A method for constructing and applying an artificial Pichia pastoris cell factory for CO2 bioconversion.

Abstract

This invention discloses a method for constructing and applying an artificial Pichia pastoris cell factory for CO2 bioconversion. It involves introducing genes related to the Calvin cycle and reduced glycine pathway into the host Pichia pastoris genome, and knocking out either formate dehydrogenase Fdh or alcohol oxidase Aox1 in Pichia pastoris. The invention also proposes a method for constructing and applying this genetically engineered strain. This invention utilizes synthetic biology methods to design and introduce the Calvin cycle and reduced glycine pathway into Pichia pastoris, constructing an artificial carbon-fixing Pichia pastoris with the ability to recover CO2 and assimilate formate, resulting in a 17.69%–34.07% reduction in carbon emissions per unit biomass.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and specifically relates to the construction of an artificial Pichia pastoris cell factory utilizing CO2 bioconversion and its application in improving methanol assimilation and achieving CO2 fixation. Background Technology

[0002] With rapid industrial development, global carbon dioxide (CO2) emissions reached a new record in 2022. Excessive CO2 emissions are exacerbating the greenhouse effect and extreme weather events, including typhoons, torrential rains, and droughts. These environmental problems sound an alarm for humanity: the necessity of achieving a low-carbon economy. CO2 recycling has become a key strategy for building an eco-friendly economy and society. However, achieving CO2 resource utilization still faces enormous challenges. While natural biological carbon sequestration is green and environmentally friendly, it is inefficient and slow, making it difficult to meet the needs of industrial production; physicochemical carbon sequestration is highly efficient, but energy consumption is high and products are limited. Therefore, how to combine the advantages of biological, physical, and chemical technologies to utilize CO2 as a raw material for bioconversion is a pressing scientific and technological problem that needs to be solved. In recent years, significant breakthroughs have been made in the technology of synthesizing methanol through CO2 hydrogenation. Converting CO2 into C1 compounds (methanol, formic acid, etc.) can provide raw materials for bioconversion and is a key factor in developing a carbon-neutral society and achieving a sustainable carbon economic cycle.

[0003] Introducing CO2 into the central carbon metabolism of industrial microorganisms can generate autotrophic or polytrophic microorganisms with the potential to improve carbon efficiency. Many natural methyltrophic bacteria, such as *Methanobacterium* and *Bacillus methanolii*, have recently been identified for the production of single-cell proteins, PHB, or amino acids. Furthermore, some model organisms, such as *Escherichia coli*, *Saccharomyces cerevisiae*, and *Corynebacterium glutamicum*, have also been engineered for methanol assimilation. *Pichia pastoris* (also known as *Komagatella phaffii*), as a representative of methyltrophic yeasts, has advantages over other methyltrophic bacteria, including strong methanol metabolism, high levels of exogenous protein expression, and high-density culture. With the development of synthetic biology, its genetic modification tools have gradually matured, and in recent years, the synthesis of chemicals from methanol to α-farnesene, heparin, fatty acids, and their derivatives has been achieved. However, the natural methanol metabolic pathway in *Pichia pastoris* has low assimilation efficiency and poor carbon economy. Studies have shown that when methanol is used as a carbon source, as much as 50%-70% of carbon atoms are lost through the methanol dissimilatory pathway. Therefore, how to enhance the methanol assimilation efficiency of Pichia pastoris and reduce the loss of carbon atoms caused by dissimilation pathways is a major bottleneck problem in the development of microbial cell factories based on Pichia pastoris. Summary of the Invention

[0004] Purpose of the invention: To address the above-mentioned problems, the purpose of this invention is to provide an artificial carbon-fixing Pichia pastoris cell factory and its application that utilizes synthetic biology methods to systematically optimize the methanol utilization pathway in Pichia pastoris, and on this basis constructs CO2 and formic acid utilization pathways, thereby enhancing methanol assimilation efficiency by recovering carbon atoms lost through methanol dissimilation.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] An engineered Pichia pastoris strain with artificial carbon fixation, wherein the strain is obtained by introducing Calvin cycle-related genes into the host genome; the Calvin cycle-related genes are the ribulose 1,5-bisphosphate carboxylase-oxygenase gene RuBisCO, the phosphoriboskinase gene PRK, two molecular chaperone genes groEL and groES, the formaldehyde lyase gene FLS, the phosphoglycerate kinase gene PGK1, the glyceraldehyde-3-phosphate dehydrogenase gene TDH3, and the triphosphate isomerase gene TPI1 from *Escherichia coli*.

[0007] The construction method includes the following steps:

[0008] 1) The Calvin cycle-related genes were cloned into the vector BB1_23, thereby obtaining the recombinant plasmids BB1_23_RuBisCO, BB1_23_PRK, BB1_23_groEL, BB1_23_groES, BB1_23_FLS, BB1_23_PGK1, BB1_23_TDH3 and BB1_23_TPI1;

[0009] 2) Recombinant plasmids containing each gene expression cassette were constructed using Golden Gate Assembly. After the reaction, the reaction system was directly transformed into *E. coli* DH5α. *E. coli* carrying the recombinant plasmids were screened using LB-AmpR plates. Similarly, recombinant plasmids BB2_BC-PRK, BB2_CD-groEL, BB2_DE-groES, BB2_EF-FLS, BB2_FG-PGK1, BB2_GH-TDH3, and BB2_HI-TPI1 were obtained. The CBB cyclic expression plasmid BB3rN_AI-CBB was also constructed using Golden Gate Assembly. 3) After the reaction, the reaction system was directly transformed into *E. coli* DH5α. *E. coli* carrying the recombinant plasmids were screened using LB-NTC plates. The constructed plasmid BB3rN_AI-CBB was linearized by Asc I digestion, purified using a gel extraction kit, electroporated into *Pichia pastoris* PPGS115, and screened using YPD-NTC plates to obtain recombinant *Pichia pastoris* PPC000.

[0010] An engineered Pichia pastoris strain with artificial carbon fixation, wherein the strain is obtained by introducing genes related to the reducing glycine pathway of Yersinia lipolytica into the host genome; the genes derived from the reducing glycine pathway of Yersinia lipolytica are c-1-tetrahydrofolate synthase (MIS1), aminomethyltransferase (GcvT), glycine cleavage system P protein (GcvP), and glycine cleavage system H protein (GcvH).

[0011] The construction method was as follows: expression plasmid BB3eH_AE-YlrGly was constructed, and recombinant plasmid was constructed using Golden Gate Assembly. Plasmid BB3eH_AE-YlrGly was linearized by Hpa I restriction enzyme digestion, purified by gel extraction kit, electroporated into Pichia pastoris PPGS115, and screened by YPD-Hph plates to obtain recombinant Pichia pastoris PP0YG0.

[0012] An engineered Pichia pastoris strain with artificial carbon fixation, wherein the strain was obtained by introducing genes related to the Calvin cycle and the reducing glycine pathway from Yersinia lipophila into the host genome;

[0013] The Calvin cycle pathway includes the ribulose 1,5-bisphosphate carboxylase-oxygenase gene RuBisCO, the phosphoriboskinase gene PRK, two molecular chaperone genes groEL and groES from Escherichia coli, the formaldehyde lyase gene FLS, the phosphoglycerate kinase gene PGK1, the glyceraldehyde-3-phosphate dehydrogenase gene TDH3, and the triphosphate isomerase gene TPI1 from Denitrifying Thiobacillus.

[0014] The genes derived from the reductive glycine pathway of Yersinia lipolytica include c-1-tetrahydrofolate synthase (MIS1), aminomethyltransferase (GcvT), glycine cleavage system P protein (GcvP), and glycine cleavage system H protein (GcvH).

[0015] The construction method is as follows: plasmid BB3eH_AE-YlrGly was linearized by Hpa I restriction enzyme digestion, purified by gel extraction kit, electroporated into recombinant Pichia pastoris PPC000, and screened by YPD-Hph plates to obtain recombinant Pichia pastoris PPCYG0.

[0016] The recombinant strains PP0YG0 and PPCYG0 were able to tolerate high concentrations of formate and grow normally in formate environments of 100 mM and above, indicating that the expression of the YlrGly pathway played a role in formate detoxification and restored normal cell growth.

[0017] An engineered Pichia pastoris strain with artificial carbon fixation is obtained by introducing genes related to the Calvin cycle and the reducing glycine pathway from Yersinia lipolytica into the host genome, and then knocking out formate dehydrogenase Fdh and alcohol oxidase Aox1 in Pichia pastoris. Knocking out formate dehydrogenase Fdh and alcohol oxidase Aox1 in Pichia pastoris avoids carbon flux loss, thereby improving the carbon atom utilization efficiency of Pichia pastoris for methanol.

[0018] The host strain is Pichia pastoris GS115 (Komagataella phaffii GS115).

[0019] The NCBI-GeneIDs for the following proteins are as follows: c-1-tetrahydrofolate synthase (MIS1) 2907923; aminomethyltransferase (GcvT) 2907779; glycine cleavage system P protein (GcvP) 2905991; glycine cleavage system H protein (GcvH) 2912864; ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) 75501182; phosphoriboskinase (PRK) 110791203; and molecular chaperone genes. The NCBI-GeneIDs for (groEL) and molecular chaperone genes (groES) are 948666 and 948655, respectively; the PDB for formaldehyde lyase (FLS) is 4QQ8; the NCBI-GeneID for phosphoglycerate kinase (PGK1) is 8197742; the NCBI-GeneID for glyceraldehyde-3-phosphate dehydrogenase (TDH3) is 8198905; the NCBI-GeneID for triphosphate isomerase (TPI1) is 64573071; the NCBI-GeneID for formate dehydrogenase (Fdh) is 2911506; and the NCBI-GeneID for alcohol oxidase (Aox1) is 8201223.

[0020] More preferably, an engineered Pichia pastoris strain that utilizes carbon dioxide bioconversion for artificial carbon fixation is obtained by introducing genes related to the Calvin cycle and the reducing glycine pathway from Yersinia lipolytica into the host Pichia pastoris genome, and then knocking out formate dehydrogenase Fdh and alcohol oxidase Aox1 in Pichia pastoris; this avoids carbon flux loss and thus improves the carbon atom utilization efficiency of Pichia pastoris for methanol.

[0021] The method for constructing the recombinant Pichia pastoris engineered strain using carbon dioxide bioconversion includes the following steps:

[0022] Step 1 proposes a recombinant Pichia pastoris engineered strain utilizing carbon dioxide biotransformation. This strain designs CBB pathway-related genes in the genome of the host Pichia pastoris, including the ribulose 1,5-bisphosphate carboxylase-oxygenase gene RuBisCO from *Thiobacillus denitrifyingus*, the phosphoriboskinase gene PRK, two molecular chaperone genes groEL and groES from *Escherichia coli*, the formaldehyde lyase gene FLS, the phosphoglycerate kinase gene PGK1, the glyceraldehyde-3-phosphate dehydrogenase gene TDH3, and the triphosphate isomerase gene TPI1.

[0023] Step 2: Systematically optimize the methanol utilization pathway in Pichia pastoris in the host strain Pichia pastoris. The main operation is to express c-1-tetrahydrofolate synthase (MIS1), aminomethyltransferase (GcvT), glycine cleavage system P protein (GcvP), and glycine cleavage system H protein (GcvH) derived from the reducing glycine pathway of Yersinia lipolytica and Pichia pastoris, to obtain strains PPC000, PP0YG0, PP0PG0, PPCYG0 and PPCPG0, respectively.

[0024] Step 3: Using homologous recombination, formate dehydrogenase Fdh and alcohol oxidase Aox1 in strains PPGS115, PPC000 and PPCYG0 were knocked out to obtain recombinant Pichia pastoris PPGS115-ΔAOX1, PPC000-ΔAOX1 and PPCYG0-ΔAOX1.

[0025] Specifically, in step 1, the CBB cycle expression plasmid BB3rN_AI-CBB is constructed using the GoldenPiCS Kit based on Golden Gate Assembly. Specifically, the eight genes required for constructing the CBB cycle are codon-optimized and synthesized at Genscript Biotech Inc., and directly cloned into the vector BB1_23, thereby obtaining the recombinant plasmids BB1_23_RuBisCO, BB1_23_PRK, BB1_23_groEL, BB1_23_groES, BB1_23_FLS, BB1_23_PGK1, BB1_23_TDH3, and BB1_23_TPI1. Subsequently, the recombinant plasmids containing each gene expression cassette are constructed using Golden Gate Assembly.

[0026] Taking BB2_AB-RuBisCO containing the RuBisCO expression cassette as an example, the reaction system of the Golden Gate Assembly is as follows:

[0027] Table 1. Golden Gate Assembly Reaction System for Constructing BB2-AB-RubisCO Plasmid

[0028]

[0029] The reaction conditions are:

[0030] Table 2. Reaction procedure for constructing BB2-AB-RubisCO plasmid using Golden Gate Assembly.

[0031]

[0032]

[0033] After the reaction was completed, the reaction system was directly transformed into *E. coli* DH5α, and the *E. coli* carrying the recombinant plasmids were screened using LB-AmpR plates. Similarly, the recombinant plasmids BB2_BC-PRK, BB2_CD-groEL, BB2_DE-groES, BB2_EF-FLS, BB2_FG-PGK1, BB2_GH-TDH3, and BB2_HI-TPI1 were obtained.

[0034] The CBB cyclic expression plasmid BB3rN_AI-CBB was also constructed using Golden Gate Assembly. The reaction system was as follows:

[0035] Table 3. Reaction system for constructing BB3rN-AI-CBB plasmid using Golden Gate Assembly

[0036]

[0037] The reaction conditions are:

[0038] Table 4. Golden Gate Assembly reaction procedure for constructing BB3rN-AI-CBB plasmid.

[0039]

[0040] After the reaction was completed, the reaction system was directly transformed into *E. coli* DH5α, and the *E. coli* carrying the recombinant plasmid were screened using LB-NTC plates. The constructed plasmid BB3rN_AI-CBB was linearized by Asc I digestion, purified by a gel extraction kit, electroporated into *Pichia pastoris* PPGS115, and screened using YPD-NTC plates to obtain recombinant *Pichia pastoris* PPC000;

[0041] In step 2, expression plasmids BB3eH_AE-YlrGly and BB3eH_AE-PprGly were constructed. The recombinant plasmids were also constructed using Golden Gate Assembly; the construction process will not be detailed here. Plasmids BB3eH_AE-YlrGly and BB3eH_AE-PprGly were linearized by Hpa I digestion, purified using a gel extraction kit, and electroporated into Pichia pastoris PPGS115. Screening was performed using YPD-Hph plates to obtain recombinant Pichia pastoris PP0YG0 and PP0PG0. The linearized BB3eH_AE-YlrGly and BB3eH_AE-PprGly were electroporated into Pichia pastoris PPC000 to obtain recombinant Pichia pastoris PPCYG0 and PPCPG0.

[0042] In step 3, gene knockout was performed based on homologous recombination. The knockout plasmid pPICZB-ΔFDH, which had been constructed in the laboratory previously (the construction process of this plasmid can be found in F. Guo, ZXDai, WFPeng, SJZhang, J.Zhou, JFMa, WLDong, FXXin, WMZhang, M.Jiang, Metabolic engineering of Pichiapastoris for malic acid production from methanol, Biotechnology and Bioengineering 118(1)(2021)357-371.), was used to knock out formate dehydrogenase Fdh. Using plasmid pPICZB-ΔFDH as a template, the Fdh knockout cassette FDH1-FRT-AOX1p-FLP-CYCt-KanR-AOXt-FRT-FDH was amplified by PCR. The fragment purified by the gel extraction kit was electroporated into Pichia pastoris PPGS115 and screened by YPD-G418 plates to obtain PPGS115-ΔFDH1 with Fdh knockout.

[0043] The Aox1 knockout cassette was constructed using the Fdh1 knockout cassette as its backbone. Specifically, the upper and lower homologous arm fragments AOX1 up and AOX1 down were amplified using the Pichia pastoris genome as a template, and the fragment FRT-AOX1p-FLP-CYCt-KanR-AOXt-FRT containing the genomycin selection marker was amplified using the plasmid pPICZB-ΔFDH as a template. The purified fragment was used to construct the knockout cassette using a one-step cloning kit and ligated into the pPICZB vector. E. coli carrying the recombinant plasmid were screened using LB-Zeocin plates to obtain the knockout plasmid pPICZB-ΔAOX. The Aox1 knockout cassette AOX1-FRT-AOX1p-FLP-CYCt-KanR-AOXt-FRT-AOX1 was amplified using the pPICZB-ΔAOX plasmid as a template. The fragments purified by the gel extraction kit were electroporated into strains PPGS115, PPC000, and PPCYG0, and screened using YPD-G418 plates to obtain recombinant Pichia pastoris PPGS115-ΔAOX1, PPC000-ΔAOX1, and PPCYG0-ΔAOX1 with Aox1 knocked out.

[0044] The application of the artificial carbon-fixing Pichia pastoris strain in CO2 bioconversion.

[0045] The application of the artificial carbon-fixing Pichia pastoris strain in CO2 fixation.

[0046] The artificial carbon-fixing Pichia pastoris strain introduces both formic acid and CO2 utilization pathways, endowing the artificial Pichia pastoris with the ability to utilize formic acid and recover CO2. By recovering carbon atoms lost through methanol dissociation, the CO2 utilization capacity is increased by 71.84%. Simultaneously, the ability to utilize methanol for growth is increased by 20.13%, and formaldehyde accumulation is reduced by 68.24%, further improving carbon atom utilization efficiency.

[0047] Beneficial effects:

[0048] Compared with existing technologies, this invention provides an engineered Pichia pastoris strain that utilizes CO2 bioconversion for artificial carbon fixation. It systematically optimizes the methanol utilization pathway in the host strain Pichia pastoris and introduces formic acid and CO2 utilization pathways, endowing the artificial Pichia pastoris with the ability to utilize formic acid and recover CO2. By recovering carbon atoms lost through methanol dissimilatory processes, CO2 utilization efficiency is increased by 71.84%. Simultaneously, the ability to utilize methanol for growth is increased by 20.13%, and formaldehyde accumulation is reduced by 68.24%, further improving carbon atom utilization efficiency.

[0049] The engineered strain constructed in this invention overexpresses the Calvin cycle, enabling CO2 fixation and thus allowing Pichia pastoris to acquire CO2 fixation capabilities. In minimal sodium bicarbonate medium, the OD600 of PPC000 increased by 43.75%. Compared to the original strain, the introduction of the CBB cycle increased the strain's ability to assimilate CO2 for growth by 71.84%. Compared to the original strain PPGS115, the OD600... 600 It increased by 20.13%, and the formaldehyde concentration in PPC000 was only 27.15 μM / OD. 600 The expression of the CBB pathway decreased by 68.24%. This demonstrates that the expression of the CBB pathway not only does not affect the normal methanol metabolism of the strain, but also works synergistically with methanol assimilation to promote the growth of the strain.

[0050] The reductive glycine pathway-related genes overexpressed in this invention were cloned from *Pichia pastoris* (PprGly) and *YlrGly*. Comparison of reductive glycine pathways from different sources demonstrated that the YlrGly pathway exhibits better carbon fixation efficiency. The introduction of the YlrGly pathway endowed the engineered carbon-fixing *Pichia pastoris* strain with the ability to assimilate formic acid and CO2. Sole expression of the YlrGly pathway and co-expression of the YlrGly pathway with the CBB cycle reduced biomass emissions per unit area by 34.07% and 17.69%, respectively, in the engineered carbon-fixing *Pichia pastoris* strain.

[0051] The modified strain constructed in this invention knocked out its catabolism pathway and successfully blocked carbon loss caused by the oxidation of formic acid to CO2. However, the accumulation of formic acid caused a decrease in cell biomass, proving that blocking the catabolism pathway still requires the establishment of formic acid tolerance and sufficient intracellular energy supply. Attached Figure Description

[0052] Figure 1 The expression of the CBB pathway in Pichia pastoris according to the present invention;

[0053] Figure 2 Screening and testing of the reducing glycine pathway in Pichia pastoris; (A) Schematic diagram of the introduction of the reducing glycine pathway in Pichia pastoris; (B) Schematic diagram of the construction of YlrGly and PprGly expression plasmids by Goldengate Assembly.

[0054] Figure 3 CO2 emission detection by artificial carbon-fixing strains;

[0055] Figure 4 The average amino acid content of artificially carbon-fixed Pichia pastoris engineered strains 13 C-methanol labeling level. Detailed Implementation

[0056] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0057] All technologies not mentioned in the embodiments are conventional technologies in the art, and the technical solutions of the present invention will be described in detail below through specific embodiments. Furthermore, unless otherwise specified, all materials and reagents used in the following embodiments are commercially available.

[0058] Example 1: Constructing a CBB loop

[0059] Plasmids constructed using the GoldenPiCS Kit based on Golden Gate Assembly

[0060] In this invention, the eight genes required for constructing the CBB cycle were codon-optimized and synthesized at Genscript Biotech Inc., and directly cloned into the vector BB1_23, thereby obtaining the recombinant plasmids BB1_23_RuBisCO, BB1_23_PRK, BB1_23_groEL, BB1_23_groES, BB1_23_FLS, BB1_23_PGK1, BB1_23_TDH3, and BB1_23_TPI1. Subsequently, the recombinant plasmids containing each gene expression cassette were constructed using Golden Gate Assembly. The strains, plasmids, and primers used in this invention are shown in Tables 5-6.

[0061] Table 5. Sequences of the 8 genes in the CBB cycle after codon optimization in this invention.

[0062]

[0063]

[0064]

[0065]

[0066] Table 6. Strains and plasmids used in this invention

[0067]

[0068]

[0069]

[0070] Table 7 Primers used in this invention

[0071]

[0072]

[0073] [1] M.Valli, NETatto, A.Peymann, C.Gruber, N.Landes, H.Ekker, GGThallinger, D.Mattanovich, B.Gasser, ABGraf, Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function, Fems Yeast Res 16(6)(2016).

[0074] Taking BB2_AB-RuBisCO, which contains the RuBisCO expression cassette, as an example, the reaction system and procedure using Golden Gate Assembly are shown in Table 1-2. Similarly, recombinant plasmids BB2_BC-PRK, BB2_CD-groEL, BB2_DE-groES, BB2_EF-FLS, BB2_FG-PGK1, BB2_GH-TDH3, and BB2_HI-TPI1 were obtained. The CBB cyclic expression plasmid BB3rN_AI-CBB was also constructed using Golden Gate Assembly; the reaction system and procedure are shown in Table 3-4. The constructed plasmid BB3rN_AI-CBB was electroporated into PPGS115 to obtain recombinant Pichia pastoris PPC000.

[0075] Example 2: Construction of the reduced glycine pathway

[0076] Similarly, using Golden Gate Assembly, the MIS1, GcvT, GcvP, and GcvH genes expressing the reducing glycine pathway from *Yersinia lipolytica* and *Pichia pastoris* were cloned into the vector BB1-23, thereby obtaining two sets of recombinant plasmids from lipolytica and Pichia pastoris, namely BB1-23-MISI, BB1-23-GcvT, BB1-23-GcvP, and BB1-23-GcvH. The experimental methods and operations were the same as in Example 1, and finally, the plasmid BB3eH_AE-YlrGly from lipolytica and the plasmid BB3eH_AE-PprGly from Pichia pastoris were constructed. The recombinant plasmids were also constructed using Golden Gate Assembly, and the construction process was the same as in Example 1. Plasmids BB3eH_AE-YlrGly and BB3eH_AE-PprGly were linearized by Hpa I enzyme digestion, purified by gel extraction kit, electroporated into Pichia pastoris PPGS115, and screened by YPD-Hph plates to obtain recombinant Pichia pastoris PP0YG0 and PP0PG0.

[0077] Linearized BB3eH_AE-YlrGly and BB3eH_AE-PprGly were electroporated into Pichia pastoris PPC000 to obtain recombinant Pichia pastoris PPCYG0 and PPCPG0.

[0078] Example 3: Construction of an artificial carbon-fixing Pichia pastoris strain with knockout methanol dissimilatory pathway

[0079] Gene knockout was performed based on homologous recombination. The knockout plasmid pPICZB-ΔFDH (construction process of which can be found in F. Guo, ZXDai, WFPeng, SJZhang, J. Zhou, JFMa, WLDong, FXXin, WMZhang, M. Jiang, Metabolic engineering of Pichia pastoris for malic acid production from methanol, Biotechnology and Bioengineering 118(1)(2021)357-371.) was used to knock out formate dehydrogenase Fdh. Using plasmid pPICZB-ΔFDH as a template, the Fdh knockout cassette FDH1-FRT-AOX1p-FLP-CYCt-KanR-AOXt-FRT-FDH was amplified by PCR. The fragment purified by the gel extraction kit was electroporated into Pichia pastoris PPGS115 and screened by YPD-G418 plates to obtain PPGS115-ΔFDH1 with Fdh knockout.

[0080] The Aox1 knockout cassette was constructed using the Fdh1 knockout cassette as its backbone. Specifically, the upper and lower homologous arm fragments AOX1 up and AOX1 down were amplified using the Pichia pastoris genome as a template, and the fragment FRT-AOX1p-FLP-CYCt-KanR-AOXt-FRT containing the genomycin selection marker was amplified using the plasmid pPICZB-ΔFDH as a template. The purified fragment was used to construct the knockout cassette using a one-step cloning kit and ligated into the pPICZB vector. E. coli carrying the recombinant plasmid were screened using LB-Zeocin plates to obtain the knockout plasmid pPICZB-ΔAOX. The Aox1 knockout cassette AOX1-FRT-AOX1p-FLP-CYCt-KanR-AOXt-FRT-AOX1 was amplified using the pPICZB-ΔAOX plasmid as a template. The fragments purified by the gel extraction kit were electroporated into strains PPGS115, PPC000, and PPCYG0, and screened using YPD-G418 plates to obtain recombinant Pichia pastoris PPGS115-ΔAOX1, PPC000-ΔAOX1, and PPCYG0-ΔAOX1 with Aox1 knocked out.

[0081] Example 4: Analytical Detection Method

[0082] (1) Detection of bacterial cell density

[0083] Using a pipette, take 2 mL of fermentation broth from the clean bench and dilute it to the appropriate concentration (the measured value should be within the instrument's accurate range: 0.2–0.8). Measure the absorbance of the diluted solution at 600 nm using a UV spectrophotometer. The final OD value... 600 = A600 × dilution factor.

[0084] (2) Plate dilution coating method

[0085] Yeast strains were cultured overnight at 30°C in their respective media. The saturated yeast strains were diluted 10,000 times with water, and 12 μL of the diluted culture was plated onto various types of media to be tested. The plates were incubated at 30°C for 2 days until clear colonies were observed.

[0086] (3) Formaldehyde accumulation test

[0087] Formaldehyde accumulation was detected using the Nash reagent method. The detection principle is that formaldehyde reacts with Nash reagent to produce a yellow-green soluble substance with a maximum absorption peak at 412 nm. The specific detection method is as follows: 2 mL of bacterial culture was collected, and the OD value was accurately measured. 600The bacterial culture was then centrifuged at 8000 rpm, and 1 mL of the supernatant was collected. A solution was prepared by mixing Nash reagent and sample at a 1:1 ratio, incubated at 58°C for 5 minutes, and the absorbance was measured at 412 nm. The same method was used to detect 1×10⁻⁶ samples. -4 -15×10 -4 A standard curve was plotted using formaldehyde standard M to calculate the amount of formaldehyde accumulated in the fermentation broth.

[0088] (4) Determination of methanol concentration

[0089] The concentration of methanol in the fermentation broth was determined using a high-performance gas chromatograph (Agilent Technologies 7890). The methanol concentration in the fermentation broth was diluted to 0-1 g / L, and then the diluted solution was filtered through a 0.22 μm diameter aqueous filter membrane to remove impurities such as bacterial cells and proteins. Specifically, nitrogen gas was passed through a DB-624UI column (Agilent Technologies, 0.32 mm × 30 m × 0.25 mm) at a constant pressure of 19.082 psi. The thermal cycling consisted of the following stages: an initial temperature of 45 °C for 1 minute, followed by a temperature increase to 150 °C at a rate of 20 °C / min, then a temperature increase to 240 °C at a rate of 45 °C / min, and finally a holding time of 1 minute.

[0090] (5) Determination of formic acid concentration

[0091] The concentration of formic acid in the fermentation broth was determined by high performance liquid chromatography (HPLC) using a UitiMate 3000 HPLC system (Dionex, USA). The specific detection method was as follows: 1.5 mL of fermentation broth was centrifuged at 13000 × g for 3 min. The supernatant was diluted appropriately and then filtered through a 0.22 μm aqueous filter membrane to remove impurities such as proteins. The chromatographic column used was a Bio Rad Aminex HPX-87H column, the mobile phase was 0.25 mM dilute sulfuric acid, the flow rate was 0.5 mL / min, the column temperature was 55℃, the UV detection wavelength was 215 nm, and the sample injection volume was 20 μL. All standard samples used in the detection were freshly prepared and a standard curve was plotted.

[0092] (6) Measurement of carbon dioxide emissions

[0093] The carbon dioxide content in the anaerobic flask was determined using a gas chromatograph / TDC detector. The specific detection method is as follows: a 1.5m × 3mm stainless steel column packed with 60 / 80 mesh silica gel; column temperature: 50℃; detector temperature: 50℃; bridge current: 181mA; helium: 25mL / min. 1mL of gas from the anaerobic flask was injected. The standard samples used for detection must be freshly prepared and used immediately.

[0094] Example 5: Phenotypic Verification of Artificial Carbon-Fixed Pichia pastoris Engineered Strains

[0095] 1. The effect of knockout of the methanol dissimilatory pathway on methanol utilization in Pichia pastoris

[0096] The methanol utilization performance of the knockout strain was verified using a minimal methanol medium, with 1% methanol added every 12 h during cultivation. The methanol utilization performance of the knockout strain was essentially consistent with that of the original strain, consuming 282.92 mM and 269.87 mM of methanol at 144 h, respectively. However, the cell growth of the knockout strain was significantly inhibited, and its OD... 600 The value decreased by 51.61%. Cell growth restriction may be influenced by two factors: Fdh knockout leads to formic acid accumulation, directly toxic to cells; it also reduces intracellular energy supply, limiting cell proliferation. These results indicate that while Fdh knockout does prevent carbon atoms from being wasted in the form of CO2, it does not improve methanol assimilation efficiency. Aox1 knockout significantly reduced the methanol consumption rate, weakened formaldehyde production, and significantly reduced formaldehyde accumulation. However, it also led to a decrease in strain biomass, demonstrating that the biomass contribution from methanol assimilation accounts for a large proportion in knockout strains. In conclusion, although Aox1 knockout does weaken methanol utilization, it did not achieve the expected increase in biomass contribution from formic acid and CO2.

[0097] 2. Validation of Calvin cycle expression in Pichia pastoris

[0098] All expressed genes were localized to peroxisomes via the PTS signal peptide. The constructed Pichia pastoris was named PPC000, and its one-carbon metabolic pathway is as follows: Figure 1 As shown. The growth performance of PPGS115 and PPC000 bacterial sludge, after methanol induction, was examined by transferring them to a minimal sodium bicarbonate medium. Neither the original strain PPGS115 nor the recombinant strain PPC000 could grow in the control medium without NaHCO3, and their biomass continuously decreased over 120 h. However, in the minimal sodium bicarbonate medium, the original strain PPGS115 achieved a biomass increase of 25.46%, and its OD... 600 The OD value increased from an initial 1.58 to 1.99 after 96 hours. In contrast, the OD value of the recombinant strain PPC000...600 The concentration increased from an initial 1.72 to 2.47 after 96 hours, representing a growth of 43.75%. Compared to the original strain, the introduction of the CBB cycle increased the strain's ability to assimilate CO2 for growth by 71.84%.

[0099] In minimal medium supplemented with 2% methanol, PPC000 showed a lower OD value than the control strain PPGS115 in the first 48 hours. 600 It increased to 4.62, while the OD of strain PPC000 600 The biomass increased to 5.93, a 20.13% increase. More significantly, as methanol was depleted after 48 hours, the biomass of the original strain PPGS115 almost ceased to grow; the OD of strain PPC000... 600 The value continued to increase, rising from 5.93 at 48 h to 7.78 at 120 h. These results indicate that the expression of the CBB cycle not only does not affect the strain's normal methanol metabolism, but also synergistically promotes methanol assimilation.

[0100] 3. Screening and construction of the reducing glycine pathway in Pichia pastoris

[0101] The heterologous reducing glycine (YlrGly) pathway from *Yersinia lipolytica* and the endogenous reducing glycine (PprGly) pathway from *Pichia pastoris* were expressed in PPGS115 strains, which were named PP0YG0 and PP0PG0, respectively. Figure 2 The growth capacity of recombinant strains PPGS115, PP0YG0, PP0PG0, PPCYG0, and PPCPG0 was characterized by dilution spotting in minimal medium plates supplemented with 10 mM glucose and 30 mM formate. There was no significant difference in growth between the original strain PPGS115 and the artificial carbon-fixing strain PP0YG0, while the growth performance of strain PP0PG0 was slightly inferior to that of the original strain PPGS115. Except for strain PPCPG0, the growth performance and formate utilization capacity of the other strains were basically the same. However, the OD of the recombinant strain PPCPG0, which simultaneously contains both the CBB cycle and the PprGly pathway, was significantly different. 600 Instead, it decreased by 22.31%. Compared to the endogenous PprGly pathway, the heterologous YlrGly pathway showed better compatibility with the chassis and better formic acid utilization. Therefore, subsequent research on recombination will be based on recombinant strains PP0YG0 and PPCYG0 expressing the YlrGly pathway.

[0102] 4. Verification of formic acid utilization ability of artificially carbon-fixed Pichia pastoris strains

[0103] The strain PP0YG0 was tested by adding different concentrations of formate solution to minimal glucose medium. Formate has a significant toxic effect on cell growth; even the addition of 1 mM formate significantly affects cell growth, while formate at 100 mM and above completely inhibits cell growth. Compared to PPGS115 and PPC000, recombinant strains PP0YG0 and PPCYG0 can grow normally, indicating that the expression of the YlrGly pathway plays a role in formate detoxification and restores normal cell growth. However, comparing strains PP0YG0 and PPCYG0, PP0YG0, which expresses only the YlrGly pathway, grows significantly better than PPCYG0, which expresses both the CBB cycle and the YlrGly pathway.

[0104] 5. Verification of the methanol consumption capacity of recombinant strains

[0105] Recombinant strains PPGS115, PPC000, PP0YG0, and PPCYG0 were grown by dilution and spotting on minimal media containing only methanol, methanol + NaHCO3, and methanol + NaHCO3 + formate, respectively. Expression of the YlrGly pathway significantly inhibited growth, with strains PP0YG0 and PPCYG0 showing significantly slower growth than PPGS115 and PPC000. However, comparing strains PP0YG0 and PPCYG0, PPCYG0 expressing the CBB cycle showed superior growth compared to PP0YG0. This further demonstrates that CBB cycle expression does not affect methanol utilization by the strains but rather has a synergistic promoting effect.

[0106] 6. Detection of carbon emissions from artificially carbon-fixing Pichia pastoris strains

[0107] Carbon-fixing strains PPGS115, PPC000, PP0YG0, and PPCYG0 were inoculated into anaerobic flasks containing minimal methanol medium (the anaerobic flasks contained air required for normal fermentation, not CO2). The cultures were incubated at an initial OD600 of 1.0 for 96 h, and CO2 emissions were measured. The original strain PPGS115 exhibited the highest CO2 emissions at 5.30 mg / L, while strains PPC000, PP0YG0, and PPCYG0 emitted 4.07, 3.53, and 3.35 mg / L, respectively. This indicates that the introduction of the CBB cycle, the YlrGly pathway, and the CBB cycle + YlrGly pathway reduced CO2 emissions by 23.22%, 33.32%, and 36.67%, respectively. However, comparing the growth performance of the four strains, PPGS115, PPC000, PP0YG0, and PPCYG0 achieved OD values ​​of 0.44, 0.47, 0.43, and 0.29, respectively. 600Growth. The carbon emission per unit biomass of strain PPGS115 was 8.43 μg / mg CDW, while those of PPC000, PP0YG0, and PPCYG0 were 6.61, 5.56, and 6.94 μg / mg CDW, respectively, representing decreases of 21.59%, 34.07%, and 17.69%. Figure 3 ).

[0108] The relevant culture medium formulation is as follows:

[0109] (1) LB medium: yeast extract 5 g / L; peptone 10 g / L; sodium chloride 10 g / L. 2% agar powder was added to the solid medium. The medium was sterilized at 121℃ for 15 min. LB-Amp was LB medium with an additional 100 mg / L ampicillin; LB-Hph was LB medium with an additional 50 mg / L hygromycin.

[0110] (2) YPD medium: yeast extract 10 g / L; peptone 20 g / L; glucose 20 g / L. 2% agar powder was added to the solid medium. The medium was sterilized at 115℃ for 20 min. YPD-Hph is YPD medium with an additional 50 mg / L hygromycin.

[0111] (3) Minimal culture medium: Ammonium sulfate 7.5 g / L; Potassium dihydrogen phosphate 11.93 g / L; Dipotassium hydrogen phosphate 2.14 g / L; Magnesium sulfate heptahydrate 0.5 g / L; Vitamin solution 1 ml / L; Trace element solution 1 ml / L; Add 20 mg / L uracil as needed. For minimum methanol culture, add an additional 2% methanol; for minimum yeast extract culture medium, add an additional 0.1% yeast extract; for minimum methanol yeast extract culture medium, add an additional 2% methanol and 0.1% yeast extract; for minimum sodium bicarbonate culture medium, add an additional 10 mM NaHCO3.

[0112] (4) LB-KanR is LB medium with an additional 50 mg / L kanamycin sulfate.

[0113] (5) LB-NTC is an LB medium supplemented with 100 mg / L Norrilskine.

[0114] (6) YPD-G418 is a YPD medium with an additional 350 mg / L of genetic mycotoxin.

[0115] (7) YPD-NTC is YPD medium with an additional 100 mg / L Norsin.

[0116] Example 6: Artificially Carbon-Fixed Pichia pastoris Strain 13 Metabolic flux analysis of C isotopes

[0117] For all artificially carbon-fixing strains at the minimum 13 Amino acids in C methanol medium 13 C labeling levels were detected. In minimal methanol medium, except... 13 Besides methanol, CO2 in the air is the only usable carbon source. Therefore, 13 A lower C-label level indicates that more biomass originates from CO2, meaning the strain has a stronger carbon fixation capacity. The strain was inoculated into 5 mL of YPD liquid medium and cultured overnight at 30°C and 200 rpm. The cultured bacterial suspension was then resuspended by centrifugation at an initial OD600 of 0.2 and inoculated into 10 mL of fresh M9 liquid medium, with the addition of 1% methanol and 20 Mn... 13 C-NaHCO3 was incubated at 30℃ and 200 rpm for approximately 144 hours before gas chromatography-mass spectrometry (GC-MS) analysis. Figure 4 of 13 The C-methanol labeling results showed that the introduction of the YlrGly pathway significantly increased the carbon fixation capacity of the strain. (The remaining text appears to be incomplete and contains errors.) 13 The levels of C-labeled amino acids all decreased to some extent, with the most significant decreases observed in serine, threonine, and leucine, which dropped from 94.7%, 92.3%, and 91.8% to 92.4%, 90.7%, and 89.2%, respectively. Interestingly, although the expression of the CBB cycle alone failed to demonstrate effective carbon fixation performance, the co-expression of the CBB cycle and the YlrGly pathway led to increased carbon fixation in strain PPCYG0. 13 C-labeled levels further decreased. In particular, levels of glycine, leucine, aspartic acid, and glutamic acid... 13 The C-labeling levels were 90.1%, 87.7%, 84.8%, and 83.2%, respectively, representing decreases of 3.7%, 4.1%, 2.6%, and 5.4% compared to the original strain PPGS115. A comparison of the eight amino acids showed that alanine and valine... 13 The C-labeling level remained consistently high across the four strains, indicating that their synthesis primarily stemmed from methanol assimilation and was largely unaffected by heterologous carbon fixation pathways. These results suggest that PP0YG0 and PPCYG0... 13 The C-methanol labeling level was significantly lower than that of the original strain PPGS115, proving that CO2 can be fixed through the reconstructed reductive glycine pathway, and further indicating that this pathway was successfully opened.

[0118] In summary, this invention has for the first time constructed a Pichia pastoris cell factory with high carbon atom utilization and low carbon emissions. We attempted (1) knocking out the methanol dissimilatory pathway in Pichia pastoris to block carbon atom loss; and (2) designing and introducing an artificial carbon fixation pathway to recover carbon atom. By expressing the Calvin cycle and the reducing glycine pathway derived from Yersinia lipolytica in Pichia pastoris, we successfully constructed an artificial carbon-fixing cell with formate and CO2 assimilation capabilities. This endows Pichia pastoris with the ability to utilize CO2 and formic acid, and further improves its methanol assimilation and CO2 utilization efficiency, laying the foundation for the future production of high value-added products using Pichia pastoris, which has far-reaching significance.

[0119] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A Pichia pastoris engineered strain with artificial carbon fixation, characterized in that, The strain was obtained by introducing genes related to the Calvin cycle pathway and the reducing glycine pathway from Yersinia lipolytica into the host genome; The genes related to the Calvin cycle pathway include the ribulose 1,5-bisphosphate carboxylase-oxygenase gene RuBisCO from *Thiobacillus denitrifyingus*, the phosphoriboskinase gene PRK, two molecular chaperone genes groEL and groES from *Escherichia coli*, the formaldehyde lyase gene FLS, the phosphoglycerate kinase gene PGK1, the glyceraldehyde-3-phosphate dehydrogenase gene TDH3, and the triphosphate isomerase gene TPI1. The genes related to the reducing glycine pathway from *Yarrowia lipolytica* include C1-tetrahydrofolate synthase MIS1, aminomethyltransferase GcvT, glycine cleavage system P protein GcvP, and glycine cleavage system H protein GcvH. The genes related to the reducing glycine pathway from Yersinia lipolytica are expressed by the BB3eH_AE-YlrGly expression cassette, which is a complete expression cassette with BB3eH_AE as the vector backbone and the ENO promoter integrated within the backbone to regulate YlrGly. The NCBI-GeneID of the C1-tetrahydrofolate synthase MIS1 is 2907923; the NCBI-GeneID of the aminomethyltransferase GcvT is 2907779; the NCBI-GeneID of the glycine cleavage system P protein GcvP is 2905991; the NCBI-GeneID of the glycine cleavage system H protein GcvH is 2912864; the sequence of the ribulose 1,5-bisphosphate carboxylase-oxygenase gene RuBisCO is shown in SEQ ID NO:1; the sequence of the phosphoriboskinase gene PRK is shown in SEQ ID NO:2; the sequence of the molecular chaperone gene groEL is shown in SEQ ID NO:3; the sequence of the molecular chaperone gene groES is shown in SEQ ID NO:4; the sequence of the formaldehyde lyase gene FLS is shown in SEQ ID NO:5; the sequence of the phosphoglycerate kinase gene PGK1 is shown in SEQ ID NO:6; and the sequence of the glyceraldehyde-3-phosphate dehydrogenase gene TDH3 is shown in SEQ ID NO:

6. As shown in NO:7; the sequence of the triphosphate isomerase gene TPI1 is shown in SEQ ID NO:8; The host strain is Pichia pastoris GS115.

2. The method for constructing the artificial carbon-fixing Pichia pastoris engineered strain according to claim 1, characterized in that, Includes the following steps: 1) Genes related to the Calvin cycle pathway were cloned into the vector BB1_23, thereby obtaining recombinant plasmids BB1_23_RuBisCO, BB1_23_PRK, BB1_23_groEL, BB1_23_groES, BB1_23_FLS, BB1_23_PGK1, BB1_23_TDH3 and BB1_23_TPI1; 2) Recombinant plasmids containing each gene expression cassette were constructed using Golden Gate Assembly. After the reaction, the reaction system was directly transformed into E. coli DH5α. E. coli carrying the recombinant plasmids were screened using LB-AmpR plates to obtain the recombinant plasmids BB2_AB-RuBisCO, BB2_BC-PRK, BB2_CD-groEL, BB2_DE-groES, BB2_EF-FLS, BB2_FG-PGK1, BB2_GH-TDH3, and BB2_HI-TPI1. The CBB cyclic expression plasmid BB3rN_AI-CBB was also constructed using Golden Gate Assembly. 3) After the reaction was completed, the reaction system was directly transformed into Escherichia coli DH5α. The E. coli carrying the recombinant plasmid was screened by LB-NTC plates. The constructed plasmid BB3rN_AI-CBB was linearized by Asc I restriction enzyme digestion, purified by gel extraction kit, electroporated into Pichia pastoris PPGS115, and screened by YPD-NTC plates to obtain recombinant Pichia pastoris PPC000. 4) The plasmid BB3eH_AE-YlrGly was linearized by Hpa I restriction enzyme digestion, purified by a gel extraction kit, electroporated into recombinant Pichia pastoris PPC000, and screened by YPD-Hph plates to obtain recombinant Pichia pastoris PPCYG0. The plasmid BB3eH_AE-YlrGly is a complete expression cassette of YlrGly, with BB3eH_AE as the vector backbone and the ENO promoter integrated within the backbone to regulate YlrGly.

3. The application of the engineered Pichia pastoris strain with artificial carbon fixation as described in claim 1 in CO2 bioconversion.

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