Engineering pichia kudriavzevii and application thereof

Abstract

The invention relates to the technical field of microorganisms and biosynthesis, and discloses engineered pichia kudriavzevii and an application of the engineered pichia kudriavzevii. Based on the engineering strain, succinic acid can be efficiently produced in a low-pH environment, no bacterial contamination risk exists in the fermentation process, the addition amount of a neutralizer (such as ammonia water) is low, downstream product extraction and purification processes can be effectively simplified, the later environmental protection pressure is reduced, the problem of pollutant treatment in the succinic acid production and separation process is solved, the cost is saved, and the method is suitable for industrial production. The method has great industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of microbial and biosynthetic technology, and in particular to an engineered Pichia kudriaz yeast and its applications. Background Technology

[0002] In existing technologies, there have been several studies involving the construction of engineered strains for succinic acid production. For example, one study disclosed an acid- and heat-resistant genetically engineered strain using *Issa mesasula* as a host, with a sugar-acid conversion rate of 0.58 g / g and a fermentation cycle of 96 hours. Although this technology uses *Issa mesasula* in its modification strategy, its sugar-acid conversion rate and fermentation cycle are relatively low, failing to meet the principles of efficient industrial production.

[0003] Another study disclosed an acid-tolerant strain of Kluyveromyces martensii, which yielded 9.73 g / L of succinic acid after 96 hours of fermentation, with a sugar-acid conversion rate of only 0.24 g / g. The fermentation performance was low, and it required the addition of 15 g / L CaCO3 as a neutralizing agent, which increased the preparation cost and made the process relatively complex.

[0004] Another study reported a genetically engineered Escherichia coli strain that can simultaneously utilize glucose and xylose to produce succinic acid, but the initial glucose concentration in its fermentation medium cannot exceed 15 g / L, which is insufficient to meet the requirements for industrial-scale production.

[0005] Therefore, there is an urgent need in this field for an engineered strain capable of high-efficiency industrial production of succinic acid, with low residual sugar content, high sugar-acid conversion rate, and low by-product generation, in order to overcome the industrial production defects of existing strains. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide an engineered *Pichia gondii* strain and its applications. Addressing the shortcomings of the prior art and the actual needs of the field, this invention provides an engineered *Pichia gondii* strain with excellent fermentation performance for the production of succinic acid. Based on this engineered strain, succinic acid can be produced efficiently under low pH conditions, with no risk of contamination during fermentation. Furthermore, the amount of neutralizing agent (such as ammonia) required is low, effectively simplifying downstream product extraction and purification processes, reducing subsequent environmental pressures, and thus mitigating pollutant treatment problems in the production and separation of succinic acid, saving costs, and possessing significant industrial application value.

[0007] In a first aspect, the present invention provides an engineered Pichia kudriaz yeast, wherein the engineered Pichia kudriaz yeast is... URA3 Genetically defective Pichia pastoris S9.

[0008] In some embodiments of the present invention, other yeasts may be used to replace the Pichia kudriaz in the present invention for engineering modification, including but not limited to: Saccharomyces cerevisiae, etc.

[0009] In this invention, "comprising," "containing," "having," or "including" means that at least the specified substance, component, element, or method step is present in the product, article, or method, but does not exclude the presence of other substances, components, elements, or method steps, even if other such substances, components, elements, or method steps have the same function as the specified ones.

[0010] In some embodiments of the present invention, the URA3 The gene-deficient Pichia kudriaz S9 was obtained by screening Pichia kudriaz S9 on yeast-deficient SD-Ura solid medium. In this invention, it was obtained through screening. URA3 The genetically defective Pichia kudriaz S9 was used as a chassis strain and a series of modifications were made to obtain engineered Pichia kudriaz S9.

[0011] In some embodiments of the present invention, the URA3 The dicarboxylic acid transporter gene was knocked out in the genetically defective Pichia pastoris S9. JEN2-2 and expresses the succinic acid transporter gene. SpMAE .

[0012] In this invention, the gene knockout method and the heterologous expression vector method used are both conventional and known methods in the art. Those skilled in the art can choose any method to achieve the knockout and introduction of a specific gene based on actual needs.

[0013] In some embodiments of the present invention, the gene knockout method used is gene knockout based on the CRISPR-Cas9 technology.

[0014] In some embodiments of the present invention, the dicarboxylic acid transporter gene is targeted. JEN2-2 The sgRNA is shown in SEQ ID NO:94.

[0015] In this invention, the design of sgRNA is a conventional design in the art. Those skilled in the art can design sgRNA with known target sites, including but not limited to the sgRNA shown in SEQ ID NO:94.

[0016] In some embodiments of the present invention, the method of heterologous expression is homologous recombination.

[0017] In some embodiments of the present invention SpMAE The recombinant expression plasmid was based on the pUC19 plasmid.

[0018] In some embodiments of the present invention, the succinic acid transporter gene SpMAE The encoded sequence is shown in SEQ ID NO: 13.

[0019] In some embodiments of the present invention, the succinic acid transporter gene SpMAE The coding sequence also includes variants of the sequence shown in SEQ ID NO: 13, such as variant sequences based on codon optimization (e.g., codon optimization for species), codon degeneracy, or conventional amino acid substitution.

[0020] In some embodiments of the present invention, the variant sequence has at least 85% sequence identity with the sequence shown in SEQ ID NO: 13.

[0021] In some embodiments of the present invention, the sequence identity is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0022] In this invention, the term "sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and (if necessary) vacancies are introduced to achieve maximum sequence identity, and no conserved substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed by various methods known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art can determine appropriate parameters for determining the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0023] In some embodiments of the present invention, the variant sequence has effects comparable to or better than the sequence shown in SEQ ID NO: 13 (such as enzyme activity, catalytic activity, yield, conversion rate, etc.).

[0024] In this invention, the term "equivalent" means that the difference (absolute value) between the effect of the variant sequence and the sequence shown in SEQ ID NO: 13 is less than or equal to 5%, including ±5%, ±4%, ±3%, ±2%, ±1%, or equal.

[0025] In some embodiments of the present invention, the promoter is... TDH3 The starter and the terminator are GAL2 Termination of contract.

[0026] In some embodiments of the present invention, the URA3 The genetically defective Pichia pastoris S9 was further knocked out of glycerol-3-phosphate dehydrogenase (G3-phosphate dehydrogenase). GPD The gene was overexpressed, and the fumarate reductase gene was also overexpressed. LpFRD .

[0027] In some embodiments of the present invention, the 3-phosphoglycerate dehydrogenase ( GPD The sgRNA of the gene is shown in SEQ ID NO:95.

[0028] In some embodiments of the present invention LpFRD The recombinant expression plasmid was based on the pUC19 plasmid.

[0029] In some embodiments of the present invention, the fumarate reductase gene LpFRD The encoded sequence is shown in SEQ ID NO: 18.

[0030] In some embodiments of the present invention, the fumarate reductase gene LpFRD The coding sequence also includes variants of the sequence shown in SEQ ID NO: 18, such as variant sequences based on codon optimization (e.g., codon optimization for species), codon degeneracy, or conventional amino acid substitution.

[0031] In some embodiments of the invention, the variant sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 18. This sequence identity is as defined above.

[0032] In some embodiments of the present invention, the variant sequence has effects comparable to or better than the sequence shown in SEQ ID NO: 18 (such as enzyme activity, catalytic activity, yield, conversion rate, etc.).

[0033] In some embodiments of the present invention, the promoter is... FBA1 The starter and the terminator are TEF1 Termination of contract.

[0034] In some embodiments of the present invention, the URA3 The genetically defective Pichia pastoris S9 was further knocked out of pyruvate decarboxylase ( PDC The gene expresses L-malate dehydrogenase. ZrMDH .

[0035] In some embodiments of the present invention, the pyruvate decarboxylase is targeted ( PDCThe sgRNA of the gene is shown in SEQ ID NO:96.

[0036] In some embodiments of the present invention ZrMDH The recombinant expression plasmid was based on the pUC19 plasmid.

[0037] In some embodiments of the present invention, the L-malate dehydrogenase gene ZrMDH The encoded sequence is shown in SEQ ID NO: 33.

[0038] In some embodiments of the present invention, the L-malate dehydrogenase gene ZrMDH The coding sequence also includes variants of the sequence shown in SEQ ID NO: 33, such as variant sequences based on codon optimization (e.g., codon optimization for species), codon degeneracy, or conventional amino acid substitution.

[0039] In some embodiments of the invention, the variant sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 33. This sequence identity is as defined above.

[0040] In some embodiments of the present invention, the variant sequence has effects comparable to or better than the sequence shown in SEQ ID NO: 33 (such as enzyme activity, catalytic activity, yield, conversion rate, etc.).

[0041] In some embodiments of the present invention, the promoter is... CYC1 The starter and the terminator are CYC1 Termination of contract.

[0042] In some embodiments of the present invention, the URA3 The genetically defective Pichia pastoris S9 was further knocked out of lactate dehydrogenase ( LDH The gene expresses pyruvate carboxylase gene. PfPYC .

[0043] In some embodiments of the present invention, the lactate dehydrogenase ( LDH The sgRNA of the gene is shown in SEQ ID NO:97.

[0044] In some embodiments of the present invention PfPYC The recombinant expression plasmid was based on the pUC19 plasmid.

[0045] In some embodiments of the present invention, the pyruvate carboxylase gene PfPYCThe encoded sequence is shown in SEQ ID NO: 48.

[0046] In some embodiments of the present invention, the pyruvate carboxylase gene PfPYC The coding sequence also includes variants of the sequence shown in SEQ ID NO: 48, such as variant sequences based on codon optimization (e.g., codon optimization for species), codon degeneracy, or conventional amino acid substitution.

[0047] In some embodiments of the invention, the variant sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 48. This sequence identity is as defined above.

[0048] In some embodiments of the present invention, the variant sequence has effects comparable to or better than the sequence shown in SEQ ID NO: 48 (such as enzyme activity, catalytic activity, yield, conversion rate, etc.).

[0049] In some embodiments of the present invention, the promoter is... SED1 The starter and the terminator are ION1 Termination of contract.

[0050] In some embodiments of the present invention, the URA3 The genetically defective Pichia pastoris S9 was further knocked out of alcohol dehydrogenase ( ADH1 The gene was overexpressed, and the fumarate gene was also overexpressed. PkFUM .

[0051] In some embodiments of the present invention, the alcohol dehydrogenase ( ADH1 The sgRNA of the gene is shown in SEQ ID NO:98.

[0052] In some embodiments of the present invention PkFUM The recombinant expression plasmid was based on the pUC19 plasmid.

[0053] In some embodiments of the present invention, the fumarate gene PkFUM The encoded sequence is shown in SEQ ID NO: 63.

[0054] In some embodiments of the present invention, the fumarate gene PkFUM The coding sequence also includes variants of the sequence shown in SEQ ID NO: 63, such as variant sequences based on codon optimization (e.g., codon optimization for species), codon degeneracy, or conventional amino acid substitution.

[0055] In some embodiments of the invention, the variant sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 63. This sequence identity is as defined above.

[0056] In some embodiments of the present invention, the variant sequence has effects comparable to or better than the sequence shown in SEQ ID NO: 63 (such as enzyme activity, catalytic activity, yield, conversion rate, etc.).

[0057] In some embodiments of the present invention, the promoter is... GPM1 The starter and the terminator are FBA1 Termination of contract.

[0058] In some embodiments of the present invention, the overexpression includes overexpression by at least one of the following methods: using a strong promoter or enhancing promoter activity, increasing gene copy number, or using at least one of an inducible expression system.

[0059] In some embodiments of the present invention, the overexpression is achieved by increasing the gene copy number.

[0060] In some embodiments of the present invention, the gene copy number is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0061] In some embodiments of the present invention, the gene copy number is increased by 1-10.

[0062] In some embodiments of the present invention, the gene copy number is increased by 1.

[0063] In some embodiments of the invention, further insertion is made at another point. PkFUM and LpFRD Encoded sequence.

[0064] In some embodiments of the present invention, the insertion site for the overexpressed gene includes II12. It should be understood that other insertion sites or known safe insertion sites may also be selected in the present invention to achieve successful expression, wherein the safe insertion site includes, but is not limited to, II12 described above.

[0065] A second aspect of the present invention provides a composition comprising: (1) The engineered Pichia kudriaz or its culture described above; (2) Proteins isolated from (1); In some embodiments of the present invention, the engineered Kudriaz Pichia pastoris includes at least one of engineered live Kudriaz Pichia pastoris and engineered inactivated Kudriaz Pichia pastoris.

[0066] In some embodiments of the present invention, the culture comprises: engineered Pichia kudriaz yeast culture, metabolites, fermentation products, or their supernatants, inactivated products, concentrated or dried products.

[0067] In some embodiments of the invention, the composition is provided in liquid or solid form.

[0068] In some embodiments of the invention, the composition is in the form of a liquid, a powder (e.g., lyophilized powder), or a gel.

[0069] In some embodiments of the present invention, the composition also contains other excipients.

[0070] In some embodiments of the present invention, the excipients are rationally selected based on factors such as the product form of the composition, the intended use, and the fermentation method, and include, but are not limited to: buffers, coenzymes, enzyme protectants, metal ions, catalysts, defoamers, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0071] A third aspect of the invention provides the use of the engineered Pichia kudriaz yeast and / or compositions described above in the biosynthesis of organic compounds.

[0072] In some embodiments of the present invention, the organic compound includes glycerol, ethanol, acetic acid, lactic acid, and succinic acid.

[0073] In some embodiments of the present invention, strains S9-UB, SA01, and SA02 can all produce glycerol.

[0074] In some embodiments of the present invention, strains S9-UB, SA01, and SA02 can all produce ethanol.

[0075] In some embodiments of the present invention, SA03 can produce lactic acid.

[0076] In some embodiments of the present invention, SA04 can generate acetic acid.

[0077] In some embodiments of the present invention, strains SA01, SA02, SA03, SA04, SA05, and SA06 can all produce succinic acid.

[0078] In some embodiments of the present invention, strains SA05 and SA06 produce succinic acid but do not produce glycerol, ethanol, acetic acid, or lactic acid.

[0079] In some embodiments of the present invention, the malic acid content in the synthesized product is less than or equal to 1 g / L.

[0080] In some embodiments of the present invention, the malic acid content in the synthesized product is less than or equal to 0.5 g / L.

[0081] In some embodiments of the present invention, the malic acid content in the synthesized product is less than or equal to 0.3 g / L.

[0082] In some embodiments of the present invention, the substrate for the biosynthesis includes glucose with a pH less than 3.

[0083] In some embodiments of the present invention, the glucose concentration is greater than or equal to 50 g / L, preferably greater than or equal to 100 g / L.

[0084] In some embodiments of the present invention, pH is less than or equal to 2.8.

[0085] In some embodiments of the present invention, the conversion rate of succinic acid is greater than 70 wt%, 71 wt%, 72 wt%, 73 wt%, 74 wt%, 75 wt%, 76 wt%, 77 wt%, 78 wt%, 79 wt%, and 80 wt%.

[0086] In this invention, conversion rate refers to glucose-acid conversion rate. In some embodiments of this invention, conversion rate refers to the mass ratio (wt%) of the amount of succinic acid produced to the amount of glucose consumed.

[0087] In some embodiments of the present invention, the engineered Kudriaz Pichia pastoris and / or composition described above achieves a succinic acid conversion rate greater than 70 wt%, 71 wt%, 72 wt%, 73 wt%, 74 wt%, 75 wt%, 76 wt%, 77 wt%, 78 wt%, 79 wt%, or 80 wt% during a fermentation time of 48 h.

[0088] In some embodiments of the present invention, the engineered Kudriaz Pichia pastoris and / or composition described above achieves a succinic acid conversion rate greater than 70 wt%, 71 wt%, 72 wt%, 73 wt%, 74 wt%, 75 wt%, 76 wt%, 77 wt%, 78 wt%, 79 wt%, or 80 wt% over a fermentation period of 60 h.

[0089] A fourth aspect of the present invention provides a method for preparing succinic acid, comprising the following steps: The product is obtained by fermentation of the engineered Pichia kudriaz and / or the composition described above on a sugar-containing yeast culture medium.

[0090] In some embodiments of the present invention, the sugar-containing yeast culture medium includes at least one of: YPD or YEPD medium, potato dextrose agar (PDA) medium, synthetic glucose (SD) basal medium, and corn steep liquor / starch hydrolysate medium.

[0091] In some embodiments of the present invention, the sugar-containing yeast culture medium is YPD or YEPD culture medium.

[0092] In some embodiments of the present invention, the sugar content in the yeast culture medium is greater than or equal to 50 g / L, preferably greater than or equal to 100 g / L.

[0093] In some embodiments of the present invention, the sugar includes at least one of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0094] In some embodiments of the present invention, the engineered Pichia kudriaz described above and / or the compositions described above are capable of producing succinic acid using the sugar as a substrate.

[0095] In some embodiments of the present invention, the sugar includes glucose.

[0096] In some embodiments of the present invention, the sugar is glucose.

[0097] In some embodiments of the present invention, the fermentation time is 24-100 h. Preferably, it is 24-80 h, more preferably 48-80 h, and even more preferably 55-65 h.

[0098] In some embodiments of the present invention, the preparation method further includes: separating, extracting and purifying the fermentation broth obtained from fermentation to obtain purified succinic acid.

[0099] In some embodiments of the present invention, the separation, extraction and purification steps can all adopt conventional succinic acid separation, extraction and purification steps in the art, and those skilled in the art can adjust them according to the application scenario and usage requirements.

[0100] The beneficial effects of this invention are: 1. The engineered Pichia kudriaz in this invention can grow and reproduce under low pH conditions, making it more suitable for succinic acid production. The low pH conditions reduce the risk of contamination during fermentation, decrease the amount of neutralizing agent required, simplify downstream extraction and purification processes, reduce environmental pressure, and consequently lower the costs of succinic acid production, separation, and wastewater treatment, thus possessing significant industrial application value.

[0101] 2. In this invention, an optimal enzyme combination was obtained through screening from a large number of enzymes from different species, which significantly enhances the succinic acid production capacity of *Pichia gondii*. *Pichia gondii* modified based on this combination exhibits lower residual sugar, higher sugar-acid conversion rate, fewer byproducts, and stronger fermentation performance in the later stages of fermentation. Furthermore, it demonstrates good stability and tolerance, reducing production costs and maintenance difficulty, further enhancing its competitiveness in practical industrial applications.

[0102] 3. The engineered Pichia kudriaz in this invention can achieve a succinic acid yield of 81.06 g / L and a sugar-acid conversion rate of 0.82 g / g in a 5 L fermenter at pH 2.8 and 30℃ for 60 h; and a yield of up to 40.96 g / L and a sugar-acid conversion rate of 0.82 g / g in shake flask fermentation at 30℃ for 48 h. Attached Figure Description

[0103] Figure 1 The expression of different transformants is shown in the figure. M is the marker, lanes 1-5 are URA3-deficient strains screened in the examples, and lane 6 is the S9 strain. Detailed Implementation

[0104] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0105] In this invention, the composition of the culture medium used in the following embodiments is as follows: LB liquid medium: composed of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, based on the final concentration.

[0106] LB solid medium is obtained by adding agar powder to the above-mentioned LB liquid medium to a final concentration of 2 wt%.

[0107] Yeast Extract Peptone Glucose (YPD) Liquid Culture Medium: Composed of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose, based on the final concentration.

[0108] YPD solid medium is obtained by adding agar powder to the above-mentioned YPD liquid medium to a final concentration of 2 wt%.

[0109] The shake flask fermentation medium used in the shake flask fermentation culture was YPD liquid medium with the final glucose concentration adjusted to 50 g / L.

[0110] Yeast-deficient SD-Ura solid medium: per 1 L, 8 g of commercially available SD-Ura solid medium (purchased from Solarbio Science & Technology Co., Ltd.) is dissolved in water with 20 g of glucose and 20 g of agar and then sterilized at high temperature.

[0111] Fermentation tank culture medium: based on final concentration, it consists of 100 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract, pH 7.0.

[0112] 5-FOA solid medium: Weigh 0.1g of 5-FOA per 100mL, add 2g of glucose, 2g of agar powder, 2g of peptone, and 1g of yeast powder, dissolve thoroughly in water, and then sterilize at high temperature.

[0113] In the following embodiments, the *Pichia gondii* S9 (accession number GDMCC No: 65148) used is disclosed in the applicant's prior Chinese patent CN 119614400 A, which is incorporated herein by reference. In the following embodiments, the editing plasmid template pIO-CAS1.0 used is disclosed in the applicant's prior Chinese patent CN 118910116 A, which is incorporated herein by reference.

[0114] Example 1 In this embodiment, a Kudriaz Pichia pastoris S9 mutant was constructed.

[0115] The specific construction method is as follows: Pichia kudriaz S9 was cultured overnight in YPD liquid medium. Then, 100 μL of the diluted bacterial suspension was taken and diluted with sterile water or physiological saline at different ratios (10x, 100x, 1000x, and 10000x) and spread onto 5-FOA (Thermo Fisher Scientific) medium. After incubation at 30°C for 2-3 days, single colonies were picked and streaked onto yeast-deficient SD-Ura solid medium and YPD solid medium for further cultivation. Strains that could not grow on SD-Ura solid medium but could grow on YPD solid medium were screened out; these strains were identified as URA3-deficient strains (i.e., Pichia kudriaz S9 mutants).

[0116] Using the obtained strain genome as a template, PCR amplification was performed using the identification primers corresponding to the URA gene. Pichia gondii S9 was used as a control.

[0117] The primers for identifying the URA gene are: URA3-JD-F: 5'-ctaagcaaaatttcaggtgacctgtac-3' (SEQ ID NO: 100); URA3-JD-R: 5'-gaagaataactggtatagag-3' (SEQ ID NO: 101).

[0118] The results are as follows Figure 1 As shown.

[0119] It can be observed that the corresponding fragment can be amplified in Pichia kudriaz S9, but the corresponding fragment cannot be amplified in the Pichia kudriaz S9 mutant. This indicates that the Pichia kudriaz S9 mutant does not contain URA3. The URA3-deficient strain was successfully constructed and named Pichia kudriaz S9-URA (abbreviated as S9-URA strain).

[0120] Example 2 In this embodiment, a Cas9-gRNA fusion plasmid pIO-CAS9- for the CRISPR / Cas9 gene editing system was constructed. WATER 3. This plasmid is suitable for the S9-URA strain, which contains... URA3 Screening markers can be used to obtain positive transformants through auxotrophic screening.

[0121] The specific construction method is as follows: Using the editing plasmid pIO-CAS1.0 disclosed in the applicant's prior Chinese patent CN 118910116 A as a template, the vector fragment was amplified using primers pTEF1-F and tTEF1-R. Using S9 genomic DNA as a template, the URA3 gene fragment was amplified using primers URA3-F and URA3-R.

[0122] The primer information is shown in the table below.

[0123] Table 1 Primer Information

[0124] The amplified vector fragment and URA3 gene fragment were purified using a PCR product purification kit. Then, the purified vector fragment and URA3 gene fragment were subjected to homologous recombination using a one-step cloning kit (Vazyme) to obtain the homologous recombination product. 10 μL of the homologous recombination product was added to 100 μL of DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42 ℃ for 90 s, and then 1 mL of LB liquid medium was added. The cells were cultured at 37 ℃ with shaking for 1 h. The bacterial culture was then spread onto LB solid medium resistant to Amp (final working concentration: 100 mg / L) and incubated upside down at 37 ℃ until single colonies appeared. Five single colonies were picked and cultured in LB liquid medium with shaking. After positive PCR detection, sequencing was performed for confirmation. The plasmid from the confirmed strain was extracted and preserved; the resulting plasmid is pIO-CAS9- WATER 3.

[0125] The following examples use a defective strain (URA3) - Gene editing was performed on the chassis (in this embodiment, the aforementioned S9-URA strain), specifically using the following method: Single colonies of S9-URA cultured on YPD solid medium were inoculated into YPD liquid medium and incubated overnight at 30°C and 220 rpm. The bacterial culture was then inoculated into 50 mL of YPD liquid medium, with an initial OD600 of 0.3. The culture was incubated at 30°C and 200 rpm until the OD600 reached 1.2–1.5. 40 mL of the bacterial culture was collected by centrifugation, washed twice with sterile deionized water, and resuspended in LDST solution (1 L of solution is prepared by dissolving 182 g sorbitol, 1.21 g Tris, 1.54 g dithiothreitol, and 6.6 g lithium acetate in water and filtering through a 0.22 μM filter). The suspension was incubated at 30°C for 30 min. After incubation, the cells were centrifuged at 5000 rpm for 3 min in a refrigerated centrifuge. The supernatant was discarded, and the cells were washed three times with 5 mL of sterile water (5000 rpm, 3 min). After the final wash, the bacterial cells were resuspended in 400 μL of sorbitol and aliquoted into 1.5 mL EP tubes (80 μL / tube) to obtain competent S9-URA cells. The corresponding editing plasmid and donor DNA fragment were added to the competent S9-URA cells, gently mixed, and transferred to pre-ice-bathed electroporation cuvettes, then placed on ice for 5 min. The electroporator was set to 1.5 kV, 200 Ω, and 25 mF, with the electroporation mode set to fungal mode, and the mixture was electroporated. 1 M of pre-chilled sorbitol was added, and the mixture was transferred to sterile 1.5 mL EP tubes and incubated at 30 °C for 1.5 h. After centrifugation, a portion of the supernatant was discarded. The bacterial cells were then resuspended using a disposable spreader and spread onto yeast-deficient SD-Ura solid medium for screening for positive transformants.

[0126] Example 3 In this embodiment, an engineered S9-URA strain was constructed. This engineered S9-URA strain had its dicarboxylic acid transporter gene knocked out. JEN2-2 The succinic acid transporter gene was introduced. SpMAE The encoded sequence.

[0127] The specific construction method is as follows: (1) Construction of donor DNA fragments for homologous recombination: Genomic DNA was extracted from *Pichia gondii* S9 using the TAKARA yeast genome extraction kit. Using *Pichia gondii* S9 genomic DNA as a template, amplification was performed using primers JEN2-2-up-F / R, JEN2-2-down-F / R, P-TDH3-F / R, and T-GAL-F / R, respectively. JEN2-2The upper and lower homologous arms, as well as the promoter and terminator fragments used by the transport protein MAE, were amplified using pUC19-SpMAE plasmid (synthesized by Qingke Biotechnology Co., Ltd.) as a template and primers SpMAE-F / R. MEE The coding sequence (as shown in SEQ ID NO: 13, or can be directly synthesized in solid phase based on this sequence) was used as a template. A linear pUC19 vector fragment was amplified using primers pUC19 F and pUC19 R. The amplification primers were recovered, and their concentrations were determined using a NanoDrop Lite Plus microspectrophotometer. The amplified... TDH3 promoter, MEE Encoded sequence, GAL2 The terminator was used to fuse fragments using primers P-TDH3-F / T-GAL2-R, and the target band (fusion sequence) was recovered using electrophoresis gel. The fusion sequence was then fused using the homologous recombinase C115 (purchased from Novizan). JEN2-2 The homologous arms and linear pUC19 vector fragment were ligated and transformed into DH5α competent cells. Single colonies with normal morphology and size were selected, cultured and propagated by shaking, and then sequenced for verification. After successful sequencing verification, JEN2-2 DNA was amplified using primers JEN2-2-up-F / JEN2-2-down-R and recovered by electrophoresis gel for later use.

[0128] The primer information is shown in the table below.

[0129] Table 2 Primer Information

[0130] in, SpMAE The encoded sequence is shown in Uniprot number P50537, and the specific sequence is as follows:

[0131] (2) Used for editing JEN2-2 Construction of the Cas9-gRNA fusion plasmid pIO-CAS9-jen: The previously constructed editing plasmid contained Cas9 and sgRNA elements. For different targets, only the sgRNA target sequence needed to be changed. The sgRNA sequence for the jen target was designed using the CRISPRscan: CRISPRs in vivo website (https: / / www.crisprscan.org / ): 5'-ACGTCAAGGACATTACATGG-3' (SEQ ID NO:94), and its editing plasmid pIO-CAS9-jen was constructed through homologous recombination.

[0132] Specifically, using pIO-CAS9-URA3 as a template, PCR amplification was performed using primers JEN-sg-F / R (Phanta enzyme was used for DNA polymerase) to obtain the amplification product. The amplification conditions were: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 58℃ annealing for 30 s, 68℃ extension for 60 s, repeated 30 times; 68℃ extension for 7 min; and storage at 12℃. The obtained amplification product was digested with Dpn I enzyme (37℃, 2 h). 10 μL of the digested product was transformed into DH5α competent cells. Single colonies with normal morphology and size were selected, cultured, and then sequenced for verification. After successful sequencing verification, the plasmid was extracted using a DNA extraction kit (purchased from Aowei) to obtain the Cas9-gRNA fusion plasmid pIO-CAS9-jen. The JEN2-2 DNA and pIO-CAS9-jen plasmid obtained in step (1) above were simultaneously transformed into S9-URA (electroporation method as described in the above example). Positive transformants were obtained through screening and further identified using primers JEN2-2-JD-F / R. Transformants that were confirmed to be positive were named strain SA01 (i.e., knockout strain). JEN2-2 And overexpression SpMAE (S9-URA strain).

[0133] The primer information is shown in the table below.

[0134] Table 3 Primer Information

[0135] Example 4 In this embodiment, an engineered *Pichia kudriaz* yeast was constructed. This engineered *Pichia kudriaz* yeast was based on the SA01 strain, with the glycerol-3-phosphate dehydrogenase gene (GPD) knocked out and overexpressed. Leptomonas pyrrhocorisKudriaz Pichia pastoris modified with the fumarate reductase gene FRD (LpFRD).

[0136] The specific construction method is as follows: (1) Selection of fumarate reductase (FRD): In this embodiment, samples were selected from Caenorhabditis elegans (Caenorhabditis elegans). Caenorhabditis elegans CeFRD (Uniprot ID: Q09567) is derived from *Geobacterium thioreducingans* (…). Geobacter sulfurreducens The GsFRD (Uniprot number: Q74DY8) is derived from Leptomonas pyrrhocoris LpFRD (Uniprot ID: Q8NN33), derived from Diplonema papillata The DpFRD (NCBI ID: KAJ9473120.1) is derived from the jumping gyrfalcon ( Bodo dancing BsFRD (Uniprot ID: A0A0S4IZL1.), derived from *Micrococcus pseudocarpa* ( Nannochloropsis from Cadiz The NgtFRD (Uniprot ID: W7U4A5) of the enzyme was tested. After codon optimization (for Pichia kudriaz) of these fumarate reductases from different sources, they were then compared with the enzymes described in the above examples. FBA1 promoters and Tgal2 Fragment fusion was performed using terminators. These recombinant expression vectors were introduced into *Saccharomyces cerevisiae* SA01 via homologous recombination (HE) technology and expressed. Fermentation was carried out in YPD medium, and high-activity FRDs were screened based on the increase in succinic acid content after 36 h of fermentation. Transformants obtained from blank plasmids without the inserted FRD coding sequence were used as controls.

[0137]

[0138] The results are shown in the table below.

[0139] Table 4. Effects of fumarate reductase fermentation from different sources on succinic acid content in the system.

[0140] It can be observed that the succinic acid content increases significantly after LpFRD overexpression, indicating that the FRD has higher catalytic activity for fumaric acid.

[0141] (2) Construct LpFRD overexpression vectors and transformants containing these expression vectors based on the optimized LpFRD: Constructing donor DNA fragments for homologous recombination: Genomic DNA was extracted from Pichia pastoris S9 using a commercially available DNA extraction kit. Using this DNA as a template, the upper and lower homologous arms of GPD, the FBA1 promoter, and the TEF1 terminator fragment were amplified using primers GPD-up-F / R, GPD-down-F / R, P-FBA1-F / R, and T-TEF1-F / R, respectively. Using the LpFRD recombinant expression vector (pUC57-LpFRD) prepared in step (1) as a template, the optimized LpFRD fragment was obtained by amplification using primer LpFRD-F / R. Using the pUC19 plasmid as a template, the pUC19 plasmid fragment was obtained by amplification using primers pUC19 F and pUC19 R (SEQ ID NO: 1 and 2). The FBA1 promoter, TEF1 terminator fragment, and optimized LpFRD fragment were fused using primers P-FBA1-F / T-TEF1-R. The target band was recovered by electrophoresis, yielding the FBA1-LpFRD-TEF1 fusion fragment. Then, the FBA1-LpFRD-TEF1 fusion fragment was inserted into the pUC19 plasmid fragment using the homologous recombinase C115 (purchased from Novizan), resulting in an LpFRD overexpression vector. This vector was transformed into DH5α competent cells, and single colonies with normal morphology and size were selected, cultured, and sequenced for verification. Verified transformants were amplified using primers GPD-up-F / R, and recovered by electrophoresis, yielding the GPD donor DNA (containing homologous arms at both ends of the GPD site and the LpFRD expression cassette fragment to be integrated).

[0142] The primer information is shown in the table below.

[0143] Table 5 Primer Information

[0144] Construct the Cas9-gRNA fusion plasmid pIO-CAS9-gpd for editing the GPD gene: The sgRNA sequence targeting the GPD gene (5'-CTAATCACTACCTGTGCCGG-3' (SEQ ID NO:95)) was designed using the same method. WATER Using template 3, amplification was performed using primers GPD-sg-F / R. The DNA polymerase used in the amplification system was Phantadine. The amplification program was as follows: 95 °C pre-denaturation for 2 min; 95 °C denaturation for 10 s, 58 °C annealing for 30 s, 68 °C extension for 60 s, repeated 30 times; 68 °C extension again for 7 min. The product was stored at 12 °C for later use. The amplification product was recovered and digested with Dpn I (37 °C, 2 h) to obtain the digested product. 10 μL of the digested product was transformed into DH5α competent cells. Single colonies with normal morphology and size were selected, cultured in a shaking environment, and sequenced for verification. After confirming the inserted sequence was correct, the plasmid was extracted to obtain the Cas9-gRNA fusion plasmid pIO-CAS1.0-gpd.

[0145] The GPD donor DNA and Cas9-gRNA fusion plasmid pIO-CAS9-gpd obtained in the above steps were co-transformed into the SA01 strain. Positive transformants were screened using SD-URA medium and further identified using primers GPD-JD-F / R. The transformed strains that were confirmed to be positive were named the SA02 strain (i.e., the SA01 strain with GPD knocked out and LpFRD overexpressed).

[0146] The primer information is shown in the table below.

[0147] Table 6 Primer Information

[0148] Example 5 In this embodiment, an engineered *Pichia kudriaz* yeast was constructed. This engineered *Pichia kudriaz* yeast was based on the SA02 strain, with the pyruvate decarboxylase gene (PDC) knocked out and overexpressed. Zygosaccharomyces rouxii Kudriaz Pichia pastoris modified from the L-malate dehydrogenase gene MDH (ZrMDH).

[0149] The specific construction method is as follows: (1) Selection of malate dehydrogenase (MDH): In this embodiment, fungi derived from *Thiophanate-methyl* (Oval-shaped fungus) were selected. Gongronella butleri The GbMDH (NCBI ID: KAI8059483.1) is derived from... Jimgerdemannia flammicoronaJfMDH (Uniprot ID: A0A433QZX9) is derived from Ramanella spp. ( Umbelopsis ramannian The UrMDH (Uniprot ID: A0AAD5EJ06) originates from... Cokeromyces recurvatus The CrMDH (NCBI ID: XP_051387069.1) is derived from... Zygosaccharomyces rouxii ZrMDH (Uniprot ID: C5DSS7) is derived from Aspergillus oryzae ( Aspergillus oryzae AoMDH (Uniprot ID: Q2U9I9) is derived from Rhizopus oryzae ( Rhizopus rice The RoMDH (Uniprot ID: A0A9P7CB77) originates from... Bifiguratus adelaidea BaMDH (Uniprot ID: A0A261XVJ7) was tested. After codon optimization (for Pichia kudriaz) of these L-malate dehydrogenases from different sources, they were then coupled with the methods described in the above examples. FBA1 promoters and Tgal2 Fragment fusion was performed using terminators, and these recombinant expression vectors were introduced into *S. sarcodactylis* SA02 via homologous recombination (HE) technology for expression. Fermentation was carried out in YPD medium, and high-activity MDH was screened based on the increase in malic acid content after 24 h of fermentation. Transformants obtained from blank plasmids without inserted MDH coding sequences were used as controls.

[0150]

[0151] The results are shown in the table below.

[0152] Table 7. Effects of fermentation with L-malate dehydrogenase from different sources on malic acid content in the system.

[0153] It can be observed that the malic acid content increased significantly after ZrMDH overexpression (within 24 h), indicating that this MDH has higher catalytic activity for oxaloacetic acid.

[0154] (2) Constructing ZrMDH overexpression vectors and transformants containing these expression vectors: Constructing donor DNA fragments for homologous recombination: Genomic DNA was extracted from Pichia pastoris S9 using a commercially available DNA extraction kit. Using this DNA as a template, the upper and lower homologous arms of the PDC (pyruvate decarboxylase gene), the CYC1 promoter, and the CYC1 terminator fragment were amplified using primers PDC-up-F / R, PDC-down-F / R, P-CYC1-F / R, and T-CYC1-F / R, respectively. Using the ZrMDH recombinant expression vector (pUC57-ZrMDH) prepared in step (1) as a template, the optimized ZrMDH fragment was amplified using primer ZrMDH-F / R. Using the pUC19 plasmid as a template, the pUC19 plasmid fragment was amplified using primers pUC19 F and pUC19 R (SEQ ID NO: 1 and 2). The CYC1 promoter, CYC1 terminator fragment, and optimized ZrMDH fragment were fused using primers P-CYC1-F / T-CYC1-R. The target band was recovered by electrophoresis, yielding the CYC1-ZrMDH-CYC1 fusion fragment. Then, the CYC1-ZrMDH-CYC1 fusion fragment was inserted into the pUC19 plasmid fragment using the homologous recombinase C115 (purchased from Novizan), resulting in a ZrMDH overexpression vector. This vector was transformed into DH5α competent cells, and single colonies with normal morphology and size were selected, cultured, and sequenced for verification. Verified transformants were amplified using primers PDC-up-F / PDC-down-R, and recovered by electrophoresis, yielding PDC donor DNA (PDC donor refers to the expression cassette fragment containing the two homologous arms of the PDC site and the MDH to be integrated).

[0155] The primer information is shown in the table below.

[0156] Table 8 Primer Information

[0157] A Cas9-gRNA fusion plasmid, pIO-CAS9-pdc, for editing the PDC gene was constructed. The method was the same as in the previous embodiment, except that the sgRNA sequence targeting PDC was 5'-GAAGGATGAAGCATTCAACA-3' (SEQ ID NO:96). Specifically, pIO-CAS9- WATER Using 3 as a template, amplification was performed using primers PDC-sg-F / R.

[0158] The PDC donor DNA and Cas9-gRNA fusion plasmid pIO-CAS9-pdc obtained in the above steps were co-transformed into the SA02 strain. Positive transformants were screened using SD-URA medium and further identified using primers PDC-JD-F / R. The transformed strains that were confirmed to be positive were named the SA03 strain (i.e., the SA02 strain with PDC knocked out and ZrMDH overexpressed).

[0159] The primer information is shown in the table below.

[0160] Table 9 Primer Information

[0161] Example 6 In this embodiment, an engineered *Pichia kudriaz* was constructed. This engineered *Pichia kudriaz* was developed based on strain SA03, with the lactate dehydrogenase gene (LDH) knocked out and *Pseudococcus feijiensis* overexpressed. Pseudocercospora fijiensis Kudriaz Pichia pastoris was modified from the pyruvate carboxylase gene PYC (PfPYC) derived from ).

[0162] The specific construction method is as follows: (1) Selection of pyruvate carboxylase gene PYC: The specific method is the same as in the above embodiments. The difference is that, in this embodiment, the ingredients are selected from... Exophial mesophiles EmPYC (Uniprot ID: A0A0D1WXX3) is derived from *Pseudococcus fijiensis*. Pseudocercospora Fijian PfPYC (Uniprot ID: M3AR28) is derived from Neohortaea acidophila The NaPYC (Uniprot ID: A0A6A6PL78) is derived from *Brucea buddingis* (…). Aureobasidium sprouting ApPYC (Uniprot ID: A0A074YDM1) is derived from Penicillium rubrum (… Penicillium malachiteum The PmPYC (Uniprot ID: A0AAD6HTJ1) is derived from the chondrion flagellate ( Salpingoeca rosettaThe SrPYC (Uniprot ID: F2UEH3) is derived from... Naegleria gruberi NgPYC (Uniprot ID: D2W2J6) is derived from Aspergillus oryzae ( Aspergillus oryzae AoPYC (Uniprot ID: Q2UGL1) is derived from Toxoplasma gondii (…). Toxoplasma gondii The TgPYC (Uniprot ID: S8F846) of these pyruvate decarboxylases from different sources was tested. After codon optimization (for Pichia kudriaz), these pyruvate decarboxylases were then coupled with the methods described in the above examples. FBA1 promoters and Tgal2 Fragment fusion was performed using terminators, and these recombinant expression vectors were introduced into the serogroup SA03 via homologous recombination (HE) technology to induce expression. Fermentation was carried out in YPD medium, and highly active PYC was screened based on the increase in malic acid content in the system after 24 h of fermentation.

[0163]

[0164] The results are shown in the table below.

[0165] Table 10 Effects of pyruvate carboxylase fermentation from different sources on malic acid content in the system

[0166] It can be observed that the malic acid content increases significantly after PfPYC overexpression (within 24 h), indicating that the PYC has the best catalytic activity.

[0167] (2) Construct the PfPYC overexpression vector and the transformants containing the expression vector: The method for constructing the donor DNA fragment for homologous recombination is the same as in the above examples. The difference lies in the primers used: when using the genomic DNA of *Pichia pastoris* S9 as a template, the primers used for amplification are LDH-up-F / R, LDH-down-F / R, P-SED1-F / R, and T-ION1-F / R, and the amplification products are the upper and lower homologous arms of LDH (lactate dehydrogenase gene), the SED1 promoter, and the ION1 terminator fragment, respectively. When using the PfPYC recombinant expression vector as a template, the primers used for amplification are PfPYC-F / R, and the amplification product is the optimized PfPYC fragment. When using the pUC19 plasmid as a template, the primers used for amplification are pUC19 F / R (SEQ ID NO: 1 and 2), and the amplification product is the pUC19 plasmid fragment. Finally, the donor DNA fragment is amplified using primers LDH-up-F / LDH-down-R.

[0168] The primer information is shown in the table below.

[0169] Table 11 Primer Information

[0170] A Cas9-gRNA fusion plasmid, pIO-CAS9-ldh, for editing the LDH gene was constructed. The method was the same as in the previous embodiment, except that the sgRNA sequence targeting LDH was: 5'-GAAGGCCATCTTTGTCACTG-3' (SEQ ID NO: 97). pIO-CAS9- WATER When using 3 as a template, amplification is performed using primers LDH-sg-F / R.

[0171] The LDH donor DNA and Cas9-gRNA fusion plasmid pIO-CAS9-ldh obtained in the above steps were co-transformed into the SA03 strain. Positive transformants were screened using SD-URA medium and further identified using primers LDH-JD-F / R. The transformed strains that were confirmed to be positive were named the SA04 strain (i.e., the SA03 strain with LDH knocked out and PfPYC overexpressed).

[0172] The primer information is shown in the table below.

[0173] Table 12 Primer Information

[0174] Example 7 In this embodiment, an engineered *Pichia kudriaz* was constructed. This engineered *Pichia kudriaz* was derived from strain SA04 by knocking out the alcohol dehydrogenase gene (ADH1) and overexpressing the original (*Pichia kudriaz*) fumarate gene FUM (PkFUM).

[0175] The specific construction method is as follows: (1) Selection of fumarate gene FUM: The specific method is the same as in the above embodiments. The difference is that, in this embodiment, yeast derived from brewing yeast ( Saccharomyces cerevisiae The fumarate scFUM (NCBI ID: AAA66909.1) is derived from Rhizopus oryzae ( Rhizopus rice The fumarate RoFUM (NCBI number: ADG65260.1) was derived from the original fumarate gene FUM (PkFUM) (Uniprot number: A0A099P3E7) for enzyme activity testing (the enzyme activity assay method is the same as the prior Chinese patent CN 118064415 A).

[0176]

[0177] The results are shown in the table below.

[0178] Table 13 Effects of pyruvate carboxylase fermentation from different sources on succinic acid content in the system

[0179] By calculating the relative intracellular enzyme activity of different overexpression transformants, it can be found that, compared with the control (S9-UB), the relative enzyme activity of the original FUM after overexpression is as high as 451%, indicating that the FUM has the best catalytic activity.

[0180] (2) Constructing the PkFUM overexpression vector and transformants containing the expression vector: The method for constructing the donor DNA fragment for homologous recombination is the same as in the above examples. The difference lies in the primers used: when using the genomic DNA of *Pichia pastoris* S9 as a template, the primers used for amplification are ADH1-up-F / R, ADH1-down-F / R, P-GPM1-F / R, and T-FBA1-F / R, and the amplification products are the upper and lower homologous arms of ADH1, the GPM1 promoter, and the FBA1 terminator fragment, respectively. When using the PkFUM recombinant expression vector as a template, the primers used for amplification are PkFUM-F / R, and the amplification product is the optimized PkFUM fragment. When using the pUC19 plasmid as a template, the primers used for amplification are pUC19 F / R (SEQ ID NO: 1 and 2), and the amplification product is the pUC19 plasmid fragment. Finally, the donor DNA fragment is amplified using primers ADH1-up-F / ADH1-up-R.

[0181] The primer information is shown in the table below.

[0182] Table 14 Primer Information

[0183] A Cas9-gRNA fusion plasmid, pIO-CAS9-adh1, for editing the ADH1 gene was constructed. The method was the same as in the previous embodiment, except that the sgRNA sequence targeting ADH1 was: 5'-TGTGTCTTTTACGAAAACGG-3' (SEQ ID NO:98). Specifically, pIO-CAS9- WATER Using 3 as a template, amplification was performed using primers ADH1-sg-F / R.

[0184] The ADH1 donor DNA and Cas9-gRNA fusion plasmid pIO-CAS9-adh1 obtained in the above steps were co-transformed into the SA04 strain. Positive transformants were screened using SD-URA medium and further identified using primers ADH1-JD-F / R. The transformed strains that were confirmed to be positive were named the SA05 strain (i.e., the SA04 strain with ADH1 knocked out and PkFUM overexpressed).

[0185] The primer information is shown in the table below.

[0186] Table 15 Primer Information

[0187] Example 8 In this embodiment, an engineered *Pichia kudriaz* was constructed. This engineered *Pichia kudriaz* was developed based on strain SA05, through 2-copy overexpression of fumarate and fumarate reductase, specifically by simultaneously integrating PkFUM and LpFRD into site II-12 of the *S. kudriaz* genome. The genomic integration site is site II-12 of the *Pichia kudriaz* genome, with homologous arms consisting of 1000 bp genomic DNA fragments on either side. Specific information is as follows: The upstream homologous arm sequence of II-12 is as follows:

[0188] The downstream homologous arm sequence of II-12 is as follows:

[0189] The specific construction method is as follows: (1) Construction of donor DNA fragments for homologous recombination: The method is the same as in the above examples. The difference is that when using the genomic DNA of Pichia pastoris S9 as a template, the primers used for amplification are II12-up-F / R and II12-down-F / R, and the amplification product is the upper and lower homologous arms of the II12 site. Lyse the SA02 and SA05 strains in the above examples respectively. Amplify the SA05 strain genome (using the Tiangen Genome Extraction Kit) as a template, using the primers pPGMP-II12-F / tFBA1-TEF1-R, and the amplification product is a PkFUM fragment with a promoter and a terminator. Amplify the SA02 strain lysate as a template, using the primers tTEF1-tFBA1-F / pFBA1-II12-R, and the amplification product is an LpFRD fragment with a promoter and a terminator. When using pUC19 plasmid as a template, the amplification primers are pUC19 F / R (SEQ ID NO: 1 and 2), and the amplification product is the pUC19 plasmid fragment. Finally, the donor DNA fragment is amplified using primers II12-up-F / II12-down-R.

[0190] The primer information is shown in the table below.

[0191] Table 16 Primer Information

[0192] A Cas9-gRNA fusion plasmid, pIO-CAS9-II12, for editing the II12 gene was constructed. The method was the same as in the previous embodiment, except that the sgRNA sequence targeting II12 was: 5'-AGCTGTGTAGCCACGTTTAC-3' (SEQ ID NO:99). Specifically, pIO-CAS9- WATER Using 3 as a template, amplification was performed using primers II12-sg-F / R.

[0193] The II12 donor DNA and Cas9-gRNA fusion plasmid pIO-CAS9-II12 obtained in the above steps were co-transformed into the SA05 strain. Positive transformants were screened using SD-URA medium and further identified using primers II12-JD-F / R. The transformed strain that was confirmed to be positive was named the SA06 strain (i.e., the SA05 strain that overexpresses 2 copies of fumarate and fumarate reductase).

[0194] The primer information is shown in the table below.

[0195] Table 17 Primer Information

[0196] Test Example 1 In this test example, the SA01-SA06 strains prepared in the above examples were used to conduct shake flask fermentation and 5 L fermenter fermentation experiments, respectively, to detect the actual fermentation effect of each strain.

[0197] The steps for the shake-flask fermentation experiment are as follows: After activating strains SA01-SA06 on YPD solid medium, single colonies were picked and inoculated into YPD liquid medium (YPD solid medium without agar) at a rate of 1 single colony / 10 mL. The culture was incubated at 30℃ and 200 rpm for 15 h to obtain the seed culture. The seed culture was adjusted to OD600 = 0.1 and then inoculated into YPD liquid medium (containing 50 g / L glucose) at a rate of 10 mL / 50 mL. The culture was then incubated at 30℃ and 200 rpm for 48 h. After incubation, 1 mL of fermentation broth was taken and the OD600 value was measured. Then, the contents of glycerol, ethanol, acetic acid, lactic acid, and succinic acid in the remaining fermentation broth were determined by HPLC to calculate the yield and conversion rate of succinic acid. S9-UB was used as a control.

[0198] The chromatographic column was a Bio-Rad Aminex HPX-87H (300 × 7.8 mm), and the liquid chromatography detection method was as follows: The autosampler was set to autosample with an injection volume of 10 μL, using 2.5 mM H2SO4 as the mobile phase, maintaining a flow rate of 0.6 mL / min, and keeping the column oven temperature at 60℃ for 25 min. The detector operating temperature was 40℃, and the sensitivity was 32 mL / min.

[0199] Standard curve establishment method: The mixed standard sample stock solution (containing 10 g / L glucose, malic acid, succinic acid, lactic acid, glycerol, acetic acid and ethanol) was serially diluted to 10 g / L, 8 g / L, 5 g / L, 2.5 g / L, 1.25 g / L, 0.5 g / L and 0.1 g / L respectively to establish the standard curve.

[0200] Take the fermentation broth, centrifuge at 12000 rpm for 2 min, collect the supernatant, filter it through a 0.22 μm aqueous filter membrane to remove impurities, and then perform sample analysis and detection according to the above-mentioned program. Calculate the contents of glucose, glycerol, ethanol, acetic acid, lactic acid, and succinic acid in the sample based on the recorded target peak area and standard curve. The fermentation experiment steps for a 5 L fermenter are as follows: After activating the test strain, a single colony was picked and inoculated into 50 mL of YPD liquid medium. The culture was carried out at 30°C and 200 rpm for 16 h to obtain seed culture 1. 20 mL of seed culture 1 was inoculated into 100 mL of YPD liquid medium containing 50 g / L glucose and cultured at 30°C and 200 rpm for 8 h to obtain seed culture 2. The OD600 of seed culture 2 was adjusted to 0.5, and then it was inoculated into a 5 L fermenter (containing YPD liquid medium containing 100 g / L glucose). Fermentation was carried out at 30°C, 0.4–0.5 L / min aeration, and 330 rpm for 60 h. During fermentation, the pH was maintained at 2.8 using a 20 wt% NaOH solution.

[0201] After fermentation, the succinic acid content was determined by HPLC, and the yield, conversion rate and production intensity of succinic acid were calculated.

[0202] The results are shown in the table below.

[0203] Table 18. Products of each strain after 48 h of conventional fermentation

[0204] The conversion rate refers to the glucose-acid conversion rate, which is the amount of succinic acid produced (g) / the amount of glucose consumed (g).

[0205] Table 19. Products of SA06 strain after 60 h of fermentation in a fermenter

[0206] The results above show that, based on the engineering modifications of this invention, the succinic acid yield of each engineered strain was significantly improved compared to S9-UB, and the yield and conversion rate were positively correlated based on the intensity of the modification.

[0207] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An engineered Pichia pastoris, characterized in that, The engineered Pichia kudriazia yeast is URA3 Genetically defective Pichia pastoris S9; Among them, the URA3 The dicarboxylic acid transporter gene was knocked out in the genetically defective Pichia pastoris S9. JEN2-2 3-Glycerol dehydrogenase ( GPD ) gene, pyruvate decarboxylase ( PDC ) gene, lactate dehydrogenase ( LDH ) gene and alcohol dehydrogenase ( ADH1 Genes, and express succinate transporter gene. SpMAE L-malate dehydrogenase gene ZrMDH and pyruvate carboxylase gene PfPYC Overexpression of fumarate reductase gene LpFRD and fumarate gene PkFUM ; Among them, sgRNA is used to target and knock out the corresponding gene, and the corresponding gene is expressed or overexpressed by introducing the coding sequence of the corresponding gene; Targeting the dicarboxylic acid transporter gene JEN2-2 The sgRNA is shown in SEQ ID NO:94; The succinic acid transporter gene SpMAE The encoded sequence is shown in SEQ ID NO: 13; Targeting the 3-phosphoglycerate dehydrogenase ( GPD The sgRNA of the gene is shown in SEQ ID NO:95; The fumarate reductase gene LpFRD The encoded sequence is shown in SEQ ID NO: 18; Targeting the pyruvate decarboxylase ( PDC The sgRNA of the gene is shown in SEQ ID NO:96; The L-malate dehydrogenase gene ZrMDH The encoded sequence is shown in SEQ ID NO: 33; Targeting the lactate dehydrogenase ( LDH The sgRNA of the gene is shown in SEQ ID NO:97; The pyruvate carboxylase gene PfPYC The encoded sequence is shown in SEQ ID NO: 48; Targeting the alcohol dehydrogenase ( ADH1 The sgRNA of the gene is shown in SEQ ID NO:98; The fumarate gene PkFUM The encoded sequence is shown in SEQ ID NO:

63.

2. The engineered Pichia pastoris according to claim 1, characterized in that, The overexpression includes overexpression through at least one of the following methods: Use a strong promoter or enhance promoter activity, increase gene copy number, or use at least one of the following inducible expression systems; Insertion sites for overexpressed genes include: II12.

3. A composition, characterized in that, The composition comprises: (1) The engineered Pichia kudriaz or its culture as described in any one of claims 1-2; (2) Proteins isolated from (1); The engineered Kudriaz Pichia pastoris includes at least one of engineered live Kudriaz Pichia pastoris and engineered inactivated Kudriaz Pichia pastoris. The cultures include engineered Pichia kudriaz yeast culture, metabolites, ferments, or their supernatants, inactivated products, concentrates, or dried products.

4. The use of the engineered Pichia kudriaz according to any one of claims 1-2 and / or the composition according to claim 3 in the biosynthesis of organic compounds; in, The organic compound includes: succinic acid; The substrates for the biosynthesis include glucose; The glucose concentration is greater than or equal to 50 g / L.

5. The application according to claim 4, characterized in that, The pH of a system for biosynthesizing organic matter is less than 3.

6. The application according to claim 4, characterized in that, It does not produce glycerol, ethanol, acetic acid, or lactic acid during the biosynthesis of organic matter.

7. The application according to claim 4, characterized in that, The malic acid content in the synthesized product is less than or equal to 1 g / L.

8. The application according to claim 4, characterized in that, The malic acid content in the synthesized product is less than or equal to 0.5 g / L.

9. A method for preparing succinic acid, comprising the following steps: The engineered Pichia kudriaz as described in any one of claims 1-2 and / or the composition as described in claim 3 are fermented on a sugar-containing yeast culture medium to obtain the product. The sugar content in the yeast culture medium is greater than or equal to 50 g / L.

10. The preparation method according to claim 9, characterized in that, The preparation method further includes: The fermentation broth obtained from fermentation was separated, extracted, and purified to obtain purified succinic acid.

11. The preparation method according to claim 9, characterized in that, The sugar-containing yeast culture medium includes at least one of the following: YPD or YEPD medium, potato dextrose agar (PDA) medium, synthetic glucose (SD) basal medium, and corn steep liquor / starch hydrolysate medium.

12. The preparation method according to any one of claims 9-11, characterized in that, The sugar includes at least one of monosaccharides, disaccharides, oligosaccharides, and polysaccharides; The engineered Pichia kudriaz as described in any one of claims 1-2 and / or the composition as described in claim 3 are capable of producing succinic acid using the sugar as a substrate.

13. The preparation method according to claim 12, characterized in that, The sugars include glucose.

14. The preparation method according to claim 9, characterized in that, Fermentation time is 24-100 h.

15. The preparation method according to claim 14, characterized in that, The fermentation time is 24-80 h.

Images