55 results about "Mevalonate pathway" patented technology

The present invention relates to a recombinant yeast with high squalene production, a construction method and a use thereof, wherein the recombinant yeast is a saccharomyces cerevisiae that expresses or overexpresses HMGR gene, ADA gene, LEU2 gene, MVA pathway gene and squalene synthase gene ERG9. The present invention integrates acetaldehyde dehydrogenase gene ADA and HMG-CoA reductase gene tHMGR in multiple copies in saccharomyces cerevisiae, integrates the screened high-activity phosphoketolase gene PK and phosphotransacetylase gene PTA, weakens epoxysqualene synthesis, strengthens the mevalonate pathway, overexpresses yeast endogenous squalene synthase gene, etc. to obtain a recombinant yeast with high squalene production, so that the production in the shake flask stage of the production process exceeds 5g / L, and the production in the fed batch fermentation stage exceeds 50g / L, breaking through the current bottleneck of microbial synthesis of squalene production, and is suitable for practical promotion and application.

The invention discloses a high-yield delta-tocotrienol genetically engineered bacterium and application thereof, and belongs to the technical field of gene recombination. The invention discloses a genetically engineered bacterium for high yield of delta-tocotrienols, which is characterized in that yarrowia lipolytica ATCC MYA-2613 is used as an original strain, and a ku70 gene is knocked out; the gene is used for expressing and coding 4-hydroxyphenylpyruvate dioxygenase, the gene is used for coding uromelanic acid phytate transferase and the gene is used for coding tocopherol cyclase; intensifying a shikimic acid pathway; a mevalonic acid pathway is strengthened; an isopentenol path is introduced; carrying out protein fusion on the HPD and the HPT; truncating a signal peptide of a gene encoding a tocopherol cyclase and back-filling a leucine defect. The genetically engineered bacterium constructed by the invention has industrial stability, can be amplified by more than 10 times in a 2L fermentation tank level compared with a shake flask level, can produce 616.45 mg / L of delta-tocotrienol after being cultured for 240 hours, and has industrial application prospects.

The invention relates to the technical field of bioengineering, in particular to a saccharomyces cerevisiae engineering strain for producing caryophyllene and application of the saccharomyces cerevisiae engineering strain. The invention provides a saccharomyces cerevisiae engineering strain which has enhanced expression and / or activity of mevalonic acid pathway enzyme and one or more modifications selected from the following (1)-(2): (1) weakened expression and / or activity of sterol synthesis pathway enzyme; (2) attenuated expression and / or activity of fatty acid synthetic pathway enzymes; compared with an original strain, the saccharomyces cerevisiae engineering strain has the advantage that the sesquiterpene yield is increased. Compared with an original strain, the saccharomyces cerevisiae engineering strain has the advantages that the production capacity of the sesquiterpenes such as the caryophyllene is obviously improved, the sesquiterpenes such as the caryophyllene can be efficiently synthesized, and the saccharomyces cerevisiae engineering strain has better application value.

The invention belongs to the technical field of bioengineering, and relates to a construction method and application of recombinant yarrowia lipolytica for synthesizing plant antitoxin Kauralexin A1. The method is realized by constructing recombinant yarrowia lipolytica. The construction method of the recombinant yarrowia lipolytica for synthesizing the Kauralexin A1 comprises the following steps: overexpressing a codon optimized antitoxin Kauralexin A1 biosynthetic pathway in yarrowia lipolytica, screening adaptive P450 reductase to improve the electron transfer efficiency in a P450 system, and constructing a metabolic pathway scaffold-free multi-enzyme complex to enhance cascade biological catalysis and metabolic flux, so as to obtain the recombinant yarrowia lipolytica for synthesizing the Kauralexin A1. The expression of the mevalonic acid pathway is enhanced, so that a sufficient GGPP precursor is provided for the biosynthesis of the Kauralexin A1. The construction method of the yarrowia lipolytica for biosynthesizing the antitoxin Kauralexin A1, provided by the invention, is simple to operate, and the constructed yarrowia lipolytica can be used for efficiently producing the Kauralexin A1 and has relatively high production and application values.

The present invention relates to the field of bioengineering, and in particular to an engineering bacterium with high yield of astaxanthin and its construction method and application. The present invention starts with wild-type Yarrowia lipolytica, introduces four genes of carB, carRP, crtZ and crBKT through a non-homologous recombination strategy, further overexpresses the key enzyme encoding gene in the mevalonate pathway (MVA) pathway to increase the yield of astaxanthin, and realizes the efficient production of the natural product astaxanthin. At the same time, the recombinant Yarrowia lipolytica provided by the present invention uses copper ion induction expression promoter MT-2 to control the expression of β-carotene ketolase crBKT gene, so that it keeps spatiotemporal differential expression with β-carotene hydroxylase gene crtZ, and when high concentration of intracellular zeaxanthin accumulates, copper ions are added to induce the expression of β-carotene ketolase crBKT gene, reduce the production of intermediate metabolic byproducts, and promote the efficient production of astaxanthin.

The invention belongs to the technical field of bioengineering, and particularly relates to an engineering bacterium for de novo biosynthesis of cycloastragenol as well as a construction method and application of the engineering bacterium. A mevalonic acid (MVA) pathway is reconstructed in a saccharomyces cerevisiae strain peroxidase body by utilizing a cell region compartment strategy, a key enzyme gene of the MVA pathway is over-expressed by using a strong promoter, and the cycloastragenol is obtained. The key enzyme genes are used as expression elements of cycloastragenol, are introduced into the chassis saccharomyces cerevisiae through a CRISPR-Cas9 technology, and are integrated on a saccharomyces cerevisiae genome through homologous recombination, so that a saccharomyces cerevisiae engineering bacterium CycZ23 and an engineering bacterium CAG-1 for efficiently synthesizing cycloastragenol are constructed, and de novo synthesis of plant triterpenoid natural products is realized; the highest titer of the generated cycloastragenol is 236 mg / L; the synthesis method is economical, efficient, green, environmentally friendly and easy to operate, and has important application value.

The invention discloses a key enzyme farnesyl pyrophosphate synthase of a black fungus terpenoid synthetic route, and belongs to the technical field of protein. The invention aims to provide a key enzyme gene of a mevalonic acid pathway synthesis pathway in black fungus. The invention provides an FPPS protein of black fungus. The amino acid sequence of the FPPS protein is as shown in SEQ ID NO. 2. A solid foundation is laid for deeply exploring functions of key genes for triterpene synthesis, and meanwhile important data support is provided for systematically explaining specific effects of the genes in terpene synthesis routes.

The invention belongs to the technical field of microorganisms and fermentation engineering, and particularly relates to yarrowia lipolytica engineering bacteria for producing zeaxanthin as well as a construction method and application of the yarrowia lipolytica engineering bacteria. Specifically, the yarrowia lipolytica is subjected to genetic engineering modification based on a genetic engineering technology, a zeaxanthin biosynthetic pathway is constructed by expressing beta-carotene hydroxylase in an original strain, and meanwhile, a zeaxanthin biosynthetic pathway is constructed through a multiple metabolic engineering modification strategy. The method comprises the steps of key enzyme PaCrtZ multi-copy integration, oxidation-reduction balance regulation and control, upstream mevalonic acid pathway enhancement through membrane-free organelles and the like, and the yield of zeaxanthin is further increased. Meanwhile, by optimizing the culture conditions and applying the optimal fermentation conditions to perform tank fermentation, the yarrowia lipolytica can continuously generate more zeaxanthin, so that the yarrowia lipolytica has excellent practical application value and application prospect.

The present invention relates to host cells comprising genes of the mevalonate and Nudix pathways, engineered fusion proteins of enzymes of the mevalonate and Nudix pathways, methods as well as kits for producing geraniol and geranyl acetate.

The present invention provides a recombinant microorganism for simultaneously producing sesquiterpenes and primary metabolites, wherein the primary metabolites include at least one of pantothenic acid, squalene, fatty acids and vitamin B2; the strain contains the mevalonate pathway, the β-farnesene synthesis gene and genes related to the synthesis of primary metabolites; the genes related to the mevalonate pathway include ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1 and ERG20 genes, and the copy numbers are ERG10:ERG13:tHMG1:ERG12:ERG8:MVD1:IDI1:ERG20 = 2:2:X:2:2:2:2:2, where X is an integer greater than or equal to 1; the β-farnesene synthesis gene includes bFS, and the sequence is as shown in SEQ ID NO.1. The present invention realizes high yield of primary metabolites while producing sesquiterpene compounds.

The invention belongs to the technical field of enzyme engineering, and discloses a high-activity isopentenyl phosphokinase mutant and application thereof. The high-activity isopentenyl phosphokinase mutant is AtIPKH274P, and the amino acid at the 274 site of wild type AtIPK is mutated from histidine H to proline P. The invention further discloses a preparation method of the high-activity isopentenyl phosphokinase mutant. According to the present invention, the AtIPKH274P, the SmDAGKS47A and the L124A can form the efficient isopentenol utilization pathway so as to effectively convert the prenol into the DMAPP; an engineering strain with improved isopentenyl modification capability can be constructed by utilizing the approach, and the strain has more advantages compared with a traditional engineering strain based on a mevalonic acid approach.

The invention discloses a multi-gene tandem recombinant strain as well as a construction method and application thereof. The invention also discloses a method for synthesizing lycopene based on an isopentenol utilization approach. The strain comprises a recombinant strain of seven key genes, namely mtipk, thim, ispA, idi, crtE, crttheB and crtI, for synthesizing lycopene in an IUP (Inhibitory Urinary Protein) way. According to the invention, the isopentenol is introduced into the escherichia coli in a way to synthesize the lycopene; according to the approach, only two-step phosphorylation reaction is used for replacing the original 2-C-methyl-D-erythritol-4-phosphoric acid approach and mevalonic acid approach which need more than seven steps to synthesize precusor substances pentadienyl pyrophosphoric acid and dimethyl allyl pyrophosphate ester of lycopene, so that the metabolic pathway is greatly shortened, and competition with central carbon metabolism in cells is avoided; the toxicity caused by accumulation of intermediate products in the metabolic process is reduced, and lycopene can be efficiently synthesized by using a cheap substrate.

This invention belongs to the field of microbial metabolic engineering and biotechnology applications, specifically relating to an engineered bacterium for synthesizing monoterpenoid compounds, its construction method, and its applications. The construction method involves using *Hansenula polymorpha* as a host, regulating the mevalonate pathway in its cytoplasm, and overexpressing monoterpene synthase to obtain the engineered bacterium; or, overexpressing monoterpene synthase and the mevalonate pathway in peroxisomes to obtain the engineered bacterium. The engineered bacterium constructed in this application can efficiently synthesize monoterpenoid compounds such as geraniol, β-myrcene, and dextrorotatory borneol.

The invention discloses yarrowia lipolytica genetically engineered bacteria for producing linalool as well as a construction method and application of the yarrowia lipolytica genetically engineered bacteria. According to the invention, an optimized linalool synthase gene AaLIS from kiwi fruit is assembled by using polypeptide interaction tags RIAD-RIDD and ERG20WW, the assembled gene is converted into a mevalonic acid pathway enhanced chassis strain Yl-19, the Yl19 is used as an original strain, and the linalool synthase is obtained by overexpressing a key rate-limiting enzyme in a metabolic pathway, knocking out a key enzyme in a side reaction pathway and a multi-copy strategy. And the metabolic flux in the biosynthetic pathway of the yarrowia lipolytica alcoholysis is improved. The shake flask fermentation yield of the constructed engineering strain Li-6 can reach 41.8 mg / L, the shake flask fermentation yield of Li-27 reaches up to 178.4 mg / L and can be further increased to 665.8 mg / L by optimizing fermentation conditions, and the method is suitable for large-scale industrial production of linalool.

The invention discloses application of an epidermal growth factor receptor inhibitor combined with a mevalonic acid pathway inhibitor in preparation of a medicine for treating KRAS mutation colorectal cancer, and belongs to the technical field of biological medicine. A lipid metabolism pathway-mevalonic acid pathway participates in a drug resistance process of KRAS mutation colorectal cancer to an EGFR (epidermal growth factor receptor) inhibitor, and a plurality of KRAS mutation colorectal cancer cells and a mouse in-vivo model prove that the drug resistance of the KRAS mutation colorectal cancer to the EGFR inhibitor can be effectively overcome by inhibiting the mevalonic acid pathway. Therefore, by combining the EGFR inhibitor and the mevalonic acid pathway inhibitor, synergistic interaction can be achieved, the treatment effect of EGFR is improved, and precise treatment of KRAS mutation colorectal cancer is achieved.

According to the present disclosure, there is provided a method for producing stem cells, which includes applying a phosphate to blood cells and inducing stem cells from the blood cells. The phosphate may be an intermediate product or a final product in a non-mevalonate pathway. The phosphate may be (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate.

The invention discloses a method for preparing a laying hen feed additive by fermenting tea-seed oil deodorized distillate, which is characterized in that the tea-seed oil deodorized distillate is used as a raw material, and efficient utilization of all components of resources is achieved by coupling yeast-paecilomyces cicadae two-step sequential fermentation and endogenous mevalonic acid (MVA) pathway cascade regulation and control technology. The final product is rich in ergosterol, vitamin E and probiotics, can improve the laying rate of laying hens and enhance the eggshell strength after being applied to laying hen breeding, realizes zero wastewater discharge in the process, and achieves the dual goals of resource utilization of grease byproducts and accurate matching of laying hen nutrition.

Disclosed are techniques for monoterpenes biosynthesis in engineered microorganisms having a mevalonate pathway, where the microorganisms express an engineered enzyme. The engineered enzyme may include one or more peptides that are selected to alter the active site pocket volume of the engineered enzyme as compared to a wild-type farnesyl pyrophosphate synthase. The engineered enzyme may include a first peptide (such as a linker peptide) fused to a C-terminus of a farnesyl pyrophosphate synthase. Optionally, a second peptide (such as a Myc-tag or a HA-tag) be fused to the first peptide. The method may include exposing the engineered microorganism to a carbon source (such a glucose). The method may include allowing the engineered microorganism to generate (i) a first product from the mevalonate pathway and (ii) the engineered enzyme. The method may include forming a monoterpene by allowing the first product and the engineered enzyme to interact.

The invention discloses kluyveromyces marxianus genetically engineered bacteria and application thereof, and belongs to the technical field of bioengineering. The invention discloses a kluyveromyces marxianus genetically engineered bacterium. The kluyveromyces marxianus genetically engineered bacterium is prepared by the following steps: firstly, screening to obtain a kluyveromyces marxianus strain with high ergosterol yield; on the basis, inducing and separating a MATa haploid strain; then, through the introduction of heterologous DHCR24 and DWF5 genes, the weakening of a competitive pathway gene ERG6, the improvement of the metabolic flux of a squalene pathway in a sterol synthesis process, the compartment engineering of a peroxisome region of a mevalonic acid pathway, the knockout of an hypoxia inhibition transcription factor ROX1, the improvement of cofactor supply, the optimization of a yeast lipid pathway and other metabolic engineering optimization reconstructions, the expression of the yeast lipid pathway is improved; and an engineering strain with high cholesterol yield is constructed and obtained. Through fed-batch fermentation, efficient and stable synthesis of cholesterol is achieved, the yield reaches 7.64 g / L, and a foundation is laid for biological manufacturing of cholesterol.

The invention belongs to the technical field of microbial fermentation. The invention relates to saccharomyces cerevisiae, in particular to saccharomyces cerevisiae with high yield of farnesene and squalene and application of the saccharomyces cerevisiae. The yeast-like yeast is constructed by the following steps: providing yeast-like host bacteria; an exogenous farnesene synthase coding gene is introduced into the yeast-like host bacteria, so that the farnesene synthase coding gene is expressed in a host; transforming a genome of the yeast-like host bacterium so as to improve the expression activity of endogenous squalene synthase of the yeast-like host bacterium; strengthening the mevalonic acid pathway of the yeast-like host bacteria; and screening to obtain an engineering strain capable of simultaneously accumulating farnesene and squalene, thereby obtaining the saccharomyces-like strain capable of producing farnesene and squalene at high yield. The genetic stability of the saccharomyces cerevisiae meets the condition that more than 90% of farnesene and squalene producing capacity is still maintained after continuous passage for 50 generations in a culture medium without selective pressure.

The invention relates to a genetically engineered bacterium for producing (-)-limonene, (-)-carvol and (-)-carvone and application of the genetically engineered bacterium. According to the method, endogenous ATP-citrate lyase genes ACL and CarYB genes are knocked out, endogenous mevalonic acid pathway key genes HMGR, ACCT and IDI are overexpressed, a mevalonic acid kinase gene MmMK sourced from methanosarcina martensii and mevalonic acid pathway key genes EfMvaS and EfMvaE sourced from enterococcus faecalis are introduced, and the (-)-limonene, (-)-carvol and (-)-carvone are synthesized into the genetically engineered strain. The method is realized in a platform strain for regulating and controlling the balance of the cofactor NADPHP. And the yield of the (-)-limonene in a bioreactor of 500-mL reaches 608.5 mg / L. The yield of the (-)-carveol reaches 667.6 mg / L when the (-)-carveol is fermented in a 500-mL bioreactor. The fermentation yield of the recombinant strain for producing the (-)-carvone in a 500-mL bioreactor reaches 67.1 mg / L.

The invention discloses yarrowia lipolytica genetically engineered bacteria capable of producing decaisopentenol and application of the yarrowia lipolytica genetically engineered bacteria, and belongs to the technical field of bioengineering. The yarrowia lipolytica genetically engineered bacterium is obtained by cloning a decene diphosphate synthase gene into yarrowia lipolytica, overexpressing related genes of an endogenous acetyl coenzyme A synthetic pathway and a mevalonic acid pathway of the yarrowia lipolytica, weakening a squalene synthetic pathway and knocking out endogenous ylCOQ1 enzyme. Experiments prove that the yarrowia lipolytica genetically engineered bacterium can significantly improve the yield of decyl isopentenol, and the product serves as an important precursor for chemical synthesis of coenzyme Q10, so that the engineered bacterium and the product thereof can be applied to biochemical synthesis of coenzyme Q10. The method is green and efficient, and has important industrial application value.

The invention discloses a construction method of a perillyl alcohol production strain and application of the perillyl alcohol production strain. The construction method comprises the following steps: transferring a plasmid pESKKDI carrying a mevalonic acid pathway, and a plasmid pNLsP450Fdx carrying a tomato Solanum lypersicum sourced NPP synthetase, a lemon Citrus limon sourced gene Ls and a Mycobacterium sp gene P450 gene into a gene knockout defective bacterium delta YqhD of wild type escherichia coli BL21 (DE3); the obtained perillyl alcohol production strain is used for production of perillyl alcohol, the yield of perillyl alcohol is improved and generation of a by-product perillyl aldehyde is reduced by further over-expressing electron transfer protein (Fdx) and optimizing the concentration of initial glucose in a fermentation culture medium, and a favorable guidance direction is provided for production of perillyl alcohol by escherichia coli.

The invention discloses a Yarrowia lipolytica strain for increasing the yield of beta-carotene. On the basis of introducing a synthetic pathway, the production of beta-carotene is effectively enhanced by increasing the copy number of crtB, crtI, and crtY genes and overexpression of genes GGS1 and HMG related to the mevalonic acid (MVA) pathway, as well as the optimization of the promoter. The Yarrowia lipolytica strain of the invention accumulates about 507.7 mg / L beta-carotene in a shake flask culture, and the yield of beta-carotene obtained in a 5L fermenter reaches 5.5 g / L.

The present application relates to a kind of rapid detection method of multiple target protein of engineering strain methylmalonic acid (MVA) pathway based on multiple reaction monitoring technology (MRM).Firstly, the MRM acquisition method is established with the target peptide segment;The target peptide segment standard is prepared into at least 5 different concentrations, and data is collected using liquid chromatography-mass spectrometry technology and MRM method, with concentration value and peak area as horizontal and vertical coordinates to draw standard curve;Engineering strain sample is obtained by using conventional proteomics sample pretreatment process Enzymolysis peptide segment solution, mass spectrometry data is collected using liquid chromatography-mass spectrometry technology and MRM method, and then external standard method is used for protein quantification.The present application is based on traditional external standard method quantification, constructs target protein detection method based on short chromatographic column, microliter liquid chromatography system, short separation gradient and MRM quantification strategy, and it is used for the rapid quantitative analysis of the five proteins of engineering strain MVA pathway.

This invention relates to a recombinant photosynthetic bacterial strain with high tocopherol production, its construction method, and its applications. The recombinant strain knocks out the photosynthetic gene repressor transcription factor PpsR and integrates the mevalonate MEV pathway gene from *Paragonimococcus zeatans*, the p-hydroxyphenylpyruvate dioxygenase (HPPD) gene from *Pseudomonas putida*, the urokinase chlorophyllase (HPT) gene from *Synostemma pentaphyllum*, the tocopherol cyclase (TC) gene from *Arabidopsis thaliana*, and feedback-resistant 3-deoxy-D-arabinohepenoyl-7-phosphate synthase (AroG*). D146N Genes and prephenyl acid dehydrogenase TyrA* M53I Genes. This recombinant photosynthetic bacterium strain achieves highly efficient heterologous synthesis of tocopherol by knocking out regulatory genes and integrating multiple metabolic modules, with a fermentation yield of up to 3.51 g / L, showing broad prospects for industrial application.

The invention relates to a photosynthetic bacterium recombinant strain with high yield of tocopherol as well as a construction method and application of the photosynthetic bacterium recombinant strain. The photosynthetic bacterium recombinant strain knocks out a photosynthetic gene inhibition transcription factor PpsR of photosynthetic bacteria; a mevalonic acid MEV pathway gene for producing paracoccus zeatin, a p-hydroxyphenylpyruvate dioxidase HPPD gene from pseudomonas putida, a uromelanic acid chlorophyllin transferase HPT gene from synechocystis, a tocopherol cyclase TC gene from arabidopsis thaliana, a recombinant plasmid and a recombinant plasmid are integrated; the invention also discloses a 3-deoxy-D-arabinoheptulose-7-phosphate synthase AroG * D146N gene resistant to feedback and a pre-benzoate dehydrogenase TyrA * M53I gene resistant to feedback. According to the photosynthetic bacterium recombinant strain, efficient heterologous synthesis of tocopherol is achieved by knocking out regulation genes and integrating multi-way metabolism modules, the fermentation yield can reach 3.51 g / L, and the photosynthetic bacterium recombinant strain has wide industrial application prospects.

The present disclosure provides a structurally related family of pro-gelling decarboxylase homologues for use in commercial foods and cosmetics. The GPDH protein is expressed in cells in plants, animals and eukaryotic microorganisms in a trace form, and plays a catalytic role in a mevalonic acid pathway. Members of the GPDH family are structurally associated with one another through a series of strictly conserved amino acid sequence motifs. The ability to promote gelation when GPDH is used as a commercial product ingredient has been unclear yet before. The low gelation onset temperature (about 50 DEG C) and very low critical gelation concentration (4%) make the GPDH proteins of the present disclosure particularly suitable as functional protein substitutes in food and personal care products compared to most commonly used plant proteins and plant protein isolates.

The invention discloses a genetically engineered bacterium for high yield of bisabolol as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. Saccharomyces cerevisiae engineering bacteria in an overexpression mevalonic acid pathway are used as starting bacteria, a fusion protein expression cassette of farnesyl pyrophosphate synthetase and bisabolol synthetase is integrated, endogenous transcription factors and transporter of the saccharomyces cerevisiae are overexpressed, an original promoter of squalene synthetase is replaced by an inducible promoter, and the recombinant squalene synthetase is obtained. And the genetically engineered bacterium for high-yield production of bisabolol is constructed. According to the present invention, the bisabolol synthase gene is introduced to achieve the heterologous synthesis of the bisabolol in the Saccharomyces cerevisiae, the rate-limiting enzyme fusion expression and the transport protein overexpression are integrated so as to alleviate the metabolism pressure of the Saccharomyces cerevisiae cells, and the competition pathway is down-regulated so as to enable the substrate farnesyl pyrophosphate to flow to the metabolism pathway of the bisabolol; the bisabolol production capability of the strain is improved. The invention provides a method for realizing high yield of bisabolol, which is superior to reports in the prior art and has a good application prospect.

This invention discloses a genetically engineered strain of *Kluyveromyces martensii* and its applications, belonging to the field of bioengineering technology. The invention discloses a genetically engineered *Kluyveromyces martensii* strain, firstly obtained through screening a strain that produces high levels of ergosterol; based on this, it induces and isolates... Eye A haploid strain was constructed and then metabolically optimized through the introduction of heterologous DHCR24 and DWF5 genes, weakening of the competitive pathway gene ERG6, increasing the metabolic flux of the post-squalene pathway during sterol synthesis, peroxisome compartmentalization engineering of the mevalonate pathway, knockout of the hypoxia-inhibiting transcription factor ROX1, improved cofactor supply, and optimization of the yeast lipid pathway, resulting in a high-cholesterol-producing engineered strain. Through fed-batch fermentation, efficient and stable cholesterol synthesis was achieved, with a yield of 7.64 g / L, laying the foundation for cholesterol biomanufacturing.