核苷酸序列经过密码子优化后，委托通用生物(安徽)股份有限公司合成优化后的延胡索酸酶编码基因EcFumY的核苷酸序列如SEQ ID NO.5所示。
6、根据NCBI上提供的来自S.pombe的C4二羧酸转运蛋白编码基因SpMae(序列号SPAPB8E5.03)的核苷酸序列经过密码子优化后，委托通用生物(安徽)股份有限公司合成优化后的C4二羧酸转运蛋白编码基因SpMaeY的核苷酸序列如SEQ ID NO.6所示。
7、根据NCBI上提供的Yarrowia lipolytica苹果酸脱氢酶基因YlMdh1(序列号YALI0D16753p，Gene ID:2910208)和YlMdh2(序列号YALI0E14190p)以及6-磷酸葡萄糖酸内酯酶基因Pgl(序列号YALI0C19085p，Gene ID:2909945)核苷酸序列，从Yarrowia lipolytica基因组中PCR获得。
(二)质粒的构建
1、质粒pKi-TbFrd的构建
根据经密码子优化以后的TbFrdY基因序列(SEQ ID NO.1)，以及表达质粒pKi-1序列设计引物：
Figure PCTCN2022143484-appb-000001
以密码子优化合成的TbFrdY基因序列为模板，使用TbFrd-F/TbFrd-R引物PCR(聚合酶链式反应)扩增得到携带相应末端同源序列的组装片段。PCR反应条件：97℃预变性5min，94℃变性60s，56℃退火30s，72℃延伸3min，30个循环后72℃延伸10min，4℃保存。
pKi-1通过Bsp119I内切酶消化后，回收纯化。利用Gibson组装克隆试剂盒(New England Biolabs(NEB),England)将上述酶切片段和PCR产物连接，构建完成pKi-TbFrd质粒。
2、质粒113-YlMdh1-EcFum的构建
根据经密码子优化以后的EcFumY基因序列(SEQ ID NO.5)，YlMdh1基因序列(YALI0D16753p)以及表达质粒113-GPD-TEF序列设计引物：
Figure PCTCN2022143484-appb-000002
分别以解脂耶氏酵母W29基因组和密码子优化合成的EcFumY基因序列为模板，使用YlMdh1-F/YlMdh1-R和EcFum-F/EcFum-R引物PCR扩增得到携带相应末端同源序列的组装片段。
113-GPD-TEF通过SmiI和SalI内切酶消化后，回收纯化。利用Gibson组装将上述酶切片段和PCR产物连接，构建完成113-YlMdh1-EcFum质粒。
3、Pgl G75S突变片段的构建
根据NCBI中的解脂耶氏酵母6-磷酸葡萄糖酸内酯酶编码基因Pgl(YALI0C19085p)及其上下游序列，以及质粒JMP113序列设计引物：
Figure PCTCN2022143484-appb-000003
Figure PCTCN2022143484-appb-000004
分别以解脂耶氏酵母W29基因组和JMP113质粒为模板，使用上述引物PCR扩增得到携带相应末端重叠序列的融合片段，随后通过重叠延伸PCR扩展得到第75位甘氨酸突变为丝氨酸的Pgl G75S片段。
4、质粒pKi-mTbFrd的构建
根据经密码子优化以后的TbFrdY基因序列(SEQ ID NO.1)，细胞色素c氧化酶5b亚基线粒体定位序列(SEQ ID NO.9)以及表达质粒pKi-1序列设计引物：
Figure PCTCN2022143484-appb-000005
分别以解脂耶氏酵母W29基因组和密码子优化合成的TbFrdY基因序列为模板，使用UT8-MLS-F/MLS-R和mTbFrd-F/TbFrd-R引物PCR扩增得到携带相应末端重叠序列的融合片段，随后通过重叠延伸PCR扩展得到组装片段。
pKi-1通过Bsp119I内切酶消化后，回收纯化。利用Gibson组装将上述酶切片段和PCR产物连接，构建完成pKi-mTbFrd质粒。
5、质粒pKi-mScOsm1的构建
根据经密码子优化以后的ScOsm1Y基因序列(SEQ ID NO.2)，细胞色素c氧化酶5b亚基线粒体定位序列(MFALRRSLLSAGRIARPQQVARF，SEQ ID NO.9)以及表达质粒pKi-1序列设计引物：
Figure PCTCN2022143484-appb-000006
Figure PCTCN2022143484-appb-000007
分别以解脂耶氏酵母W29基因组和密码子优化合成的ScOsm1Y基因序列为模板，使用UT8-MLS-F/MLS-R和mScOsm1-F/mScOsm1-R引物PCR扩增得到携带相应末端重叠序列的融合片段，随后通过重叠延伸PCR扩展得到组装片段。
pKi-1通过Bsp119I内切酶消化后，回收纯化。利用Gibson组装将上述酶切片段和PCR产物连接，构建完成pKi-mScOsm1质粒。
6、质粒pKi-mSfFrd的构建
根据经密码子优化以后的SfFrdY基因序列(SEQ ID NO.3)，细胞色素c氧化酶5b亚基线粒体定位序列(SEQ ID NO.9)以及表达质粒pKi-1序列设计引物：
Figure PCTCN2022143484-appb-000008
分别以解脂耶氏酵母W29基因组和密码子优化合成的SfFrdY基因序列为模板，使用UT8-MLS-F/MLS-R和mSfFrd-F/mSfFrd-R引物PCR扩增得到携带相应末端重叠序列的融合片段，随后通过重叠延伸PCR扩展得到组装片段。
pKi-1通过Bsp119I内切酶消化后，回收纯化。利用Gibson组装将上述酶切片段和PCR产物连接，构建完成pKi-mSfFrd质粒。
7、质粒113-GPD-YlMdh1的构建
根据NCBI中的YlMdh1基因序列(YALI0D16753p)，以及表达质粒113-GPD-TEF序列设计引物：
Figure PCTCN2022143484-appb-000009
以解脂耶氏酵母W29基因组为模板，使用YlMdh1-F/YlMdh1-R引物PCR扩增得到携带相应末端同源序列的组装片段。
113-GPD-TEF通过SalI内切酶消化后，回收纯化。利用Gibson组装克隆试剂盒(New England Biolabs(NEB),England)将上述酶切片段和PCR产物连接，构建完成113-GPD-YlMdh1质粒。
8、质粒113-GPD-YlMdh2的构建
根据NCBI中的YlMdh2基因序列(YALI0E14190p)，以及表达质粒113-GPD-TEF序列设计引物：
Figure PCTCN2022143484-appb-000010
以解脂耶氏酵母W29基因组为模板，使用YlMdh2-F/YlMdh2-R引物PCR扩增得到携带相应末端同源序列的组装片段。
113-GPD-TEF通过SalI内切酶消化后，回收纯化。利用Gibson组装克隆试剂盒(New England Biolabs(NEB),England)将上述酶切片段和PCR产物连接，构建完成113-GPD-YlMdh2质粒。
9、质粒113-GPD-CgMdh的构建
根据经密码子优化以后的CgMdh基因序列(SEQ ID NO.4)，以及表达质粒113-GPD-TEF序列设计引物：
Figure PCTCN2022143484-appb-000011
以解脂耶氏酵母W29基因组为模板，使用CgMdh-F/CgMdh-R引物PCR扩增得到携带相应末端同源序列的组装片段。
113-GPD-TEF通过SalI内切酶消化后，回收纯化。利用Gibson组装克隆试剂盒(New England Biolabs(NEB),England)将上述酶切片段和PCR产物连接，构建完成113-GPD-CgMdh质粒。
10、质粒113-YlMdh2-mEcFum的构建
根据经密码子优化以后的EcFumY基因序列(SEQ ID NO.5)，细胞色素c氧化酶5b亚基线粒体定位序列(SEQ ID NO.9)以及表达质粒113-GPD-YlMdh2序列设计引物：
Figure PCTCN2022143484-appb-000012
分别以解脂耶氏酵母W29基因组和密码子优化合成的SfFrdY基因序列为模板，使用UT8-MLS-F/MLS-R和mEcFum-F/mEcFum-R引物PCR扩增得到携带相应末端重叠序列的融合片段，随后通过重叠延伸PCR扩展得到组装片段。
113-GPD-YlMdh2通过SmiI内切酶消化后，回收纯化。利用Gibson组装克隆试剂盒(New England Biolabs(NEB),England)将上述酶切片段和PCR产物连接，构建完成113-YlMdh2-mEcFum质粒。
11、质粒pKi-hyg-SpMae的构建
根据经密码子优化以后的SpMaeY基因序列(SEQ ID NO.6)，以及表达质粒pKi-hyg序列设计引物：
Figure PCTCN2022143484-appb-000013
以密码子优化合成的SpMaeY基因序列为模板，使用SpMae-F/SpMae-R引物PCR扩增得到携带相应末端同源序列的组装片段。
pKi-hyg通过Bsp119I内切酶消化后，回收纯化。利用Gibson组装将上述酶切片段和PCR产物连接，构建完成pKi-hyg-SpMae质粒。
12、质粒YLEP-hrGFP的构建
根据经密码子优化以后的hrGFP基因序列(SEQ ID NO.7，见序列表seq_7)，以及游离表达载体YLEP-leu序列设计引物：
Figure PCTCN2022143484-appb-000014
以密码子优化合成的hrGFP基因序列为模板，使用hrGFP-F/hrGFP-R引物PCR扩增得到携带相应末端同源序列的组装片段。
YLEP-leu通过Bsp119I内切酶消化后，回收纯化。利用Gibson组装将上述酶切片段和PCR产物连接，构建完成YLEP-hrGFP质粒。
13、质粒YLEP-MLS-mCherry的构建
根据经密码子优化以后的mCherry基因序列(SEQ ID NO.8，见序列表seq_8)，细胞色素c氧化酶5b亚基线粒体定位序列(SEQ ID NO.9)以及游离表达载体YLEP-leu序列设计引物：
Figure PCTCN2022143484-appb-000015
分别以解脂耶氏酵母W29基因组和密码子优化合成的mCherry基因序列为模板，使用UT8-MLS-F/MLS-R和mCherry-F/mCherry-R引物PCR扩增得到携带相应末端重叠序列的融合片段，随后通过重叠延伸PCR扩展得到组装片段。
YLEP-leu通过Bsp119I内切酶消化后，回收纯化。利用Gibson组装将上述酶切片段和PCR产物连接，构建完成YLEP-MLS-mCherry质粒。
实施例2延胡索酸还原酶的线粒体定位及其对琥珀酸合成影响
本实施例采用非同源依赖的重组(NHEJ)转化方式，将含有TbFrd、YlMdh1和EcFum基因表达盒的线性化质粒pKi-TbFrd和113-YlMdh1-EcFum导入工程菌株Yarrowia lipolytica PGC91中，并通过同源重组将6-磷酸葡萄糖酸内酯酶Pgl第75位甘氨酸突变为丝氨酸，得到高产琥珀酸的出发重组菌Mbmr-SA0。
具体方法如下：(1)Yarrowia lipolytica PGC91于YPG液体培养基(含有2％蛋白胨、1％酵 母提取物和2％甘油)中过夜培养后，采用常规酵母醋酸锂感受态制备方法制备感受态细胞。(2)向40μL感受态细胞中分别加入2μL pKi-TbFrd和113-YlMdh1-EcFum质粒片段，再加入2μL鲑鱼精DNA，30℃水浴培养15min。(3)向上述体系中加入350μL PEG 4000-Lithium Acetate(0.1M pH 6.0)及16μL 1MDTT(40mM)，30℃水浴静止培养1h。(4)向上述体系中加40μLDMSO(终浓度约40％)，39℃热击10min。(5)加600μL Lithium Acetate(0.1M pH6.0)，室温放置15min。(6)取200μL上述混合物涂布SD筛选平板，30℃培养2-3天。(6)随机挑选转化子，摇瓶发酵并对发酵液中的琥珀酸进行检测。
琥珀酸的发酵和检测方法，简言之：将上述平板上长出的单菌落接入装有10mL的YPG液体培养基(2％蛋白胨，1％酵母粉，2％甘油)的50mL三角瓶中，30℃，220rpm转速振荡培养活化。分别取1mL活化的培养液转接到装有50ml发酵培养基(2％蛋白胨，1％酵母粉，6％葡萄糖)的250mL三角瓶中，30℃，120rpm培养96h，发酵过程中不外源添加酸碱剂维持发酵液中性pH值。发酵过程中，每间隔12-24h取样，然后再600nm波长下检测菌液的光吸收值。即取1mL菌液10,000-12,000rpm转速离心2min。弃去上清，用等体积的H 2O重悬，稀释至合适倍数后使用分光光度计检测其光吸收值。发酵液中的碳源和有机酸等代谢物采用高压液相色谱仪(HPLC)分析。具体方法为取1mL发酵液室温下12,000rpm离心2min，取上清，然后用孔径为0.22μm的微孔滤膜过滤，用高效液相色谱检测有机酸和葡萄糖浓度。检测条件为：色谱柱为HPX-87H(BioRad Labs,300mm×7.8mm)，检测器为示差折光检测器RID-10A，柱温为65℃，流动相为5mM H 2SO 4溶液，流速为0.6ml/min。(7)挑选琥珀酸产量和转化率较高的转化子，将pUB4-CRE质粒导入该高产菌株。YPG-hyg培养基中30℃培养1-2d，CRE重组酶组成型表达。利用接种环蘸取菌液在YPG-hyg筛选平板上划线，将长出的单克隆同时转入SD筛选平板和YPG-hyg平板上培养，在YPG-hyg平板上生长而在SD平板上不生长的筛选标记基因已被CRE重组酶删除。将得到的阳性重组子在YPG培养基中连续传代2-3d，筛选出去除pUB4-CRE质粒的工程菌株。(8)将Pgl G75S突变片段转化上述去除筛选标记的琥珀酸高产菌株，涂布SD筛选平板。挑选阳性转化子，利用引物F：5’-ACGACTCAACAAGACACAAAG-3’和R：5‘-ACATGATGGACTCTTCATTG-3’验证。
2、利用在线预测工具MITOPROT(https://ihg.gsf.de/ihg/mitoprot.html)鉴定出解脂耶氏酵母线粒体基质蛋白细胞色素c氧化酶5b亚基的线粒体定位序列(Mitochondrial localization sequence，MLS)。为了验证MLS序列的功能，将含有23个氨基酸的该定位序列(MFALRRSLLSAGRIARPQQVARF)融合至红色荧光蛋白mCherry的N端，转化解脂耶氏酵母野生型菌株Po1f并分析其胞内荧光分布。
具体方法如下：(1)Yarrowia lipolytica Po1f于YPD液体培养基(含有2％蛋白胨、1％酵母提取物和2％葡萄糖)中过夜培养后，采用常规酵母醋酸锂感受态制备方法制备感受态细胞。(2)将YLEP-hrGFP和YLEP-MLS-mCherry质粒转化Po1f感受态，涂布SD筛选平板，30℃培养2-3天。(3)随机挑选转化子，在含有相应SD筛选液体培养基的24孔深孔板中培养24h并检测荧光表达强度。(4)挑选荧光强度较高的培养物进行荧光显微镜检察。由如图2可知携带线粒体定位信号的mCherry表达状况良好，并且荧光分布比较聚集符合线粒体定位的特征，而作为对照的绿色荧光蛋白hrGFP表达后呈现均匀分布。说明了N端MLS信号序列可以将目的蛋白靶向线粒体基质。
3、将线粒体靶向信号MLS添加至三种不同类型的可溶性延胡索酸还原酶TbFrd(T.brucei来源NAD依赖)、ScOsm1(酿酒酵母来源FAD依赖)和SfFrd(希瓦氏菌来源电子传递依赖)。采用非同源重组转化(NHEJ)方式，将线性化的pKi-mTbFrd、pKi-mScOsm1和pKi-mSfFrd质粒分别导入Mbmr-SA0菌株，对比分析琥珀酸合成与细胞生长情况。如图3所示，TbFrd的线粒体定位能够显著提高解脂耶氏酵母菌株Mbmr-SA1的琥珀酸合成能力，琥珀酸产量和产率分别提高至30.4g/L和0.84g/g葡萄糖。mScOsm1过表达菌株的琥珀酸转化率提高至0.75g/g葡萄糖，但是琥珀酸产量相比对照下降18.3％。尽管mSfFrd的过表达能够使得琥珀酸产量提高至22.8g/L，琥珀酸转化率却有所下降。以上实验结果表明，T.brucei来源TbFrd能够利用线粒体中氧化TCA循环产生的NADH进行丁二酸合成。
实施例3还原TCA途径相关基因的组合过表达
为了进一步提高菌株琥珀酸产量，本实施例分别将线性化质粒113-GPD-YlMdh1、113-GPD-YlMdh2和113-GPD-CgMdh整合至mTbFRD过表达菌株Mbmr-SA1中，YPD摇瓶发酵结果如图4所示。结果显示三种苹果酸脱氢酶的单独过表达均对Mbmr-SA1的琥珀酸产量有提高作用，其中YlMdh2基因过表达Mbmr-SA2菌株的琥珀酸产量提高了32.9％，约为40.4g/L。本发明进一步在工程菌株Mbmr-SA2中过表达线粒体定位的延胡索酸酶编码基因mEcFum获得菌株Mbmr-SA3。经筛选挑取转化子进行摇瓶发酵，结果如图4所示，Mbmr-SA3菌株的琥珀酸产量和转化率均相较对照有所下降，分别为39.8g/L和0.84g/g葡萄糖。
增强终产物的胞外分泌不仅可以减少其过量积累造成的细胞毒性，而且有助于解除代谢途径中的反馈抑制。因此，本发明在在Mbmr-SA3菌株基础上进一步过表达了二羧酸转运蛋白SpMae1。由图4可知，SpMae1过表达菌株Mbmr-SA4的丁二酸产量、产率和OD 600分别达到最高水平，约为46.7g/L、0.86g/g葡萄糖和11.3。琥珀酸产量和转化率已达到目前报道的最高摇瓶水平，具有工业化应用前景。
实施例4 Mbmr-SA4菌株的连续补料发酵培养
将实施例2中所述基因工程构建的高产琥珀酸的解脂耶氏酵母Mbmr-SA4在1L发酵罐中进行连续补料发酵实验，本实施例选用的培养基为YPD培养基(2％胰蛋白胨，1％酵母提取物，6％葡萄糖)，初始体积为0.6L。从甘油保种管中取出菌液在YPDG固体平板上划线，培养36h。将长起来的单克隆接种到1瓶含有50mL YPDG培养基的摇瓶中，30℃，220rpm培养24h，将此种子接种到发酵罐中。发酵罐的设定温度为30℃，通气量为1.0vvm，搅拌速度500rpm，不进行pH调节。每个6小时取样检测发酵罐中葡萄糖浓度，当葡萄糖浓度低于10g/L时，补糖至60g/L。同时对样品的琥珀酸以及菌体的生物量进行检测。图5显示发酵结果，相比摇瓶发酵，前54h丁二酸生产速率较快，琥珀酸生产力可达0.8g/h/L。在发酵96h时，菌体OD 600为12.5，琥珀酸的产量和转化率分别为50.1g/L和0.75g/g葡萄糖。
本发明未尽事宜为公知技术。
以上所述仅为本发明的优选实施例，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株，其特征在于，所述工程菌株其表达延胡索酸还原酶，且所述延胡索酸还原酶过表达并重定位至线粒体基质，同时，该工程菌株过表达琥珀酸合成相关酶和二羧酸转运蛋白。
  2. 如权利要求1所述的酵母工程菌株，其特征在于，所述酵母工程菌株其出发菌株包括Candida sonorensis、Kluyveromyces marxianus、Kluyveromyces thermotolerana、Issatchenkia orientalis、Candida methanesobosa、Candida lambica、Candida sorboxylosa、Saccharomyces bayanus、Kluyveromyces lactis、Pichia jadinii、Pichia anomala、Zygosaccharomyces lentus、Candida zemplinina、Candida geochares、Pichia membranifaciens、Saccharomyces cerevisiae、Pichia pastoris；进一步的，所述出发菌株优选解脂耶氏酵母Yarrowia lipolytica；更进一步的，所述出发菌株为解脂耶氏酵母菌株(Yarrowia lipolytica)Mbmr-SA0，其基因型为MatA,xpr2-322,axp-2,leu2-270,ura3-302,ΔSdh5::loxP,ΔAch1::loxP,YlPyc,TbFrd,EcFum,YlMdh1和Pgl G75S。
  3. 如权利要求1所述的酵母工程菌株，其特征在于，所述延胡索酸还原酶为外源酶，其来源包括布氏锥虫Trypanosoma brucei、酿酒酵母Saccharomyces cerevisiae和希瓦氏菌Shewanella frigidimarina，优选为布氏锥虫Trypanosoma brucei；更具体的，编码所述布氏锥虫延胡索酸还原酶的基因序列如SEQ ID NO.1所示。
  4. 如权利要求1所述的酵母工程菌株，其特征在于，所述琥珀酸合成相关酶包括苹果酸脱氢酶，所述苹果酸脱氢酶编码基因为苹果酸脱氢酶基因YlMdh2，其基因号为YALI0E14190p。
  5. 如权利要求1所述的酵母工程菌株，其特征在于，所述二羧酸转运蛋白编码基因SpMae1来自粟酒裂殖酵母，其核苷酸序列如SEQ ID NO.6所示。
  6. 权利要求1-5任一项所述具有线粒体定位还原TCA途径的高产琥珀酸酵母工程菌株的构建方法，其特征在于，所述构建方法包括：
    以酵母菌株作为出发菌株，过表达线粒体定位的延胡索酸酶和内源的苹果酸脱氢酶，然后过表达二羧酸转运蛋白；
    优选的，所述酵母菌株包括Candida sonorensis、Kluyveromyces marxianus、Kluyveromyces thermotolerana、Issatchenkia orientalis、Candida methanesobosa、Candida lambica、Candida sorboxylosa、Saccharomyces bayanus、Kluyveromyces lactis、Pichia jadinii、Pichia anomala、Zygosaccharomyces lentus、Candida zemplinina、Candida geochares、Pichia membranifaciens、Saccharomyces cerevisiae、Pichia pastoris；进一步的，所述出发菌株优选解脂耶氏酵母Yarrowia lipolytica。
  7. 如权利要求6所述的构建方法，其特征在于，所述构建方法包括：
    以解脂耶氏酵母菌株(Yarrowia lipolytica)Mbmr-SA0作为出发菌株，将外源线粒体定位延胡索酸酶TbFrd编码基因mTbFrd导入所述出发菌株中，然后依次导入苹果酸脱氢酶编码基因YlMdh2和二羧酸转运蛋白SpMae1，即得解脂耶氏酵母(Yarrowia lipolytica)Mbmr-SA4；
    优选的，所述解脂耶氏酵母菌株(Yarrowia lipolytica)Mbmr-SA0，其基因型为MatA,xpr2-322,axp-2,leu2-270,ura3-302,ΔSdh5::loxP,ΔAch1::loxP,YlPyc,TbFrd,EcFum,YlMdh1和Pgl G75S；其是以解脂耶氏酵母菌株PGC91(MatA,xpr2-322,axp-2,leu2-270,ura3-302,ΔSdh5::loxP,ΔAch1::loxP,YlPyc)通过基因工程技术构建获得，更具体的，所述Mbmr-SA0的构建方法包括：将TbFrd(SEQ ID NO.1)、YlMdh1(序列号YALI0D16753p，Gene ID:2910208)和EcFum(SEQ ID NO.5)基因导入解脂耶氏酵母菌株(Yarrowia lipolytica)PGC91中，同时将6-磷酸葡萄糖酸内酯酶(序列号YALI0C19085p，Gene ID:2909945)Pgl第75位甘氨酸突变为丝氨酸即得；
    优选的，mTbFrd基因序列是将解脂耶氏酵母线粒体基质蛋白细胞色素c氧化酶5b亚基的线粒体定位序列(如SEQ ID NO.9所示)与可溶性延胡索酸还原酶TbFrd(如SEQ ID NO.1所示)融合获得；进一步优选的，线粒体定位肽序列位于融合蛋白的N端；
    优选的，所述苹果酸脱氢酶编码基因为苹果酸脱氢酶基因YlMdh2，其基因号为YALI0E14190p；
    优选的，所述二羧酸转运蛋白编码基因SpMae1来自粟酒裂殖酵母，其核苷酸序列如SEQ ID NO.6所示。
  8. 一种琥珀酸的工业化生产方法，其特征在于，所述工业化生产方法包括：对权利要求1-5任一项所述酵母工程菌株进行培养发酵。
  9. 如权利要求8所述工业化生产方法，其特征在于，所述培养发酵方法包括：将所述酵母工程菌株置于YPD培养基进行发酵培养；
    优选的，所述发酵培养方法包括：发酵培养阶段，温度控制为25-35℃(优选为30℃)，通气量为0.5-5vvm(优选为1.0vvm)，搅拌速度控制为100-800rpm(优选为500rpm)；
    优选的，所述发酵培养方法中，还包括对发酵液中的葡萄糖浓度进行检测，当葡萄糖浓度低于10g/L时，进行补糖，补至50～60g/L；所述培养方法中，无需对pH进行调节；
    优选的，所述YPD培养基其包括：2％胰蛋白胨，1％酵母提取物，6％葡萄糖。
  10. 权利要求1-5任一项所述酵母工程菌株或权利要求8或9所述工业化生产方法在化工、可降解材料、食品及医药工业领域中的应用。
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