Construction and application of a pichia pastoris strain with high efficiency of CO2 fixation and methanol conversion to produce single-cell protein

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to construction and application of a high-efficiency CO2 fixation and methanol conversion Pichia pastoris for producing single-cell protein. The present application obtains a synthesis module for converting CO2 into acetyl coenzyme A by path introduction, and further obtains a methanol high-tolerance strain MMEC-3 / 5 for synthesizing single-cell protein by using CO2 and methanol as substrates through a series of gene editing. The strain can be used for green biosynthesis of single-cell protein by fermentation catalysis method with methanol as substrate, and provides a new method for industrialized production of high-quality protein such as food-grade and feed-grade.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the construction and application of a Pichia pastoris strain that efficiently converts CO2 into methanol to produce single-cell proteins. Background Technology

[0002] The concentration of CO2 in the global atmosphere is increasing at an alarming rate, and climate change and the greenhouse effect are impacting human survival and development. The increasingly severe environmental crisis exacerbates the need to shift from a traditional fossil fuel economy to sustainable raw materials. Microbial assimilation of C1 compounds has emerged as a promising method for mitigating climate change. Methanol, due to its wide availability, low price, convenient transportation and storage, and renewable nature, is considered one of the important alternative raw materials for future biomanufacturing. Because it can be produced not only from low-grade coal but also in large quantities through electrocatalytic or photocatalytic CO2 and H2 (electrolysis of water), it is considered one of the most promising methods for achieving global carbon neutrality. Constructing efficient microbial cell factories to convert methanol into chemicals has become a research hotspot in the green biomanufacturing industry.

[0003] To meet global demand for animal-derived protein, the world needs to produce 1.25 billion tons of meat and dairy products annually. However, due to the low efficiency of converting feed into meat and dairy products, the ever-increasing human demand for protein cannot be sustainably met by simply increasing meat and dairy production. Although cultured meat and plant-based meat alternatives are rapidly developing, their higher production costs and less appealing taste make them less competitive than animal protein produced through traditional livestock farming. Therefore, finding and developing new protein resources is essential.

[0004] Single-cell protein (SCP), first proposed by MIT in 1968, replaced the previously used terms "microbial protein" and "petroleum protein." Today, it mostly refers to the cell protein of bacteria (non-pathogenic), microalgae, or fungi obtained through large-scale artificial fermentation and cultivation using agricultural and petroleum waste in various substrates and suitable environments. SCP is not a single protein, but rather a cytoplasmic mass composed of various nutrients, such as nucleic acids, carbohydrates, fats, proteins, and other nitrogenous substances. Therefore, it contains abundant nutrients such as proteins, amino acids, vitamins, nucleic acids, and various polysaccharides. With the increasing demand for high-quality food-grade and feed-grade proteins in my country, and the growing market potential, domestic production processes should develop towards a green, environmentally friendly, efficient, and energy-saving sustainable direction. Strengthening the research and development of biosynthetic methods and utilizing methanol as a substrate for SCP production in Pichia pastoris cell factories for industrial applications could become a new solution to the feed protein gap.

[0005] Currently, methanol assimilation pathways can be constructed in model microorganisms by creating heterologous and artificial utilization pathways, enabling the conversion of methanol into central metabolism. However, this approach suffers from limitations, including the inability to use methanol as the sole carbon source and low methanol metabolism rates and product synthesis efficiency. Furthermore, natural methyltrophic microorganisms exhibit low methanol utilization rates due to their complex methanol metabolism pathways and the toxicity of intracellular formaldehyde. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a methanol-tolerant strain that utilizes CO2 and methanol as co-substrate to biosynthesize single-cell proteins.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a Pichia pastoris engineered strain, which is obtained by genetic modification of a methanol-tolerant Pichia pastoris strain GS-1 as the starting strain. The engineered strain overexpresses carbon monoxide dehydrogenase / acetyl-CoA synthase complex CODH / ACS, phosphatidylethanolamine N-methyltransferase 2, phosphatidylserine synthase PSS, ribulose-5-phosphate-3-isomerase RPE, dihydroxyacetone synthase DAS, and glutamine synthase GLN1. The methanol-tolerant strain Pichia pastoris GS-1 has the accession number GDMCC 68746 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 28, 2026.

[0008] This invention achieves efficient synthesis of single-cell proteins using CO2 and methanol as substrates by combining two modules: one that converts CO2 and methanol into acetyl-CoA, and another that modifies the cell wall structure to convert nitrogen sources into single-cell proteins via an ammonia cycle module. Further modification of the cell membrane structure enhances the strain's tolerance to methanol.

[0009] To utilize CO2 to synthesize single-cell proteins in Pichia pastoris, this invention, through MetaCyc and ATLAS database mining and literature review, constructs a CO2 utilization pathway in Pichia pastoris based on the natural Wood-Ljungdahl pathway, such as... Figure 1-2 As shown: This approach is divided into the following two branches and the core assembly path: (1) Methyl branch: Reduces CO2 to methyl (-CH3). The goal of this branch is to reduce one molecule of CO2 to a high-energy methyl (-CH3). In this process, tetrahydrofolate (H4F) is used as a carrier to undergo a series of transformations from formyl to methylene and then to methyl.

[0010] (2) Carbonyl pathway: CO2 is reduced to CO. This pathway uses carbon monoxide dehydrogenase (CODH) to directly reduce the second molecule of CO2 to CO. This is the most thermodynamically difficult step in the entire pathway, requiring a strong reducing electron donor.

[0011] (3) Core assembly pathway: CH3-CoFeSP and CO generated by the two branches are transported to the active center of the same enzyme complex CODH / ACS, where the final assembly reaction takes place on the A cluster of the ACS subunit.

[0012] This process synthesizes the final product acetyl-CoA through three key steps. In addition to being used for the synthesis of cellular substances (converted into the cellular carbon skeleton through other pathways), the generated acetyl-CoA is also used to produce acetic acid, and in the process, ATP is generated.

[0013] Preferably, the carbon monoxide dehydrogenase / acetyl-CoA synthase complex (CODH / ACS) is derived from Clostridium acetylcholine (Clostridium diethylcarbamate). Clostridium formicaceticum The phosphatidylethanolamine N-methyltransferase 2, phosphatidylserine synthase PSS, ribulose-5-phosphate-3-isomerase RPE, dihydroxyacetone synthase DAS, and glutamine synthase GLN1 are derived from Pichia pastoris. (Pichia pastoris) .

[0014] Preferably, the amino acid sequence of the carbon monoxide dehydrogenase / acetyl-CoA synthase complex CODH / ACS is shown in SEQ ID NO.1; the amino acid sequence of the phosphatidylethanolamine N-methyltransferase 2 is shown in SEQ ID NO.2; the amino acid sequence of the phosphatidylserine synthase PSS is shown in SEQ ID NO.3; the amino acid sequence of the ribulose-5-phosphate-3-isomerase RPE is shown in SEQ ID NO.4; the amino acid sequence of the dihydroxyacetone synthase DAS is shown in SEQ ID NO.5; and the amino acid sequence of the glutamine synthase GLN1 is shown in SEQ ID NO.6.

[0015] Secondly, the present invention provides a method for constructing the above-mentioned engineered Pichia pastoris strain, comprising the following steps: (1) Using the methanol-tolerant Pichia pastoris strain GS-1 as the starting strain, the carbon monoxide dehydrogenase / acetyl-CoA synthase complex CODH / ACS, phosphatidylethanolamine N-methyltransferase 2, phosphatidylserine synthase PSS, ribulose-5-phosphate-3-isomerase RPE, and dihydroxyacetone synthase DAS were knocked into the strain using gene editing technology. (2) Knockout using gene editing technology PAS_chr4_0305 The Pichia pastoris engineered strain was obtained by knocking in the gene and inserting glutamine synthase GLN1.

[0016] This invention knocks out the PAS_chr4_0305 gene and knocks in glutamine synthase GLN1 at that site. PAS_chr4_0305 knockout can maximize the flux of methanol to SCP in the cellular metabolic network, and the growth regulation caused by cell wall metabolism enhances the directed conversion of methanol-protein metabolic flux, improves the efficiency of carbon-nitrogen co-conversion, and reduces carbon loss.

[0017] Thirdly, the present invention provides the application of the above-mentioned engineered Pichia pastoris strain in the production of single-cell proteins.

[0018] Fourthly, the present invention provides a method for producing single-cell protein using the above-mentioned engineered Pichia pastoris strain, wherein the engineered Pichia pastoris strain is expanded and cultured, and then transferred to a fermentation medium for fermentation culture to produce single-cell protein.

[0019] Preferably, the engineered Pichia pastoris strain uses methanol and CO2 as substrates.

[0020] Preferably, the fermentation conditions are: 30℃, stirring speed 200-800 rpm, and air introduction at a gas velocity of 3-5 m / s. 3 / h, when the glucose concentration in the fermentation broth is detected to be 0, methanol is added for fermentation. Wait for the dissolved oxygen (DO) to rise back to 100% (v / v), then start feeding methanol continuously. Maintain the DO concentration at no less than 20% (v / v) during the culture process. The fermentation time is 72h. Preferably, the fermentation medium comprises the following components at the following mass concentrations: ammonium sulfate 5-7.5 g / L, potassium dihydrogen phosphate 3-5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 24 g / L, trace metal solution 1 mL / L, and vitamin solution 1 mL / L.

[0021] Preferably, the trace metal solution comprises the following components at the following mass concentrations: EDTA-Na2 13.00-16.00 g / L, ZnSO4·7H2O 3.50-5.50 g / L, MnCl2·4H2O 0.50-2.00 g / L, CoCl2·6H2O 0.25-0.45 g / L, CuSO4·5H2O 0.20-0.40 g / L, NaMoO4·2H2O 0.25-0.45 g / L, CaCl2·2H2O 0.25-0.45 g / L, FeSO4·7H2O 2.00-4.00 g / L, H3BO3 0.50-2.00 g / L, and KI 0.10-0.50 g / L.

[0022] Preferably, the vitamin solution comprises the following components in the indicated mass concentrations: biotin 0.05-0.25 g / L, calcium pantothenate 1-3 g / L, thiamine hydrochloride 1-5 g / L, pyridoxine hydrochloride 1-5 g / L, nicotinic acid 1-4 g / L, 4-aminobenzoic acid 0.2-2.0 g / L, and inositol 25-30 g / L.

[0023] The beneficial effects of this invention are as follows: Pichia pastoris can utilize methanol as a carbon source for growth in its natural environment, thus its metabolic pathway has a certain natural basis. This invention, based on Pichia pastoris, introduces the Wood-Ljungdahl metabolic pathway from Clostridium acetate and integrates key genes, transforming Pichia pastoris, which can only use methanol as a substrate, into one that can simultaneously use methanol and CO2 as substrates. This significantly expands the yeast's diet, increases substrate conversion efficiency, and can also be applied to large-scale carbon reduction scenarios. By enhancing methanol tolerance, methanol utilization, and nitrogen metabolism pathways, and integrating related genes, substrate conversion efficiency and yield are further increased. Attached Figure Description

[0024] Figure 1 This diagram illustrates the conversion of CO2 into formic acid via the Wood-Ljungdahl pathway (WL pathway), followed by a series of enzymatic reactions that convert formic acid into acetyl-CoA.

[0025] Figure 2 This diagram illustrates the process of converting methanol into formaldehyde via alcohol oxidase, followed by a series of enzymatic reactions via the XuMP pathway to acetyl-CoA.

[0026] Figure 3 A schematic diagram of the pPICZ-Cas9-gRNA editing plasmid structure.

[0027] Figure 4 This is the result of evaluating the strain's tolerance to methanol; in each subplot, the bacterial concentration is 1, 10, etc. -1 10 -2 10 -3 10 -4 .

[0028] Figure 5 The results show the growth capacity of Pichia pastoris MMEC-3 / 5 during fermentation. Detailed Implementation

[0029] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products.

[0031] The culture medium formulations involved in the following examples are as follows: LB medium (g / L): yeast extract 5 g / L, NaCl 10 g / L, peptone 10 g / L; bring to the appropriate volume with ddH2O, sterilize at 121℃ for 20 min before use; solid medium requires the addition of about 2% agar powder before sterilization.

[0032] YPD medium (g / L): 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose; bring to the appropriate volume with ddH2O, sterilize at 115℃ for 15 min before use; solid medium requires the addition of approximately 2% agar powder before sterilization.

[0033] L-His solution (40 g / L): 40 g / L L-His solid dissolved in ddH2O. No pH adjustment is required. Use a 0.22 μm pore size aqueous filter membrane for filtration and sterilization in a clean bench. Store at 4℃ for later use (prepare immediately before use).

[0034] Sorbitol solution (1 M): 18.217 g / L sorbitol solid, dissolved in ddH2O, filtered and sterilized using a 0.22 μm pore size aqueous filter membrane in a clean bench, and stored at 4℃ for later use (prepare immediately before use).

[0035] YNB medium (g / L): 6.67 g / L yeast extract, adjusted to the appropriate volume with ddH2O, sterilized at 121℃ for 20 min before use; solid medium requires the addition of approximately 2% agar powder before sterilization.

[0036] YNBM medium (g / L): 6.67 g / L yeast extract, diluted to the appropriate volume with ddH2O, sterilized at 21℃ for 20 min, and stored at room temperature until use. When using, add the appropriate volume fraction of sterile methanol and add L-His solution according to the growth requirements of the strain.

[0037] Fermentation medium formula: ammonium sulfate 5-7.5 g / L, potassium dihydrogen phosphate 3-5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 24 g / L, trace metal solution 1 mL / L, vitamin solution 1 mL / L.

[0038] Trace metal solutions (g / L): EDTA-Na2 13.00-16.00 g / L, ZnSO4·7H2O 3.50-5.50 g / L, MnCl2·4H2O 0.50-2.00 g / L, CoCl2·6H2O 0.25-0.45 g / L, CuSO4·5H2O 0.20-0.40 g / L, NaMoO4·2H2O 0.25-0.45 g / L, CaCl2·2H2O 0.25-0.45 g / L, FeSO4·7H2O 2.00-4.00 g / L, H3BO3 0.50-2.00 g / L, KI 0.10-0.50 g / L. First, completely dissolve EDTA-Na2 and ZnSO4·7H2O in ddH2O, then adjust the pH to 6.0. After dissolving each drug, adjust the pH to 6.0. Once all drugs are dissolved, adjust the pH to 4.0 with 1 M HCl solution. Sterilize in a high-temperature and high-pressure autoclave at 121℃ for 20 min, protect from light, and store at 4℃ for later use (do not exceed 2 months after opening).

[0039] Vitamin solution: Biotin 0.05-0.25 g / L, calcium pantothenate 1-3 g / L, thiamine hydrochloride 1-5 g / L, pyridoxine hydrochloride 1-5 g / L, nicotinic acid 1-4 g / L, 4-aminobenzoic acid 0.2-2.0 g / L, inositol 25-30 g / L, dissolved in double-distilled water (ddH2O), no pH adjustment required, filtered and sterilized using a 0.22μm pore size aqueous filter membrane in a clean bench, stored at 4℃ for later use (prepare immediately).

[0040] ClonExpress one-step directional cloning kit and gel extraction kit were purchased from Vazyme Biotech (Nanjing). Plasmid extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. All analytical grade reagents were purchased from Sinopharm Group. All enzymes used in this invention were obtained through expression using genetic engineering methods.

[0041] The carbon monoxide dehydrogenase / acetyl-CoA synthase complex (CODH / ACS) involved in the following examples is derived from Clostridium acetylcholine (Clostridium perfringens) Clostridium formicaceticum ), EC number 1.2.7.4, amino acid sequence as shown in SEQ ID NO.1; Phosphatidylethanolamine N-methyltransferase 2 (PEAMT2) is derived from Pichia pastoris. (Pichia pastoris) EC number 2.1.1.17, amino acid sequence as shown in SEQ ID NO.2; Phosphatidylserine synthase (PSS) is derived from Pichia pastoris. (Pichia pastoris) The amino acid sequence of EC2.7.8.8 is shown in SEQ ID NO.3. Ribulose-5-phosphate-3-isomerase (RPE) is derived from Pichia pastoris. (Pichia pastoris) EC number 5.1.3.1, amino acid sequence as shown in SEQ ID NO.4; Dihydroxyacetone synthase (DAS) is derived from Pichia pastoris. (Pichia pastoris) EC number 2.2.1.3, amino acid sequence as shown in SEQ ID NO.5; Glutamine synthase (GLN1) is derived from Pichia pastoris. (Pichia pastoris) EC number 6.3.1.2, amino acid sequence as shown in SEQ ID NO.6.

[0042] Example 1: Laboratory adaptive evolution enhances the methanol tolerance of bacterial strains Starting with Pichia pastoris GS115, the methanol tolerance of the strain was improved through laboratory adaptive evolution, and the evolved strain was named GS-1.

[0043] The specific method is as follows: (1) Pasteurella GS115 (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) stored at -80℃ was spread on YPD solid plates and cultured in a 30℃ incubator for 48h to obtain single colonies. (2) Pick a single colony from the YPD plate and incubate it overnight in YNBM + 2% methanol medium, and set up 3 replicates; (3) These three strains were subjected to initial OD 600 nm = 0.2 Transfer to a 250 mL shake flask containing 100 mL YNBM + 6% methanol, and incubate at 30 °C and 220 rpm for 24 h; (4) After 24 h, the strain was transferred again to a strain with the same initial OD. 600nm They were placed in a medium of fresh YNBM + 6% methanol and then incubated under the same conditions. (5) Repeat steps (3)-(4) above approximately 100 times until a high-density strain is obtained in methanol medium, which is named strain GS-1. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC 68746.

[0044] This evolved strain was able to grow normally in YNB medium with a maximum volume fraction of 8% methanol as the sole carbon source.

[0045] Example 2: Construction of engineered strain MMEC-3 / 5 1. Design of Clostridium acetate Clostridium formicaceticum Carbon monoxide dehydrogenase / acetyl-CoA synthase complex (CODH / ACS), Pichia pastoris Pichia pastorisPrimer sequences for the gene integration frames of endogenous phosphatidylethanolamine N-methyltransferase 2 (PEAMT2), phosphatidylserine synthase (PSS), endogenous ribulose-5-phosphate-3-isomerase (RPE), dihydroxyacetone synthase (DAS), and endogenous glutamine synthase (GLN1).

[0046] Using Pichia pastoris GS-1 as the starting strain, a knock-in gene was constructed. CODH / ACS, PEAMT2, PSS, RPE, DAS and GLN1 An engineered strain (GLN1 knocked into the PAS_chr4_0305 gene locus, with the integration frame containing the homologous arms above and below PAS_chr4_0305 and the target gene; PAS_chr4_0305 knockout (a gene directly related to cell wall thickness under methanol stress) maximizes the flux of methanol to SCP in the cellular metabolic network, regulates growth caused by cell wall metabolism, enhances the directed conversion of methanol-protein metabolic flux, improves carbon-nitrogen co-conversion efficiency, and reduces carbon loss) was named MMEC-3 / 5. All gene editing operations were performed using CRISPR-Cas9 gene editing technology.

[0047] Taking the knock-in of the RPE gene as an example, the specific method is as follows: (1) Using the genome of Pichia pastoris GS-1 strain as a template, the upper and lower homologous arms of the neutral site were amplified using primers F1 and R1, F3 and R3, and the target gene was amplified using primers F2 and R2. Finally, the dDNA integration frame was assembled using F1 and R3. (2) pPICZ-Cas9-gGUT1 (from the team of Zhou Yongjin at the Dalian Institute of Chemical Physics, whose construction method is described in the "Materials and Methods" section of the literature "Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia PSStoris") was used as the plasmid backbone for CRISPR-Cas9 gene editing. The gRNA sequences synthesized in Table 3 were inserted into the plasmid (the insertion sites are detailed in the appendix). Figure 3 ), construct the pPICZ-Cas9-gRNA plasmid.

[0048] The constructed pPICZ-Cas9-gRNA plasmid was introduced into E. coli. E. coli In DH5α competent cells, Escherichia coli was cultured at 37°C for 24 hours for plasmid amplification; then, the synthesized pPICZ-Cas9-gRNA plasmid was extracted from the E. coli cells for transformation of the target microorganism.

[0049] (3) Target microbial transformation: The verified pPICZ-Cas-gRNA plasmid and dDNA were electroporated into Pichia pastoris GS-1, and then plated on YPD plates containing 100 ng / μL bleomycin and cultured at 30°C for 2-3 days. The specific steps for target microbial transformation are as follows: (3.1) Bacterial culture: Before electroporation, Pichia pastoris GS-1 was cultured to the logarithmic growth phase, and its concentration was OD 600 Around 0.8-1; (3.2) Pretreatment of cells: The cultured bacteria are centrifuged and washed to obtain an appropriate number of bacteria and bring them to their optimal state; (3.3) Plasmid preparation: Extract the constructed pPICZ-Cas9-gRNA; (3.4) Electroporation: The prepared pPICZ-Cas9-gRNA plasmid, dDNA and bacterial cells were mixed, and then the mixture was placed in an electroporator to introduce the plasmid into the cells by electric field pulses; The conditions for electroconversion were: electrocup: 2mm, pre-cooled; electroconversion parameters: Eppendorf electroconversion instrument, 2KV, 4.3ms. Recovery conditions after transformation: After electroporation, the bacteria need to recover in an appropriate culture medium (0.5 mL 1M sorbitol + 0.5 mL YPD medium). The culture conditions during recovery can be optimized according to the characteristics of the microorganism (e.g., incubation at 30°C in a shaker for 1-2 hours for recovery).

[0050] (4) Screening: Colony PCR was performed using validation primers F4 and R4, and correct transformants were obtained by sequencing. The correct transformants were transferred into YPD medium without bleomycin and cultured at 30°C to eliminate the pPICZ-Cas-gRNA plasmid. The correct transformants were then screened by streaking on plates. (5) Iterate through the steps above. CODH / ACS, PEAMT2, PSS, DAS and GLN1 Genes get G S115-CODH / ACS, PEAMT2, PSS, DAS, ΔPAS_chr4_0305::GLN1 The strain was named MMEC-3 / 5.

[0051] 2. Study on the co-culture of strains GS115 and MMEC-3 / 5 under methanol and CO2: YPD medium was selected for culturing microorganisms.

[0052] Two types of Pichia pastoris were cultured separately in sealed bottles with a total volume of 250 mL. One bottle contained 100 mL of liquid culture medium with 16 g / L methanol, 6.67 g / L yeast extract (amino acid-free), and 40 mg / L histidine. The liquid surface was filled with CO2 at a pressure of 1.5 atmospheres (1.5 kg). The culture was carried out under strictly anaerobic conditions in sealed plasma bottles for 72 hours. The shaker was set to 30℃ and 220 rpm. The CO2 pressure, methanol concentration, and bacterial concentration (OD) were measured immediately after inoculation (0 h). These parameters were also measured at the end of the 72-hour experiment.

[0053] Table 1 The results are shown in Table 1. After 72 hours of fermentation, the original Pichia pastoris strain GS115 showed an increase in gas pressure, indicating the generation of CO2. At the same time, it consumed 9.5g of methanol and generated 4.9 OD of microorganisms, with a yield of approximately 0.537 OD / g methanol.

[0054] After 72 hours of fermentation, strain MMEC-3 / 5 showed a decrease in gas pressure, indicating CO2 consumption, and also consumed 13 grams of methanol, producing a microbial output of 7.7 OD, with a yield of approximately 0.612 OD / g methanol. Therefore, this invention successfully constructed strain MMEC-3 / 5 capable of producing single-cell proteins using both methanol and CO2 as substrates.

[0055] The primer sequences and gRNA sequences designed in this embodiment are shown in Tables 2 and 3.

[0056] Table 2: Primer sequences Table 3: gRNA Sequences Example 3: Validation of tolerance of MMEC-3 / 5 strains On YNB solid plates with different concentrations of methanol (32 g / L, 40 g / L, 48 g / L, 56 g / L and 64 g / L, respectively) as the sole carbon source, different concentration gradients (1, 10) were inoculated. -1 10 -2 10 -3 10 -4 Tolerance assessment was conducted on the bacterial culture.

[0057] The results of the evaluation of the strain's ability to tolerate methanol are as follows: Figure 4 As shown, with increasing methanol concentration, the growth of the control strain GS115 slowed down, and almost no colonies were observed on the YNB plate with a methanol concentration of 48 g / L. The modified MMEC-3 / 5 strain grew well, indicating that this strain acquired a high tolerance to methanol.

[0058] Example 4: Validation of single-cell protein growth catalyzed by fermentation of MMEC-3 / 5 strain 1. Primary Seed Culture: The isolated and purified Pichia pastoris MMEC-3 / 5 was streaked onto a YPD agar plate and incubated statically at 30℃ for 2 days. Single colonies grown on the plate were picked and inoculated into 4 mL test tubes, incubated overnight at 30℃ and 220 rpm until OD reached the target growth rate. 600nm The concentration is 2-6, and a first-grade seed solution is obtained.

[0059] 2. Secondary seed culture: Inoculate 1 mL of primary seed culture into a 50 mL YPD medium shake flask, and simultaneously transfer to 6 flasks. Incubate overnight at 30°C on a shaker at 220 rpm until OD reaches the target value. 600nm The ratio is 3-10, and secondary seed solution is obtained.

[0060] 3. Fermentation culture: Inoculate 300 mL of secondary seed culture into 5 L of fermentation medium, incubate at 30℃, with stirring speed of 300 rpm and air introduced at a velocity of 3 m / s. 3 Aerobic fermentation was carried out under conditions of / h. As dissolved oxygen (DO) decreased, the gas rate and stirring were increased to maintain DO at no less than 20% (v / v). Biomass, wet weight, and products were measured at different time points. When the glucose concentration in the fermentation broth was detected to be 0, pure methanol carbon source was added for fermentation 1 h later. Initially, 250 mL was added for fermentation. After waiting for DO to rise back to 100% (v / v), methanol was added at a flow rate of 25 mL / h, increasing the methanol gradient by 10 mL per hour until the final flow rate reached 150 mL / h. Mass transfer was increased (stirring, tank pressure, and gas rate) to control dissolved oxygen at 20% (v / v). Fermentation lasted for 72 h.

[0061] OD of fermentation broth at different times 600nm The dry weight was tested, and the results were as follows: Figure 5 As shown, Pichia pastoris MMEC-3 / 5 exhibits good growth ability when using glucose, methanol, and CO2 as a mixed carbon source. The OD value can reach over 200, and the dry weight is 120 g / L.

[0062] After 72 hours of fermentation, all fermentation broth was collected, centrifuged and dried to obtain the bacterial protein product. The crude protein content was finally determined to be 60 g / L using the Kjeldahl method.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Pichia pastoris engineered strain, characterized in that, The engineered strain was obtained by genetic modification of the methanol-tolerant Pichia pastoris strain GS-1. The engineered strain overexpresses carbon monoxide dehydrogenase / acetyl-CoA synthase complex CODH / ACS, phosphatidylethanolamine N-methyltransferase 2, phosphatidylserine synthase PSS, ribulose-5-phosphate-3-isomerase RPE, dihydroxyacetone synthase DAS, and glutamine synthase GLN1. The methanol-tolerant strain GS-1 has the accession number GDMCC 68746 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection.

2. The engineered Pichia pastoris strain as described in claim 1, characterized in that, The carbon monoxide dehydrogenase / acetyl-CoA synthase complex CODH / ACS is derived from Clostridium acetylcholine (Clostridium perfringens). Clostridium foricaceticum The phosphatidylethanolamine N-methyltransferase 2, phosphatidylserine synthase PSS, ribulose-5-phosphate-3-isomerase RPE, dihydroxyacetone synthase DAS, and glutamine synthase GLN1 are derived from Pichia pastoris. (Pichiapastoris) .

3. The engineered Pichia pastoris strain as described in claim 1, characterized in that, The amino acid sequence of the carbon monoxide dehydrogenase / acetyl-CoA synthase complex CODH / ACS is shown in SEQ ID NO.1; the amino acid sequence of the phosphatidylethanolamine N-methyltransferase 2 is shown in SEQ ID NO.2; the amino acid sequence of the phosphatidylserine synthase PSS is shown in SEQ ID NO.3; the amino acid sequence of the ribulose-5-phosphate-3-isomerase RPE is shown in SEQ ID NO.4; the amino acid sequence of the dihydroxyacetone synthase DAS is shown in SEQ ID NO.5; and the amino acid sequence of the glutamine synthase GLN1 is shown in SEQ ID NO.

6.

4. The method for constructing Pichia pastoris engineered strains as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Using the methanol-tolerant Pichia pastoris strain GS-1 as the starting strain, the carbon monoxide dehydrogenase / acetyl-CoA synthase complex CODH / ACS, phosphatidylethanolamine N-methyltransferase 2, phosphatidylserine synthase PSS, ribulose-5-phosphate-3-isomerase RPE, and dihydroxyacetone synthase DAS were knocked into the strain using gene editing technology. (2) Knockout using gene editing technology PAS_chr4_0305 The Pichia pastoris engineered strain was obtained by knocking in the gene and inserting glutamine synthase GLN1.

5. The use of the engineered Pichia pastoris strain as described in any one of claims 1-3 in the production of single-cell protein.

6. A method for producing single-cell protein using Pichia pastoris engineered strains as described in any one of claims 1-3, characterized in that, After the Pichia pastoris engineered strain was cultured on a large scale, it was transferred to a fermentation medium for fermentation to produce single-cell protein.

7. The method as described in claim 6, characterized in that, The engineered Pichia pastoris strain uses methanol and CO2 as substrates.

8. The method as described in claim 6, characterized in that, The fermentation conditions are: 30℃, stirring speed 200-800 rpm, air introduction, and air velocity 3-5 m / s. 3 / h, when the glucose concentration in the fermentation broth is detected to be 0, methanol is added for fermentation. After waiting for DO to rise to 100% (v / v), methanol is added continuously. During the culture process, DO is kept at no less than 20% (v / v) for 72 hours.

9. The method as described in claim 6, characterized in that, The fermentation medium comprises the following components at the following mass concentrations: ammonium sulfate 5-7.5 g / L, potassium dihydrogen phosphate 3-5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 24 g / L, trace metal solution 1 mL / L, and vitamin solution 1 mL / L.

10. The method as described in claim 9, characterized in that, The trace metal solution comprises the following components at the following mass concentrations: EDTA-Na₂ 13.00-16.00 g / L, ZnSO₄·7H₂O 3.50-5.50 g / L, MnCl₂·4H₂O 0.50-2.00 g / L, CoCl₂·6H₂O 0.25-0.45 g / L, CuSO₄·5H₂O 0.20-0.40 g / L, NaMoO₄·2H₂O 0.25-0.45 g / L, CaCl₂·2H₂O 0.25-0.45 g / L, FeSO₄·7H₂O 2.00-4.00 g / L, H₃BO₃ 0.50-2.00 g / L, and KI 0.10-0.50 g / L; the vitamin solution comprises the following component at the following mass concentration: biotin 0.05-0.25 g / L. g / L, calcium pantothenate 1-3 g / L, thiamine hydrochloride 1-5 g / L, pyridoxine hydrochloride 1-5 g / L, nicotinic acid 1-4 g / L, 4-aminobenzoic acid 0.2-2.0 g / L, inositol 25-30 g / L.

Images