23 results about "Mevalonate pathway" patented technology

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.

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 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.

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 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.

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.

A genetically engineered microbe capable of producing isoprene from glycerol includes at least a native nucleic acid sequence encoding at least a native enzyme capable of catalyzing one or more steps of a conversion from at least a carbon source to acetyl coenzyme A (A-CoA), at least a first heterologous nucleic acid sequence encoding at least a first enzyme of a mevalonate (MVA) pathway, and at least a second heterologous nucleic acid sequence encoding at least a second enzyme capable of catalyzing at least a terpene-producing chemical reaction, wherein the at least a carbon source includes glycerol.

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.

The invention discloses a key enzyme isopentene diphosphate delta isomerase in a black fungus terpene 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 IDI protein of black fungus. The amino acid sequence of the IDI 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 discloses a construction method and application of squalene-producing saccharomyces cerevisiae, and belongs to the technical field of biology. According to the invention, an auxotroph strain of S.cerevisiae BY4742 is taken as an original strain, and by optimizing an intracellular mevalonic acid pathway, the expression level of a key enzyme is enhanced and the metabolic bottleneck is relieved, so that the flux of intermediate metabolites is remarkably improved; by introducing an exogenous NADH dependent HMGR and strengthening an Acetyl-CoA supply approach, supply and demand of cofactors are effectively balanced, the availability of a precursor is improved, the yield of the constructed engineering strain AG15 fermented for 240 hours in fermentation at a 5-L fermentation tank level reaches 20.9 g / L, the production efficiency of squalene and the applicability of a carbon source are greatly improved, and the method has important industrial application potential and economic advantages.

The application provides a method for biosynthesis of tetraenemenaquinone by Yarrowia lipolytica. The method is achieved by constructing recombinant Yarrowia lipolytica, and the construction method of the recombinant Yarrowia lipolytica for synthesizing tetraenemenaquinone comprises the following steps: intensively expressing a mevalonate pathway in Yarrowia lipolytica, and introducing a heterologous synthesis pathway of geranylgeranyl pyrophosphate to provide sufficient isoprene side chain donors for the biosynthesis of tetraenemenaquinone; on the basis, overexpressing a codon-optimized aromatic isopentenyltransferase gene with menaquinone substrate specificity in Yarrowia lipolytica. The construction method of Yarrowia lipolytica for biosynthesis of tetraenemenaquinone provided by the application is simple in operation, the constructed Yarrowia lipolytica can obtain a high-concentration tetraenemenaquinone product by using commercially available menaquinone as a substrate, the biosynthesis steps are short, and the method has high production application value.

A genetically engineered microbe capable of producing isoprene from a carbon source and method related thereto include a first nucleic acid sequence encoding a first enzyme, wherein the first enzyme is configured to catalyze one or more steps of a conversion from the carbon source to acetyl coenzyme A (A-CoA), a second nucleic acid sequence encoding a second enzyme of a mevalonate (MVA) pathway, and a heterologous nucleic acid sequence encoding a third enzyme, wherein the third enzyme is configured to catalyzing an isoprene-producing chemical reaction.

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 field of microbial metabolism engineering and biotechnology application, and particularly relates to an engineering bacterium for biosynthesis of farnesene and a construction method and application thereof. The construction method comprises the following steps: screening beta-farnesene synthase, optimizing a host strain mevalonic acid pathway related gene, increasing the copy number of a rate-limiting step gene, enhancing the supply of acetyl coenzyme A, and optimizing the expression of exogenous beta-farnesene synthase. The hansenula polymorpha chassis cell is utilized to realize high yield of beta-farnesene, and a foundation is laid for green and mild synthesis of farnesene and efficient biotransformation of methanol.

The invention provides yarrowia lipolytica for producing 7-dehydrocholesterol as well as a construction method and application of the yarrowia lipolytica, and relates to the technical field of microorganisms. In order to solve the problem that the yield of 7-dehydrocholesterol produced by using yarrowia lipolytica in the prior art is low, a genome of the yarrowia lipolytica is modified, 24-dehydrocholesterol reductase from chicken is expressed, a mevalonic acid pathway and a sterol pathway are strengthened, and the yield of 7-dehydrocholesterol is increased. The Yarrowia lipolytica strain has the advantages that the Yarrowia lipolytica strain is transformed, the possible influence on the growth of the strain is solved while a by-product way is knocked out, the shake flask yield of the transformed Yarrowia lipolytica is 340.7 mg / L, the amplified production yield of a 5L fermentation tank reaches 1.4 g / L, the yield is greatly increased, a new thought is provided for transforming Yarrowia lipolytica by metabolic engineering, the construction method is simple, the metabolic pathway is clear, and the Yarrowia lipolytica strain is suitable for industrial production. The method has a good application prospect and is beneficial to promoting industrial production of 7-dehydrocholesterol.

The present invention discloses a method for synthesizing sclareol using recombinant Saccharomyces cerevisiae, a recombinant Saccharomyces cerevisiae strain for producing sclareol, the strain comprising: a modified mevalonic acid (MVA) pathway for enhancing the supply of farnesyl pyrophosphate (FPP); a taxadiene synthase (TS) coding gene is introduced in an exogenous manner; the coding gene of taxadiene-5alpha-hydroxylase (T5H) is introduced in an exogenous manner; and a sclareol synthase (SSS) coding gene is introduced from an external source. The method realizes de novo synthesis of sclareol in saccharomyces cerevisiae, does not depend on plant extraction, significantly improves path efficiency and product yield through multi-gene coordinated regulation and organelle compartment, is environment-friendly and sustainable in the production process, is easy to amplify to an industrial scale, and has a wide application prospect. And a referential general strategy is provided for expressing a complex P450 enzyme system in yeast.

The invention discloses an antibody coupling medicine for targeting colorectal cancer cells and a preparation method of the antibody coupling medicine, and belongs to the technical field of biological medicine. The antibody coupling drug is formed by connecting cetuximab, atorvastatin or a derivative thereof and a specific connexon through covalent bonds and is constructed by adopting a site specific coupling technology, the average drug-antibody ratio is 3.13, and the monomer purity is greater than or equal to 97%. The connexon can be broken in response to acidity, high reducibility or high cathepsin expression conditions of a tumor microenvironment, so that accurate release of the effective load is realized. The invention also discloses a preparation method of the antibody coupling drug, which comprises the steps of antibody reduction, active intermediate preparation, coupling, quenching and purification, and the yield is greater than or equal to 85%. The antibody coupling drug can specifically target EGFR high-expression KRAS mutant colorectal cancer cells, reverses the drug resistance of tumors to an EGFR inhibitor by inhibiting a mevalonic acid pathway, remarkably reduces the systemic toxicity of atorvastatin, and has excellent curative effect and safety.

This invention discloses a high-astaxanthin-producing *Pseudomonas putida* strain, its construction method, and its applications, belonging to the fields of synthetic biology and microbial metabolic engineering. Using the industrial microorganism *Pseudomonas putida* KT2440 as the substrate bacterium, this invention utilizes genome site-specific integration technology to stably integrate key gene clusters of the mevalonate pathway from *Enterococcus faecalis* and *Saccharomyces cerevisiae* into the substrate bacterium genome, providing a sufficient and stable precursor supply for terpene synthesis. Simultaneously, this invention constructs two independent and compatible broad-host expression plasmids, including plasmid pVLT31- crtEBIY - idi The plasmid pBbB5k- is responsible for efficiently converting the upstream precursor into the key intermediate lycopene. crtZW This method is responsible for sequentially hydroxylating and ketolizing lycopene to ultimately generate astaxanthin. The construction method provided by this invention not only ensures the stability of precursor supply but also achieves flexible regulation and balance of metabolic flux in the synthetic pathway through modular expression with dual plasmids, effectively avoiding the accumulation of intermediate products and cytotoxicity issues. This provides a novel cell factory with great application potential for the green, efficient, and large-scale biomanufacturing of astaxanthin.