A high-efficiency carbon dioxide fixing strain for improving succinic acid conversion rate and a construction method thereof

Abstract

The application belongs to the technical field of biology, and discloses a strain for efficiently fixing carbon dioxide and improving succinic acid conversion rate and a construction method thereof. The application discloses a genetically engineered bacterium, which is obtained by overexpressing carbonic anhydrase genes, phosphoenolpyruvate carboxylase genes and phosphoenolpyruvate carboxykinase genes in a succinic acid-producing strain. ‑ The carbonic anhydrase can enhance the conversion efficiency of CO2 to HCO3 , and the phosphoenolpyruvate carboxylase and the phosphoenolpyruvate carboxykinase can realize efficient fixation of C3 and CO2 into C4 intermediates. Experiments prove that the genetically engineered bacterium can effectively produce succinic acid by using glucose, and has a relatively high sugar acid conversion rate (up to 79.80%).

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain that efficiently fixes carbon dioxide to improve succinic acid conversion rate and its construction method. Background Technology

[0002] Succinic acid (Succinic acid) is an important bio-based platform compound with wide applications in pharmaceuticals, biodegradable plastics (such as polybutylene succinate PBS), and food additives. Traditional chemical synthesis methods rely on petroleum resources, resulting in high energy consumption, high pollution, and unsustainability. Microbial fermentation, using renewable resources (such as glucose, cassava, and industrial waste gas CO2) as raw materials, offers advantages such as being environmentally friendly, energy-efficient, and capable of CO2 fixation, making it a research hotspot. Examples include the synthesis of succinic acid using microbial CO2 fixation. However, current bio-fermentation technologies still face challenges such as low CO2 fixation efficiency and low conversion rates. Many microbial strains have insufficient CO2 assimilation capacity, leading to low succinic acid yields; uneven carbon metabolic flux distribution results in the accumulation of byproducts (such as acetic acid, ethanol, and lactic acid). Therefore, recent research has focused on metabolic engineering, immobilized fermentation, and CO2 co-utilization technologies, but further optimization is still possible. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. It provides a genetically engineered strain that improves succinic acid synthesis efficiency by enhancing the CO2 fixation pathway, optimizing carbon metabolic flux allocation, and increasing the strain's environmental tolerance.

[0004] The first objective of this invention is to provide a genetically engineered bacterium.

[0005] The second objective of this invention is to provide an acid-resistant genetically engineered bacterium.

[0006] The third aspect of the present invention is to provide a Kudriaz Pichia pastoris.

[0007] The fourth aspect of this invention is to provide a method for preparing succinic acid.

[0008] The fifth aspect of this invention aims to provide the use of the genetically engineered bacteria of the first aspect of this invention, the acid-resistant genetically engineered bacteria of the second aspect of this invention, or the Pichia kudriaz of the third aspect of this invention in the preparation of succinic acid or the preparation of products containing succinic acid.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a genetically engineered bacterium obtained by overexpressing a carbonic anhydrase gene, a phosphoenolpyruvate carboxylase gene, and a phosphoenolpyruvate carboxylkinase gene in a succinic acid-producing strain.

[0011] In some embodiments of the present invention, the overexpression of the carbonic anhydrase gene, the phosphoenolpyruvate carboxylase gene, and the phosphoenolpyruvate carboxylkinase gene includes the following methods:

[0012] A) Increase the copy number of the genes encoding carbonic anhydrase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxylkinase; and / or,

[0013] B) Alter the regulatory sequences of the carbonic anhydrase gene, phosphoenolpyruvate carboxylase gene, and phosphoenolpyruvate carboxylkinase gene.

[0014] In some embodiments of the present invention, A) includes integrating homologous or heterologous carbonic anhydrase genes, phosphoenolpyruvate carboxylase genes and phosphoenolpyruvate carboxylkinase genes into the genome of the strain, or transforming the strain into an expression vector carrying homologous or heterologous carbonic anhydrase genes, phosphoenolpyruvate carboxylase genes and phosphoenolpyruvate carboxylkinase genes.

[0015] In some embodiments of the present invention, B) includes optimizing the nucleic acid sequence of the regulatory sequence, or increasing the number of promoters and / or enhancers.

[0016] In some embodiments of the present invention, the carbonic anhydrase gene is derived from 蓝细菌 , 嗜氨热弧菌 , 嗜热甲烷八叠球菌 and 硫氢基菌属 阿佐尔菌 Any one of them.

[0017] In some embodiments of the present invention, the phosphoenolpyruvate carboxykinase gene is derived from... 酿酒酵母 , 硕大利什曼原虫 , 嗜热栖热放线菌 , 拟南芥 and 克氏锥虫 Any one of them.

[0018] In some embodiments of the present invention, the phosphoenolpyruvate carboxylase gene is derived from... 东北南星 , 甘蓝型油菜 , 天蓝色链霉菌 , 柄杆菌属 新种 and 三角褐指藻 Any one of them.

[0019] In some embodiments of the present invention, the nucleotide sequence of the carbonic anhydrase gene is as shown in any one of SEQ ID NO:1-SEQ ID NO:4; or a nucleotide sequence that has at least 70% similarity to and is functionally identical or similar to the nucleotide sequence shown in any one of SEQ ID NO:1-SEQ ID NO:4.

[0020] In some embodiments of the present invention, the nucleotide sequence of the carbonic anhydrase gene is as shown in SEQ ID NO:1; or a nucleotide sequence having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to the nucleotide sequence shown in SEQ ID NO:1 and having the same or similar function.

[0021] In some embodiments of the present invention, the nucleotide sequence of the phosphoenolpyruvate carboxylase gene is as shown in any one of SEQ ID NO:17-SEQ ID NO:21; or a nucleotide sequence that has at least 70% similarity to and is functionally identical or similar to the nucleotide sequence shown in any one of SEQ ID NO:17-SEQ ID NO:21.

[0022] In some embodiments of the present invention, the nucleotide sequence of the phosphoenolpyruvate carboxylase gene is as shown in SEQ ID NO:17; or a nucleotide sequence having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to the nucleotide sequence shown in SEQ ID NO:17 and having the same or similar function.

[0023] In some embodiments of the present invention, the nucleotide sequence of the phosphoenolpyruvate carboxykinase gene is as shown in any one of SEQ ID NO:34-SEQ ID NO:38; or a nucleotide sequence that has at least 70% similarity to and is functionally identical or similar to the nucleotide sequence shown in any one of SEQ ID NO:34-SEQ ID NO:38.

[0024] In some embodiments of the present invention, the nucleotide sequence of the phosphoenolpyruvate carboxykinase gene is as shown in SEQ ID NO:34; or a nucleotide sequence having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to the nucleotide sequence shown in SEQ ID NO:34 and having the same or similar function.

[0025] In some preferred embodiments of the present invention, expression cassettes for carbonic anhydrase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxylkinase, as well as their upstream and downstream homologous arms, are constructed respectively. The expression cassettes and their upstream and downstream homologous arms are transformed into the strains using electroporation. After culturing and screening, genetically engineered strains expressing the carbonic anhydrase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxylkinase genes heterologously are obtained.

[0026] In some embodiments of the present invention, the carbonic anhydrase gene, the phosphoenolpyruvate carboxylase gene, and / or the phosphoenolpyruvate carboxylkinase gene are integrated into the genome of the strain using a CRISPR / Cas gene editing system.

[0027] In some preferred embodiments of the present invention, the carbonic anhydrase gene, the phosphoenolpyruvate carboxylase gene, and / or the phosphoenolpyruvate carboxylkinase gene are sequentially integrated into the genome of the strain using the following method:

[0028] Taking the integration of the carbonic anhydrase gene as an example, using the genome of Pichia kudriaz S9 (disclosed in CN119614400A, accession number GDMCC No: 65148) as a template, gene fragments pPGK1 and tTDH3 were amplified; the gene fragments pPGK1, carbonic anhydrase gene (CA), and tTDH3 were subjected to overlap extension PCR to obtain the targeted fragment;

[0029] Using the genome of Pichia kudriaz S9 as a template, the upstream and downstream homologous arm fragments of the carbonic anhydrase gene expression cassette were amplified.

[0030] The target fragment, upstream homologous arm fragment, downstream homologous arm fragment, and CAS9-gRNA fusion plasmid were mixed and transformed into the strain using electroporation to obtain a genetically engineered strain that heterologously expresses the carbonic anhydrase gene.

[0031] Using the same method described above, the phosphoenolpyruvate carboxylase gene and the phosphoenolpyruvate carboxylkinase gene were sequentially integrated into the strain.

[0032] In some embodiments of the present invention, the strain includes *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). 酵母属 酿酒酵母 ), Escherichia coli ( 大肠杆菌 ), Succinic acid-producing Actinobacillus ( 放线杆菌属 产琥珀酸放线杆菌 ), Mannheim-derived succinic acid bacteria ( 产琥珀酸曼海姆氏菌 ), succinic acid-producing anaerobic spirochetes ( 产琥珀酸厌氧螺菌 Klebsiella pneumoniae ( 克雷伯氏菌属 ), Yarrowia lipolytica ( 解脂耶氏酵母 ) and Pichia pastoris ( 季也蒙毕赤酵母 At least one of the following.

[0033] In some embodiments of the present invention, the strain is Pichia kudriaz S9-4, with preservation number GDMCC No: 67259.

[0034] In some embodiments of the present invention, the genetically engineered bacteria are derived from heterologous expression of Pichia pastoris S9-4 in *Pichia kudriaz*. 蓝细菌 The carbonic anhydrase gene, derived from 东北南星 Phosphoenolpyruvate carboxylase gene and derived from 酿酒酵母 The phosphoenolpyruvate carboxylkinase gene was obtained, that is, the genetically engineered bacterium is Pichia kudriaz S9-4 containing the carbonic anhydrase gene shown in SEQ ID NO:1, the phosphoenolpyruvate carboxylase gene shown in SEQ ID NO:17, and the phosphoenolpyruvate carboxylkinase gene shown in SEQ ID NO:34.

[0035] In a second aspect, the present invention provides an acid-resistant genetically engineered bacterium, wherein the acid-resistant genetically engineered bacterium is obtained by acid domestication of the genetically engineered bacterium of the first aspect of the present invention.

[0036] In some embodiments of the present invention, the acid domestication process is as follows: the genetically engineered bacteria of the first aspect of the present invention are cultured and passaged sequentially in a culture medium containing succinic acid of varying concentrations to obtain acid-resistant genetically engineered bacteria.

[0037] In some embodiments of the present invention, the genetically engineered bacteria of the first aspect of the present invention are activated before domestication, specifically including: streaking the genetically engineered bacteria on a solid culture medium, picking single colonies and culturing them in a liquid culture medium at 25-40℃ (such as any value or a range formed by any two of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40℃) and 150-300 rpm (such as any value or a range formed by any two of 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 rpm) until OD. 600 6-8.

[0038] In some embodiments of the present invention, the activated genetically engineered bacteria are sequentially placed in liquid culture media containing different concentrations of succinic acid (from low to high), cultured, and the number of passages for each gradient is adjusted according to the growth of the strains to screen for acid-resistant genetically engineered bacteria.

[0039] In some embodiments of the present invention, the culture medium includes media commonly used for culturing Pichia kudriaz, such as YM medium (containing 10 g / L glucose, 5 g / L peptone, 3 g / L yeast extract and 3 g / L malt extract; solid medium also contains 20 g / L agar), YPD medium, PYG medium (containing 1 g / L glucose, 10 g / L peptone and 5 g / L yeast extract, pH 6.8-7.0±0.2; solid medium also contains 15 g / L agar), etc.

[0040] By acclimatizing genetically engineered bacteria to acid tolerance, they can maintain good growth and fermentation performance in a lower pH environment, thereby effectively consuming glucose and fermenting it to produce succinic acid under lower pH conditions, thus improving the sugar-acid conversion rate.

[0041] A third aspect of the present invention provides a *Pichia kudriaz* yeast, the preservation number of which is GDMCC No:66847.

[0042] The *Pichia kudriaz* yeast is deposited at the Guangdong Provincial Microbial Culture Collection Center, under the name... 毕赤酵母属 季也蒙毕赤酵母 TSA 16, deposited on August 15, 2025, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0043] A fourth aspect of the present invention provides a method for preparing succinic acid, comprising the following steps: fermentation using the genetically engineered bacteria of the first aspect of the present invention, the acid-resistant genetically engineered bacteria of the second aspect of the present invention, or the Pichia kudriaz of the third aspect of the present invention as the generating strain.

[0044] In some embodiments of the present invention, the carbon source used for fermentation includes at least one selected from glucose, xylose, glycerol, sucrose, maltose, molasses, fructose, rhamnose, arabinose, and sorbitol; preferably glucose.

[0045] In some embodiments of the present invention, the nitrogen source used for fermentation includes one or more of yeast extract, peptone, yeast powder, corn steep liquor, urea, ammonia, ammonium sulfate, potassium nitrate, and ammonium nitrate; preferably yeast extract or peptone.

[0046] In some embodiments of the present invention, the pH value of the fermentation system is 2.5-5, such as any value or a range formed by any two of the following: 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.7, 3.9, 4.1, 4.3, 4.5, 4.7, 4.9, 5.

[0047] In some embodiments of the present invention, the fermentation temperature is 25-34°C, such as any value of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34°C or a range formed by any two of them.

[0048] In some embodiments of the present invention, the fermentation speed is 150-350 rpm, such as any value or a range formed by any two of the following: 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 rpm.

[0049] In some embodiments of the present invention, the fermentation time is 10-100h, such as any value or a range formed by any two of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100h.

[0050] In some embodiments of the present invention, during the fermentation process, gas is introduced into the system at a flow rate of 0.1-1 L / min, such as any value or a range formed by any two of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 L / min.

[0051] In some embodiments of the present invention, the gas is sterile air.

[0052] In some embodiments of the present invention, in the fermentation system, the initial OD of the generating strain is... 600 It is a range of 0.05-0.3, such as any value or any combination of 0.07, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3.

[0053] In some embodiments of the present invention, before fermentation, the generating strain is activated to obtain a seed culture, including the following steps: streaking the generating strain on a solid culture medium, picking single colonies and culturing them in a liquid culture medium at 25-40℃ (such as any value or a range formed by any two of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40℃) and 150-300 rpm (such as any value or a range formed by any two of 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 rpm) until OD. 600 4-8.

[0054] In some embodiments of the present invention, the culture medium includes media commonly used for culturing Pichia kudriaz, such as YM medium (containing 10 g / L glucose, 5 g / L peptone, 3 g / L yeast extract and 3 g / L malt extract; solid medium also contains 20 g / L agar), YPD medium, PYG medium (containing 1 g / L glucose, 10 g / L peptone and 5 g / L yeast extract, pH 6.8-7.0±0.2; solid medium also contains 15 g / L agar), etc.

[0055] A fifth aspect of the present invention provides the use of the genetically engineered bacteria of the first aspect of the present invention, the acid-resistant genetically engineered bacteria of the second aspect of the present invention, the Pichia kudriaz of the third aspect of the present invention, or the method of the fourth aspect of the present invention in the preparation of succinic acid or the preparation of products containing succinic acid.

[0056] The beneficial effects of this invention are:

[0057] This invention provides a genetically engineered bacterium that heterologously expresses the encoding genes for carbonic anhydrase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxylkinase, wherein the carbonic anhydrase can enhance the conversion of CO2 to HCO3. -The conversion efficiency was improved by using phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylkinase to achieve efficient fixation of the C3-to-C4 intermediate from CO2. Experiments demonstrated that this genetically engineered bacterium can effectively utilize glucose to produce succinic acid, exhibiting a high glucose-to-acid conversion rate (up to 79.80%).

[0058] During succinic acid production, the pH value of the system decreases, which affects the effective growth of the strain and the yield of succinic acid in low pH environments. To solve this problem, this invention further acid-acclimated the aforementioned genetically engineered bacteria to obtain acid-resistant genetically engineered bacteria. These acid-resistant bacteria can adapt to low pH environments (e.g., pH 2.7) and grow and ferment normally at pH 2.7, thus achieving efficient glucose consumption and effective fermentation to produce succinic acid at lower pH values. Experiments show that compared to the unacclimated state, the rate of glucose consumption and the yield of succinic acid in the fermentation system can be significantly increased (glucose-acid conversion rate can reach 80.5%). There was no significant difference in fermentation intensity and conversion rate between pH 2.7 and pH 3.1 conditions for these acid-resistant genetically engineered bacteria. Attached Figure Description

[0059] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0060] Figure 1 The results show the yield of succinic acid in the fermentation system of Pichia pastoris strains S9-4, S9-CYCA, S9-CYCA-SRPPC, S9-CYCA-SRPPC-SCPCK and SA-S9-CYCA-SRPPC-SCPCK under pH 3.1 (NaOH as neutralizing agent).

[0061] Figure 2 The glucose consumption of Pichia pastoris strains S9-4 and S9-CYCA-SRPPC-SCPCK during fermentation at pH 3.1 (with NaOH as a neutralizing agent) is shown.

[0062] Figure 3 The results show the yield of succinic acid during fermentation of Pichia pastoris strains S9-4 and S9-CYCA-SRPPC-SCPCK at pH 3.1 (with NaOH as a neutralizing agent).

[0063] Figure 4 The glucose consumption of Pichia pastoris strains S9-CYCA-SRPPC-SCPCK and SA-S9-CYCA-SRPPC-SCPCK during fermentation at pH 2.7 (with NaOH as a neutralizing agent) is shown.

[0064] Figure 5The results show the yield of succinic acid during fermentation of Pichia pastoris strains S9-CYCA-SRPPC-SCPCK and SA-S9-CYCA-SRPPC-SCPCK at pH 2.7 (with NaOH as a neutralizing agent).

[0065] Figure 6 The content of byproducts such as ethanol, glycerol, malic acid and lactic acid in the fermentation system of Pichia pastoris strains S9-4 and SA-S9-CYCA-SRPPC-SCPCK under pH 3.1 (NaOH as neutralizing agent) was determined. Detailed Implementation

[0066] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0067] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0068] YPD liquid medium: Weigh 50g glucose, 20g peptone, and 10g yeast extract, and bring the volume to 1L with pure water. Autoclave at 115℃ for 20 minutes. For YPD solid medium (plates), add 2% agar powder and pour into plates to solidify. Store the prepared medium and plates at 4℃ for later use.

[0069] In this embodiment of the invention, succinic acid, glucose, glycerol, ethanol, malic acid, and lactic acid were all quantitatively analyzed by high-performance liquid chromatography (HPLC). A Waters 2695 HPLC system was used, with a Milford RI-2414 differential refractive index detector and a Bio-Rad Aminex HPX-87H column (300 × 7.8 mm). The mobile phase was 2.5 mM dilute sulfuric acid.

[0070] HPLC detection method. The specific method is as follows: set the autosampler program, the injection volume is 10 μL, the mobile phase flow rate is 0.6 mL / min, the column oven temperature is 40℃, and the analysis time is 25 min.

[0071] Preparation of the mobile phase. The mobile phase was 2.5 mM dilute sulfuric acid. Measure 2 L of ultrapure water (18.2 MΩ) into a reagent bottle, take a small amount (about 20 mL) of ultrapure water into a beaker, add 280 μL of concentrated acid, filter it through a 0.22 μm filter membrane to remove bacteria, add it to the ultrapure water, and sonicate to degas for 30 min.

[0072] Construction of standard curves. Standard curves for succinic acid, glucose, glycerol, ethanol, malic acid, and lactic acid were prepared: mixed standard solutions with concentration gradients of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 g / L were prepared using the mobile phase and filtered into chromatographic vials using a 0.22 μm filter membrane. After liquid chromatography, the peak areas of succinic acid, glucose, glycerol, ethanol, malic acid, and lactic acid were recorded, and the corresponding standard curves were plotted.

[0073] Sample preparation. Take 200 μL of the fermentation broth into a 1.5 mL centrifuge tube, add 800 μL of mobile phase, mix well, filter through a 0.22 μm filter membrane into a chromatographic vial, and then perform HPLC analysis.

[0074] The inventive concept of this invention is as follows: In a bacterial strain (such as *Pichia gondii*), the synthesis of succinic acid from glucose involves a reduced TCA cycle, requiring glycolysis to generate phosphoenolpyruvate, which then generates pyruvate. Pyruvate is then converted to oxaloacetate via CO2 fixation by pyruvate carboxylase, followed by a three-step reaction to produce succinic acid. The carbon fixation step is the rate-limiting step in this pathway, and its reaction flux is crucial for improving the synthesis and conversion rate of succinic acid. However, the activity of pyruvate carboxylase is relatively low, and the conversion of phosphoenolpyruvate to pyruvate and then to oxaloacetate requires two steps. Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylkinase can directly catalyze the carbon fixation of phosphoenolpyruvate to oxaloacetate, and their activity is higher than that of pyruvate carboxylase. Therefore, introducing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylkinase into the strain can not only shorten the reaction steps but also enhance the CO2 fixation module. In addition, the direct substrate of phosphoenolpyruvate carboxylase is HCO3-. - Strains that heterologously express carbonic anhydrase can increase the reaction of CO2 molecules with HCO3. - The rate of ion conversion within the cell allows CO2 molecules that enter the cell via free diffusion to be converted into HCO3 more quickly. - Ions enhance HCO3 - The increased supply of ions leads to a higher yield of succinic acid. However, the increased succinic acid concentration and decreased pH during the later stages of fermentation in succinic acid-producing strains negatively impact strain activity and growth. Therefore, acid tolerance acclimation allows the strain to ferment and produce succinic acid normally under low pH conditions, significantly improving succinic acid synthesis efficiency.

[0075] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0076] Example 1

[0077] This embodiment uses different sources (including) 蓝细菌 , 嗜氨热弧菌 , 嗜热甲烷八叠球菌 and 阿佐尔硫氢基菌 The carbonic anhydrase (CA) gene in Pichia pastoris (Kudriaz pichia) 季也蒙毕赤酵母 Different recombinant strains were obtained by overexpressing carbonic anhydrase from different sources, and the effects of heterologous expression of carbonic anhydrase from different sources on succinic acid production by Pichia kudriaz.

[0078] 1. Constructing *Pichia pastoris* strains heterologously expressing carbonic anhydrases from different sources.

[0079] 蓝细菌 , 嗜氨热弧菌 , 嗜热甲烷八叠球菌 and 阿佐尔硫氢基菌 The nucleotide sequences of the derived carbonic anhydrase gene are shown in SEQ ID NO:1-SEQ ID NO:4.

[0080] With cyanobacteria ( 蓝细菌 Taking carbonic anhydrase from this source as an example, it is found in *Pichia pastoris* S9-4 (which has been preserved at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) under the name...). 季也蒙毕赤酵母 S9-4, taxonomic name is 季也蒙毕赤酵母 The document, with accession number GDMCC No:67259, accession date November 10, 2025, and accession location at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, has been expressed in the following manner:

[0081] (1) Construction of carbonic anhydrase gene expression cassette

[0082] A synthetically produced carbonic anhydrase gene derived from cyanobacteria, controlled by the pPGK1 promoter and tTDH3 terminator (SEQ ID NO:1). Using the genome of *Pichia pastoris* S9 (disclosed in CN119614400A, accession number GDMCC No:65148) as a template, PCR was performed using primers V7-pPGK1-F (SEQ ID NO:5) and CA-pPGK1-R (SEQ ID NO:6) to obtain the pPGK1 gene fragment (1000 bp). The obtained PCR product was purified using a PCR purification kit (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: D0033).

[0083] Using the genome of Pichia kudriaz S9 as a template, PCR was performed with primers CA-tTDH3-F (SEQ ID NO:7) and tTDH3-V7-R (SEQ ID NO:8) to obtain the gene fragment tTDH3 (500bp), and the PCR product was purified.

[0084] Using the artificially synthesized CA gene as a template, PCR was performed with primers pPGK1-CA-F (SEQ ID NO:9) and tTDH3-CA-R (SEQ ID NO:10) to obtain the gene fragment CA (783bp), and the obtained PCR product was purified using a PCR purification kit.

[0085] The fragments pPGK1, CA and tTDH3 were subjected to overlap extension PCR using primers V7-pPGK1-F (SEQ ID NO:5) and tTDH3-V7-R (SEQ ID NO:8) to obtain the targeted fragment pPGK1-CA-tTDH3. The obtained PCR product was then purified using a PCR purification kit.

[0086] (2) Construction of upstream and downstream homologous arms of carbonic anhydrase gene expression cassette

[0087] Using the genome of Pichia kudriaz S9 as a template, PCR was performed with primers V7-F (SEQ ID NO:11) and pPGK1-V7-R (SEQ ID NO:12) to obtain the upstream homologous arm fragment 5-V7 (1000bp).

[0088] Using the genome of Pichia kudriazis S9 as a template, PCR was performed with primers tTDH3-V7-F (SEQ ID NO:13) and V7-R (SEQ ID NO:14) to obtain the downstream homologous arm fragment 3-V7 (1000bp). The obtained PCR products 5-V7 and 3-V7 were purified using a PCR purification kit.

[0089] (3) Transformation

[0090] The obtained targeting fragment pPGK1-CA-tTDH3, upstream homologous arm 5-V7, downstream homologous arm 3-V7, and CAS9-gRNA fusion plasmid (the CAS9-gRNA fusion plasmid and its construction method were described in CN118910116A) were mixed (the components were mixed in a molar ratio of 1:1:1:1) and transformed into the high-succinic acid-producing Pichia kudriaz strain S9-4 (accession number GDMCC:67259). The transformed bacterial solution was plated on SD / -Ura plates and incubated upside down at 30℃ for 2-4 days. The positive transformants were verified by colony PCR using seq-V7-F (SEQ ID NO:15) and seq-V7-R (SEQ ID NO:16). The PCR product size was 2Kb, which is the obtained Pichia kudriaz strain overexpressing the carbonic anhydrase gene from cyanobacteria, denoted as S9-CYCA.

[0091] The steps of electroconversion are as follows:

[0092] 1) Preparation of electrocompetent cells. The starting strain (Pichia gondii S9-4) was streaked onto YPD solid plates for strain activation. The streaked plates were incubated at 30℃ for 2-3 days until single colonies grew. Single colonies were selected and inoculated into YPD liquid medium, and incubated overnight at 30℃ and 250 rpm. The overnight culture was then divided according to the initial OD... 600 0.3 mg / L was transferred to 100 mL of YPD liquid medium and incubated at 30°C and 250 rpm for approximately 3.5-4.5 h until OD was reached. 600 To achieve a pH of 1.1-1.3; centrifuge the cultured bacterial solution at 5000 rpm for 3 min at room temperature, discard the supernatant and collect the bacterial cells; add 40 mL of freshly prepared and filtered sterilized LDST mixture (100 mM lithium acetate, 10 mM dithiothreitol, 10 mM Tris and 1 M sorbitol, adjusted to pH 7.5, filtered through a sterile membrane) to each centrifuge tube, gently shake to suspend the bacterial cells, and then incubate at 30°C for 30 min; centrifuge at 5000 rpm for 3 min at 4°C, discard the supernatant and collect the bacterial cells; wash the bacterial cells with 1 mL of ice-treated 1 M sorbitol, then centrifuge at 3000 rpm for 3 min at 4°C, discard the supernatant and collect the bacterial cells; add 400 μL of ice-treated 1 M sorbitol, suspend and mix the bacterial cells evenly, then quickly dispense 100 μL / tube, and immediately store the dispensed competent cells at -80°C for later use.

[0093] 2) Electroporation. Add approximately 0.5-1 μg of linearized DNA (i.e., the targeting fragment pPGK1-CA-tTDH3, upstream homologous arm 5-V7, and downstream homologous arm 3-V7) to the prepared yeast competent cells. Gently mix the linearized DNA and yeast competent cells thoroughly and transfer to a pre-ice-baked electroporation cuvette. Incubate on ice for 5 min. Set the electroporation mode to fungal mode and select PIC mode. Electroporate the mixture. Add 1M pre-chilled sorbitol to the electroporation cuvette and transfer the bacterial mixture to a sterile 1.5 mL EP tube using a pipette. Incubate at 30°C for 1.5 h. Centrifuge at 5000 rpm for 1-2 min, discard a portion of the supernatant, leaving approximately 200 μL of liquid. Resuspend the bacterial cells and plate them onto the appropriate SD / -Ura plate (SD / -Ura with...). Agar (purchased from coolaber, catalog number: PM2272) SD / -Ura plates are used to screen for uracil auxotrophic strains. The plates are inverted and incubated in a 30°C incubator for 2-4 days.

[0094] The PCR amplification system used in this embodiment is as follows: 25 μL of 2×PhantaMax Buffer (Vazyme), 1 μL of dNTPs (10 mM each), 20 ng of DNA template, 2 μL each of forward and reverse primers (10 μM), 1 μL of PhantaMax Super-Fidelity DNA polymerase (2.5 U / μL), and 20 μL of distilled water, for a total volume of 50 μL. The PCR amplification program (hereinafter the same) is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 5 min.

[0095] The amplification system for overlap extension PCR in this embodiment is as follows: 25 μL of 2×Phanta Max Buffer (Vazyme), 1 μL of dNTPs (10 mM each), 20 ng of DNA template, 2 μL of primers (10 μM each), 1 μL of Phanta Max Super-Fidelity DNA polymerase (2.5 U / μL), and 20 μL of distilled water, for a total volume of 50 μL. The amplification conditions are: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, and 72℃ extension for 1 min, for 30 cycles; and 72℃ extension for 5 min.

[0096] Using the above method, heterologous expression was constructed sequentially. 嗜氨热弧菌 , 嗜热甲烷八叠球菌 or 阿佐尔硫氢基菌The strains of Pichia kudriaz that derived the carbonic anhydrase gene are designated as S9-TACA, S9-MTCA, and S9-SACA, respectively.

[0097] 2. Succinic acid production of Pichia kudriazioides

[0098] The constructed Pichia pastoris strains S9-4, S9-CYCA, S9-TACA, S9-MTCA, and S9-SACA were streaked onto YPD solid plates and incubated upside down at 30°C for 2 days. Single colonies of each strain were then picked and transferred to test tubes containing 5 mL of YPD liquid medium and incubated at 30°C and 200 rpm until OD500 was reached. 600 5-8, Control the initial OD 600 The concentration was set to 0.05, and the culture medium was transferred to a 500 mL Erlenmeyer flask containing 100 mL of synthetic medium. The culture was then incubated at 30 °C and 220 rpm for 24 h to obtain the seed culture. The seed culture was inoculated into a 5 L fermenter containing 3 L of synthetic medium at a 5% (v / v) inoculation rate. The pH of the fermentation broth was adjusted to 5.5 with 20% NaOH, the shaking speed was set to 300 rpm, and the aeration (sterile air) was 0.4 L / min. Fermentation was carried out for 70 h. The fermentation broth was centrifuged (12000 rpm for 2 min), and the supernatant was collected. The succinic acid and glucose concentrations were determined using HPLC, and the sugar-acid conversion rate was calculated using the following formula: Sugar-acid conversion rate = (70 h succinic acid concentration - 0 h succinic acid concentration) / (0 h glucose concentration - 70 h glucose concentration).

[0099] Preparation of the above-mentioned synthetic culture medium:

[0100] The first part is the basic carbon and nitrogen source: Weigh 150.0 g / L glucose, 2 g / L sodium glutamate, 4 g anhydrous potassium dihydrogen phosphate, and 3 g anhydrous magnesium sulfate, and add distilled water to a final volume of 1 L. After preparation, autoclave at 115°C for 20 min.

[0101] The second part is the trace element solution: Weigh 2.80g ferrous sulfate, 2.90g calcium chloride, 0.48g sodium molybdate, 0.47g cobalt chloride, 5.75g zinc sulfate, 0.32g manganese chloride, 0.50g copper sulfate, and 15g EDTA, add distilled water to make up to 1L, and filter through a 0.22μm filter membrane in a clean bench to remove bacteria.

[0102] The third part is a vitamin solution: 0.05g biotin, 1g calcium pantothenate, 1g niacin, 25g inositol, 1g thiamine hydrochloride, 1g pyridoxine hydrochloride, 1g para-aminobenzoic acid, add distilled water to a final volume of 1L, filter in a clean bench and store at 4°C.

[0103] Before use, add 1 mL of filtered trace element solution and 1.2 mL of vitamin solution to the first part of the basic carbon and nitrogen source (1 L).

[0104] The results are shown in Table 1. Heterologous expression in Pichia pastoris S9-4 蓝细菌 , 嗜氨热弧菌 , 嗜热甲烷八叠球菌 , 硫氢基菌属 阿佐尔菌 Carbonic anhydrase genes from various sources can all enhance the succinic acid production capacity of *Pichia gondii* to some extent. Among them, heterologous expression... Cyanobacteria The derived carbonic anhydrase gene showed the best effect in increasing succinic acid production and sugar-acid conversion rate in *Pichia pastoris* S9-4. Subsequent experiments all used... Cyanobacteria The source of the carbonic anhydrase gene.

[0105] Table 1. Succinic acid yield and sugar-acid conversion rate of different Pichia kudriaz strains

[0106]

[0107] Example 2

[0108] This embodiment, based on Example 1, further involves heterologous overexpression of different sources (including) in Pichia pastoris S9-CYCA. Symplocarpus renifolius , Brassica napus , Streptomyces sky color , Ancylobacter novus and Phaeodactylum tricornutus The effects of phosphoenolpyruvate carboxylase (PPC) genes from different sources on succinic acid production by Pichia kudriaz.

[0109] The methods used in this embodiment for PCR, overlap extension PCR, electroporation, and HPLC detection are the same as those in Example 1.

[0110] 1. Constructing *Pichia pastoris* strains heterologously expressing phosphoenolpyruvate carboxylases from different sources.

[0111] Symplocarpus renifolius , Brassica napus , Streptomyces coelicolor , Ancylobacter novus and Phaeodactylum tricornutus The nucleotide sequences of the phosphoenolpyruvate carboxylase gene are shown in SEQ ID NO:17-SEQ ID NO:21.

[0112] by Symplocarpus renifoliusTaking the phosphoenolpyruvate carboxylase from this source as an example, it was overexpressed in Pichia pastoris S9-CYCA, and the specific method is as follows:

[0113] (1) Construction of phosphoenolpyruvate carboxylase gene expression cassette

[0114] Artificially synthesized pGPM1 promoter and tFBA1 terminator under the control of Symplocarpus renifolius The source of the enolpyruvate carboxylase gene (SEQ ID NO:17) was obtained. Using the genome of Pichia pastoris S9 as a template, PCR was performed with primers III-12-pGPM1-F (SEQ ID NO:22) and PPC-pGPM1-R (SEQ ID NO:23) to obtain the gene fragment pGPM1 (1000 bp), and the obtained PCR product was purified using a PCR purification kit.

[0115] Using the genome of Pichia kudriaz S9 as a template, PCR was performed with primers PPC-tFBA1-F (SEQ ID NO:24) and III-12-tFBA1-R (SEQ ID NO:25) to obtain the gene fragment tFBA1 (500bp), and the obtained PCR product was purified using a PCR purification kit.

[0116] Using the artificially synthesized PPC gene as a template, PCR was performed with primers pGPM1-PPC-F (SEQ ID NO:26) and tFBA1-PPC-R (SEQ ID NO:27) to obtain the gene fragment PPC (2652bp), and the obtained PCR product was purified using a PCR purification kit.

[0117] The fragments pGPM1, PPC and tFBA1 were extended by overlapping PCR using primers III-12-pGPM1-F (SEQ ID NO:22) and III-12-tFBA1-R (SEQ ID NO:25) to obtain the targeted fragment pGPM1-PPC-tFBA1. The obtained PCR product was then purified using a PCR purification kit.

[0118] (2) Construction of upstream and downstream homologous arms of phosphoenolpyruvate carboxylase gene expression cassette

[0119] Using the genome of *Pichia kudriaz* S9 as a template, PCR was performed with primers III-12-F (SEQ ID NO:28) and pGPM1-III-12-R (SEQ ID NO:29) to obtain the upstream homologous arm fragment 5-III-12 (1000 bp). Using the genome of *Pichia kudriaz* S9 as a template, PCR was performed with primers tFBA1-III-12-F (SEQ ID NO:30) and III-12-R (SEQ ID NO:31) to obtain the downstream homologous arm fragment 3-III-12 (1000 bp). The obtained PCR products 5-III-12 and 3-III-12 were purified using a PCR purification kit.

[0120] (3) Transformation

[0121] The obtained target fragment pGPM1-PPC-tFBA1, upstream homologous arm 5-III-12, downstream homologous arm 3-III-12, and CAS9-gRNA fusion plasmid (the CAS9-gRNA fusion plasmid and its construction method are described in CN118910116A) were mixed using electroporation (the components were mixed in a molar ratio of 1:1:1:1) and transformed into the above-mentioned Pichia kudriaz S9-CYCA. The transformed bacterial solution was plated on SD / -Ura plates and incubated upside down at 30℃ for 2-4 days. The obtained positive transformants were verified by colony PCR using the primers seq-III-12-F (SEQ ID NO:32) and seq-III-12-R (SEQ ID NO:33). The PCR product size was 2Kb, and a Pichia kudriaz strain (denoted as S9-CYCA-SRPPC) heterologously expressing phosphoenolpyruvate carboxylase from cyanobacteria was obtained.

[0122] Using the above method, heterologous expression was constructed sequentially. Brassica napus , Streptomyces sky color , Ancylobacter novus and Phaeodactylum tricornutus The *Pichia pastoris* strains from which the phosphoenolpyruvate carboxylase gene originated are designated as S9-CYCA-BNPPC, S9-CYCA-SCPPC, S9-CYCA-ANPPC, and S9-CYCA-PTPPC, respectively.

[0123] 2. Succinic acid production of Pichia kudriazioides

[0124] The constructed *Pichia pastoris* strains S9-CYCA, S9-CYCA-BNPPC, S9-CYCA-SCPPC, S9-CYCA-ANPPC, and S9-CYCA-PTPPC were streaked onto YPD solid plates and incubated upside down at 30°C for 2 days. Single colonies of each strain were then picked and transferred to test tubes containing 5 mL of YPD liquid medium and incubated at 30°C and 200 rpm until OD500 was reached. 600 5-8, Control the initial OD 600 The concentration was set to 0.05, and the culture medium was transferred to a 500 mL Erlenmeyer flask containing 100 mL of synthetic medium. The culture was then incubated at 30 °C and 220 rpm for 24 h to obtain the seed culture. The seed culture was inoculated at a rate of 5% (v / v) into a 5 L fermenter containing 3 L of synthetic medium (same as in Example 1). The pH of the fermentation broth was adjusted to 5.5 with 20% NaOH, the fermentation speed was set to 300 rpm, and the aeration rate was 0.4 L / min. Fermentation was carried out for 70 h. The fermentation broth was centrifuged (12000 rpm for 2 min), and the supernatant was collected. The succinic acid and glucose concentrations were determined using HPLC, and the sugar-acid conversion rate was calculated using the following formula: Sugar-acid conversion rate = (70 h succinic acid concentration - 0 h succinic acid concentration) / (0 h glucose concentration - 70 h glucose concentration).

[0125] The results are shown in Table 2. Heterologous expression originated from... Symplocarpus renifolius Streptomyces coelicolorAncylobacter novellus The phosphoenolpyruvate carboxylase gene can increase succinic acid production in *Pichia pastoris* S9-CYCA; however, heterologous expression originates from... Brassica napus, Phaeodactylum tricornutus However, the presence of a phosphoenolpyruvate carboxylase gene from a different source reduced the succinic acid production of this strain. This indicates that there are significant differences in how phosphoenolpyruvate carboxylase genes from different sources enhance the strain's ability to produce succinic acid.

[0126] Table 2. Succinic acid yield and sugar-acid conversion rate of different Pichia kudriaz strains

[0127]

[0128] Example 3

[0129] This embodiment, based on Example 2, further heterologously expresses different sources (including) in Pichia pastoris S9-CYCA-SRPPC. Saccharomyces cerevisiae , Leishmania major , Anoxybacillus yellow-hot , Arabidopsis thaliana and Trypanosoma cruziThe effects of phosphoenolpyruvate carboxykinase (PCK) genes from different sources on succinic acid production by *Pichia kudriaz* were investigated.

[0130] The methods used in this embodiment for PCR, overlap extension PCR, electroporation, and HPLC detection are the same as those in Example 1.

[0131] 1. Constructing *Pichia pastoris* strains heterologously expressing phosphoenolpyruvate carboxykinase from different sources.

[0132] Saccharomyces cerevisiae , Leishmania major , Anoxybacillus flavithermus , Arabidopsis thaliana and Trypanosoma cruzi The nucleotide sequences of the derived phosphoenolpyruvate carboxykinase gene are shown in SEQ ID NO:34-SEQ ID NO:38.

[0133] by Saccharomyces cerevisiae Taking the phosphoenolpyruvate carboxykinase from this source as an example, it was overexpressed in Pichia pastoris S9-CYCA-SRPPC, and the specific method is as follows:

[0134] (1) Construction of the phosphoenolpyruvate carboxykinase gene expression cassette

[0135] Under the control of artificially synthesized pENO2 promoter and tCYC1 terminator Saccharomyces cerevisiae The phosphoenolpyruvate carboxykinase gene (SEQ ID NO:34) was obtained. Using the genome of Pichia pastoris S9 as a template, PCR was performed with primers V-2-pENO2-F (SEQ ID NO:39) and PCK-pENO2-R (SEQ ID NO:40) to obtain the gene fragment pENO2 (1000bp), and the obtained PCR product was purified using a PCR purification kit.

[0136] Using the genome of Pichia kudriaz S9 as a template, PCR was performed with primers PCK-tCYC1-F (SEQ ID NO:41) and V-2-tCYC1-R (SEQ ID NO:42) to obtain the gene fragment tCYC1 (500bp).

[0137] Using the artificially synthesized PPC gene as a template, PCR was performed with primers pENO2-PCK-F (SEQ ID NO:43) and tCYC1-PCK-R (SEQ ID NO:44) to obtain the gene fragment PCK (1578bp), and the obtained PCR product was purified using a PCR purification kit.

[0138] Overlap extension PCR was performed on fragments pENO2, PCK, and tCYC1 to obtain the targeted fragment pENO2-PCK-tCYC1, and the obtained PCR product was purified using a PCR purification kit.

[0139] (2) Construction of upstream and downstream homologous arms of phosphoenolpyruvate carboxykinase gene expression cassette

[0140] Using the genome of *Pichia kudriaz* S9 as a template, PCR was performed with primers V2-F (SEQ ID NO:45) and pENO2-V2-R (SEQ ID NO:46) to obtain the upstream homologous arm fragment 5-V2 (1000 bp). Using the genome of *Pichia kudriaz* S9 as a template, PCR was performed with primers tCYC1-V2-F (SEQ ID NO:47) and V2-R (SEQ ID NO:48) to obtain the downstream homologous arm fragment 3-V2 (1000 bp). The obtained PCR products 5-V2 and 3-V2 were purified using a PCR purification kit.

[0141] (3) Transformation

[0142] The obtained target fragment pENO2-PCK-tCYC1, upstream homologous arm 5-V2, downstream homologous arm 3-V2, and CAS9-gRNA fusion plasmid (the CAS9-gRNA fusion plasmid and its construction method are described in CN118910116A) were mixed (each component was mixed in a molar ratio of 1:1:1:1) and transformed into the above-mentioned S9-CYCA-SRPPC strain using electroporation. The transformed bacterial solution was plated on SD / -Ura plates and incubated upside down at 30℃ for 2-4 days. The obtained positive transformants were verified by colony PCR using seq-V2-F (SEQ ID NO:49) and seq-V2-R (SEQ ID NO:50). The PCR product size was 2Kb, and heterologous expression was obtained. Saccharomyces cerevisiae The strain of Pichia kudriaz that yielded the phosphoenolpyruvate carboxykinase gene is designated as S9-CYCA-SRPPC-SCPCK.

[0143] Using the above method, heterologous expression was constructed sequentially. Leishmania major , Anoxybacillus yellow-hot , Arabidopsis thaliana and Trypanosoma cruziThe *Pichia gondii* strains containing the phosphoenolpyruvate carboxykinase gene were designated as S9-CYCA-SRPPC-LMPCK, S9-CYCA-SRPPC-AFPCK, S9-CYCA-SRPPC-ATPCK, and S9-CYCA-SRPPC-TCPCK, respectively.

[0144] 2. Succinic acid production of Pichia kudriazioides

[0145] The constructed *Pichia pastoris* strains S9-CYCA-SRPPC, S9-CYCA-SRPPC-SCPCK, S9-CYCA-SRPPC-LMPCK, S9-CYCA-SRPPC-AFPCK, S9-CYCA-SRPPC-ATPCK, and S9-CYCA-SRPPC-TCPCK were streaked onto YPD solid plates and incubated upside down at 30°C for 2 days. Single colonies of each strain were then picked and transferred to test tubes containing 5 mL of YPD liquid medium and incubated at 30°C and 200 rpm until OD500. 600 5-8, Control the initial OD 600 The concentration was set to 0.05, and the culture medium was transferred to a 500 mL Erlenmeyer flask containing 100 mL of synthetic medium. The culture was then incubated at 30 °C and 220 rpm for 24 h to obtain the seed culture. The seed culture was inoculated at a rate of 5% (v / v) into a 5 L fermenter containing 3 L of synthetic medium (same as in Example 1). The pH of the fermentation broth was adjusted to 5.5 with 20% NaOH, the fermentation speed was set to 300 rpm, and the aeration (sterile air) was 0.4 L / min. Fermentation was carried out for 70 h. The fermentation broth was centrifuged (12000 rpm for 2 min), and the supernatant was collected. The succinic acid and glucose concentrations were determined using HPLC, and the sugar-acid conversion rate was calculated using the following formula: Sugar-acid conversion rate = (70 h succinic acid concentration - 0 h succinic acid concentration) / (0 h glucose concentration - 70 h glucose concentration).

[0146] The results are shown in Table 3. Heterologous expression originated from... Saccharomyces cerevisiae , Leishmania older , Arabidopsis thaliana and Trypanosoma cruzi The phosphoenolpyruvate carboxykinase gene derived from this source can increase succinic acid production in *Pichia pastoris* S9-CYCA-SRPPC. Specifically, the expression of this gene is derived from... Saccharomyces cerevisiae The phosphoenolpyruvate carboxykinase gene showed the best effect; however, heterologous expression derived from... Anoxybacillus flavithermus However, the presence of the phosphoenolpyruvate carboxykinase gene from different sources reduced the succinic acid production of this strain. This indicates that there are significant differences in the ability of phosphoenolpyruvate carboxykinase genes from different sources to enhance the succinic acid production capacity of strains.

[0147] Table 3. Succinic acid yield and sugar-acid conversion rate of different Pichia kudriaz strains

[0148]

[0149] Example 4

[0150] During the succinic acid production process, as the succinic acid yield increases, the pH value of the system decreases, which in turn affects the strain's effective growth and succinic acid production at low pH values. To address this issue, this embodiment further domesticates the *Pichia gondii* S9-CYCA-SRPPC-SCPCK strain constructed in Example 3, enabling it to maintain its activity under low pH conditions and thus normally consume glucose for fermentation to produce succinic acid.

[0151] The domestication process is as follows:

[0152] (1) Pichia pastoris S9-CYCA-SRPPC-SCPCK was streaked on YPD solid plates and incubated upside down in a 30℃ incubator for 2 days. Then, a single colony of Pichia pastoris S9-CYCA-SRPPC-SCPCK was picked and transferred to a 250mL shake flask containing 50mL of YPD liquid medium. The culture was carried out at 30℃ and 200rpm until OD. 600 6-8 is used as seed liquid.

[0153] (2) The strains were acclimatized to acid tolerance using the Tianmu Bio-Automatic Microbial Adaptive Evolution Analyzer (EVOL cell). The above seed culture was transferred to fresh YPD liquid medium, with an initial OD... 600 The concentration is 0.2, and 8 mL of it is connected to the consumables box buffer bottle.

[0154] (3) Prepare sterile YPD medium and high-concentration succinic acid YPD medium (75 g / L) for EVOL cells. The concentration of succinic acid is automatically adjusted according to the passage parameters, and the acclimatization succinic acid concentration gradient is 40, 50, 60, and 70 g / L. The number of passages for each gradient is adjusted according to the growth of the strain. The operation of the equipment is observed regularly, and samples are taken in a timely manner for strain performance verification and preservation of superior strains to obtain high-concentration succinic acid-tolerant Pichia pastoris SA-S9-CYCA-SRPPC-SCPCK.

[0155] The strain *Pichia gondii* SA-S9-CYCA-SRPPC-SCPCK has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) under the name […]. Pichia kudriavzevii TSA 16, taxonomic name is Peach kudriavzeviiThe accession number is GDMCC No:66847, the accession date is August 15, 2025, and the accession location is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0156] Example 5

[0157] This example compares the succinic acid production capacity and acid tolerance of Pichia pastoris strains S9-4, S9-CYCA, S9-CYCA-SRPPC, S9-CYCA-SRPPC-SCPCK, and SA-S9-CYCA-SRPPC-SCPCK, as detailed below:

[0158] Pichia pastoris strains S9-4, S9-CYCA, S9-CYCA-SRPPC, S9-CYCA-SRPPC-SCPCK, and SA-S9-CYCA-SRPPC-SCPCK were streaked onto YPD solid plates. After incubation at 30°C for 2 days in an inverted incubator, single colonies of each strain were picked and transferred to test tubes containing 5 mL of YPD liquid medium. The culture was then incubated at 30°C and 200 rpm until OD500. 600 5-8, Control the initial OD 600 The concentration was set to 0.05, and the culture medium was transferred to a 500 mL Erlenmeyer flask containing 100 mL of synthetic medium. The mixture was then incubated at 30 °C and 220 rpm for 24 h on a shaker to obtain the seed culture. The seed culture was inoculated at a rate of 5% (v / v) into a 5 L fermenter containing 3 L of synthetic medium (same as in Example 1). During fermentation, 20% NaOH was used as a neutralizing agent to maintain the pH of the fermentation system at 3.1 or 2.7. The shaking speed was adjusted to 300 rpm, and the aeration (sterile air) was 0.4 L / min. Fermentation lasted for 70 h. The fermentation broth was centrifuged (12000 rpm for 2 min), and the supernatant was collected. The yield of succinic acid was determined using HPLC.

[0159] By detecting the succinic acid content in different fermentation systems (pH 3.1), the results showed that compared with *Pichia gondii* S9-4, the succinic acid production of S9-CYCA, S9-CYCA-SRPPC, S9-CYCA-SRPPC-SCPCK, and SA-S9-CYCA-SRPPC-SCPCK was increased to varying degrees, and there was no significant difference in the succinic acid production capacity between S9-CYCA-SRPPC-SCPCK and SA-S9-CYCA-SRPPC-SCPCK. This suggests that heterologous expression of the carbonic anhydrase gene, phosphoenolpyruvate carboxylase gene, and phosphoenolpyruvate carboxylkinase gene in *Pichia gondii* can enhance the CO2 fixation pathway, optimize carbon metabolic flux allocation, and improve its ability to produce succinic acid from glucose. Figure 1 ).

[0160] By statistically analyzing the glucose consumption and succinic acid production of each fermentation system (pH 3.1) at different time points during fermentation, the results showed that S9-CYCA-SRPPC-SCPCK consumed glucose at a significantly faster rate than S9-4. Figure 2 Furthermore, S9-CYCA-SRPPC-SCPCK can achieve higher yields of succinic acid in a shorter time. Figure 3 This indicates that the S9-CYCA-SRPPC-SCPCK strain constructed in this invention has good sugar-acid conversion ability.

[0161] By statistically analyzing the glucose consumption and succinic acid production in the S9-CYCA-SRPPC-SCPCK and S9-CYCA-SRPPC-SCPCK fermentation systems (pH 2.7) at different time points during fermentation, the results are as follows: Figure 4-Figure 5 As shown in Table 4, with the extension of fermentation time, S9-CYCA-SRPPC-SCPCK and SA-S9-CYCA-SRPPC-SCPCK showed significant differences in glucose consumption and succinic acid production. This indicates that the unacclimated S9-CYCA-SRPPC-SCPCK could not maintain high strain activity for a long time under low pH (2.7) conditions and could not normally consume glucose to ferment and produce succinic acid. This suggests that acclimation can enable S9-CYCA-SRPPC-SCPCK to normally consume glucose and ferment to produce succinic acid under low pH conditions, significantly improving the succinic acid synthesis efficiency. Furthermore, the SA-S9-CYCA-SRPPC-SCPCK strain grew and fermented normally under pH 2.7 conditions, with no significant difference in fermentation intensity and conversion rate compared to pH 3.1 conditions.

[0162] Table 4. Results of succinic acid production by different Pichia pastoris strains (pH 2.7)

[0163]

[0164] Example 6

[0165] This example compares the byproduct production during succinic acid production by Pichia gondii S9-4 and SA-S9-CYCA-SRPPC-SCPCK, as follows:

[0166] Pichia pastoris S9-4 and SA-S9-CYCA-SRPPC-SCPCK were streaked onto YPD solid plates and incubated upside down at 30°C for 2 days. Single colonies of each strain were then picked and transferred to test tubes containing 5 mL of YPD liquid medium and incubated at 30°C and 200 rpm until OD200. 600 5-8, Control the initial OD 600The concentration was set to 0.05, and the culture medium was transferred to a 500 mL Erlenmeyer flask containing 100 mL of synthetic medium. The mixture was then incubated at 30 °C and 220 rpm for 24 h on a shaker to obtain the seed culture. The seed culture was inoculated at a rate of 5% (v / v) into a 5 L fermenter containing 3 L of synthetic medium (same as in Example 1). NaOH was used as a neutralizing agent during fermentation to maintain the pH of the fermentation system at 3.1. The fermentation speed was set to 300 rpm, and the aeration (sterile air) was 0.4 L / min. Fermentation lasted for 70 h. The mixture was centrifuged (12000 rpm for 2 min), and the supernatant was collected. The contents of byproducts such as ethanol, glycerol, malic acid, and lactic acid were determined using HPLC.

[0167] The results are as follows Figure 6 As shown, compared with Pichia pastoris S9-4, SA-S9-CYCA-SRPPC-SCPCK can significantly reduce the generation of by-products during fermentation.

[0168] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria is *Pichia pastoris* (Kudriaz Pichia pastoris). Pichia kudriavzevii The gene encoding carbonic anhydrase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxylkinase is expressed heterologously in the same cell. The nucleotide sequence of the carbonic anhydrase gene is shown in SEQ ID NO:1; The nucleotide sequence of the phosphoenolpyruvate carboxylase gene is shown in SEQ ID NO:

17. The nucleotide sequence of the phosphoenolpyruvate carboxykinase gene is as shown in SEQ ID NO:34; The preservation number of the *Pichia kudriaz* yeast is GDMCC No: 67259.

2. An acid-resistant genetically engineered bacterium, characterized in that, The acid-resistant genetically engineered bacteria are obtained by acid domestication of the genetically engineered bacteria described in claim 1.

3. A type of Pichia pastoris (Kudriaz Pichia pastoris) Pichia kudriavzevii The *Pichia kudriaz* yeast described has the accession number GDMCC No:66847 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection. The accession name is... Pichia kudriavzevii TSA 16, deposited on August 15, 2025, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

4. A method for preparing succinic acid, comprising the following steps: Fermentation is carried out using the genetically engineered bacteria of claim 1, the acid-resistant genetically engineered bacteria of claim 2, or the Pichia kudriaz as the generating strain.

5. The method according to claim 4, characterized in that, The carbon source used in the fermentation includes at least one of glucose, xylose, glycerol, sucrose, maltose, molasses, fructose, rhamnose, arabinose, and sorbitol.

6. The method according to claim 5, characterized in that, The pH value of the fermentation system is 2.5-5.

7. The method according to claim 4 or 5, characterized in that, The nitrogen source used in the fermentation includes one or more of the following: yeast extract, peptone, yeast powder, corn steep liquor, urea, ammonia, ammonium sulfate, potassium nitrate, and ammonium nitrate.

8. The method according to claim 4 or 5, characterized in that, The fermentation temperature is 25-34℃.

9. The use of the genetically engineered bacteria of claim 1, the acid-resistant genetically engineered bacteria of claim 2, the Pichia kudriaz of claim 3, or the method of any one of claims 4-8 in the preparation of succinic acid or the preparation of products containing succinic acid.

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