Candida tropicalis engineering bacteria for producing succinic acid with methanol as carbon source, application and method thereof

Abstract

The application discloses a kind of with methanol as carbon source production succinic acid tropical Candida engineering bacteria and its application and method, engineering bacteria is the starting strain CT-1 of tropical Candida, and is obtained by following modification: introduction of heterologous reductive tricarboxylic acid cycle (rTCA) carbon fixation pathway, including pyruvate carboxylase gene Anpyc, malate dehydrogenase gene Aomdh2, fumarase gene FumCcp and fumarate reductase gene Frd1cp;Knock out pyruvate decarboxylase gene PDC2;Introduction NAD-dependent methanol dehydrogenase gene MDH Bs The engineering bacteria can be fermented under anaerobic condition with methanol as sole carbon source, and the succinic acid yield reaches 4.4 g / L.The application first constructs a complete "methanol-succinic acid" metabolic route in tropical Candida, provides a new way for green low-carbon biological manufacturing of succinic acid, and has important theoretical value and industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to an engineered strain of Candida tropicalis that produces succinic acid using methanol as a carbon source, and also to the application and methods of the engineered strain. Background Technology

[0002] Succinic acid, also known as succinic acid, is an important C4 platform compound with a symmetrical dicarboxyl group structure. Its molecular structure endows it with excellent chemical reactivity, making it widely applicable as an intermediate in pharmaceuticals, food, chemicals, and biomaterials. Traditional chemical synthesis of succinic acid uses petroleum-based derivatives (such as maleic anhydride) as raw materials, resulting in high energy consumption and the generation of large amounts of industrial wastewater and waste gas, which does not align with the principles of green and sustainable development. While biosynthesis can circumvent the environmental drawbacks of chemical methods by converting carbon sources into succinic acid through microbial metabolism, its mainstream technological routes still rely on food-based carbohydrates such as glucose and sucrose as carbon sources, leading to resource conflicts such as competition for food and land. Therefore, developing environmentally friendly and renewable non-food raw materials, such as agricultural waste, lignocellulose, and methanol, is of great significance for the sustainable production of biochemicals.

[0003] One-carbon (C1) compounds (such as CO2, methanol, and methane) have attracted widespread attention as feedstocks for third-generation biorefining. Methanol is an important chemical feedstock that can be produced from natural gas and CO2 using green energy sources (such as solar energy). Its inexpensive source and flexible production process reduce costs and enhance the competitiveness of methanol-based biochemicals. Therefore, achieving high-value bioconversion of methanol with a zero CO2 footprint is the core of third-generation biorefining. Methanol is easy to store and transport and has a high reducing power, which is beneficial for achieving higher product conversion rates. After being absorbed into cells, methanol is converted into formaldehyde by formaldehyde dehydrogenase or alcohol oxidase. Then, it is catalyzed by dihydroxyacetone synthase and dihydroxyacetone kinase, entering the central metabolic pathway (glycolysis), and finally entering the reductive TCA (rTCA) pathway to produce succinic acid.

[0004] Currently, the main microbial substrates used for succinic acid production include succinate-producing Actinobacillus, Escherichia coli, succinate-producing anaerobic spirochetes, Corynebacterium glutamicum, and Yersinia lipolytica. However, most studies use food-based carbon sources such as glucose as substrates, and research on using C1 compounds such as methanol as raw materials for succinic acid production remains limited. Introducing a heterologous methanol metabolic pathway in E. coli allows for the production of succinic acid using glucose and methanol as a common carbon source, but methanol consumption is only 1.1 g / L. In Yersinia lipolytica, after introducing a methanol assimilation pathway, using xylose and methanol as a mixed carbon source, the succinic acid yield is only 0.9 g / L. Most microorganisms naturally lack efficient methanol assimilation capabilities, and their metabolic flux is insufficient to support the efficient synthesis of the target product. Therefore, it is necessary to reconstruct and optimize microbial methanol utilization pathways through metabolic engineering strategies to construct efficient C1 raw material conversion cell factories.

[0005] Currently, commonly used chassis microorganisms in biomanufacturing (such as *Escherichia coli*, *Actinomyces succinate*, and *Saccharomyces cerevisiae*) still face a series of challenges, including strict anaerobic requirements, demanding fermentation environmental conditions, and high production costs. Furthermore, due to insufficient cell wall strength and high cell membrane permeability, bacteria have poor tolerance to acidic conditions, often requiring the addition of neutralizing agents to maintain a suitable pH during fermentation. Compared to traditional bacterial chassis (such as *E. coli*), yeast cells exhibit greater robustness; *Saccharomyces cerevisiae* and *Yarrowia lipolytica* have been used to produce succinic acid. Modified *E. coli*, using glucose as a carbon source, produces 32.0 g / L succinic acid under low pH (5.6) conditions, while engineered *Yarrowia lipolytica*, using glycerol as a carbon source, produces 110.7 g / L succinic acid under uncontrolled pH conditions. Compared to other yeasts, *Candida tropicalis* has a broader substrate spectrum (glycosylated carbon sources, alkanes, fatty acids, and fatty acid esters, etc.) and greater robustness (acid tolerance, substrate and product tolerance, etc.). By blocking the β-oxidation pathway, the engineered Candida tropicalis strain H5343 produces 210 g / L tetradecanoic acid using methyl myristate. Therefore, Candida tropicalis possesses unique metabolic advantages, strong robustness, and anaerobic fermentation capabilities, making it a potentially excellent substrate for the synthesis of methanol-based succinic acid.

[0006] Therefore, it is urgent to use Candida tropicalis CT-1 as the starting strain, weaken the byproduct ethanol synthesis pathway through metabolic engineering, construct a heterologous rTCA pathway, screen methanol dehydrogenases to construct an anaerobic methanol metabolism module, and finally combine shake flask fermentation optimization and fermenter scale-up culture so that the engineered bacteria can synthesize succinic acid with methanol as the sole carbon source. Summary of the Invention

[0007] In view of this, one objective of the present invention is to provide an engineered strain of Candida tropicalis for producing succinic acid using methanol as a carbon source; a second objective of the present invention is to provide the application of the engineered strain of Candida tropicalis in the fermentation production of succinic acid using methanol as a carbon source; and a third objective of the present invention is to provide a method for producing succinic acid by anaerobic fermentation using methanol as a carbon source.

[0008] To achieve the above objectives, the present invention provides the following technical solution: 1. An engineered strain of *Candida tropicalis* that produces succinic acid using methanol as a carbon source, wherein the engineered strain is obtained by modifying the original strain CT1 of *Candida tropicalis* through the following modifications: (1) Introduce the heterologous reducing tricarboxylic acid cycle (rTCA) carbon fixation pathway into the starting strain, the pathway comprising the pyruvate carboxylase gene Anpyc, the malate dehydrogenase gene Aomdh2, the fumarate gene FumCcp and the fumarate reductase gene Frd1cp. (2) Knock out the pyruvate decarboxylase gene PDC2; (3) Introducing the NAD-dependent methanol dehydrogenase gene MDH Bs .

[0009] Preferably, the nucleotide sequence of Anpyc is shown in SEQ ID NO.1, the nucleotide sequence of FumCcp is shown in SEQ ID NO.2, the nucleotide sequence of Frd1cp is shown in SEQ ID NO.3, the nucleotide sequence of Aomdh2 is shown in SEQ ID NO.4, and the nucleotide sequence of MDH is shown in SEQ ID NO.4. Bs The nucleotide sequence is shown in SEQ ID NO.6.

[0010] 3. The engineered Candida tropicalis strain according to claim 1 or 2, characterized in that: the method for knocking out the pyruvate decarboxylase gene PDC2 is to construct the knockout vector pGAPH-ARS2-cas9-tRNA. Ala -sgRNA (PDC2) was then transformed together with donor into the starting strain CT1 for gene knockout.

[0011] Preferably, the present invention involves the introduction of the NAD-dependent methanol dehydrogenase gene MDH. Bs The method is to use the MDH shown in SEQ ID NO:6 Bs The gene was ligated into the pBARGPE1nat vector to obtain pBARGPE1MDH Bs The nat was then transformed into the starting strain CT1.

[0012] In a preferred embodiment of the present invention, the method for introducing the heterologous reducing tricarboxylic acid cycle carbon fixation pathway is to construct the expression vector pBARGPE1AnpycnatAomdh2FumCcpFrd1cp by linking the Anpyc, Aomdh2, FumCcp and Frd1cp genes into the pBARGPE1nat vector, and then transforming it into the starting strain CT1.

[0013] 2. Application of the engineered tropical Candida albicans in the fermentation production of succinic acid using methanol as a carbon source.

[0014] 3. A method for producing succinic acid by anaerobic fermentation using methanol as a carbon source, comprising: under anaerobic conditions, using methanol as a carbon source, culturing the engineered strain of Candida tropicalis as described in any one of claims 15, and fermenting to obtain succinic acid.

[0015] Preferably, the initial concentration of methanol in this invention is 1030 g / L, and more preferably 20 g / L.

[0016] Preferably, the fermentation medium of the present invention further contains yeast extract, and the concentration of the yeast extract is 13 g / L.

[0017] Preferably, the fermentation of the present invention adopts a two-stage fermentation mode: the first stage is a high-density culture under aerobic conditions using glucose as a carbon source, and the second stage is a synthesis of succinic acid using methanol as a carbon source under anaerobic conditions.

[0018] The beneficial effects of this invention are as follows: This invention provides an engineered strain of *Candida tropicalis* that produces succinic acid using methanol as a carbon source, which has the following advantages: 1) A Candida tropicalis engineered strain that synthesizes succinic acid using methanol as the sole carbon source was constructed for the first time. Through metabolic engineering, a complete "methanol-succinic acid" metabolic route was successfully constructed in Candida tropicalis, realizing the anaerobic biotransformation from a one-carbon compound to a C4 platform chemical, providing a new pathway for the green and low-carbon biomanufacturing of succinic acid.

[0019] 2) Significantly improved succinic acid yield and conversion efficiency. When the engineered strain CT7 fermented under anaerobic conditions with methanol as the sole carbon source, the succinic acid yield reached 0.6 g / L. Through optimization of fermentation conditions, in a 5 L fermenter with fed-batch fermentation, the succinic acid yield was further increased to 4.4 g / L, with a production rate of 0.05 g / L·h and an anaerobic stage yield of 0.13 g / g methanol, which is significantly higher than the existing technology for anaerobic synthesis of succinic acid using methanol as a carbon source.

[0020] 3) The challenge of anaerobic methanol metabolism in Candida tropicalis was successfully solved. A methanol dehydrogenase (MDH) with high catalytic efficiency under anaerobic conditions was obtained through screening. BsThis enables engineered bacteria to efficiently utilize methanol for growth and metabolism under anaerobic conditions, providing important technical support for the production of high-value-added chemicals from carbon resources.

[0021] 4) Effective regulation of carbon metabolic flux was achieved. By weakening the ethanol synthesis pathway and knocking out the PDC2 gene, ethanol production was reduced by 56.0%, and carbon metabolic flux was successfully redirected from the byproduct ethanol to the succinic acid synthesis pathway. Succinic acid production was increased by 3.8 times compared with the starting strain, significantly improving carbon conversion efficiency.

[0022] 5) It has good prospects for industrial application. Candida tropicalis has excellent characteristics such as acid resistance and a broad carbon source spectrum. The engineered strain adopts a two-stage fermentation mode. In the growth stage, it uses glucose to rapidly accumulate biomass, and in the acid production stage, it directly uses methanol to synthesize succinic acid. The process is simple and easy to scale up, laying the foundation for industrial production. Attached Figure Description

[0023] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 To illustrate the synthesis of succinic acid from glucose by Candida tropicalis under anaerobic conditions (A: Growth curve of Candida tropicalis under anaerobic conditions with glucose as the sole carbon source; B: Metabolic enhancement diagram of the rTCA pathway in Candida tropicalis; C: Fermentation results of strains CT-1 and CT-2).

[0024] Figure 2 To investigate the effect of knocking out the PDC2 gene on succinic acid synthesis; Figure 3 For the screening of heterologous anaerobic methanol dehydrogenases (A: Growth curves of *Candida tropicalis* with methanol as the sole carbon source under aerobic and anaerobic conditions; B: Schematic diagram of methanol oxidation reaction; C: Anaerobic metabolism of methanol by different methanol dehydrogenase strains; D: Growth curves of different methanol dehydrogenase strains). Figure 4 To synthesize succinic acid using methanol as a carbon source (A: metabolic pathway for producing succinic acid using methanol; B: fermentation results of strains CT-3 and CT-7).

[0025] Figure 5 The effects of different concentrations of carbon and nitrogen sources on succinic acid synthesis (A: fermentation results of different concentrations of methanol; B: fermentation results of different concentrations of yeast extract).

[0026] Figure 6 Batch feeding fermentation of Candida tropicalis CT-7 5-L fermenter. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0028] LB medium (g / L): peptone 10, yeast extract 5, NaCl 10, solid medium with 2% agar powder.

[0029] YPD medium (g / L): 20g peptone, 10g yeast extract, 20g glucose, solid medium with 2% agar powder.

[0030] PTM1 Trace Metal Elements (g / L): Accurately weigh CuSO4·5H2O 6 6, NaI 0.088, MnSO4·H2O 3, Na2MoO4·2H2O 0.2, Boric acid 0.02, CoCl2 0.5, ZnCl2 20, FeSO4·7H2O 65, Biotin 0.2, and 5 mL concentrated sulfuric acid.

[0031] 100× Concentrated Vitamin Solution (g / L): Calcium pantothenate 0.1, Niacin 0.1, Inositol 2.5, Thiamine hydrochloride 0.1, Pyridoxine hydrochloride 0.1, Para-aminobenzoic acid 0.02.

[0032] Fermentation medium: 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 1 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L NaCl, 0.01 g / L CaCl2·2H2O, 0.1 g / L yeast extract, 4 mL / L PTM1 trace metal element, vitamin solution, and different carbon sources added as needed. Except for the trace metal element and concentrated vitamin solution, all media must be sterilized in a high-temperature steam sterilizer at 115 ℃ for 30 min, and carbon and nitrogen sources should be sterilized separately.

[0033] Example 1: Construction of recombinant plasmids and knockout plasmids (1) Construction of recombinant plasmids The exogenous enzyme genes used in this study were: fumocinase gene fumC from *Candida krusei* and methanol dehydrogenase gene MDH from *Bacillus stearothermophilus*. Bs And the methanol dehydrogenase gene mdh from the insecticidal copper-loving bacterium Cupriavidus necator CnAll genes were synthesized by Sangon Biotech Co., Ltd., including the pyruvate carboxylase gene Anpyc from Aspergillus niger, the malate dehydrogenase gene Aomdh2 from Aspergillus oryzae, the fumarate reductase gene frd1 and the alcohol dehydrogenase gene ADH from Saccharomyces cerevisiae. Sc The genomes of the enzymes were amplified separately from their respective strains. The mitochondrial signal peptides of heterologous fumarate enzyme (FumC) and fumarate reductase (Frd1) were predicted and truncated using the online tools DeepLoc 2.1 and TargetP-2.0, and named FumCcp and Frd1cp, respectively. Anpyc, FumCcp, Frd1cp, Aomdh2, and MDH were among the enzymes identified. Cn MDH Bs and ADH Sc The nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0034] First, the bar resistance gene on the pBARGPE1(P0303) plasmid was replaced with the norsinolytic resistance gene nat to obtain the plasmid pBARGPE1-nat.

[0035] The nat gene expression cassette was obtained by amplification using plasmid pAMEXlox-1 (accession number: MH453956) as a template and with the following primers: nat-F: 5'-aggattcaatcttaagaaacattaccttcatgcatccatcggac-3'; nat-R: 5'-aggtagttctggtccattggaattcgggggatctggattttagtact-3'; The plasmid pBARGPE1-nat was linearized by PCR using P1-F / P1-R primers.

[0036] P1-F: 5'-gatcctctagagtcgaggtcgac-3'; P1-R: 5'-gaattcgatatcaagcttatcgatacc-3'; Using the Aspergillus niger genome as a template, the pyruvate carboxylase gene Anpyc was amplified using primers Anpyc-F / Anpyc-R; Anpyc-F: 5'-cgtcgacctcgactctagaggatcctctcattcctctctgtccatc-3'; Anpyc-R: 5'-atcgataagcttgatatcgaattcgttttgaaatcacttgtcattcgt-3'; Using the Aspergillus oryzae genome as a template, the malate dehydrogenase gene Aomdh2 derived from Aspergillus oryzae was amplified using primers Aomdh2-F1 / Aomdh2-R1; Aomdh2-F1: 5'-gacctcgactctagaggatccatggtcaaagctgcggtacttg-3'; Aomdh2-R1: 5'-gataagcttgatatcgaattcttactttggtggtgggttcttaac-3'; Using the Saccharomyces cerevisiae genome as a template, primers Frd-F1 / R1 and ADH were used respectively. Sc -F / R amplification of fumarate reductase gene Frd1cp and alcohol dehydrogenase gene adh Sc ; Frd-F1: 5'-cgacctcgactctagaggatcatggaattggttaacaagtataacatccctgtaaccatcc-3'; Frd-R1: 5'-gataagcttgatatcgaattttacttgcggtcattggcaatagattc-3'; ADHSc-F: 5'-acctcgactctagaggatccatgtcttatcctgagaaatttgaaggt-3'; ADHSc-R: 5'-tcctgcagcccgggggatccctacaacttggaggtcatggact-3'; Using primers FumC-F1 / R1 and MDH respectively Bs -F / R and MDH Cn -F / R amplified codons optimized for FumCcp and mdh Bs and mdh Cn Gene.

[0037] FumC-F1: 5'-acctcgactctagaggatccatgattgctaagagaattgaaaaagat-3'; FumC-R1: 5'-ataagcttgatatcgaattcctaatcttttggaccaatcatattttcaggt-3'; MDH Bs -F: 5'-acctcgactctagaggatccatgaaggccgctgttgttaac-3'; MDH Bs -R: 5'-tcctgcagcccgggggatccctacaacttggaggtcatggactt-3'; MDH Cn -F: 5'-acctcgactctagaggatccatgacccacctgaacatcgctaa-3'; MDH Cn -R: 5'-tcctgcagcccgggggatccctacaacttggaggtcatggac-3'; The above genes were ligated into the linearized vector pBARGPE1-nat using the Gibson Assembly method to obtain recombinant plasmids pBARGPE1-Anpyc-nat, pBARGPE1-Aomdh2-nat, pBARGPE1-Frd1cp-nat, and pBARGPE1-adh. Sc -nat, pBARGPE1-FumCcp-nat, pBARGPE1-mdh Bs -nat and pBARGPE1-mdh Cn -nat.

[0038] Using pBARGPE1-Aomdh2-nat and pBARGPE1-Frd1cp-nat plasmids as templates, the Aomdh2 and FumCcp gene expression cassettes were amplified using primers Aomdh2-F / R and FumC-F / R, respectively, and then ligated into the NheI restriction endonuclease-linearized vector pBARGPE1-Anpyc-nat to obtain the recombinant plasmid pBARGPE1-Anpyc-nat-Aomdh2-FumCcp.

[0039] Aomdh2-F: 5'-aggagtttacgtccagccaagctagctccccagcaggcagaagtatg-3'; Aomdh2-R: 5'-tccacaccctaactgacacacattccacccatctcataaataacgtcatgc-3'; FumC-F: 5'-gtggaatgtgtgtcagttagggtg-3'; FumC-R: 5'-ctacaatgacccgattcttgctagctgttacatgcgtacacgcgc-3'; Using the pBARGPE1-Frd1cp-nat plasmid as a template, the Frd1cp gene expression cassette was amplified with primers Frd-F2 / R2 and ligated into the NdeI restriction endonuclease-linearized vector pBARGPE1-Anpyc-nat-Aomdh2-FumCcp to obtain the recombinant plasmid pBARGPE1-Anpyc-nat-Aomdh2-FumCcp-Frd1cp.

[0040] Frd-F2: 5'-gcggtatttcacaccgcatatggcatgcggagagacggac-3'; Frd-R2: 5'-gtactgagagtgcaccatatggcattgcagatgagctgtatctgg-3'; pBARGPE1-Frd1cp-nat and pBARGPE1-mdh Bs -nat is the template, and primers Spe-frd-F / R and MDH are used respectively. Bs -F1 / R1 amplification of Frd1cp and mdh Bs The gene expression cassette was ligated into the SpeI restriction endonuclease linearized vector pBARGPE1-Anpyc-nat-Aomdh2-FumCcp to obtain the recombinant plasmid pBARGPE1-Anpyc-nat-Aomdh2-FumCcp-Frd1cp-MDH Bs .

[0041] Spe-frd-F: 5'-agattttatgtttagatccactagtgcatgcggagagacggac-3'; Spe-frd-R: 5'-agcctggggactttccacaccgcattgcagatgagctgtatctgg-3'; MDH Bs -F1:5'-ggtgtggaaagtccccagg-3'; MDH Bs -R1: 5'-gcatagtaccgagaaactagtgcattgcagatgagctgtatctg-3'; (2) Construction of knockout plasmids First, using reverse PCR (primers: QC-Ala / GlyPDC2.1-F / QC-AlaPDC2.1-R), the N20 on plasmid pCas9-gRNA (Int-S1) (Nan C, et al. De Novo Biosynthesis of Sabinene from Methanol by Multiple Engineered Pichia pastoris. J. Agric. Food Chem., 2026,74(6): 5405-5416) was replaced with the N20 of the PDC2 gene (5'-tgatcttggtgtaatcagag-3') (designed by the online website CHOPCHOP), and the self-splicing ribozyme 5'-hammerhead enzyme was replaced with tRNA. Ala (tRNA) Ala From tRNA Ala -F and tRNA Ala -R is the primer, obtained by amplification using plasmid pCT-tRNA (#133813, Addgene) as a template. QC-Ala / GlyPDC2.1-F: 5'-tgatcttggtgtaatcagaggttttagagctagaaatagcaagttaaaata-3'; QC-AlaPDC2.1-R:5'-atagttgttcaattgattgaaataggg-3'; tRNA Ala -F:5'-ttcaatcaattgaacaactataaacaaagagcttaaaatgggcgtgtggcgtag-3'; tRNA Ala -R: 5'-ctctgattacaccaagatcaacgagataagaatcgaactcatgacc-3'; The recombinant plasmid was linearized by PCR using primers pGAP-F1 / pGAP-R1. The hyg resistance gene expression cassette was then ligated into the linearized vector (the hyg resistance gene was amplified using hyg-PF and hyg-PR primers and plasmid pK2-hygR (Zhang Y, et al. CRISPR / Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans. Gene, 2019, 709:8-16) as a template), resulting in the knockout plasmid pGAPH-ARS2-cas9-tRNA. Ala -sgRNA(PDC2).

[0042] pGAP-F1:5'-gcttccttttcatcacgtgctat-3'; pGAP-R1: 5'-cagtatagcgaccagcattcaca-3'; hyg-PF: 5'-tgaatgctggtcgctatactgtcgacagaagatgacattgaagg-3'; hyg-TR: 5'-agcacgtgatgaaaaggaagcgcattgcagatgagctgtatctg-3'; The recombinant plasmid was linearized by PCR using primers pCas9-F (SEQ ID NO.44) / pCas9-R (SEQ ID NO.45), and the replicon ARS2 was ligated into the linearized vector (ARS2 was obtained by amplification of plasmid pCT-tRNA using primers ARS2-F (SEQ ID NO.46) and ARS2-R (SEQ ID NO.47) as a template). pCas-F: 5'-gcgctgtgagcaaaaggcca-3'; pCas-R: 5'-gaaaaggaagcgcattgcaga-3'; ARS2-F: 5'-tctgcaatgcgcttccttttcgagaggcggtttgcgtattg-3'; ARS2-R: 5'-ctggccttttgctcacagcgcctgcagctacgaatgttagagaca-3'; Using pUC57-Kna (P0089) plasmid as a template, PCR linearization was performed using primers pUC-F / pUC-R. The upstream and downstream donors of the PDC2 gene were amplified by PCR using the Candida tropicalis CT-1 genome as a template (primers pUC-PDC2-UP700-F / PDC2-nat-R and PDC2-nat-F / pUC-pdc2-down588-R, respectively). The norovirus resistance gene nat expression cassette was amplified using plasmid pAMEXlox-1 as a template (primers loxp-nat-F / loxp-nat-R). This cassette was ligated into the vector pUC57-Kna using the Gibson Assembly method to obtain pUC57-natloxp-PDC2. The PDC2 knockout donor DNA was then amplified by PCR (primers PDC2-UP700-F / PDC2-down588-R).

[0043] pUC-F: 5'-tcaagcggttgaataaatccc-3'; pUC-R: 5'-cttcaatacaggaacagtggcttt-3'; pUC-PDC2-UP700-F: 5'-aaagccactgttcctgtattgaaggaacaatgagataatcgtgcctactctg-3'; PDC2-nat-R: 5'-agcatacattatacgaacggtagaaatggaccagattgaagcctcatc-3'; PDC2-nat-F: 5'-tgtatgctatacgaacggtaggttgcattcaagaaatttttgatagaagaa-3'; pUC-pdc2-down588-R:5'-gggatttattcaaccgcttgaggaaaaccgccacaaaaacggagaac-3'; loxp-nat-F: 5'-taccgttcgtataatgtatgctatacgaagttatattacc-3'; loxp-nat-R: 5'-taccgttcgtatagcatacattatacgaagttattaattcg-3'; PDC2-UP700-F: 5'-gaacaatgagataatcgtgcctactctg-3'; PDC2-down588-R: 5'-gaaaaccgccacaaaaacggag-3'.

[0044] Example 2: Anaerobic synthesis of succinic acid by Candida tropicalis using glucose as a carbon source Succinic acid is mainly synthesized via the reducing TCA (rTCA) pathway. Therefore, *Candida tropicalis* was first cultured under anaerobic conditions to test its ability to grow under anaerobic conditions. Figure 1 As shown in Figure A, this strain can effectively utilize glucose as a carbon source for growth and metabolism under anaerobic conditions. This lays the foundation for its use as an anaerobic fermentation chassis for the production of succinic acid.

[0045] First, a heterologous carbon fixation rTCA pathway was designed and constructed in Candida tropicalis. Figure 1 B). The recombinant plasmid pBARGPE1-Anpyc-nat-Aomdh2-FumCcp-Frd1cp was transformed into strain CT-1 to obtain strain CT-2. Anaerobic fermentation using glucose as the carbon source significantly enhanced the succinic acid synthesis capacity of the engineered strain CT-2. Figure 1 (C). The succinic acid yield of CT-2 reached 2.6 g / L, which was 2.2 times higher than that of wild-type CT-1 (1.2 g / L), indicating that the key enzyme of the heterologous rTCA pathway was functionally expressed in *Candida tropicalis*, promoting succinic acid synthesis. However, the byproduct ethanol yield of CT-2 reached 13.8 g / L, indicating a significant diversion of intracellular carbon metabolic flux. During anaerobic metabolism, a large amount of pyruvate flowed to the ethanol synthesis pathway, which limited further improvement in succinic acid yield.

[0046] Example 3: Effect of weakening the ethanol synthesis pathway on succinic acid synthesis To direct more carbon towards the target product succinic acid, the competitive byproduct ethanol synthesis pathway was weakened. Two pyruvate decarboxylases (PDC1 and PDC2) exist in *Candida tropicalis*, responsible for catalyzing the conversion of pyruvate to acetaldehyde, which in turn produces ethanol. The absence of PDC1 severely impacts yeast growth and metabolism. Therefore, PDC2 was knocked out to weaken the ethanol synthesis pathway.

[0047] Knockout vector pGAPH-ARS2-cas9-tRNA Ala-sgRNA (PDC2) and donor were simultaneously transformed into CT-1 strain to knock out PDC2, and mutant strains with the PDC2 gene successfully knocked out in the CT-1 strain genome were screened. The knockout plasmid was eliminated by antibiotic-free passaging and then transformed into pAMCRE-1 (Wang K, et al. A novel PMA synthetase is the keyenzyme for polymalate biosynthesis and its gene is regulated by a calciumsignaling pathway in Aureobasidium melanogenum ATCC62921. Int. J. Biol. Macromol., 2020, 156: 1053-1063) to eliminate the nat resistance tag on the genome. Then, the recombinant plasmid pBARGPE1-Anpyc-nat-Aomdh2-fumCcp-frd1cp was introduced to obtain engineered strain CT-3. The shake-flask fermentation results are as follows: Figure 2 As shown, the ethanol yield of CT-3 was 4.8 g / L, a decrease of 56.0% compared to the starting strain CT-1. Succinic acid yield was significantly increased, reaching 3.4 g / L, a 3.8-fold increase compared to the starting strain CT-1. These results indicate that knocking out the PDC2 gene effectively reduced ethanol synthesis, successfully redirecting the carbon metabolic flux from the byproduct ethanol to the succinic acid pathway, thus significantly improving the conversion efficiency of succinic acid. Therefore, based on the CT-3 strain, a methanol-metabolizing strain will be constructed.

[0048] Example 4: Screening of heterologous anaerobic methanol dehydrogenase Candida tropicalis was cultured under both aerobic and anaerobic conditions using methanol as the sole carbon source. The results showed that Candida tropicalis exhibits methanol metabolism under aerobic conditions and utilizes methanol for growth. After 48 hours, the OD... 600 The level reached 6. However, under anaerobic conditions, the strain lacked the ability to metabolize methanol, and therefore could not grow. Figure 3 A). To achieve the goal of replacing glucose with methanol as a carbon source using a one-carbon substrate, it is necessary to address the problem of the natural lack of anaerobic methanol metabolism in *Candida tropicalis*. The first step in methanol metabolism is the oxidation of methanol to formaldehyde catalyzed by methanol dehydrogenase (MDH) or methanol oxidase (Aox). Figure 3(B). However, since Aox catalysis requires an oxygen supply, and biochemical reactions using PQQ as a cofactor are rare in microorganisms, PQQ-dependent MDH is difficult to use for biosynthesis in microorganisms. In contrast, NAD-dependent MDH can assimilate methanol under anaerobic conditions and also provide NADH for succinic acid synthesis. Therefore, NAD-dependent MDH was chosen to construct the methanol metabolism pathway in Candida tropicalis.

[0049] This study selected three NAD-dependent methanol dehydrogenases from different sources, namely MDH. Bs MDH Cn and ADH Sc The recombinant plasmids pBARGPE1-MDH were derived from B. stearothermophilus, C. necator, and S. cerevisiae, respectively. Bs -nat, pBARGPE1-MDH Cn -nat and pBARGPE1-ADH Sc -nat was transformed into CT-1 strain to obtain recombinant strain CT-4 (ADH). Sc ), CT-5 (MDH) Cn ) and CT-6 (MDH) Bs Anaerobic fermentation was performed using methanol as the sole carbon source, and the methanol consumption capacity and growth of these strains were evaluated. Figure 3 (C and D). The results showed that expression of MDH Cn and ADH Sc Strains CT-5 and CT-4 exhibited low methanol metabolism capacity, consuming only 0.4 g / L and 0.08 g / L of methanol, respectively, resulting in almost no growth. Conversely, strains expressing MDH... Bs The CT-6 strain exhibits a high methanol metabolism capacity, meaning it can utilize methanol for growth and metabolism, and its OD... 600 The concentration reached 2.9. CT-6 consumed 1.9 g / L of methanol, which was 4.8 times and 23.8 times that of CT-5 and CT-4, respectively. These results demonstrate that, under anaerobic conditions, MDH derived from *B. stearothermophilus*... Bs It exhibits optimal catalytic efficiency in Candida tropicalis and was therefore selected as a key enzyme for constructing a methanol metabolism module.

[0050] Example 5: Anaerobic synthesis of succinic acid using methanol as a carbon source The recombinant plasmid pBARGPE1-Anpyc-nat-Aomdh2-FumCcp-Frd1cp-MDH was used. BsTransformation into CT-3 strain yielded engineered strain CT-7. This strain theoretically possesses the ability to synthesize succinic acid from methanol under anaerobic conditions, such as... Figure 4 As shown in A, methanol is oxidized by methanol dehydrogenase to formaldehyde, which is then catalyzed by dihydroxyacetone synthase (DAS), dihydroxyacetone kinase (DAK), and triose phosphate isomerase (TPI) to enter the glycolysis pathway, and subsequently enters the rTCA cycle to synthesize succinic acid.

[0051] Shake-flask anaerobic fermentation using methanol as the sole carbon source, such as... Figure 4 As shown in Figure B, compared with strain CT-3, strain CT-7 exhibits a stronger methanol metabolism capacity and can metabolize methanol to produce succinic acid. Methanol consumption was 9.1 g / L, and succinic acid production reached 0.6 g / L, indicating that strain CT-7 can synthesize succinic acid using methanol as the sole carbon source. This result marks the first time a complete "methanol-succinic acid" metabolic pathway has been constructed in *Candida tropicalis*, achieving anaerobic biotransformation from a one-carbon compound to a C4 platform chemical.

[0052] Example 6: Optimization of Fermentation Conditions When the engineered strain CT-7 uses methanol as a carbon source, the yield of succinic acid remains relatively low. Therefore, the concentrations of the fermentation substrate and nitrogen source were optimized. First, the effects of different initial methanol concentrations (10 g / L, 20 g / L, 30 g / L) were evaluated. Figure 5 (A). The results showed that when 10 g / L methanol was used as the carbon source, almost 100% of the methanol was consumed, producing 0.6 g / L succinic acid. When the methanol concentration was increased to 20 g / L, 10.8 g / L methanol was consumed, producing 0.7 g / L succinic acid, an increase of 16.7% compared to adding 10 g / L methanol. However, when the methanol concentration was increased to 30 g / L, compared to adding 20 g / L methanol, both methanol consumption (9.0 g / L) and succinic acid concentration (0.6 g / L) decreased, possibly because the toxic effect of high-concentration methanol inhibits cell growth and metabolism, thereby reducing the product concentration. Therefore, 20 g / L methanol was chosen as the carbon source next.

[0053] Yeast extract, as an organic nitrogen source, can be used as a readily available nitrogen source and growth factor supplement for microbial growth and metabolism. When methanol is used as a carbon source, yeast extract can promote methanol metabolism and product partitioning. Therefore, this study evaluated the effect of different concentrations of yeast extract on the production of succinic acid from methanol. Figure 5As shown in Figure B, the addition of yeast extract significantly promoted the metabolism of methanol and the synthesis of its products. When 1 g / L of yeast extract was added, succinic acid production increased to 1.1 g / L. Methanol consumption and succinic acid concentration increased with increasing yeast extract concentration. When the yeast extract concentration increased to 3 g / L, methanol consumption increased dramatically to 15.93 g / L, and succinic acid production increased to 1.64 g / L, representing increases of 65.6% and 45.5%, respectively, compared to the addition of 1 g / L yeast extract. These results indicate that supplementing with abundant organic nitrogen sources is a key strategy for promoting methanol metabolism and succinic acid synthesis.

[0054] Example 7: Production of succinic acid by batch-fed fermentation in a 5-L fermenter To further verify the performance of the engineered strain in synthesizing succinic acid using methanol as a carbon source under scale-up conditions, fed-batch fermentation was performed in a 5-L fermenter. The specific steps are as follows: The initial liquid volume of the 5-L fermenter was 1.372 L. 0.4 L of seed culture was inoculated into the fermenter, and 0.2 L of 500 g / L glucose, 8 mL of PTM1 trace metal element and 20 mL of vitamin solution were added at the same time.

[0055] Because *Candida tropicalis* grows rapidly and has a high biomass under aerobic conditions, a two-stage fed-batch fermentation method was adopted to achieve high-density fermentation. The initial fermentation conditions for the first stage were controlled as follows: 30 °C, 600 r / min, aeration rate of 5 L / min, and fermentation time of 12 h. Under aerobic conditions, biomass was accumulated using glucose as the carbon source. After 12 h of cultivation, the OD... 600 The cell density rapidly increased from 0.4 to 82.1, achieving high-density cell accumulation. At this point, glucose was completely depleted, effectively avoiding the inhibitory effect of glucose on subsequent methanol metabolism. Then, the air was replaced with CO2, and the rotation speed was adjusted to 250 r / min, with an aeration rate of 0.8 L / min. Methanol was added to maintain its concentration at 20 g / L. This process utilized methanol to produce succinic acid under anaerobic conditions. Throughout the fermentation process, the pH of the fermentation broth was controlled at 6.5 by automatically adding 20% ​​NaOH solution. At 84 h of fermentation, the succinic acid yield and production rate were 4.4 g / L and 0.05 g / L·h, respectively, with a yield of 0.13 g / g methanol in the anaerobic stage. During the anaerobic acid production stage, the cell OD value increased slightly and remained around 95, indicating that the strain maintained high activity during methanol metabolism. Continued fermentation could further increase the succinic acid yield. Figure 6 ).

[0056] Succinic acid is the most valuable C4 platform compound in the rTCA cycle, serving as a precursor for compounds such as 1,4-butanediol, tetrahydrofuran, and polybutylene succinate. Currently, the chemical synthesis of succinic acid relies on petroleum-based feedstocks, is energy-intensive, and causes severe environmental pollution. Therefore, researchers have conducted extensive work attempting to replace traditional chemical synthesis methods with microbial fermentation. However, current mainstream technologies still use grain-based carbohydrates such as glucose and sucrose as carbon sources, leading to resource conflicts such as competition with food production and land use. Therefore, developing succinic acid synthesis technologies based on non-grain carbon sources (such as methanol) has become a new breakthrough.

[0057] Based on this, this study utilized metabolic engineering to introduce a heterologous rTCA carbon fixation pathway into *Candida tropicalis*, significantly improving succinic acid production under anaerobic conditions. By weakening the ethanol synthesis pathway and altering the carbon flow, the engineered strain increased succinic acid production by 3.8 times when using glucose as a substrate. Furthermore, a methanol dehydrogenase (MDH) with high catalytic efficiency under anaerobic conditions was obtained through screening. Bs This study solved the problem of anaerobic methanol metabolism in chassis cells. By integrating the methanol metabolism module with the succinic acid synthesis module, de novo synthesis of succinic acid using methanol as the sole carbon source was successfully achieved in *Candida tropicalis*. Through optimization of fermentation conditions, it was found that supplementing with yeast extract could promote both methanol metabolism and succinic acid synthesis. Finally, in a 5-L fed-batch fermentation tank, the succinic acid yield reached 4.4 g / L.

[0058] Currently, microbial production of succinic acid mainly relies on bacteria (such as *Escherichia coli*) and yeast. However, most bacteria can only grow under neutral pH conditions, while yeast has good robustness, especially its acid-resistant properties. Therefore, using yeast as a cell factory to produce succinic acid has become a research hotspot. In *Yarrowia lipolytica*, some studies have achieved succinic acid production using methanol as a carbon source through metabolic engineering and xylose as a common carbon source, but the yield was only 0.9 g / L. *Candida tropicalis*, as an unconventional yeast, has excellent dicarboxylic acid production capacity, strong acid resistance, and a broad carbon source spectrum (it can utilize non-grain carbon sources, such as alkanes and fatty acids), making it a potential host for succinic acid production. The engineered *Candida tropicalis* strain constructed in this study produced 4.4 g / L of succinic acid using methanol as the sole carbon source, which is higher than the current level of anaerobic succinic acid synthesis using methanol as a carbon source. The two-stage fermentation model fully utilizes the advantages of high-density fermentation. During the growth stage, biomass is rapidly accumulated using glucose, and then the anaerobic acid production stage is directly entered, which can fully convert methanol into the final product.

[0059] Although the production of methanol-based succinic acid has been successfully achieved, industrialization is still some distance away. How to further improve methanol metabolism and carbon conversion efficiency via the rTCA pathway are key issues to be addressed in methanol-based succinic acid production. After methanol is oxidized to formaldehyde intracellularly, if it cannot enter downstream metabolism in a timely manner, formaldehyde accumulation will cause significant toxicity to cells. Therefore, future research will accelerate formaldehyde detoxification and conversion by heterologously expressing formaldehyde lyase, while simultaneously enhancing the XuMP pathway (…). Figure 4 The expression of key enzymes (A) increases the flux of methanol into the central metabolic process, reducing the inhibition of cell growth and metabolism caused by metabolic bottlenecks and intermediate product accumulation. The free expression system currently used in this study has certain limitations: as the number of heterologous genes increases, the construction of multi-gene recombinant plasmids becomes more difficult; simultaneously, free plasmids are easily lost intracellularly, requiring continuous antibiotic addition to maintain stable inheritance, which not only increases production costs but also burdens industrial scale-up. Therefore, future work will focus on optimizing the CRISPR system or using the PiggyBac transposon system to integrate key heterologous genes into the genome, constructing stable expression strains that do not rely on antibiotic selection, thereby improving the genetic stability and industrial applicability of the strains.

[0060] In summary, this study successfully constructed the first tropical Candida cell factory capable of synthesizing succinic acid using methanol as the sole carbon source. This work not only provides a promising new route for the green and low-carbon biomanufacturing of succinic acid, but also offers important theoretical basis and technical support for non-model yeasts to utilize one-carbon resources to produce high-value-added chemicals.

[0061] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. An engineered strain of *Candida tropicalis* that produces succinic acid using methanol as a carbon source, characterized in that... The engineered strain was obtained from the original strain CT1 of *Candida tropicalis* through the following modifications: (1) Introduce the heterologous reducing tricarboxylic acid cycle (rTCA) carbon fixation pathway into the starting strain, the pathway comprising the pyruvate carboxylase gene Anpyc, the malate dehydrogenase gene Aomdh2, the fumarate gene FumCcp and the fumarate reductase gene Frd1cp. (2) Knock out the pyruvate decarboxylase gene PDC2; (3) Introducing the NAD-dependent methanol dehydrogenase gene MDH Bs .

2. The engineered Candida tropicalis strain according to claim 1, characterized in that, The nucleotide sequence of Anpyc is shown in SEQ ID NO.1, the nucleotide sequence of FumCcp is shown in SEQ ID NO.2, the nucleotide sequence of Frd1cp is shown in SEQ ID NO.3, the nucleotide sequence of Aomdh2 is shown in SEQ ID NO.4, and the nucleotide sequence of MDH is shown in SEQ ID NO.

4. Bs The nucleotide sequence is shown in SEQ ID NO.

6.

3. The engineered Candida tropicalis strain according to claim 1 or 2, characterized in that: The method for knocking out the pyruvate decarboxylase gene PDC2 is to construct the knockout vector pGAPH-ARS2-cas9-tRNA. Ala -sgRNA (PDC2) was then transformed together with donor into the starting strain CT1 for gene knockout.

4. The engineered Candida tropicalis strain according to claim 1 or 2, characterized in that: The introduction of the NAD-dependent methanol dehydrogenase gene MDH Bs The method is to use the MDH shown in SEQ ID NO:6 Bs The gene was ligated into the pBARGPE1nat vector to obtain pBARGPE1MDH Bs nat.

5. The engineered Candida tropicalis strain according to claim 1 or 2, characterized in that: The method for introducing the heterologous reducing tricarboxylic acid cycle carbon fixation pathway is to construct the expression vector pBARGPE1AnpycnatAomdh2FumCcpFrd1cp by ligating the Anpyc, Aomdh2, FumCcp and Frd1cp genes into the pBARGPE1nat vector, and then transforming it into the starting strain CT1.

6. The application of the engineered tropical Candida albicans according to any one of claims 1 to 5 in the fermentation production of succinic acid using methanol as a carbon source.

7. A method for producing succinic acid by anaerobic fermentation using methanol as a carbon source, characterized in that, include: Under anaerobic conditions, using methanol as a carbon source, the engineered strain of Candida tropicalis as described in any one of claims 15 is cultured and fermented to obtain succinic acid.

8. The method according to claim 7, characterized in that, The initial concentration of methanol is 1030 g / L, preferably 20 g / L.

9. The method according to claim 6 or 7, characterized in that, The fermentation medium also contains yeast extract at a concentration of 13 g / L.

10. The method according to any one of claims 1, characterized in that, The fermentation adopts a two-stage fermentation mode: the first stage is a high-density culture under aerobic conditions using glucose as a carbon source, and the second stage is the synthesis of succinic acid under anaerobic conditions using methanol as a carbon source.

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