A method for constructing a reductive tricarboxylic acid cycle in vivo in pichia kudriavzevii and applications thereof

Abstract

The application discloses a method for constructing a reductive tricarboxylic acid cycle (rTCA cycle) in vivo of Pichia kudriavzevii and application. Zygosaccharomyces rouxii The application integrates pyruvate carboxylase (ZrPYC) from Zygosaccharomyces rouxii Ustilago trichophora and malate dehydrogenase (UtMDH) from Ustilago tritici into the genome of Pichia kudriavzevii by metabolic engineering, constructs a recombinant strain with a high-efficiency rTCA cycle, and applies the recombinant strain to the fermentation production of L-malic acid. Compared with a control strain for constructing the rTCA cycle in vivo of Pichia kudriavzevii by using other conventional enzyme sources, the yield of L-malic acid of the recombinant strain provided by the application is increased by more than 30%. The application widens the selection range of enzyme sources for constructing the rTCA cycle, provides a new technical scheme for the biological manufacturing of C4 dicarboxylic acid, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of metabolic engineering and microbial fermentation technology, specifically relating to a method for constructing a reduced tricarboxylic acid cycle (rTCA cycle) pathway in Pichia kudricazvichia, and the application of this method in the production of L-malic acid. Background Technology

[0002] The reductive tricarboxylic acid cycle (rTCA cycle) is the reverse metabolic pathway of the tricarboxylic acid cycle (TCA cycle). In the classic TCA cycle, acetyl-CoA condenses with oxaloacetate to form citrate, which undergoes a series of oxidative decarboxylation reactions to ultimately produce CO2, generating a large amount of reducing power (NADH) and energy (ATP). The rTCA cycle, however, utilizes NADH and ATP to fix CO2, synthesizing C4 dicarboxylic acids by reversing key steps of the TCA cycle. Specifically, the rTCA cycle begins with pyruvate carboxylase (PYC) catalyzing the fixation of CO2 from pyruvate to oxaloacetate, which is then reduced to L-malate by malate dehydrogenase (MDH). L-malate can then be further catalyzed by other enzymes to produce fumarate or succinate. The catalytic efficiency of the two key enzymes, PYC and MDH, directly affects the overall flux of the rTCA cycle. Therefore, the rTCA cycle is not only an ideal pathway for synthesizing C4 dicarboxylic acids but also a crucial strategy for achieving CO2 biofixation.

[0003] Kudria zwibichi yeast ( Pichia kudriavzevii This is a non-model yeast strain with extremely strong acid resistance, capable of growing normally at pH 1.5, demonstrating its potential as an excellent chassis cell for organic acid fermentation. Previous studies have reported the overexpression of pyruvate carboxylase and malate dehydrogenase in *Pichia kudrica* to construct the rTCA pathway for C4 dicarboxylic acid production, but the PYC and MDH introduced in current studies are still mainly derived from *Aspergillus oryzae*. Aspergillus oryzae ) or Aspergillus flavus ( Aspergillus flavus The selection of enzyme sources is relatively limited, lacking enzyme sources from specific ecological niches or with unique catalytic properties. Therefore, the discovery and screening of diverse sources, especially novel high-activity PYC and MDH enzyme sources suitable for the *Pichia goudryana* expression system, is of great significance for expanding the enzyme toolbox of the rTCA cycle and constructing an efficient rTCA cycle to increase the yield of C4 dicarboxylic acids. This invention is the first to utilize enzyme sources derived from *Zygosacchariformis rouxeri* (…). Zygosaccharomyces rouxii PYC and derived from Ustilago maydis ( Ustilago trichophora The MDH combination was applied to the construction of the rTCA cycle in Pichia kudrica, providing a new enzyme source and combination for the biosynthesis of C4 dicarboxylic acids. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies in constructing the reductive tricarboxylic acid cycle (rTCA cycle) in *Pichia kudriezvichi*, which suffers from limited enzyme source selection and insufficient catalytic efficiency. To this end, this invention provides a set of pyruvate carboxylases (ZrPYC) derived from *Zygosaccharidus rouxii* and malate dehydrogenases (UtMDH) derived from *Ustilago maydis*, and a method for constructing an efficient rTCA cycle by co-expressing these two enzymes in *Pichia kudriezvichi*. Furthermore, this invention also provides genes, recombinant strains, and their applications in L-malic acid production for the above method.

[0005] This invention starts with an acid-tolerant yeast strain, *Pichia kudrica*, purchased from the China Industrial Microbial Culture Collection Center (CICC), with accession number CICC 33427. Through genetic engineering, the strain is made to express ZrPYC and UtMDH, and an rTCA cycle pathway is constructed for the fermentation production of L-malic acid.

[0006] In a first aspect, the present invention provides a method for constructing a reduced tricarboxylic acid cycle in Pichia pastoris, comprising the following steps: The gene encoding pyruvate carboxylase or a conserved mutant of *Zygomyces rouxii* was integrated into the genome of *Pichia kudrica*. The gene encoding malate dehydrogenase derived from Ustilago maydis or a conserved mutant thereof was integrated into the genome of Pichia kudrica. Genes were expressed in yeast to obtain recombinant strains with the reduced tricarboxylic acid cycle pathway.

[0007] Furthermore, the amino acid sequence of the pyruvate carboxylase derived from *Zygosaccharidus rouxii* is shown in SEQ ID NO:1; and the amino acid sequence of the malate dehydrogenase derived from *Ustilago maydis* is shown in SEQ ID NO:2.

[0008] Furthermore, the conserved mutant of pyruvate carboxylase derived from *Zygosaccharidus rouxii* satisfies the following characteristics: Its amino acid sequence has at least 99% sequence identity with SEQ ID NO:1; The amino acid difference between it and SEQ ID NO:1 is the conservative substitution of 1 to 12 amino acid residues, and the difference only occurs in the non-active center region; After integrating the gene encoding the mutant and the gene encoding malate dehydrogenase SEQ ID NO:2 or its conserved mutant into the genome of *Kudriazwibichthys*, the resulting recombinant strain produced L-malic acid at a yield not less than 80% of that of the control strain obtained after integrating the gene encoding the enzyme shown in SEQ ID NO:1 and the gene encoding the enzyme shown in SEQ ID NO:2 into the genome of *Kudriazwibichthys*.

[0009] Furthermore, the conserved mutant of malate dehydrogenase derived from Ustilago maydis satisfies the following characteristics: Its amino acid sequence has at least 99% sequence identity with SEQ ID NO:2; The amino acid difference between it and SEQ ID NO:2 is a conservative substitution of 1 to 4 amino acid residues, and the difference only occurs in the non-active center region; After integrating the gene encoding the mutant and the gene encoding pyruvate carboxylase SEQ ID NO:1 or its conserved mutant into the genome of *Kudriazwibichthys*, the resulting recombinant strain has an L-malic acid yield of not less than 80% of that of the control strain obtained after integrating the gene encoding the enzyme shown in SEQ ID NO:1 and the gene encoding the enzyme shown in SEQ ID NO:2 into the genome of *Kudriazwibichthys*.

[0010] Furthermore, the gene encoding pyruvate carboxylase and the gene encoding malate dehydrogenase are operatively linked to a promoter and a terminator, respectively, and then introduced into the yeast; the promoter is a constitutive strong promoter, and the terminator is a strong terminator.

[0011] Preferably, the promoter is a PGPD promoter and the terminator is a TGAL terminator.

[0012] Furthermore, the gene encoding pyruvate carboxylase and the gene encoding malate dehydrogenase are integrated into non-essential gene sites in the yeast genome.

[0013] Preferably, the gene encoding pyruvate carboxylase is integrated into the genome of *Pichia goudryanazvi*. PkPDC1 At the site; the gene encoding malate dehydrogenase was integrated into the genome of *Pichia gondii*. PkGPD1 In the locus.

[0014] Secondly, the present invention provides genes encoding pyruvate carboxylase derived from Zygomyces rouxii and malate dehydrogenase derived from Ustilago maydis.

[0015] Furthermore, the nucleotide sequence of the gene encoding pyruvate carboxylase is shown in SEQ ID NO:3; the nucleotide sequence of the gene encoding malate dehydrogenase is shown in SEQ ID NO:4.

[0016] Furthermore, the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 are codon-optimized sequences for *Pichia kudria*.

[0017] Thirdly, the present invention provides a recombinant strain of *Kudelazvibichisis*.

[0018] Furthermore, the recombinant strain is constructed by the method described in the first aspect, or contains the genes described in the second aspect.

[0019] Furthermore, the genome of the recombinant strain integrates the gene encoding pyruvate carboxylase and the gene encoding malate dehydrogenase as described in the second aspect.

[0020] Fourthly, the present invention provides the application of the method described in the first aspect, the gene described in the second aspect, or the recombinant strain described in the third aspect in the production of L-malic acid.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) Expanding the range of enzyme sources. Existing technologies for constructing the rTCA cycle in *Pichia kudria* primarily utilize pyruvate carboxylase and malate dehydrogenase derived from conventional strains such as *Aspergillus oryzae*, *Aspergillus flavus*, *Rhizopus oryzae*, or *Saccharomyces cerevisiae*. There are no reports of co-expressing pyruvate carboxylase from *Zygosacchariformis* and malate dehydrogenase from *Ustilago maydis* in *Pichia kudria* to construct the rTCA cycle. This invention is the first to apply the combination of ZrPYC and UtMDH to the construction of the rTCA cycle in *Pichia kudria*, providing a new enzyme source and its combination for the biosynthesis of C4 dicarboxylic acids.

[0022] (2) Significantly increased L-malic acid production. ZrPYC and UtMDH were co-expressed in Pichia kudrica to construct an rTCA cycle. Compared with the control strain using conventional enzyme sources, the recombinant strain obtained could increase L-malic acid production by more than 30% in the same fermentation cycle, showing a higher overall flux of the rTCA cycle and good prospects for industrial application. Attached Figure Description

[0023] Figure 1 The L-malic acid yield and cell density (OD) of the Pk004 series recombinant strains in Example 5. 600 Comparison chart.

[0024] Figure 2 The L-malic acid yield and cell density (OD) of the Pk006 series recombinant strains in Example 6. 600 Comparison chart. Detailed Implementation

[0025] To make the technical solution and beneficial effects of the present invention clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All equivalent transformations or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0026] The bacterial strains and plasmids involved in the experiments of this invention are as follows: Kudria zwibichi yeast ( Pichia kudriavzevii CICC 33427: The chassis cells used in the construction of rTCA cycles and the production of L-malic acid in this invention were purchased from the China Industrial Microbial Culture Collection Center (CICC), with accession number CICC 33427.

[0027] Escherichia coli ( Escherichia coli DH5α: Used as a gene cloning host for the construction and amplification of plasmids in this invention, purchased from Beijing Qingke Biotechnology Co., Ltd.

[0028] pBKcas9 plasmid: A tool plasmid for CRISPR / Cas9 gene editing, used in *Pichia pastoris* for gene editing. This plasmid is based on published literature. [1] The pVT36b plasmid map reported in the previous article was constructed as follows: A DNA fragment containing the Cas9 gene expression cassette, sgRNA transcription cassette, yeast replicon, and URA3 selection marker was obtained through gene synthesis. A DNA fragment containing the E. coli replicon and ampicillin resistance selection marker was amplified by PCR from the pUC57 plasmid (a commercially available universal plasmid) using primers pUC57-Amp-ori-S (5'-catgtgagcaaaaggccagc-3') and pUC57-Amp-ori-A (5'-gtggcacttttcggggaaat-3'). After purification of the synthesized and PCR-amplified fragments, recombination ligation was performed according to the instructions of the seamless cloning kit, and the reaction product was transformed. E. coli DH5α competent cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. Positive clones were picked, plasmids were extracted, and after sequence confirmation, the correct plasmid was named pBKcas9.

[0029] The above lists the basic bacterial strains and basic plasmids used in this invention. The construction methods for the remaining derived plasmids and recombinant strains are described in specific embodiments. It should be noted that the construction methods provided in the embodiments of this invention are exemplary and not restrictive. Those skilled in the art can also use any other technical means to achieve the final gene editing purpose. Other raw materials, reagents, consumables, and instruments involved in this invention, unless otherwise specified, can be purchased commercially.

[0030] The present invention will be further explained and illustrated below through specific embodiments.

[0031] Example 1: Construction of a uracil auxotrophic yeast strain This embodiment aims to knock out Pichia pastoris CICC 33427 using CRISPR / Cas9 technology. PkURA3 Genes were used to construct uracil auxotrophic strains. PkURA3 The gene encodes orotate nucleoside-5'-phosphate decarboxylase (EC4.1.1.23), an enzyme that converts 5-fluoroorotate (5-FOA) into a toxic substance that leads to cell death. Therefore, successful knockout of this enzyme was achieved. PkURA3 The gene-producing strains can be grown on plates containing 5-FOA, enabling positive selection. The constructed uracil auxotrophic strains can be used to screen positive transformants for auxotrophic complementation using the URA3 marker carried on the pBKcas9 plasmid, and can be used as host strains for subsequent gene editing experiments.

[0032] 1.1 Construction of gene editing plasmid pBKcas9-PkURA3 Template-free PCR was performed using PkURA3-UF / UR and PkURA3-DF / DR primers (see Table 1). The primers extended by annealing to their complementary 3' ends, yielding primers containing the target... PkURA3 The sgRNA prototype spacer sequence of the gene and PkURA3 Homologous arms of 50 bp each upstream and downstream of the gene were obtained. The PCR product was precipitated with anhydrous ethanol, appropriately diluted, and then subjected to overlap extension PCR. The resulting fusion fragment was recovered from the gel and then assembled with the pBKcas9 plasmid using Golden Gate PCR (following the NEBridge® Golden Gate Assembly Kit (BsaI-HF® v2) instructions). The reaction product was transformed... E. coli DH5α competent cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C (approximately 14 hours). Single colonies were picked and colony PCR was performed using the BKURA3-CES / CEA primers (see Table 1). Positive clones were sent for sequencing confirmation, and the correct plasmid was named pBKcas9-PkURA3.

[0033] 1.2 Yeast transformation and screening of positive transformants Single colonies of *Pichia pastoris* CICC 33427 were picked and inoculated into YPD liquid medium, cultured at 30°C with shaking until the logarithmic growth phase. The cells were collected by centrifugation, washed with sterile water, and resuspended in 100 mM lithium acetate solution to obtain competent cells. The pBKcas9-PkURA3 plasmid was transformed into the competent cells using the lithium acetate transformation method. The transformed cells were plated on YPD plates containing 1 mg / mL 5-FOA and cultured at 30°C for 2-3 days. PkURA3 Only strains whose genes have been successfully knocked out can grow on plates containing 5-FOA.

[0034] 1.3 Validation of positive transformants Single colonies grown from transformation plates were picked and verified by PCR using PkURA3-CES / CEA primers (see Table 1). The PCR reaction solution was then sent for sequencing confirmation. PkURA3 The gene has been successfully knocked out. The verified positive transformant has been named Pk001 (genotype: CICC 33427, Δ). PkURA3 ).

[0035] Table 1. Primers used in Example 1 Example 2: Construction of a yeast strain overexpressing the C4 dicarboxylic acid transporter SpMAE1 To enhance the efflux efficiency of L-malic acid in *Pichia kudrica*, this embodiment first integrated the code for *Schizosaccharomyces kudrica* into its genome before constructing a strain expressing pyruvate carboxylase (PYC) and malate dehydrogenase (MDH). Schizosaccharomyces pombe The gene for SpMAE1, a C4 dicarboxylic acid transporter, is used. SpMAE1 can transport intracellularly synthesized L-malic acid to the extracellular space, thereby reducing the concentration of intracellular products and mitigating feedback inhibition. This allows the differences in L-malic acid production caused by enzyme activity differences among recombinant strains expressing various PYC and MDH to be fully amplified in terms of extracellular accumulation, facilitating comparison and screening in subsequent fermentation experiments.

[0036] 2.1 Construction of gene editing plasmid pBKcas9-PkMAE1 Using pBKcas9 plasmid as a template, primers BKPkMAE1-S1 / A1 and BKPkMAE1-S2 / A2 (see Table 2) were used to amplify plasmids containing pBKcas9. PkMAE1 A plasmid backbone fragment containing the gene-specific sgRNA prototype spacer sequence. The PCR product was validated by agarose gel electrophoresis, purified, and ligated using a seamless cloning kit before transformation. E. coliDH5α competent cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C (approximately 14 hours). Single colonies were picked and colony PCR was performed using the BKPkMAE1-CES / CEA primers (see Table 2). Positive clones were sent for sequencing confirmation, and the correct plasmid was named pBKcas9-PkMAE1.

[0037] 2.2 Construction of donor fragment PkMAE1-SpMAE1 Based on the amino acid sequence of the Schizosaccharide C4 dicarboxylic acid transporter SpMAE1 from the UniProt database (SEQ ID NO:5, UniProt database search number: P50537), codon optimization was performed to adapt it to the Pichia kudriezvichia expression system. The optimized nucleotide sequence is shown in SEQ ID NO:6 and was synthesized by a commercial gene synthesis service provider. Using the genomic DNA of Pichia kudriezvichia CICC 33427 as a template, primers PkMAE1-UF / UR and PkMAE1-DF / DR were used to amplify the protein. PkMAE1 Upstream and downstream homologous arm fragments at the site; the PGPD promoter fragment was amplified using PGPD-SpMAE1-S / A primers (see Table 2); the TGAL terminator fragment was amplified using TGAL-SpMAE1-S / A primers (see Table 2). PkMAE1 upstream homologous arm fragment, PGPD promoter fragment, and synthesized SpMAE1 Gene fragments, TGAL terminator fragments and PkMAE1 Downstream homologous arm fragments were sequentially ligated via overlap extension PCR to complete the construction of the donor fragment PkMAE1-SpMAE1.

[0038] 2.3 Yeast transformation and screening of positive transformants Competent cells of *Pichia pastoris* strain Pk001 were prepared by co-transforming the pBKcas9-PkMAE1 plasmid and the PkMAE1-SpMAE1 donor fragment into competent cells using the lithium acetate transformation method. The transformed cells were then plated on SD-URA plates and cultured at 30°C for 3-4 days. The pBKcas9-PkMAE1 plasmid carries the URA3 selection marker. Pk001 is a uracil-deficient strain; only transformants successfully transformed with the pBKcas9-PkMAE1 plasmid could grow on uracil-deficient SD-URA plates.

[0039] 2.4 Validation of positive transformants Single colonies grown on SD-URA plates were picked and PCR was performed using PkMAE1-CES / CEA primers (see Table 2) for verification. Successful PCR was achieved. PkMAE1 Site integration SpMAE1The transformants of the gene expression cassette should amplify a 4670 bp band, while the Pk001 control strain amplified a 2225 bp band. The PCR reaction solution was sent for sequencing to further confirm this. SpMAE1 The gene expression cassette has been correctly integrated. The verified positive transformant has been named Pk002 (genotype: Pk001). PkMAE1∷PGPD-SpMAE1- TGAL ).

[0040] Table 2. Primers used in Example 2 Example 3: Construction of a yeast strain overexpressing pyruvate carboxylase PYC PYC is the first key enzyme in the rTCA cycle pathway, catalyzing the fixation of pyruvate into CO2 to oxaloacetate. In this embodiment, based on the Pk002 strain, the gene encoding the pyruvate carboxylase ZrPYC from *Zygosaccharidus rouxeris* was integrated into the yeast genome. PkPDC1 The site is expressed by the constitutive strong promoter PGPD, with TGAL as the terminator. Simultaneously, knockout... PkPDC1 The gene can block the metabolic pathway of pyruvate decarboxylation to ethanol, causing more carbon source and NADH to be diverted to the rTCA cycle. In addition, this embodiment also constructed a control strain overexpressing AoPYC from Aspergillus oryzae, which will be used to construct a complete control rTCA cycle in subsequent combination with AoMDH.

[0041] 3.1 Construction of gene editing plasmid pBKcas9-PkPDC1 Using pBKcas9 plasmid as a template, primers BKPkPDC1-S1 / A1 and BKPkPDC1-S2 / A2 (see Table 3) were used to amplify plasmids containing pBKcas9. PkPDC1 A plasmid backbone fragment containing the gene-specific sgRNA prototype spacer sequence. The PCR product was validated by agarose gel electrophoresis, purified, and ligated using a seamless cloning kit before transformation. E. coli DH5α competent cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C (approximately 14 hours). Single colonies were picked and colony PCR was performed using the BKPkPDC1-CES / CEA primers (see Table 3). Positive clones were sent for sequencing confirmation, and the correct plasmid was named pBKcas9-PkPDC1.

[0042] 3.2 Construction of donor fragments PkPDC1-ZrPYC, PkPDC1-AoPYC, and PkPDC-HA The pyruvate carboxylase ZrPYC, derived from *Zygosaccharidus rouxiensis*, has the amino acid sequence shown in SEQ ID NO:1 (UniProt database search number: A0A1Q2ZX57). Codon optimization was performed to adapt it to the *Pichia kudriezvichi* expression system, and the optimized nucleotide sequence is shown in SEQ ID NO:3. It was synthesized by a commercial gene synthesis service provider. Using *Pichia kudriezvichi* CICC 33427 genomic DNA as a template, primers PkPDC1-UF / UR and PkPDC1-DF / DR (see Table 3) were used to amplify the enzyme. PkPDC1 Upstream and downstream homologous arm fragments were identified at the locus; the PGPD-ZrPYC promoter fragment was amplified using PGPD-ZrPYC-S / A primers (see Table 3); the TGAL-ZrPYC terminator fragment was amplified using TGAL-ZrPYC-S / A primers (see Table 3). PkPDC1 upstream homologous arm fragment, PGPD-ZrPYC promoter fragment, and synthesized ZrPYC Gene fragments, TGAL-ZrPYC terminator fragments and PkPDC1 Downstream homologous arm fragments were sequentially ligated via overlap extension PCR to complete the construction of the donor fragment PkPDC1-ZrPYC.

[0043] The pyruvate carboxylase AoPYC, derived from *Aspergillus oryzae*, has the amino acid sequence shown in SEQ ID NO:7 (UniProt database search number: Q2UGL1). Codon optimization was performed to adapt it for the *Pichia kudriezvichi* expression system, and the optimized nucleotide sequence is shown in SEQ ID NO:8. It was synthesized by a commercial gene synthesis service provider. Using *Pichia kudriezvichi* CICC 33427 genomic DNA as a template, the PGPD-AoPYC promoter fragment was amplified using the PGPD-AoPYC-S / A primers (see Table 3); the TGAL-AoPYC terminator fragment was amplified using the TGAL-AoPYC-S / A primers (see Table 3). PkPDC1 upstream homologous arm fragment, PGPD-AoPYC promoter fragment, synthesized AoPYC Gene fragments, TGAL-AoPYC terminator fragments and PkPDC1 Downstream homologous arm fragments were sequentially ligated via overlap extension PCR to complete the construction of the donor fragment PkPDC1-AoPYC.

[0044] In addition, using Pichia kudrica CICC 33427 genomic DNA as a template, primers PkPDC1-UF / URa and PkPDC1-DFa / DR were used to amplify the DNA. PkPDC1 The upstream and downstream homologous arm fragments of the site were used to construct the donor fragment PkPDC-HA by overlapping extension PCR.

[0045] 3.3 Yeast transformation and screening, validation and nomenclature of positive transformants Competent cells of *Pichia pastoris* strain Pk002 were prepared. The pBKcas9-PkPDC1 plasmid and the PkPDC1-ZrPYC donor fragment were co-transformed into the competent cells using the lithium acetate transformation method. The cells were then plated on SD-URA plates and incubated at 30°C for 3-4 days. Single colonies grown on the SD-URA plates were picked and PCR was performed using PkPDC1-CES / CEA primers (see Table 3) for verification. Successful PCR was confirmed. PkPDC1 Site integration ZrPYC The transformants of the gene expression cassette should amplify a 7453 bp band, while the Pk002 control strain amplified a 2748 bp band. The PCR reaction solution was sent for sequencing to further confirm that the target gene expression cassette had been correctly integrated. The correctly validated positive transformant was named: Pk003-ZrPYC (genotype: Pk002). PkPDC1∷PGPD- ZrPYC-TGAL ).

[0046] The pBKcas9-PkPDC1 plasmid and the PkPDC1-AoPYC donor fragment were transformed into strain Pk002, and successful strains were obtained by screening using the same method described above. PkPDC1 Site integration AoPYC The positive transformant of the gene expression cassette was named Pk003-AoPYC (genotype: Pk002, PkPDC1∷PGPD-AoPYC-TGAL ).

[0047] The pBKcas9-PkPDC1 plasmid and the PkPDC1-HA donor fragment were transformed into strain Pk002, and successful knockouts were obtained by screening using the same method described above. PkPDC1 The positive transformant of the gene was named Pk003 (genotype: Pk002, Δ). PkPDC1 ).

[0048] Table 3. Primers used in Example 3 Example 4: Construction of a yeast strain overexpressing malate dehydrogenase (MDH) MDH is the second key enzyme in the rTCA cycle, catalyzing the reduction of oxaloacetate to L-malate. In this example, based on the Pk003-ZrPYC strain, the gene encoding the malate dehydrogenase UtMDH from *Ustilago maydis* was integrated into the yeast genome. PkGPD1 The site is driven by the constitutive strong promoter PGPD, terminated by TGAL, and together with ZrPYC, constitutes the complete rTCA cycle pathway. Simultaneously, knockout... PkGPD1The gene can block the glycerol synthesis pathway, causing further diversion of carbon sources and NADH to the rTCA cycle. In addition, this embodiment also constructed a control strain overexpressing AoMDH from Aspergillus oryzae, which, combined with AoPYC, forms a complete control rTCA cycle for subsequent fermentation experiments to compare the effects of different enzyme source combinations on L-malic acid production.

[0049] 4.1 Construction of gene editing plasmid pBKcas9-PkGPD1 Using pBKcas9 plasmid as a template, primers BKPkGPD1-S1 / A1 and BKPkGPD1-S2 / A2 (see Table 4) were used to amplify plasmids containing pBKcas9. PkGPD1 A plasmid backbone fragment containing the gene-specific sgRNA prototype spacer sequence. The PCR product was validated by agarose gel electrophoresis, purified, and ligated using a seamless cloning kit before transformation. E. coli DH5α competent cells were plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C (approximately 14 hours). Single colonies were picked and colony PCR was performed using the BKPkGPD1-CES / CEA primers (see Table 4). Positive clones were sent for sequencing confirmation, and the correct plasmid was named pBKcas9-PkGPD1.

[0050] 4.2 Construction of donor fragments PkGPD1-UtMDH, PkGPD1-AoMDH and PkGPD1-HA The malate dehydrogenase UtMDH, derived from *Ustilago maydis*, has the amino acid sequence shown in SEQ ID NO:2 (UniProt database search number: A0A5C3E3R7). Codon optimization was performed to adapt it to the *Pichia kudriezvichi* expression system, and the optimized nucleotide sequence is shown in SEQ ID NO:4. It was synthesized by a commercial gene synthesis service provider. Using *Pichia kudriezvichi* CICC 33427 genomic DNA as a template, primers PkGPD1-UF / UR and PkGPD1-DF / DR (see Table 4) were used to amplify the enzyme. PkGPD1 Upstream and downstream homologous arm fragments were identified at the locus. The PGPD-UtMDH promoter fragment was amplified using PGPD-UtMDH-S / A primers (see Table 4), and the TGAL-UtMDH terminator fragment was amplified using TGAL-UtMDH-S / A primers (see Table 4). PkGPD1 upstream homologous arm fragment, PGPD-UtMDH promoter fragment, and synthesized UtMDH Gene fragments, TGAL-UtMDH terminator fragments and PkGPD1 Downstream homologous arm fragments were sequentially ligated using overlapping extension PCR to complete the construction of the donor fragment PkGPD1-UtMDH.

[0051] The malate dehydrogenase AoMDH, derived from *Aspergillus oryzae*, has the amino acid sequence shown in SEQ ID NO:9 (UniProt database search number: Q2U9I9). Codon optimization was performed to adapt it to the *Pichia kudriezvichi* expression system, and the optimized nucleotide sequence is shown in SEQ ID NO:10. This sequence was synthesized by a commercial gene synthesis service provider. Using *Pichia kudriezvichi* CICC 33427 genomic DNA as a template, the PGPD-AoMDH promoter fragment was amplified using the PGPD-AoMDH-S / A primers (see Table 4), and the TGAL-AoMDH terminator fragment was amplified using the TGAL-AoMDH-S / A primers (see Table 4). PkGPD1 upstream homologous arm fragment, PGPD-AoMDH promoter fragment, and synthesized AoMDH Gene fragments, TGAL-AoMDH terminator fragments and PkGPD1 Downstream homologous arm fragments were sequentially ligated using overlapping extension PCR to complete the construction of the donor fragment PkGPD1-AoMDH.

[0052] In addition, using Pichia kudrica CICC 33427 genomic DNA as a template, primers PkGPD1-UF / URa and PkGPD1-DFa / DR were used to amplify the DNA. PkGPD1 The donor fragment PkGPD-HA was constructed by using homologous arm fragments upstream and downstream of the site through overlapping extension PCR.

[0053] 4.3 Yeast transformation and screening, validation and nomenclature of positive transformants Competent cells of *Pichia pastoris* strain Pk003-ZrPYC were prepared. The pBKcas9-PkGPD1 plasmid and the PkGPD1-UtMDH donor fragment were co-transformed into the competent cells using the lithium acetate transformation method. The cells were then plated on SD-URA plates and incubated at 30°C for 3-4 days. Single colonies grown on the SD-URA plates were picked and PCR was performed using PkGPD1-CES / CEA primers (see Table 4) for verification. Successful PCR was confirmed. PkGPD1 Site integration UtMDH The transformants of the gene expression cassette should amplify a 4865 bp band, while the Pk003-ZrPYC control strain amplified a 2746 bp band. The PCR reaction solution was sent for sequencing to further confirm that the target gene expression cassette had been correctly integrated. The correctly validated positive transformant was named: Pk004-ZrPYC-UtMDH (genotype: Pk003-ZrPYC). PkGPD1∷PGPD-UtMDH-TGAL ).

[0054] The pBKcas9-PkGPD1 plasmid and the PkGPD1-AoMDH donor fragment were transformed into the Pk003-AoPYC strain, and successful strains were obtained by screening using the same method described above. PkGPD1 Site integration AoMDH The positive transformant of the gene expression cassette was named Pk004-AoPYC-AoMDH (genotype: Pk003-AoPYC). PkGPD1∷PGPD-AoMDH-TGAL ).

[0055] The pBKcas9-PkGPD1 plasmid and the PkGPD1-HA donor fragment were transformed into strain Pk003, and successful knockouts were obtained by screening using the same method described above. PkGPD1 The positive transformant of the gene was named Pk004 (genotype: Pk003, Δ). PkGPD1 ).

[0056] Table 4. Primers used in Example 4 Example 5: Fermentation production of L-malic acid by recombinant strains To evaluate the impact of different enzyme source combinations on the overall carbon flux of the rTCA cycle, this example uses L-malic acid production as an indicator. Shake-flask fermentation was performed on three recombinant strains of Pyrrosia kudriezwiecchi constructed in Example 4: Pk004-ZrPYC-UtMDH, Pk004-AoPYC-AoMDH, and Pk004. The catalytic efficiency of PYC / MDH combinations from different species in Pyrrosia kudriezwiecchi was compared.

[0057] The three recombinant strains were inoculated into 5 mL of YPD seed culture medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) and cultured at 30℃ and 220 rpm for 12 hours. Subsequently, the seed culture was divided according to OD... 600An inoculum of 0.1 g / L was transferred to 50 mL of YPD fermentation medium (50 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 40 g / L calcium carbonate). Fermentation was carried out in shake flasks at 30°C and 220 rpm for 48 hours, with three parallel controls per group. After fermentation, the pH of the fermentation broth was adjusted to 2.5 with hydrochloric acid and allowed to stand at room temperature for 30 min to allow residual calcium carbonate and a small amount of calcium malate to dissolve completely. The acidified fermentation broth was centrifuged at 12000 rpm for 5 min to remove cells and precipitate. The supernatant was diluted 100-fold with ultrapure water, filtered through a 0.22 μm filter membrane, and analyzed by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); mobile phase: 0.02 mol / L potassium dihydrogen phosphate solution-acetonitrile (98:2, v / v) (pH adjusted to 2.5 with phosphoric acid); flow rate: 0.8 mL / min; column temperature: 30℃; detection wavelength: 210 nm; injection volume: 10 μL. Quantification was performed using the external standard method, qualitative analysis was performed using the retention time of L-malic acid standards, and the concentration of L-malic acid in the sample was calculated by substituting the peak area into the standard curve.

[0058] L-malic acid yield and cell density (OD) of the three recombinant strains after 48 hours of shake-flask fermentation 600 As shown in Table 5 and Figure 1 As shown in the figure, the control strain Pk004, lacking the rTCA cycle, produced only 5.38 g / L of L-malic acid, while the recombinant strain Pk004-ZrPYC-UtMDH, overexpressing ZrPYC and UtMDH, produced 38.87 g / L of L-malic acid. This represents a 32.6% increase compared to the control strain Pk004-AoPYC-AoMDH, overexpressing the conventional enzyme sources (Aspergillus oryzae) AoPYC and AoMDH (29.32 g / L). These results clearly demonstrate that the ZrPYC and UtMDH enzyme combination selected in this invention has higher catalytic efficiency in the rTCA cycle of Pichia kudrifolia, effectively increasing the synthesis yield of L-malic acid.

[0059] Table 5. Comparison of L-malic acid yield and cell density of Pk004 series recombinant strains Example 6: Construction of yeast strains overexpressing ZrPYC and UtMDH mutants and fermentation evaluation To demonstrate that the amino acid sequences of the pyruvate carboxylase from *Zygosaccharidus rouxii* and the malate dehydrogenase from *Ustilago maydis* protected by this invention exhibit good tolerance to amino acid substitutions in the non-active center region, and to provide experimental support for the scope of protection of "conservative mutants having at least 99% sequence identity with SEQ ID NO:1 or SEQ ID NO:2" in the claims, this embodiment constructed recombinant strains expressing ZrPYC and UtMDH mutants, respectively, and evaluated the catalytic function of each mutant by the production of L-malic acid.

[0060] 6.1 Construction of yeast strains overexpressing ZrPYC mutant 6.1.1 Delineation of the active and inactive ZrPYC regions Highly conserved regions exist in PYC enzyme proteins from different species. In the amino acid sequence of ZrPYC, positions 18-470 form the biotinylate carboxylase domain, responsible for biotin carboxylation; positions 557-824 form the pyruvate carboxyltransferase domain, responsible for pyruvate carboxylation; and positions 1094-1169 form the biotin-binding domain, responsible for biotin binding. Within these domains, highly conserved functional motifs are further present: within the N-terminal biotinylate carboxylase domain, there is a conserved sequence GGGGRG (positions 182-187), which is present in all biotinylate enzymes (R can be K) and is considered to be the ATP binding site. [2,3] Within the pyruvate carboxyltransferase domain in the middle of the sequence, there is another crucial region, EDPW (positions 615-618), where the tryptophan residue (W) at position 618 participates in the pyruvate binding process during the transcarboxylation reaction. [4] Within the C-terminal biotin-binding domain, there is a highly conserved site AMKM (positions 1133-1136), where the lysine residue (K) at position 1135 is considered the biotin binding site, and it has been confirmed that this residue can specifically covalently bind to biotin. [5,6] The three domains mentioned above, along with the key motifs within them, constitute the active site region of ZrPYC, while the remaining regions are inactive site regions.

[0061] 6.1.2 Design of ZrPYC conserved mutants Based on the above definition of active and inactive center regions, this embodiment designed two sets of conserved amino acid substitution mutants (ZrPYC1 and ZrPYC2) in the inactive center region of ZrPYC. ​​"Conserved amino acid substitution" refers to the substitution of one amino acid residue by another amino acid residue with similar side chain chemical properties (such as charge, hydrophobicity, and size), for example, the substitution between leucine (L) and isoleucine (I), the substitution between arginine (R) and lysine (K), or the substitution between aspartic acid (D) and glutamic acid (E). All of these mutation sites are located far from the ATP-binding site, pyruvate-binding site, and biotin-binding site, within the inactive center region. Specific mutation sites are shown in Table 6-1.

[0062] Table 6-1. Mutation sites of ZrPYC conserved mutants 6.1.3 Construction of ZrPYC conserved mutant expression strain Based on the mutation sites shown in Table 6-1, the amino acid sequences of conserved mutants of ZrPYC1 and ZrPYC2 (as shown in SEQ ID NO:11 and SEQ ID NO:12, respectively) were designed. Codon optimization was performed to adapt them to the *Pichia pastoris* expression system, and the corresponding gene fragments (nucleotide sequences shown in SEQ ID NO:13 and SEQ ID NO:14, respectively) were synthesized by a commercial gene synthesis service provider. The synthesized [sequences / sequences] were then used to [achieve specific gene sequences / sequences]. ZrPYC1 and ZrPYC2 Mutant gene fragment replaces wild type ZrPYC The gene was constructed using the same method as the donor fragment PkPDC1-ZrPYC in Example 3 (with... PkPDC1 The upstream and downstream homologous arms, PGPD promoter, and TGAL terminator were used as flanking elements and sequentially linked by overlap extension PCR to construct donor fragments PkPDC1-ZrPYC1 and PkPDC1-ZrPYC2.

[0063] Following the same transformation and screening methods as in Example 3, the pBKcas9-PkPDC1 plasmid was co-transformed into strain Pk002 with either the PkPDC1-ZrPYC1 or PkPDC1-ZrPYC2 donor fragments. Positive transformants were screened on SD-URA plates, and single colonies were picked for PCR verification using PkPDC1-CES / CEA primers (see Table 3). Positive transformants were then sent for sequencing confirmation. ZrPYC1 or ZrPYC2 Gene expression cassette has been correctly integrated into PkPDC1 The verified transformants were named Pk005-ZrPYC1 (genotype: Pk002, ...). PkPDC1∷PGPD-ZrPYC1-TGAL) and Pk005-ZrPYC2 (genotype: Pk002, PkPDC1∷PGPD-ZrPYC2-TGAL ).

[0064] To investigate the impact of a single conserved ZrPYC mutation on the overall rTCA cycle flux, the obtained Pk005-ZrPYC1 and Pk005-ZrPYC2 strains were further prepared into competent cells, which were then transformed with the pBKcas9-PkGPD1 plasmid and the PkGPD1-UtMDH donor fragment described in Example 4. Genotypes were verified using PkGPD1-CES / CEA primers (see Table 4) to confirm the results. UtMDH Gene expression cassette has been correctly integrated into PkGPD1 The verified transformants were named Pk006-ZrPYC1-UtMDH (genotype: Pk005-ZrPYC1, ...). PkGPD1∷PGPD-UtMDH-TGAL ) and Pk006-ZrPYC2-UtMDH (genotype: Pk005-ZrPYC2, PkGPD1∷PGPD-UtMDH-TGAL ).

[0065] 6.1.4 Design of ZrPYC active site mutants To further clarify the technical boundaries of the scope of protection of this invention, this embodiment designs active center mutants in the three active center regions of ZrPYC, and the specific design concept is as follows: The GGGGRG motif (positions 182-187): Glycine at position 183 is mutated to alanine (G183A). This motif is a glycine-rich region at the ATP-binding site. The absence of a side chain in glycine provides the necessary conformational flexibility for proper ATP binding. The introduction of alanine disrupts this conformational flexibility, preventing proper ATP binding and thus blocking the entire enzyme's catalytic process in the first stage.

[0066] EDPW motif (positions 615-618): Tryptophan at position 618 is mutated to glutamate (W618E). This tryptophan residue is involved in the pyruvate binding process in the transcarboxylation reaction, and its bulky hydrophobic side chain plays a crucial role in the substrate binding pocket. The mutation to negatively charged glutamate completely alters the hydrophobicity and charge properties of this site, with the electrostatic repulsion effect almost entirely blocking the catalytic reaction.

[0067] The AMKM motif (positions 1133-1136): The lysine residue at position 1135 is mutated to glutamate (K1135E). This lysine residue specifically covalently binds to biotin and is a conserved active site residue in the biotin carboxylase family. The mutation to negatively charged glutamate completely reverses the charge, disrupting the original electrostatic interaction and preventing biotin from binding properly, resulting in a high probability of complete inactivation of catalytic activity.

[0068] 6.1.5 Construction of ZrPYC active center mutant expression strain Using the donor fragment PkPDC1-ZrPYC constructed in Example 3 as a template, two DNA fragments were amplified using ZrPYC-G183A-S1 / A1 and ZrPYC-G183A-S2 / A2 primers (see Table 6-3). Both fragments have homologous arms containing the G183A mutation site at their ends. The two fragments were spliced ​​together by overlap extension PCR to obtain the donor fragment PkPDC1-ZrPYC(G183A) containing the G183A mutation. Using the same method, with the donor fragment PkPDC1-ZrPYC as a template, primers ZrPYC-W618E-S1 / A1 and ZrPYC-W618E-S2 / A2 (see Table 6-3), and primers ZrPYC-K1135E-S1 / A1 and ZrPYC-K1135E-S2 / A2 (see Table 6-3) were used to construct donor fragments PkPDC1-ZrPYC(W618E) and PkPDC1-ZrPYC(K1135E) containing the W618E mutation and the K1135E mutation, respectively, by overlap extension PCR.

[0069] Following the same transformation and screening methods as in Example 3, the pBKcas9-PkPDC1 plasmid was co-transformed into strain Pk002 with donor fragments of PkPDC1-ZrPYC(G183A), PkPDC1-ZrPYC(W618E), or PkPDC1-ZrPYC(K1135E), respectively. Positive transformants were screened on SD-URA plates, and single colonies were picked for PCR verification using PkPDC1-CES / CEA primers (see Table 3). Positive transformants were then sent for sequencing confirmation. ZrPYC(G183A) , ZrPYC(W618E) or ZrPYC(K1135E) Gene expression cassette has been correctly integrated into PkPDC1 The verified transformants were named Pk005-ZrPYC(G183A) (genotype: Pk002, ...). PkPDC1∷PGPD-ZrPYC(G183A)-TGAL ), Pk005-ZrPYC(W618E) (genotype: Pk002, PkPDC1∷PGPD-ZrPYC(W618E)-TGAL ) and Pk005-ZrPYC(K1135E) (genotype: Pk002, PkPDC1∷ PGPD-ZrPYC(K1135E)-TGAL ).

[0070] To investigate the effect of individual ZrPYC active site mutants on the overall rTCA cycle flux, the obtained Pk005-ZrPYC(G183A), Pk005-ZrPYC(W618E), and Pk005-ZrPYC(K1135E) strains were further prepared into competent cells, which were then transformed with the pBKcas9-PkGPD1 plasmid and the PkGPD1-UtMDH donor fragment described in Example 4. The genotype was verified using PkGPD1-CES / CEA primers (see Table 4), confirming the results. UtMDH Gene expression cassette has been correctly integrated into PkGPD1 The verified transformants were named Pk006-ZrPYC(G183A)-UtMDH (genotype: Pk005-ZrPYC(G183A), ...). PkGPD1∷PGPD-UtMDH-TGAL ), Pk006-ZrPYC(W618E)-UtMDH (genotype: Pk005-ZrPYC(W618E), PkGPD1∷PGPD-UtMDH-TGAL ) and Pk006-ZrPYC(K1135E)-UtMDH (genotype: Pk005-ZrPYC(K1135E), PkGPD1∷PGPD-UtMDH-TGAL ).

[0071] 6.2 Construction of yeast strains overexpressing the UtMDH mutant 6.2.1 Delineation of UtMDH active and inactive center regions NAD of UtMDH + The key binding motif is located at amino acids 8-14 (GAAGGIG), a characteristic sequence of the Rossmann fold responsible for binding NAD. + The phosphate groups are highly conserved in MDH. [7,8] The above motifs represent the active site region of UtMDH, while the remaining regions are inactive site regions.

[0072] 6.2.2 Design of UtMDH conserved mutants Based on the above definition of active and inactive central regions, this embodiment designed two sets of conserved amino acid substitution mutants (UtMDH1 and UtMDH2) in the inactive central region of UtMDH. "Conserved amino acid substitution" refers to the substitution of one amino acid residue by another amino acid residue with similar side-chain chemical properties (such as charge, hydrophobicity, and size). All of the above mutation sites are far from NAD. + The binding motif is located in the inactive center region. Specific mutation sites are shown in Table 6-2.

[0073] Table 6-2. Mutation sites of UtMDH conserved mutants 6.2.3 Construction of UtMDH conserved mutant expression strain Based on the mutation sites shown in Table 6-2, the amino acid sequences of conserved mutants of UtMDH1 and UtMDH2 (as shown in SEQ ID NO:15 and SEQ ID NO:16, respectively) were designed. Codon optimization was performed to adapt them to the *Pichia pastoris* expression system, and the corresponding gene fragments (nucleotide sequences shown in SEQ ID NO:17 and SEQ ID NO:18, respectively) were synthesized by a commercial gene synthesis service provider. The synthesized fragments were then used as... UtMDH1 and UtMDH2 Mutant gene fragment replaces wild type UtMDH The gene was constructed using the same method as the donor fragment PkGPD1-UtMDH in Example 4 (with... PkGPD1 The upstream and downstream homologous arms, PGPD promoter, and TGAL terminator were used as flanking elements and sequentially linked by overlap extension PCR to construct donor fragments PkGPD1-UtMDH1 and PkGPD1-UtMDH2.

[0074] To investigate the effect of a single conserved UtMDH mutant on the overall rTCA cycle flux, following the same transformation and screening methods as in Example 4, the pBKcas9-PkGPD1 plasmid was co-transformed into strain Pk003-ZrPYC with either the PkGPD1-UtMDH1 or PkGPD1-UtMDH2 donor fragments. Positive transformants were screened on SD-URA plates, and single colonies were picked for PCR verification using PkGPD1-CES / CEA primers (see Table 4). Positive transformants were then sent for sequencing confirmation. UtMDH1 or UtMDH2 Gene expression cassette has been correctly integrated into PkGPD1 The verified transformants were named Pk006-ZrPYC-UtMDH1 (genotype: Pk003-ZrPYC, ...). PkGPD1::PGPD-UtMDH1-TGAL ) and Pk006-ZrPYC-UtMDH2 (genotype: Pk003-ZrPYC, PkGPD1::PGPD-UtMDH2-TGAL ).

[0075] To investigate the effect of the combination of ZrPYC and UtMDH conserved mutants on the overall rTCA cycle flux, competent cells were prepared from strains Pk005-ZrPYC1 and Pk005-ZrPYC2, respectively, and transformed with pBKcas9-PkGPD1 plasmid and PkGPD1-UtMDH1 or PkGPD1-UtMDH2 donor fragments, respectively. Genotyping was verified using PkGPD1-CES / CEA primers (see Table 4). UtMDH1 or UtMDH2The gene expression cassette has been correctly integrated into the PkGPD1 site. The verified transformants are named as follows: Pk006-ZrPYC1-UtMDH1 (genotype: Pk005-ZrPYC1, PkGPD1::PGPD-UtMDH1-TGAL ); Pk006-ZrPYC1-UtMDH2 (genotype: Pk005-ZrPYC1, PkGPD1::PGPD-UtMDH2-TGAL ); Pk006-ZrPYC2-UtMDH1 (genotype: Pk005-ZrPYC2, PkGPD1::PGPD-UtMDH1-TGAL ); Pk006-ZrPYC2-UtMDH2 (genotype: Pk005-ZrPYC2, PkGPD1::PGPD-UtMDH2-TGAL ).

[0076] 6.2.4 Design of UtMDH active center mutants To further clarify the technical boundaries of the scope of protection of this invention, this embodiment focuses on the NAD of UtMDH. + Active site mutants were designed based on the motif. The glycine at position 8 was mutated to glutamate (G8E), a site located in NAD+. + The conserved glycine-rich region of the binding motif (GAAGGIG), where glycine has only hydrogen atoms in its side chain, provides the necessary spatial conditions for the flexibility of the main chain conformation in this region, enabling NAD... + The phosphate groups can bind correctly. After the introduction of glutamic acid, on the one hand, its large side chain occupies the original space, restricting the freedom of the main chain conformation; on the other hand, the glutamic acid side chain carries a negative charge, which binds to NAD+. + The phosphate groups in the phosphate group generate electrostatic repulsion. The combined effect of these two factors will severely reduce the catalytic activity of UtMDH.

[0077] 6.2.5 Construction of UtMDH active center mutant expression strain Using the donor fragment PkGPD1-UtMDH constructed in Example 4 as a template, two DNA fragments were amplified using primers UtMDH-G8E-S1 / A1 and UtMDH-G8E-S2 / A2 (see Table 6-3). Both fragments have homologous arms containing the G8E mutation site at their ends. The two fragments were spliced ​​together by overlap extension PCR to obtain the donor fragment PkGPD1-UtMDH(G8E) containing the G8E mutation.

[0078] To investigate the effect of a single UtMDH active site mutant on the overall rTCA cycle flux, the pBKcas9-PkGPD1 plasmid and the PkGPD1-UtMDH(G8E) donor fragment were co-transformed into the Pk003-ZrPYC strain using the same transformation and screening methods as in Example 4. Positive transformants were screened on SD-URA plates, and single colonies were picked for PCR verification using PkGPD1-CES / CEA primers (see Table 4). Positive transformants were then sent for sequencing confirmation. UtMDH(G8E) Gene expression cassette has been correctly integrated into PkGPD1 The verified transformants were named Pk006-ZrPYC-UtMDH(G8E) (genotype: Pk003-ZrPYC, ...). PkGPD1::PGPD-UtMDH(G8E)-TGAL ).

[0079] Table 6-3. Primers used in Example 6 6.3 Fermentation evaluation of yeast strains overexpressing ZrPYC and UtMDH mutants To evaluate the effects of conserved mutants of ZrPYC and UtMDH, as well as mutants of the active site, on rTCA cycling flux, this example uses L-malic acid yield as the evaluation index and conducts shake-flask fermentation experiments on the Pk006 series recombinant strains constructed above. Each strain was fermented in shake flasks under the fermentation conditions of Example 5 (the seed culture medium and fermentation medium formulations were the same as in Example 5). Pk004-ZrPYC-UtMDH (overexpressing wild-type ZrPYC and wild-type UtMDH) was used as the control strain. Three parallel controls were set up for each group. Samples were taken after 48 hours of fermentation, and the L-malic acid content was detected by HPLC. The results are shown in Tables 6-4 and 6-5. Figure 2 As shown.

[0080] The results showed that the L-malic acid yield of the eight recombinant strains overexpressing conserved mutants of ZrPYC or UtMDH (including single and combined expression) remained between 31.51 and 37.66 g / L. Compared with the L-malic acid yield (38.69 g / L) of the control strain Pk004-ZrPYC-UtMDH overexpressing wild-type ZrPYC and wild-type UtMDH, the yield of the eight recombinant strains was 81.4%-97.3% of the control strain, all not lower than 80%. Among them, the strain expressing the ZrPYC1-UtMDH2 combination had the highest yield (35.97 g / L), while the strain expressing the ZrPYC2-UtMDH1 combination had the lowest yield (31.51 g / L). The above results indicate that the conserved mutations in ZrPYC or UtMDH, whether occurring alone or in combination, have a limited impact on rTCA cycling flux, and the L-malic acid yield of the recombinant strains can be maintained at more than 80% of that of the control strains. Taking the above eight recombinant strains as examples, the amino acid sequence differences between the mutant proteins expressed by each strain and the corresponding wild-type enzymes (SEQ ID NO:1 or SEQ ID NO:2) are only 1 to 12 conserved substitutions, and all substitutions are located in the non-active center region, maintaining sequence identity of more than 99%. After integrating the genes encoding the above conserved mutants into the yeast genome, the L-malic acid yield of the obtained recombinant strains is no less than 80% of that of the control strains. The above results are highly consistent with the scope of protection of the present invention regarding "sequence identity, conserved substitutions in the non-active center region, and yield no less than 80% of the control strains".

[0081] In contrast, the L-malic acid production of the four recombinant strains carrying mutants of key residues in the active center was significantly reduced. The three strains expressing ZrPYC active center mutants (G183A, W618E, or K1135E) and simultaneously expressing wild-type UtMDH had L-malic acid production of only 7.69-8.74 g / L, which was 19.9%-22.6% of the control strain's production; the strain expressing wild-type ZrPYC and simultaneously expressing the UtMDH active center mutant (G8E) had an L-malic acid production of 11.86 g / L, which was 30.7% of the control strain's production. Furthermore, the OD of the recombinant strains carrying the active center mutants... 600The values ​​(6.02-6.41) were generally higher than those of recombinant strains carrying conserved mutants (4.87-5.23), indicating that the growth of these recombinant strains carrying active site mutants was not inhibited. The significant decrease in yield was not due to strain growth defects, but rather to the loss of catalytic activity of key enzymes in the rTCA cycle. These results suggest that mutations in key residues at the ATP-binding site (G183A), pyruvate-binding site (W618E), and biotin-binding site (K1135E) of ZrPYC, and NAD+ of UtMDH, are significant. + Mutations in key motif residues (G8E) can severely impair catalytic function, leading to a significant decrease in rTCA cycle flux. These sites are crucial for enzyme activity.

[0082] The above experimental evidence directly supports the scope of protection of the conserved mutants in the claims of this invention, and clarifies the technical boundary of loss of function caused by mutation of key residues in the active site.

[0083] Table 6-4. Comparison of L-malic acid yield and cell density of Pk006 series recombinant strains References: [1] Tran VG, Cao M, Fatma Z, et al. Development of a CRISPR / Cas9-based tool for gene deletion in Issatchenkia orientalis[J]. Msphere, 2019, 4(3): e00345-19. [2] Lim F, Morris CP, Occhiodoro F, et al. Sequence and domain structure of yeast pyruvate carboxylase[J]. Journal of Biological Chemistry, 1988, 263(23): 11493-11497. [3] Wexler I D, Du Y, Lisgaris M V, et al. Primary amino acidsequence and structure of human pyruvate carboxylase[J]. Biochimica etBiophysica Acta (BBA)-Molecular Basis of Disease, 1994, 1227(1-2): 46-52. [4] Kumar G K, Haase F C, Phillips N F B, et al. Involvement andidentification of a tryptophanyl residue at the pyruvate binding site oftranscarboxylase[J]. Biochemistry, 1988, 27(16): 5978-5983. [5] Rylatt D B, Keech D B, Wallace J C. Pyruvate carboxylase:Isolation of the biotin-containing tryptic peptide and the determination ofits primary sequence[J]. Archives of Biochemistry and Biophysics, 1977, 183(1): 113-122. [6] Leon-Del-Rio A, Gravel R A. Sequence requirements for thebiotinylation of carboxyl-terminal fragments of human propionyl-CoAcarboxylase alpha subunit expressed in Escherichia coli[J]. Journal ofBiological Chemistry, 1994, 269(37): 22964-22968. [7] Bell J K, Yennawar H P, Wright S K, et al. Structural analyses of a malate dehydrogenase with a variable active site[J]. Journal of Biological Chemistry, 2001, 276(33): 31156-31162. [8] Chapman A D M, Cortés A, Dafforn T R, et al. Structural basis of substrate specificity in malate dehydrogenases: crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, α-ketomalonate and tetrahydroNAD[J]. Journal of Molecular Biology, 1999, 285(2): 703-712. Sequence Listing SEQ ID NO: 1: Derived from Zygosaccharomyces rouxii ( Zygosaccharomyces rouxii SEQ ID NO:2: Derived from Ustilago maydis ( Ustilago trichophora The amino acid sequence of malate dehydrogenase (UtMDH) is as follows: MVKATVIGAAGGIGQPLSLLLKQSSLVTDLALYDVVNAPGVAADLSHINTPSVVTGYLPADGGLGKALKNADIVVIPAGVPRKPGMTRDDLFNINAGIVRDIAVGIAEHSPKAFVLVISNPVNSTVPIVAEVLKKKGVYDPKRLFGVTTLDVVRASTFIAEAAGKPTESLNYRIPVVGGHSGVTIVPLISQSQPPISVDQAKIEQLTNRIQFGGDEVVKAKDGAGSATLSMAYAGARFAIAVLEAASGKALANPEMGYVDLTSDAAGAKEVTAVIGNDTPFFSVPLQLGPNGVEKILPLGKLNDYESELIKKAVVDLNGNINKGVQFTANL SEQ ID NO:5: Derived from *Schizosaccharomyces cerevisiae* ( Schizosaccharomyces pombe The amino acid sequence of the C4 dicarboxylic acid transporter (SpMAE1) MGELKEILKQRYHELLDWNVKAPHVPLSQRLKHFFTWSWFACTMATGGVGLIGSPFRFYGLNTIGKIVYILQIFLFSLFGSCMLFRFIKYPSTIKDSWNHHLEKLFIA TCLLSISTFIDMLAIYAYPDTGEWMVWVIRILYYIYVAVSFIYCVMAFFTIFNNHVYTIETASPAWILPIFPPMICGVIAGAVNSTQPAHQLKNMVIFGILFQGLGFWVY LLLFAVNVLRFFTVGLAKPQDRPGMFMFVGPPAFSGLALINIARGAMGSRPYIFVGANSSEYLGFVSTFMAIFIWGLAAWCYCLAMVSFLAGFFTRAPLKFACGWFAFI FPNVGFVNCTIEIGKMIDSKAFQMFGHIIGVILCIQWILLMYLMVRAFLVNDLCYPGKDEDAHPPPKPNTGVLNPTFPPEKAPASLEKVDTHVTSTGGESDPPSSEHESV SEQ ID NO:6: Nucleotide sequence of the SpMAE1 encoding gene (artificial) SEQ ID NO:7: Derived from Aspergillus oryzae ( Aspergillus oryzae The amino acid sequence of pyruvate carboxylase (AoPYC) SEQ ID NO:8: Nucleotide sequence of the AoPYC encoding gene (artificial) SEQ ID NO:9: Derived from Aspergillus oryzae ( Aspergillus oryzae The amino acid sequence of malate dehydrogenase (AoMDH) MVKAAVLGASGGIGQPLSLLLKTCPLVEELALYDVVNTPGVAADLSHISSIAKISGFLPKDDGLKQALTGANIVVIPAGIPRKPGMTRDDLFKINAGIVRDLVKGIAEFCPKAFVLVISNPVNSTVPIAAEVLKAAGVFDPKRLFGVTTLDVVRAETFTQEFSGQ KDPSAVQIPVVGGHSGETIVPLFSKTTPAIQIPEEKYDALIHRVQFGGDEVVQAKDGAGSATLSMAYAGYRFAESVIKASKGQTGIVEPTFVYLPGIPGGDEIVKATGVEFFSTLVTLGTNGAEKASNVLEGVTEKEKKLLEACTKGLKGNIEKGIDFVKNPPPK SEQ ID NO:10: Nucleotide sequence of the AoMDH encoding gene (artificial) atggttaaggctgcagtgttgggtgcatcaggcgggattggacagccgttgtctttgctattgaaaacgtgccccctagtggaggagttagccctgtatgatgttgtgaatacacccggtgttgcagctgatttatcccacatttcttcgattgctaaaatttctggatttcttcccaaggacgacggcttaaaacaagctttaacgggggccaacattgttgtcatccctgctggaataccaaggaaaccaggtatgactagagatgatttgttcaaaatcaatgcaggcatcgtaagagacttagtaaaaggaatagcagagttttgtccaaaggcatttgtgctagtcattagtaatccagtcaactccactgttccaattgctgctgaagttttgaaggccgcgggggtttttgatccaaagaggctatttggggttactaccctcgacgtcgttcgtgcggaaaccttcacacaagaattctcgggacaaaaggatccttctgctgtccaaattcctgttgttggtggtcatagcggtgaaacaattgtgcctcttttctccaaaactacccctgcaatccaaatacctgaagaaaaatacgatgcattgattcatagagttcagtttggtggtgacgaggttgtacaggcaaaagatggtgccggatctgccacactgagtatggcatatgcaggttatagattcgcagaatctgttatcaaagcaagtaaaggtcaaactggtatagttgaaccaacatttgtttacttaccgggtataccgggaggagatgaaattgtcaaagccaccggcgttgagttcttttcaactttggtaaccttaggtacaaatggtgctgagaaagcttcaaatgtacttgaaggtgtcactgaaaaagaaaagaagcttttggaagcttgtacaaagggccttaagggaaacattgagaaggggattgattttgtcaagaacccaccacctaaataa SEQ ID NO:11: Amino acid sequence of a conserved mutant of ZrPYC1 (artificial) SEQ ID NO:12: Amino acid sequence of a conserved mutant of ZrPYC2 (artificial) SEQ ID NO:13: Nucleotide sequence of the gene encoding the ZrPYC1 conserved mutant (artificial) SEQ ID NO:14: Nucleotide sequence of the gene encoding the ZrPYC2 conserved mutant (artificial). SEQ ID NO:15: Amino acid sequence of the conserved mutant of UtMDH1 (artificial) MVKATVIGAAGGIGQPLTLLLKQSSLVTELALYDVVNAPGVAADLSHINTPSVVTGYLPADGGLGKALKNADIVVIPAGVPRKPGMTRDDLFNINAGIVRDIAVGIAEHSPKAFVLVISNPVNSTVPIVAEVLKKKGVYDPKRLFGVTTLDVVRASTFIAEAAGK PTESLNYRIPVVGGHSGVTIVPLISQSQPPISVDQAKIEQLTNRIQFGGDEVVKAKDGAGSATLSMAYAGARFAIAVLEAASGKGLANPEMGYVDLTSDAAGAKEVTAVIGNDTPFFSVPLQLGPNGVEKILPLGKLNDYESELLKKAVVDLNGNINKGVQFTANL SEQ ID NO:16: Amino acid sequence of the conserved mutant of UtMDH2 (artificial) MVKATVIGAAGGIGQPISLLLRQSSLVTDLALYDVVNAPGVAADLSHINTPSVVTGYLPADGGLGKALKNADIVVIPAGVPRKPGMTRDDLFNINAGIVRDIAVGIAEHSPKAFVLVISNPVNSTVPIVAEVLKKKGVYDPKRLFGVTTLDVVRASTFIAEAAGK PTESLNYRIPVVGGHSGVTIVPLISQSQPPISVDQAKIEQLTNRIQFGGDEVVKAKDGAGSATLSMAYAGARFAIAVLEAASGKALANPEMGYVELTSDAAGAKEVTAVIGNDTPFFSVPLQLGPNGVEKILPLGKLNDYESELIKKAVVDLNGNINKGVQFTAQL SEQ ID NO:17: Nucleotide sequence of the gene encoding the UtMDH1 conserved mutant (artificial) atggttaaagctactgtaattggtgctgcgggaggtatcgggcagcctttgacgctacttctaaaacaatcctccctagtgactgaattggctttatacgatgttgtgaatgcacctggggttgcagccgacctttcccatatcaatactccgtcagtagttacaggttatctcccagcagatgggggattaggtaaagccttgaaaaatgctgacatagtagtcattcctgctggagtaccaaggaaacctggtatgacaagagatgatttattcaatataaatgccggtattgtgcgtgatattgcggttggaatagcagaacattctcctaaagcatttgttttagtcatcagcaaccctgttaactcaaccgttcccatcgtggccgaggtattgaagaagaaaggtgtttatgatccaaagagattatttggagttaccactttggacgtggtcagagcaagtacttttattgccgaagctgcaggcaaaccaacagagagcctaaactaccgaataccggtggttggtggacactctggtgtcacgattgttccattaatttcgcagtctcaaccaccaatttctgttgatcaagccaaaattgaacaattaacaaacagaatccaatttggtggtgatgaagttgtcaaggcaaaagatggtgcaggctctgcaacactttcaatggcatatgctggcgccaggtttgcaatagctgtgttagaggcggcttcaggaaagggactcgctaatccagaaatgggttacgtcgacttgaccagtgatgcagcgggggcaaaggaagtaactgctgttattggcaatgataccccattcttttcggtccccttgcagttgggcccgaatggtgttgaaaagatactccccctaggcaagttgaatgactatgaaagtgagctgctgaaaaaggccgttgttgaccttaacggtaatatcaacaaaggggtccaattcacagcaaacttgtaa SEQ ID NO:18: Nucleotide sequence of the gene encoding the UtMDH2 conserved mutant (artificial) atggttaaagcaacagttattggagccgctggaggaattggccaaccaataagcctgctactcagacaatcatcgttagtcactgacttggccttatatgatgtggtaaacgcgccgggtgtggcagctgatttgtctcacattaatacgccgagtgttgtgactggttacctaccagcagatggaggtttaggaaaggcattgaaaaatgcagacatagtggtaattcccgcaggcgtcccaagaaaacctggtatgacaagagatgatttattcaatatcaatgccggcattgttcgtgacatagcagttggtatcgctgaacattcacctaaagcctttgttttggttatctccaatccagtcaactcaactgttcctatagttgcagaagttttgaagaaaaagggtgtttacgatccaaagcgactatttggggtcacaacattggatgttgttagggcgtccactttcatagcagaggcggctggtaaaccaaccgagtctcttaattacagaattcctgtagtaggaggacattctggcgtcaccattgtccccctgatctcacagtcgcaacctcctatttctgttgaccaagctaaaattgaacaacttacaaataggatccaatttggtggtgatgaagtcgtgaaagccaaagatggtgctgggtctgctaccctttcaatggcatatgcaggtgcaagatttgcgattgctgttttggaagctgcttcaggaaaagctttagcaaacccagaaatgggttatgttgagcttacgagtgatgcagcaggggccaaggaagtgaccgctgtcataggtaatgacactccatttttctccgtaccgttacagttaggtccaaatggcgttgaaaagattctccccttggggaagctaaacgattatgaaagtgagttgattaagaaggccgttgttgatcttaatggtaacatcaacaaaggtgtccagtttacagcgcaactctaa

Claims

1. A method for constructing a reductive tricarboxylic acid cycle in vivo in Pichia coudriavzeii, characterized by, Includes the following steps: The code is derived from *Zygosaccharomyces rouxii* ( Zygosaccharomyces rouxii The gene for pyruvate carboxylase or a conserved mutant thereof was integrated into the genome of *Pichia kudrica*. The code is derived from barnyard grass fungus (Smut fungus) Ustilago trichophora The gene for malate dehydrogenase or a conserved mutant of it was integrated into the genome of *Pichia kudrica*. Genes were expressed in yeast to obtain recombinant strains with the reduced tricarboxylic acid cycle pathway.

2. The method according to claim 1, characterized in that, The amino acid sequence of the pyruvate carboxylase derived from *Zygosaccharidus rouxii* is shown in SEQ ID NO:1; the amino acid sequence of the malate dehydrogenase derived from *Ustilago maydis* is shown in SEQ ID NO:

2.

3. The method according to claim 1, characterized in that, The conserved mutant of pyruvate carboxylase derived from *Zygosaccharidus rouxii* satisfies the following characteristics: Its amino acid sequence has at least 99% sequence identity with SEQ ID NO:1; The amino acid difference between it and SEQ ID NO:1 is a conservative substitution of 1 to 12 amino acid residues, and the difference only occurs in the non-active center region; After integrating the gene encoding the mutant and the gene encoding malate dehydrogenase SEQ ID NO:2 or its conserved mutant into the genome of *Kudriazwibichthys*, the resulting recombinant strain produced L-malic acid at a yield not less than 80% of that of the control strain obtained after integrating the gene encoding the enzyme shown in SEQ ID NO:1 and the gene encoding the enzyme shown in SEQ ID NO:2 into the genome of *Kudriazwibichthys*.

4. The method according to claim 1, characterized in that, The conserved mutant of malate dehydrogenase derived from Ustilago maydis satisfies the following characteristics: Its amino acid sequence has at least 99% sequence identity with SEQ ID NO:2; The amino acid difference between it and SEQ ID NO:2 is a conservative substitution of 1 to 4 amino acid residues, and the difference only occurs in the non-active center region; After integrating the gene encoding the mutant and the gene encoding pyruvate carboxylase SEQ ID NO:1 or its conserved mutant into the genome of *Kudriazwibichthys*, the resulting recombinant strain has an L-malic acid yield of not less than 80% of that of the control strain obtained after integrating the gene encoding the enzyme shown in SEQ ID NO:1 and the gene encoding the enzyme shown in SEQ ID NO:2 into the genome of *Kudriazwibichthys*.

5. The method according to claim 1, characterized in that, The gene encoding pyruvate carboxylase and the gene encoding malate dehydrogenase are operatively linked to a promoter and a terminator, respectively, and then introduced into the yeast; the promoter is a constitutive strong promoter, and the terminator is a strong terminator.

6. The method according to claim 5, characterized in that, The promoter is the PGPD promoter, and the terminator is the TGAL terminator.

7. The method according to claim 1, characterized in that, The gene encoding pyruvate carboxylase and the gene encoding malate dehydrogenase are integrated into non-essential gene sites in the yeast genome.

8. The method according to claim 7, characterized in that, The gene encoding pyruvate carboxylase was integrated into the genome of *Pichia gondii*. PkPDC1 At the site; the gene encoding malate dehydrogenase was integrated into the genome of *Pichia gondii*. PkGPD1 In the locus.

9. A gene encoding pyruvate carboxylase from *Zygosaccharidus rouxii* and malate dehydrogenase from *Ustilago maydis*, characterized in that... The nucleotide sequence of the gene encoding pyruvate carboxylase is shown in SEQ ID NO:3; the nucleotide sequence of the gene encoding malate dehydrogenase is shown in SEQ ID NO:

4.

10. The gene according to claim 9, characterized in that, The nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4 are codon-optimized sequences for *Kudelazvibich yeast*.

11. A recombinant strain of *Pichia kudrica*, characterized in that, The recombinant strain is constructed by the method according to any one of claims 1-8, or contains the gene according to claim 9 or 10.

12. The recombinant strain according to claim 11, characterized in that, Its genome integrates the gene encoding pyruvate carboxylase and the gene encoding malate dehydrogenase as described in claim 9 or 10.

13. The use of the method according to any one of claims 1-8, the gene according to claim 9 or 10, or the recombinant strain according to claim 11 or 12 in the production of L-malic acid.

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