A method for synthesizing natural products by using methanol yeast chassis cells and an engineered strain

Abstract

The application discloses a method for synthesizing natural products by using methanol yeast chassis cells and an engineering strain, relates to the technical field of genetic engineering, and comprises a squalene engineering strain.The construction of the squalene engineering strain comprises the following steps: cloning a rate-limiting enzyme gene in Pichia pastoris GS115 into a vector pPICZA with a PAOX1 promoter, electrically transforming a linearized recombinant plasmid into the Pichia pastoris GS115, integrating the linearized recombinant plasmid into a genomic HIS4 site, screening positive transformants, and obtaining a first strain.The first strain is synthesized, the squalene synthesis capacity is established in the chassis cells, the problem that the wild-type Pichia pastoris has a very low endogenous mevalonate pathway flow is solved, the rate-limiting bottleneck of the squalene synthesis pathway is eliminated, and the precursor supply level is improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a method and engineered strain for synthesizing natural products using methanol yeast chassis cells. Background Technology

[0002] Natural products are among the core raw materials in the pharmaceutical, cosmetic, and functional food industries. Terpenoids, such as squalene, are particularly valued for their excellent bioactivities, including antioxidant and immunomodulatory effects. Traditional methods of obtaining squalene primarily involve extraction from deep-sea shark liver and chemical synthesis. The former suffers from limited resources, difficulty in acquisition, high costs, and ecological damage; the latter is environmentally unfriendly due to its complex reaction processes, numerous byproducts, and severe pollution.

[0003] Methanol yeast, as a commonly used chassis cell, possesses characteristics such as rapid growth rate, high carbon source utilization efficiency, and strong protein expression ability. Pichia pastoris, as a chassis cell, primarily directs its carbon metabolic flow towards cell growth and basal metabolism, resulting in extremely low accumulation of the target natural product. By introducing key enzyme genes in the squalene biosynthesis pathway and precisely regulating its metabolic pathway, efficient squalene synthesis can be achieved.

[0004] Patent CN120624508B discloses a recombinant Pichia pastoris strain, its construction method, and its uses. The above patent has achieved a significant increase in the yield of p-coumaric acid, systematically optimized the synthesis pathway of p-coumaric acid, and constructed a recombinant Pichia pastoris strain that efficiently produces p-coumaric acid using methanol as a carbon source.

[0005] The aforementioned patents have addressed the significant challenges in the effective microbial synthesis of coumaric acid through a combination of factors, including optimizing metabolic flow, removing restrictions, strengthening direct precursors, reducing diversion, and increasing sources. However, there is still room for optimization in the synthesis of natural products. This application has achieved the synthesis of the natural product squalene, solving the problem of extremely low flow rates of the endogenous mevalonic acid pathway in wild-type Pichia pastoris.

[0006] Therefore, this application proposes a method and engineered strain for synthesizing the natural product squalene using methanol yeast chassis cells. Summary of the Invention

[0007] The purpose of this invention is to provide a method and engineered strain for synthesizing natural products using yeast chassis cells, in order to solve the technical problem mentioned in the background art of wild-type Pichia pastoris due to its extremely low endogenous mevalonic acid pathway flow.

[0008] To achieve the above objectives, the present invention provides the following technical solution: an engineered strain for synthesizing natural products using yeast chassis cells, including a squalene engineered strain, wherein the construction of the squalene engineered strain includes the following steps: The rate-limiting enzyme gene endogenous in Pichia pastoris GS115 was cloned into the vector pPICZA containing the PAOX1 promoter. The linearized recombinant plasmid was electroporated into Pichia pastoris GS115 and integrated into the HIS4 site of the genome. Positive transformants were screened to obtain the first strain. ERG20 and ERG9 were overexpressed sequentially on the first strain to obtain the second and third strains. Using the CRISPR-Cas9 system, a tandem expression cassette containing ERG8 and ERG10 genes was targeted and integrated into the PNSIV16 site of the third strain genome to construct the fourth strain. A tandem expression cassette containing ERG12, ERG13, and ERG19 genes was targeted and integrated into the Int6 site of the fourth strain genome to construct the fifth strain. Using the CRISPR-Cas9 system, the copy number of the rate-limiting enzyme gene was increased at different neutral sites in the fifth strain to construct the ninth strain. The TKL gene expression cassette was integrated into the PNSI2 site of the ninth strain genome to construct the tenth strain. The tandem expression cassette containing DAK and RPE was targeted and integrated into the PNSI14 site of the tenth strain genome to construct the squalene engineered strain.

[0009] Preferably, the rate-limiting enzyme gene is the 3-hydroxy-3-methylglutaryl-CoA reductase gene.

[0010] Preferably, the second strain is obtained by integrating the ERG20 gene expression cassette into the HIS4 site of the first strain's genome.

[0011] Preferably, the third strain is obtained by integrating the ERG9 gene expression cassette into the neutral site Int1 of the second strain's genome.

[0012] Preferably, the process of increasing the copy number of the rate-limiting enzyme gene at different neutral sites on the fifth strain includes the following steps: A single copy of the 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the Int12 site of the fifth strain genome to construct the sixth strain; The expression cassette of the 3-hydroxy-3-methylglutaryl-CoA reductase gene was integrated into the Int12 site of the sixth strain genome to construct the seventh strain.

[0013] Preferably, the process of increasing the copy number of the rate-limiting enzyme gene at different neutral sites on the fifth strain further includes the following steps: The single-copy 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the PNSII4 site of the seventh strain genome to construct the eighth strain; The expression cassette of the 3-hydroxy-3-methylglutaryl-CoA reductase gene was integrated into the PNSII4 site of the genome of strain 8 to construct strain 9.

[0014] Preferably, the method for screening positive transformants includes: Pichia pastoris GS115, which was electroporated with linearized recombinant plasmid, was plated on YPDZ solid plates containing the antibiotic Zeocin and incubated upside down in a 30°C incubator for 2-4 days. Single colonies growing on the YPD solid plates were used as primary screening positive clones, and positive transformants were obtained.

[0015] Preferably, the parameters of the YPD solid plate are: yeast extract 1% w / v, peptone 2% w / v, glucose 2% w / v, agar 2% w / v, and Zeocin antibiotic 100 μg / mL.

[0016] Preferably, the method for synthesizing the natural product includes the following steps: S1. Inoculate the squalene engineered strain onto YPD solid plates and incubate upside down in a 30℃ incubator for 48-72 hours. Inoculate the resulting single colonies into Erlenmeyer flasks containing YPD liquid medium and incubate with shaking at 30℃ and 250 rpm for 18-24 hours to obtain the seed culture. S2. Inoculate the seed culture into a fermenter containing MSM salt medium with 4% glycerol at an inoculation rate of 5-10%. Adjust the fermenter temperature to 30°C, adjust the pH to 5.5 with ammonia, and adjust the stirring speed and aeration rate to control dissolved oxygen at 30-40%. When dissolved oxygen rises sharply, add glycerol feed solution. S3. Adjust the fermenter temperature to 20℃, adjust the pH to 6 with ammonia water, adjust the stirring speed and aeration rate to control dissolved oxygen at 20-30%, add terbinafine, add methanol feed solution, and separate solid and liquid by centrifugation to obtain bacterial cells; S4. Add zirconium oxide beads and ethyl acetate to a cell grinding tube containing bacterial cells, place the grinding tube in a cryogenic grinder, grind at -5°C for 30 minutes, and perform separation and purification to obtain the natural product squalene.

[0017] Preferably, the glycerol feed solution and the methanol feed solution contain 1.2% v / v PTM1 solution.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention synthesizes a first strain and clones the endogenous 3-hydroxy-3-methylglutaryl-CoA reductase gene of Pichia pastoris into the vector pPICZA with the PAOX1 promoter, thereby establishing squalene synthesis capability in the chassis cells. This solves the problem of extremely low endogenous mevalonic acid pathway flow in wild-type Pichia pastoris, removes the rate-limiting bottleneck of the squalene synthesis pathway, and improves the precursor supply level. 2. This invention achieves relay enhancement of precursor supply by integrating the ERG20 gene expression cassette on the basis of the first strain and the ERG9 gene expression cassette on the basis of the second strain. This solves the problem that the enhanced HMGR activity in the first strain may not be efficiently converted into the final product precursor, solves the carbon flow splitting problem, and increases the yield of squalene. 3. This invention achieves maximum dosage of the rate-limiting enzyme by sequentially integrating multiple copies of the 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette at the Int12 and PNSII4 neutral sites, thereby solving the deep bottleneck of insufficient absolute catalytic capacity under high throughput and improving the overall flow rate and final yield of the synthetic pathway. 4. This invention constructs a tenth strain and a squalene engineered strain, which optimizes carbon flow, improves the overall efficiency of methanol carbon source assimilation into central metabolites, realizes systematic reprogramming of carbon metabolism, and improves yield and carbon conversion rate. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the process for constructing the third strain of the present invention; Figure 2 This is a schematic diagram of the process for constructing the fifth strain of the present invention; Figure 3 This is a schematic diagram of the process for constructing the seventh strain of the present invention; Figure 4 This is a schematic diagram of the process for constructing squalene-engineered strains according to the present invention; Figure 5 This is a schematic diagram of the seed liquid preparation process of the present invention; Figure 6 This is a schematic diagram of the bacterial cell preparation process of the present invention; Figure 7 This is a schematic diagram of the synthesis process of squalene, a natural product of the present invention. Figure 8 This is a schematic diagram of the YPD solid plate of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1, please refer to Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 8 An engineered strain for synthesizing natural products using yeast chassis cells, including a squalene engineered strain, wherein the construction of the squalene engineered strain includes the following steps: The endogenous 3-hydroxy-3-methylglutaryl-CoA reductase gene of Pichia pastoris GS115 was cloned into the vector pPICZA with PAOX1 promoter. The linearized recombinant plasmid was electroporated into Pichia pastoris GS115 and integrated into the HIS4 site of the genome. Positive transformants were screened to obtain the first strain. In the first strain, the ERG20 gene expression cassette was integrated into the HIS4 site of the first strain genome to obtain the second strain. In the second strain, the ERG9 gene expression cassette was integrated into the Int1 neutral site of the second strain genome to obtain the third strain. Using the CRISPR-Cas9 system, a tandem expression cassette containing ERG8 and ERG10 genes was targeted and integrated into the PNSIV16 site of the third strain genome to construct the fourth strain. A tandem expression cassette containing ERG12, ERG13, and ERG19 genes was targeted and integrated into the Int6 site of the fourth strain genome to construct the fifth strain. Using the CRISPR-Cas9 system, a single-copy 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the Int12 site of the fifth strain genome to construct the sixth strain; a double-copy 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the Int12 site of the sixth strain genome to construct the seventh strain; a single-copy 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the PNSII4 site of the seventh strain genome to construct the eighth strain; a double-copy 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the PNSII4 site of the eighth strain genome to construct the ninth strain; a TKL gene expression cassette was integrated into the PNSI2 site of the ninth strain genome to construct the tenth strain; and a tandem expression cassette containing DAK and RPE was targeted and integrated into the PNSI14 site of the tenth strain genome to construct a squalene engineered strain. The method for synthesizing natural products includes the following steps: The squalene engineered strain was inoculated onto YPD solid plates and incubated upside down in a 30°C incubator for 48 hours. The resulting single colonies were then inoculated into Erlenmeyer flasks containing YPD liquid medium and incubated with shaking at 30°C and 250 rpm for 18 hours to obtain the seed culture. The seed culture was inoculated into a fermenter containing MSM salt medium with 4% glycerol at a 10% inoculation rate. The fermenter temperature was adjusted to 30°C, the pH was adjusted to 5.5 with ammonia, and the dissolved oxygen was controlled at 40% by adjusting the stirring speed and aeration rate. When the dissolved oxygen rose sharply, glycerol feed solution containing 1.2% v / v PTM1 solution was added. Adjust the fermenter temperature to 20℃, adjust the pH to 6 with ammonia water, adjust the stirring speed and aeration rate to control dissolved oxygen at 30%, add terbinafine, and add methanol feed solution containing 1.2% v / v PTM1 solution. Separate the solid and liquid by centrifugation to obtain the bacterial cells. Zirconia beads and ethyl acetate were added to cell grinding tubes containing bacterial cells. The grinding tubes were then placed in a cryogenic grinder and ground at -5°C for 30 minutes. After separation and purification, the natural product squalene was obtained.

[0022] Furthermore, based on the fifth strain, single and double copies of the HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) gene expression cassette were sequentially integrated at the Int12 site, increasing the gene copy number and expression level of the key rate-limiting enzyme. The activity of HMGR determines the conversion rate from HMG-CoA to mevalonic acid. By adding an additional HMGR gene copy number at the new genomic site Int12, the total mRNA transcription level and protein expression level of HMGR enzyme in the cell were significantly increased. Based on the fifth strain with one basic HMGR copy, an additional HMGR copy was added at the Int12 site to enhance the initial dose of the rate-limiting step. Based on the sixth strain with two HMGR copies, a double copy of the HMGR expression cassette was integrated at the same Int12 site, inserting two consecutive HMGR genes at the Int12 site to achieve gene dose accumulation. Single and double copies of the HMGR gene expression cassette were integrated again at another neutral site, PNSII4, thereby amplifying the dose at multiple genomic sites and maximizing the rate-limiting enzyme throughput. Based on the ninth strain, the TKL gene expression cassette was integrated at the PNSI2 site to enhance the metabolic reflux of the pentose phosphate pathway. The TKL gene encodes a transketolase, which can accelerate the flux cycle of the PPP pathway, efficiently regenerate xylulose 5-phosphate, improve the assimilation rate of methanol and the overall carbon metabolism efficiency, and increase the supply of central metabolic intermediates such as glyceraldehyde-3-phosphate. Based on the tenth strain, a tandem expression cassette containing DAK and RPE was integrated at the PNSI14 site to synergistically optimize the methanol assimilation and pentose phosphate pathway. The DAK gene encodes dihydroxyacetone kinase, which is used to phosphorylate dihydroxyacetone. The RPE gene encodes ribulose phosphorylase, which is used for the isomerization of ribulose 5-phosphate. It works synergistically with TKL to maintain the efficient operation of the PPP pathway. The effect of RPE and the synergistic effect of TKL further enhance the flow efficiency of the PPP pathway, utilize and regenerate the reducing power and carbon skeleton generated by methanol metabolism, and solve the problem of insufficient precursor supply that may occur at high yield levels.

[0023] Example 2, please refer to Figure 6 , Figure 7 and Figure 8 The present invention provides an embodiment of an engineered strain that synthesizes natural products using yeast chassis cells. The construction of the first strain includes the following steps: The endogenous 3-hydroxy-3-methylglutaryl-CoA reductase gene of Pichia pastoris GS115 was cloned into the vector pPICZA containing the PAOX1 promoter. The linearized recombinant plasmid was electroporated into Pichia pastoris GS115 and integrated into the HIS4 site of the genome. The Pichia pastoris GS115 cells electroporated with the linearized recombinant plasmid were plated on YPDZ solid plates containing Zeocin antibiotic and incubated upside down in a 30°C incubator for 2-4 days. Single colonies growing on YPD solid plates were considered as positive clones for initial screening. Positive transformants were obtained by screening positive transformants to obtain the first strain. The parameters of the YPD solid plate were: yeast extract 1% w / v, peptone 2% w / v, glucose 2% w / v, agar 2% w / v, and Zeocin antibiotic 100 μg / mL. The method for synthesizing natural products includes the following steps: inoculating a first strain onto a YPD solid plate, and synthesizing the bacterial cells to obtain the natural product by the same steps as in Example 1.

[0024] Furthermore, using Pichia pastoris as a chassis, a squalene biosynthesis network was constructed through overexpression, multicopy integration, and carbon flow optimization. Fermentation was then used to provide the cellular environment and external conditions for the efficient operation of this network, while blocking product consumption. In wild-type Pichia pastoris GS115 chassis cells, the endogenous key rate-limiting enzyme gene (3-hydroxy-3-methylglutaryl-CoA reductase gene) was introduced and overexpressed, removing the natural bottleneck of the endogenous metabolic pathway and increasing precursor supply. This endowed wild-type GS115 with the ability to initially synthesize and accumulate squalene, utilizing homologous regeneration... The group integrated the endogenous HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) gene with a strong promoter into a specific site in the genome, thereby achieving a targeted enhancement of carbon flow to squalene precursors by removing the natural rate-limiting bottleneck of the MVA pathway. The first strain obtained serves as the foundational platform for subsequent expression of downstream enzymes, increasing gene copy number, and optimizing carbon flow. In the endogenous MVA pathway of Pichia pastoris, the expression level and total activity of intracellular HMGR enzyme were significantly increased by additionally integrating an HMGR gene copy driven by the strong promoter PAOX1 into the genome.

[0025] Example 3, please refer to Figure 1 , Figure 6 and Figure 7 An engineered strain for synthesizing natural products using yeast chassis cells, including a squalene engineered strain, wherein the construction of the squalene engineered strain includes the following steps: The endogenous 3-hydroxy-3-methylglutaryl-CoA reductase gene of Pichia pastoris GS115 was cloned into the vector pPICZA with PAOX1 promoter. The linearized recombinant plasmid was electroporated into Pichia pastoris GS115 and integrated into the HIS4 site of the genome. Positive transformants were screened to obtain the first strain. In the first strain, the ERG20 gene expression cassette was integrated into the HIS4 site of the first strain genome to obtain the second strain. In the second strain, the ERG9 gene expression cassette was integrated into the Int1 neutral site of the second strain genome to obtain the third strain. The method for synthesizing natural products includes the following steps: inoculating a third strain onto a YPD solid plate and synthesizing the bacterial cells to obtain the natural product through the same steps as in Example 1.

[0026] Furthermore, the ERG20 gene encodes farnesyl pyrophosphate synthase, a key downstream enzyme in the mevalonate pathway, responsible for polymerizing IPP and DMAPP to generate farnesyl pyrophosphate. FPP serves as a core precursor node for the synthesis of various products such as squalene, sterols, and ubiquinone. Overexpressing ERG20 on the basis of the first strain increases the intracellular supply of FPP, ensuring that the enhanced precursor flux of the first strain can be effectively converted into the materials required for squalene synthesis. The ERG9 gene encodes squalene synthase, a key enzyme catalyzing the condensation of FPP to squalene. Introducing and overexpressing ERG9 on the basis of the second strain establishes and strengthens the complete synthetic pathway from FPP to squalene, ensuring that sufficient precursor FPP can be efficiently converted into the target product squalene, thereby significantly increasing squalene yield. FPP is a competitive node in multiple metabolic pathways. Overexpressing ERG9 can compete with other intracellular FPP consumption pathways for substrates, guiding more FPP towards squalene synthesis, thereby significantly reducing precursor diversion loss.

[0027] Example 4, please refer to Figure 1 , Figure 2 , Figure 6 and Figure 7 An engineered strain for synthesizing natural products using yeast chassis cells, including a squalene engineered strain, wherein the construction of the squalene engineered strain includes the following steps: The endogenous 3-hydroxy-3-methylglutaryl-CoA reductase gene of Pichia pastoris GS115 was cloned into the vector pPICZA with PAOX1 promoter. The linearized recombinant plasmid was electroporated into Pichia pastoris GS115 and integrated into the HIS4 site of the genome. Positive transformants were screened to obtain the first strain. In the first strain, the ERG20 gene expression cassette was integrated into the HIS4 site of the first strain genome to obtain the second strain. In the second strain, the ERG9 gene expression cassette was integrated into the Int1 neutral site of the second strain genome to obtain the third strain. Using the CRISPR-Cas9 system, a tandem expression cassette containing the ERG8 and ERG10 genes was targeted and integrated into the PNSIV16 site of the third strain genome to construct the fourth strain. A tandem expression cassette containing the ERG12, ERG13, and ERG19 genes was targeted and integrated into the Int6 site of the fourth strain genome to construct the fifth strain.

[0028] The method for synthesizing natural products includes the following steps: inoculating the fifth strain onto a YPD solid plate, and synthesizing the bacterial cells to obtain the natural product by the same steps as in Example 1.

[0029] Furthermore, based on the third strain that already expressed HMGR, ERG20, and ERG9, a tandem expression cassette containing ERG8 and ERG10 genes was integrated at the PNSIV16 site to enhance the upstream module of the mevalonate pathway and increase the supply of the universal precursor IPP / DMAPP. The ERG8 gene encodes mevalonate kinase, which catalyzes the conversion of mevalonate to mevalonate 5-phosphate; the ERG10 gene encodes acetyl-CoA acetyltransferase, which enhances the entire upstream metabolic flux from acetyl-CoA to mevalonate and then to its phosphorylated products. Overexpression of ERG8 and ERG10 can prevent the formation of new metabolic bottlenecks due to insufficient upstream supply, ensuring that the flux enhanced by HMGR can be smoothly transferred downstream, thereby achieving a further increase in yield. Based on the fourth strain, a tandem expression cassette containing ERG12, ERG13, and ERG19 genes was integrated at the Int6 site to enhance the complete mevalonate pathway and maximize the synthesis capacity of IPP / DMAPP precursors. The ERG12 gene encodes mevalonate kinase, catalyzing the conversion of mevalonate 5-phosphate to mevalonate 5-bisphosphate; the ERG13 gene encodes HMG-CoA synthase, catalyzing the conversion of acetyl-CoA and acetyl-CoA to HMG-CoA; and the ERG19 gene encodes mevalonate bisphosphate decarboxylase, catalyzing the conversion of mevalonate 5-bisphosphate to isopentenyl pyrophosphate. Overexpression of ERG12, ERG13, and ERG19 increased the intracellular concentration of IPP / DMAPP, enabling the entire squalene synthesis pathway to operate at a higher throughput level. This resulted in a sustained increase in squalene production and solved the precursor supply problem for squalene synthesis.

[0030] Example 5, please refer to Figure 1 , Figure 2 , Figure 3 , Figure 6 and Figure 7 An engineered strain for synthesizing natural products using yeast chassis cells, including a squalene engineered strain, wherein the construction of the squalene engineered strain includes the following steps: The rate-limiting enzyme gene endogenous in Pichia pastoris GS115 was cloned into the vector pPICZA containing the PAOX1 promoter. The linearized recombinant plasmid was electroporated into Pichia pastoris GS115 and integrated into the HIS4 site of the genome. Positive transformants were screened to obtain the first strain. ERG20 and ERG9 were overexpressed sequentially on the first strain to obtain the second and third strains. Using the CRISPR-Cas9 system, a tandem expression cassette containing ERG8 and ERG10 genes was targeted and integrated into the PNSIV16 site of the third strain genome to construct the fourth strain. A tandem expression cassette containing ERG12, ERG13, and ERG19 genes was targeted and integrated into the Int6 site of the fourth strain genome to construct the fifth strain. Using the CRISPR-Cas9 system, the copy number of the rate-limiting enzyme gene was increased at different neutral sites in the fifth strain to construct the ninth strain; The method for synthesizing natural products includes the following steps: inoculating the ninth strain onto a YPD solid plate, and synthesizing the bacterial cells to obtain the natural product by the same steps as in Example 1.

[0031] Comparative Example 1: A method for synthesizing natural products using yeast chassis cells, the method comprising the following steps: S1. Inoculate the squalene engineered strain onto YPD solid plates and incubate upside down in a 30℃ incubator for 48 hours. Inoculate the resulting single colonies into Erlenmeyer flasks containing YPD liquid medium and incubate with shaking at 30℃ and 250 rpm for 18 hours to obtain the seed culture. S2. Inoculate the seed culture at a rate of 10% into a fermenter containing MSM salt medium with 4% glycerol. Adjust the fermenter temperature to 30°C, adjust the pH to 5.5 with ammonia, and control the dissolved oxygen at 40% by adjusting the stirring speed and aeration rate. When the dissolved oxygen rises sharply, add glycerol feed solution containing 1.2% v / v PTM1 solution. S3. Adjust the fermenter temperature to 20℃, adjust the pH to 6 with ammonia water, adjust the stirring speed and aeration rate to control dissolved oxygen at 30%, and add methanol feed solution containing 1.2% v / v PTM1 solution. Separate the solid and liquid by centrifugation to obtain the bacterial cells; terbinafine is not added. S4. Add zirconium oxide beads and ethyl acetate to a cell grinding tube containing bacterial cells, place the grinding tube in a cryogenic grinder, grind at -5°C for 30 minutes, and perform separation and purification to obtain the natural product squalene.

[0032] Comparative Example 2: A method for synthesizing natural products using yeast chassis cells, the method comprising the following steps: S1. Pichia pastoris GS115 was inoculated onto YPD solid plates and incubated upside down in a 30℃ incubator for 48 hours. The resulting single colonies were then inoculated into Erlenmeyer flasks containing YPD liquid culture medium and incubated with shaking at 30℃ and 250 rpm for 18 hours to obtain the seed culture. S2. Inoculate the seed culture at a rate of 10% into a fermenter containing MSM salt medium with 4% glycerol. Adjust the fermenter temperature to 30°C, adjust the pH to 5.5 with ammonia, and control the dissolved oxygen at 40% by adjusting the stirring speed and aeration rate. When the dissolved oxygen rises sharply, add glycerol feed solution containing 1.2% v / v PTM1 solution. S3. Adjust the fermenter temperature to 20℃, adjust the pH to 6 with ammonia water, adjust the stirring speed and aeration rate to control dissolved oxygen at 30%, add terbinafine, add methanol feed solution containing 1.2% v / v PTM1 solution, and separate the solid and liquid by centrifugation to obtain the bacterial cells. S4. Add zirconium oxide beads and ethyl acetate to a cell grinding tube containing bacterial cells, place the grinding tube in a cryogenic grinder, grind at -5°C for 30 minutes, and perform separation and purification to obtain the natural product squalene.

[0033] Comparative Example 3: A method for synthesizing natural products using yeast chassis cells, the method comprising the following steps: S1. Inoculate the squalene engineered strain onto YPD solid plates and incubate upside down in a 30℃ incubator for 48 hours. Inoculate the resulting single colonies into Erlenmeyer flasks containing YPD liquid medium and incubate with shaking at 30℃ and 250 rpm for 18 hours to obtain the seed culture. S2. Inoculate the seed culture into a fermenter containing MSM salt medium with 4% glycerol at a seed inoculation rate of 10%. Adjust the fermenter temperature to 30°C, adjust the pH to 5.5 with ammonia water, and adjust the stirring speed and aeration rate to control dissolved oxygen at 40%. When the dissolved oxygen rises sharply, add glycerol feed solution. S3. Adjust the fermenter temperature to 20℃, adjust the pH to 6 with ammonia water, adjust the stirring speed and aeration rate to control dissolved oxygen at 30%, add terbinafine, add methanol feed solution, and separate solid and liquid by centrifugation to obtain bacterial cells; S4. Add zirconium oxide beads and ethyl acetate to a cell grinding tube containing bacterial cells, place the grinding tube in a cryogenic grinder, grind at -5°C for 30 min, and perform separation and purification to obtain the natural product squalene; wherein, the glycerol feed solution and methanol feed solution do not contain 1.2% v / v PTM1 solution.

[0034] Performance testing Test 1, cell dry weight test: Take 10 mL of the fermentation broth from Examples 1-5 and Comparative Examples 1-3 before solid-liquid separation as a sample, and vacuum filter it through quantitative filter paper that has been pre-dried to constant weight. Place the filter paper with cells in an oven and dry it to constant weight. Weigh it accurately with an analytical balance and calculate the cell dry weight.

[0035] Test 2, Squalene titer and yield test: 10 mL of the fermentation broth from Examples 1-5 and Comparative Examples 1-3 before solid-liquid separation was taken as a sample and placed in a cell grinding tube. An equal volume of ethyl acetate and an appropriate amount of zirconia beads were added. The mixture was ground at -5°C for 30 min using a cryogenic grinder. After centrifugation, the clear ethyl acetate phase at the top was collected, filtered, and the squalene extract to be tested was obtained. Gas chromatography-flame ionization detector was used for analysis to calculate the squalene titer and squalene yield.

[0036] The performance test results are shown in Table 1 below: Table 1. Performance Test Results In summary, overexpression of the 3-hydroxy-3-methylglutaryl-CoA reductase gene increased yield from zero; introduction and overexpression of ERG20 and ERG9 nearly doubled the yield; strengthening the intact mevalonate pathway further increased yield; increasing the copy number of the rate-limiting enzyme gene further increased yield; and terbinafine and PTM1 were crucial to the yield increase.

[0037] Working principle: By integrating and overexpressing the 3-hydroxy-3-methylglutaryl-CoA reductase gene, the mevalonate pathway is strengthened, the bottleneck of precursor supply is solved, and the ERG20 and ERG9 genes are integrated and overexpressed in sequence to catalyze the generation of farnesyl pyrophosphate, the direct precursor of squalene, and the final synthesis of the precursor into squalene, forming a complete synthetic pathway. By integrating multiple copies of rate-limiting enzyme genes at multiple genomic sites using CRISPR-Cas9 technology, their expression levels are significantly increased, maximizing the synthesis throughput. By integrating and overexpressing genes such as TKL, DAK, and RPE, the methanol assimilation pathway and pentose phosphate pathway are optimized, improving the efficiency of cellular methanol utilization and directing more carbon flux to acetyl-CoA, the precursor required for squalene synthesis. Glycerol was used as a carbon source to promote high-density bacterial growth. Then, methanol was switched as both a carbon source and an inducer to activate the potent AOX1 promoter, driving the expression of all exogenous genes. During the induction phase, the specific inhibitor terbinafine was added to inhibit the activity of squalene epoxidase, thereby blocking the further metabolism of squalene by the cells. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. An engineered strain that synthesizes natural products using the chassis cells of *Saccharomyces cerevisiae*, characterized in that: Including squalene engineered strains, the construction of which includes the following steps: The rate-limiting enzyme gene endogenous in Pichia pastoris GS115 was cloned into the vector pPICZA containing the PAOX1 promoter. The linearized recombinant plasmid was electroporated into Pichia pastoris GS115 and integrated into the HIS4 site of the genome. Positive transformants were screened to obtain the first strain. ERG20 and ERG9 were overexpressed sequentially on the first strain to obtain the second and third strains. Using the CRISPR-Cas9 system, a tandem expression cassette containing ERG8 and ERG10 genes was targeted and integrated into the PNSIV16 site of the third strain genome to construct the fourth strain. A tandem expression cassette containing ERG12, ERG13, and ERG19 genes was targeted and integrated into the Int6 site of the fourth strain genome to construct the fifth strain. Using the CRISPR-Cas9 system, the copy number of the rate-limiting enzyme gene was increased at different neutral sites in the fifth strain to construct the ninth strain. The TKL gene expression cassette was integrated into the PNSI2 site of the ninth strain genome to construct the tenth strain. The tandem expression cassette containing DAK and RPE was targeted and integrated into the PNSI14 site of the tenth strain genome to construct the squalene engineered strain.

2. The engineered strain for synthesizing natural products using the chassis cells of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The rate-limiting enzyme gene is the 3-hydroxy-3-methylglutaryl-CoA reductase gene.

3. The engineered strain for synthesizing natural products using the chassis cells of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The second strain was obtained by integrating the ERG20 gene expression cassette into the HIS4 site of the first strain's genome.

4. The engineered strain for synthesizing natural products using the chassis cells of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The third strain was obtained by integrating the ERG9 gene expression cassette into the neutral site Int1 of the second strain's genome.

5. An engineered strain for synthesizing natural products using the chassis cells of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The process of increasing the copy number of the rate-limiting enzyme gene at different neutral sites on the fifth strain includes the following steps: A single copy of the 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the Int12 site of the fifth strain genome to construct the sixth strain; The expression cassette of the 3-hydroxy-3-methylglutaryl-CoA reductase gene was integrated into the Int12 site of the sixth strain genome to construct the seventh strain.

6. The engineered strain for synthesizing natural products using the chassis cells of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The process of increasing the copy number of the rate-limiting enzyme gene at different neutral sites on the fifth strain also includes the following steps: The single-copy 3-hydroxy-3-methylglutaryl-CoA reductase gene expression cassette was integrated into the PNSII4 site of the seventh strain genome to construct the eighth strain; The expression cassette of the 3-hydroxy-3-methylglutaryl-CoA reductase gene was integrated into the PNSII4 site of the genome of strain 8 to construct strain 9.

7. The engineered strain for synthesizing natural products using the chassis cells of *Saccharomyces cerevisiae* according to claim 1, characterized in that: The method for screening positive transformants includes: Pichia pastoris GS115, which was electroporated with linearized recombinant plasmid, was plated on YPDZ solid plates containing the antibiotic Zeocin and incubated upside down in a 30°C incubator for 2-4 days. Single colonies growing on the YPD solid plates were used as primary screening positive clones, and positive transformants were obtained.

8. An engineered strain for synthesizing natural products using the chassis cells of *Saccharomyces cerevisiae* according to claim 7, characterized in that: The parameters of the YPD solid plate are: yeast extract 1% w / v, peptone 2% w / v, glucose 2% w / v, agar 2% w / v, and Zeocin antibiotic 100 μg / mL.

9. A method for synthesizing natural products using *Saccharomyces cerevisiae* chassis cells, applicable to the engineered strain for synthesizing natural products using *Saccharomyces cerevisiae* chassis cells as described in any one of claims 1-8, characterized in that: The method for synthesizing natural products includes the following steps: S1. Inoculate the squalene engineered strain onto YPD solid plates and incubate upside down in a 30℃ incubator for 48-72 hours. Inoculate the resulting single colonies into Erlenmeyer flasks containing YPD liquid medium and incubate with shaking at 30℃ and 250 rpm for 18-24 hours to obtain the seed culture. S2. Inoculate the seed culture into a fermenter containing MSM salt medium with 4% glycerol at an inoculation rate of 5-10%. Adjust the fermenter temperature to 30°C, adjust the pH to 5.5 with ammonia, and adjust the stirring speed and aeration rate to control dissolved oxygen at 30-40%. When dissolved oxygen rises sharply, add glycerol feed solution. S3. Adjust the fermenter temperature to 20℃, adjust the pH to 6 with ammonia water, adjust the stirring speed and aeration rate to control dissolved oxygen at 20-30%, add terbinafine, add methanol feed solution, and separate solid and liquid by centrifugation to obtain bacterial cells; S4. Add zirconium oxide beads and ethyl acetate to a cell grinding tube containing bacterial cells, place the grinding tube in a cryogenic grinder, grind at -5°C for 30 minutes, and perform separation and purification to obtain the natural product squalene.

10. The method for synthesizing natural products using yeast chassis cells according to claim 9, characterized in that: The glycerol and methanol feed solutions contain 1.2% v / v PTM1 solution.

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