49 results about "Cytochrome P450 reductase" patented technology

The invention discloses a recombinant microorganism for preparing dharma diene and protopanoxadiol and a construction method of the recombinant microorganism. The construction method of the recombinant bacteria comprises a step of adding dharma diene synthase, protopanoxadiol synthase and nicotinamide adenine dinucleotide phosphate - cytochrome P450 reductase encoding gene into saccharomyces cerevisiae to obtain recombinant bacteria I. According to the recombinant microorganism for preparing the dharma diene and the protopanoxadiol and the construction method of the recombinant microorganism, by means of homologous recombination, the dharma diene synthase, the protopanoxadiol synthase and the nicotinamide adenine dinucleotide phosphate - the cytochrome P450 reductase encoding gene are all added into the saccharomyces cerevisiae to obtain the initial recombinant bacteria, and the effect that the initial recombinant bacteria can produce trace amount of the dharma diene and trace amount of the protopanoxadiol is discovered; tHMG1 activity of the initial recombinant bacteria is further improved, and therefore intermediate recombinant bacteria are obtained, and by means of the intermediate recombinant bacteria, the yield of the dharma diene and the yield of the protopanoxadiol are significantly increased; the activity of one or two or three of ERG1, ERG9 and ERG20 are improved on the basis of the intermediate recombinant bacteria, and the effect that the recombinant bacteria which can be used to increase the yield of the dharma diene and the yield of the protopanoxadiol are constructed is also discovered. By means of the recombinant microorganism for preparing the dharma diene and the protopanoxadiol and the construction method of the recombinant microorganism, the foundation is laid for artificially synthesizing the dammar diene and the protopanoxadiol.

The invention discloses recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose and a construction method. The construction method comprises the steps of replacing a promoter of a saccharomyces cerevisiae xylulokinase gene XKS1 with a promoter PFBA1 by virtue of a homologous recombination method, introducing xylose reductase XYL1 and a xylitol dehydrogenase XYL2 expression cassette, increasing the activities of transketolase TKL1 and transaldolase TAL1 so as to obtain recombinant bacteria 1, introducing a farnesyl-diphosphate farnesyltransferase gene ERG9, a squalene monooxygenase gene ERG1 and a dammarenediol synthase gene DS into the recombinant bacteria 1 so as to obtain recombinant bacteria 2, and introducing a nicotinamide adenine dinucleotide-hydroxymethylglutaryl coenzyme A reductase gene NADH-HMGr, farnesyl diphosphatesynthase ERG20 and a protopanoxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 into the recombinant bacteria 2, so as to obtain recombinant bacteria 3. According to the recombinant saccharomyces cerevisiae, dammarenediol and protopanoxadiol can be artificially synthesized by virtue of xylose.

The present invention relates to production of alkaloids from poppy plants and in particular togenes encoding enzymes in the alkaloid pathway, to proteins encoded by the gens, to plants transformed or transfected with the genes and to methods of altering alkaloid content or blend of poppy plants.

The present invention relates to production of alkaloids from poppy plants and in particular togenes encoding enzymes in the alkaloid pathway, to proteins encoded by the gens, to plants transformed or transfected with the genes and to methods of altering alkaloid content or blend of poppy plants.

The invention provides a method of enhancing the effectiveness of pesticides, as well as pesticidal formulations. Furthermore, it also provides the means for the de novo rational design of pesticides. The present invention also relates to a method of screening agents for potential use in insecticides, particularly against mosquitoes.

The invention provides cytochrome P450 polypeptides, including cytochrome P450 santalene oxidase polypeptides, cytochrome P450 bergamotene oxidase polypeptides and cytochrome P450 reductase polypeptides. Also provided are nucleic acid molecules encoding the cytochrome P450 polypeptides. Cells containing the nucleic acids and / or the polypeptides are provided as are methods for producing terpenes, such as santalols and bergamotols, by culturing the cells.

Provided is a nicotinamide adenine dinucleotide (NADPH)-cytochrome P450 reductase and a use thereof. Also provided is a polynucleotide encoding the NADPH-cytochrome P450 reductase, an expression vector and a host cell which express the NADPH-cytochrome P450 reductase, and a method for producing the protopanoxadiol.

The invention provides Locusta migratoria cytochrome P450 reductase gene dsRNA and an application thereof. The nucleotide sequence of the Locusta migratoria cytochrome P450 reductase gene is SEQ ID No.1, based on SEQ ID No.1, a gene fragment SEQ ID No.7 and corresponding dsRNA are designed and synthesized. The designed and synthesized dsRNA is injected into Locusta migratoria body, Locusta migratoria cytochrome P450 reductase gene expression level is reduced, cytochrome P450 reductase enzyme activity is reduced, and fatality of Locusta migratoria due to carbaryl is increased by 1.73 to 2.3 times. The cytochrome P450 gene obtained by screening is used as a molecular target for control of carbaryl resistance, and a new approach is provided for treating carbaryl resistance of Locusta migratoria.

The present invention relates to non-human transgenic animals, tissues and / or cells derived therefrom having depleted or ablated cytochrome P450 reductase (CPR) expression, methods of producing such animals, tissues and / or cells, and methods of using such animals, tissues and / or cells. Non-human transgenic animals, tissues and or cells derived therefrom of the present invention may be used for, but non exclusively, in both in vivo and in vitro screening of therapeutic agents, drug development, drug metabolism / disposition studies and studying disease states, pregnancy, fetal development, modulation of hormone function and hormone levels, and other pathways and / or substrate metabolism in which cytochrome P450 plays a role.

A bacterial cell containing a functional cytochrome P450 monooxygenase system, said cell comprising a genetic construct capable of expressing a cytochrome P450 and genetic construct capable of expressing, separately from said cytochrome P450, a cytochrome P450 reductase, wherein each of the cytochrome P450 and the cytochrome P450 reductase have at their N-terminus a bacterial signal peptide. The cytochrome P450 and the cytochrome P450 reductase may be encoded by different constructs or the same construct. A bacterial cell containing a cytochrome P450 comprising a genetic construct encoding, and capable of expressing, said cytochrome P450 wherein the cytochrome P450 has, at is N-terminal, a bacterial signal peptide. The bacterial cells are useful as, for example, bioreactors, in drug testing and mutagenicity testing and as a source of cytochrome P450.

The invention relates to stabilization of cytochrome P450 reductase. The present invention relates to a recombinant microorganism which is capable of expressing a cytochrome P450 reductase (CPR) and, optionally, a cytochrome P450 enzyme (CYP) and which is capable of overexpressing a Ice2p comprising the amino acid sequence set out in SEQ ID NO: 2 or a sequence having at least 50% sequence identity thereto. The invention further relates to use of Ice2p to stabilize expression of a cytochrome P450 reductase in a recombinant microorganism.

The invention discloses a saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone and a construction method thereof. The method comprises the following steps: introducing an optimized alpha-humulene synthase encoding gene ZSS1, an optimized cytochrome P450 enzyme encoding gene CYP71BA1, an optimized cytochrome P450 reductase encoding gene AtCPR1 and an optimized dehydrogenase encoding gene ZSD1S114A into saccharomyces cerevisiae by utilizing homologous recombination; and obtaining a saccharomyces cerevisiae recombinant bacterium 1 capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone. Experiments prove that the saccharomyces cerevisiae recombinant bacterium capable of producing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone, constructed by the invention, can produce alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone, and lays a foundation for synthesizing alpha-humulene, 8-hydroxy-alpha-humulene and zerumbone by artificial cells.

The present invention discloses a P450 enzyme of anisodus acutangulus. 4-(1-methyl-2-pyrrolidyl)-3-oxobutanoic acid may be converted into tropinone due to the combined catalysis of the P450 enzyme anda cytochrome P450 reductase. The method disclosed by the present invention is environment-friendly and mild in reaction condition and has an industrial development prospect.

A bacterial cell containing a functional cytochrome P450 monooxygenase system, said cell comprising a genetic construct capable of expressing a cytochrome P450 and a genetic construct capable of expressing, separately from said cytochrome P450, a cytochrome P450 reductase wherein the N-terminus of the cytochrome P450 and the N-terminus of the cytochrome P450 reductase are each adapted to allow functional coupling of said cytochrome P450 and said cytochrome P450 reductase within said cell. A bacterial cell containing a cytochrome P450 comprising a genetic construct encoding, and capable of expressing, said cytochrome P450 wherein the cytochrome P450 comprises an N-terminal portion which directs the cytochrome P450 to a cellular compartment of membrane of the bacterial cell. The bacterial cells are useful as, for example, bioreactors, in drug testing and mutagenicity testing and as a source of cytochrome P450.

The invention discloses a method for efficient enzyme catalytic synthesis of sanguinarine and chelerythrine. The method comprises the following steps: respectively screening optimal genes with high expression efficiency from a known protopine-6-hydroxylase gene, a dihydrobenzophen anthridine oxidase gene and a cytochrome P450 reductase gene through heterologous expression and result comparison andanalysis, and carrying out codon optimization on selected optimal genes; establishing optimal gene sequences on expression carriers, transferring into a yeast engineering bacterium, and carrying outtransformation so as to obtain a recombinant yeast engineering strain; finally feeding the recombinant yeast engineering strain with a macleaya cordata leaf raw material liquid precursor to carry outfermentation, thereby obtaining sanguinarine and chelerythrine. By adopting the method, the enzyme catalysis efficiency of the sanguinarine and the chelerythrine is improved from multiple aspects suchas gene levels and fermentation processes, fumarine and allocryptopine which are high in alkaloid content in leaves can be transformed into sanguinarine and chelerythrine with high values, and comprehensive utilization of macleaya cordata resources can be achieved.

The invention relates to a cytochrome P450 reductase and a coding gene and application thereof. The invention discloses a cytochrome P450 reductase 2 of an amaryllidaceae plant lycoris aurea for the first time, and the cytochrome P450 reductase 2 has good coenzyme specificity and can assist a cytochrome P450 enzyme to develop the catalytic activity to oxidatively modify a substrate synthetic product thereof. The invention further discloses a polynucleotide for coding the cytochrome P450 reductase, a carrier for expressing the cytochrome P450 reductase and a method for producing a caffeic acid.

The invention belongs to the technical field of genetic engineering, in particular to construction of a recombinant vector containing a reindeer NADPH-cytochrome P450 reductase gene and a method for expression, separation, purification and activity assay. The method comprises the following steps: 1) according to the NADPH-cytochrome P450 reductase gene annotated from the genome of a reindeer and E. coli codon preference, the optimized reindeer NADPH-cytochrome P450 reductase gene sequence is obtained, and the NADPH-cytochrome P450 reductase gene sequences of roe deer and sheep optimized according to the codon preference of E. coli are obtained to be as a control; and 2) the optimized reindeer, roe deer and sheep 25 hydroxyvitamin D-1 alpha hydroxylase gene are constructed into E. coli-expressed vector pET-28a by gibson connection method using the designed primers to obtain a recombinant vector. The catalytic activity of the strain containing the reindeer NADPH-cytochrome P450 reductasegene is 20 times that of the sheep and 5 times that of the roe deer.

The invention relates to cytochrome P450 reductase as well as coding gene and application thereof. The invention discloses cytochrome P450 reductase 1 of lycoris aurea as an amaryllidaceae plant for the first time; the cytochrome P450 reductase 1 has favorable coenzyme specificity and can be used for assisting a cytochrome P450 enzyme in exerting catalytic activity to modify a synthetic product of a substrate of the cytochrome P450 enzyme in an oxidative manner. The invention also discloses a polynucleotide for coding the cytochrome P450 reductase, a carrier and a host cell for expressing the cytochrome P450 reductase and a method for producing p-coumaric acid.

The invention discloses an NADH analog dependent cytochrome P450 reductase and an application thereof. An NADH analogue is used as a cofactor and reducing power to catalyze electron transfer and reduce cytochrome P450 to complete catalytic circulation. The enzyme is subjected to fusion expression with different types of cytochrome P450 enzymes to construct the NADH analog-dependent, heterozygous and self-sufficient cytochrome P450 enzyme, the obtained NADH analog-dependent cytochrome P450 enzyme can be coupled with oxidoreductases of a regenerated NADH analogue, and the NADH analog is used tocatalyze corresponding substrates of different families of cytochrome P450 to be converted into products. The NADH analog-dependent cytochrome P450 reductase can be used for constructing a biologicalorthogonal metabolic pathway independent of natural cofactor NAD(P)H to realize uncoupling of P450 enzyme-catalyzed energy consumption and endogenous energy metabolism.

The invention discloses a method for increasing the content of artemisinin in Artemisia annua. The method includes jointly transforming the Artemisia annua by the aid of amorpha fruticosa diene synthetase ADS genes, amorpha fruticosa-4, 11-diene oxidase CYP71AV1 genes, cytochrome P450 reductase CPR genes and acetaldehyde dehydrogenase ALDH1 genes; measuring the content of the artemisinin in the Artemisia annua by the aid of high-performance liquid chromatography-evaporative light-scattering detectors and acquiring transgenic Artemisia annua plants with the increased artemisinin content by means of screening. The method has the advantages that the content of the artemisinin in the transgenic Artemisia annua can be obviously increased by the aid of the method, and the maximum content of the artemisinin can reach 3.4 times the content of artemisinin in non-transformation comparison plants.

The invention relates to a construction method of an escherichia coli bacterial strain for generating lanosterol. The construction method comprises the steps of optimizing a codon of squalene epoxidase of methylococcus capsulatus according to an escherichia coli codon, and performing PCR amplification to obtain a squalene epoxidase genetic fragment; performing PCR amplification to obtain NADPH-cytochrome P450 reductase, and lanosterol synthetase genetic fragments, performing merging to obtain a DNA fragment SE-CPR-LS, cloning the SE-CPR-LS fragment to a vector pet21c by a restriction enzyme cutting connection method, and constructing an expression vector pet21c-SE-CPR-LS; cloning the squalene synthase genetic fragment to the vector pACYCduet-1 by a homologous recombination method, and constructing an expression vector pACYCduet-SS; and transforming the vectors pet21c-SE-CPR-LS and pACYCduet-SS into escherichia coli competence cells, so as to obtain the escherichia coli bacterial strainfor generating lanosterol. Through an exogenous plasmid transforming method, an escherichia coli BL21(DE3) system is improved, so that synthesis of the lanosterol can be realized; and through a codonoptimization technique, the expression level of exogenous genes in an escherichia coli system can be increased.

The invention discloses lactic acid bacterium containing an IFS-CPR fusion gene and application thereof. The recombinant bacterium provided in the invention is recombinant bacterium obtained by introducing coding genes of IFS-CPR into host bacterium, and IFS-CPR is a protein as described in 1) or 2): 1) a protein as represented by sequence 1 in a sequence table; 2) a protein derived by substituting and / or deleting and / or adding one or more amino acid residue in the amino acid residue sequence of sequence 1 in the sequence table. A recombinant lactic acid bacterial strain obtained in the invention can be used for production of isoflavone and has a great potential market, economic benefits and social benefits in the food industry.

The invention discloses a recombinant microorganism for preparing dharma diene and protopanoxadiol and a construction method of the recombinant microorganism. The construction method of the recombinant bacteria comprises a step of adding dharma diene synthase, protopanoxadiol synthase and nicotinamide adenine dinucleotide phosphate - cytochrome P450 reductase encoding gene into saccharomyces cerevisiae to obtain recombinant bacteria I. According to the recombinant microorganism for preparing the dharma diene and the protopanoxadiol and the construction method of the recombinant microorganism, by means of homologous recombination, the dharma diene synthase, the protopanoxadiol synthase and the nicotinamide adenine dinucleotide phosphate - the cytochrome P450 reductase encoding gene are all added into the saccharomyces cerevisiae to obtain the initial recombinant bacteria, and the effect that the initial recombinant bacteria can produce trace amount of the dharma diene and trace amount of the protopanoxadiol is discovered; tHMG1 activity of the initial recombinant bacteria is further improved, and therefore intermediate recombinant bacteria are obtained, and by means of the intermediate recombinant bacteria, the yield of the dharma diene and the yield of the protopanoxadiol are significantly increased; the activity of one or two or three of ERG1, ERG9 and ERG20 are improved on the basis of the intermediate recombinant bacteria, and the effect that the recombinant bacteria which can be used to increase the yield of the dharma diene and the yield of the protopanoxadiol are constructed is also discovered. By means of the recombinant microorganism for preparing the dharma diene and the protopanoxadiol and the construction method of the recombinant microorganism, the foundation is laid for artificially synthesizing the dammar diene and the protopanoxadiol.

The invention relates to cytochrome P450, a nicotinamide adenine dinucleotide-cytochrome P450 reductase and application thereof. The invention discloses the set of new cytochrome P450 (P450 GP01 and P450 GP02) which performs the effect in the terpenoid hydroxylation catalysis and synthesis of Ganoderma triterpenes, and the nicotinamide adenine dinucleotide-cytochrome P450 reductase (GLCPR) which completes hydroxylation catalysis by cooperating with the cytochrome P450. In specific, the cytochrome P450 GP01 can specifically and efficiently catalyze the hydroxylation of C-23 site of a tetracyclic triterpene compound substrate. The cytochrome P450 GP02 performs hydroxylation om ganoderic acid DM and ganoderic acid TR to generate various kinds of products. The GLCPR can be paired with the cytochrome P450, and assists the cytochrome P450 to perform the hydroxylation activity.

The invention discloses a genetic engineering bacterium for producing protopanaxadiol. The genetic engineering bacterium for producing protopanaxadiol is an engineering bacterium for expressing a squalene synthase gene, a 2,3-oxidosqualene synthase gene, a dammarenediol synthase gene, a protopanaxadiol synthase gene and a nicotinamide adenine dinucleoside phosphate-cytochrome P450 reductase gene in Escherichia coli. The protopanaxadiol is biologically synthesized by using the genetic engineering bacterium, so the genetic engineering bacterium and the method have the advantages of short fermentation time, simple medium, meeting the requirements of modern industry, and facilitation of promotion and application.

The invention discloses recombinant saccharomyces cerevisiaes, construction methods of the recombinant saccharomyces cerevisiaes and applications of the recombinant saccharomyces cerevisiaes in production of hydroxy fatty acids. Through a homologous recombination method, the recombinant fungi (1) are obtained by knocking out key gene acyl coenzyme A oxidase POX1 in a beta-oxidation pathway relatedto fatty acid catabolism in saccharomyces cerevisiaes and knocking out acyl coenzyme A activating enzymes FAA1 and FAA4, recombinant fungi (2) are obtained by introducing cytochrome P450 oxidase CYP52M1 and cytochrome P450 reductase SbCPR into the recombinant fungi (1), or the recombinant fungi (3) are obtained by introducing cytochrome P450 oxidase CYP52M1 and cytochrome P450 reductase AtCPR1 into the recombinant fungi (1), or the recombinant fungi (4) are obtained by introducing cytochrome P450 oxidase and cytochrome P450 reductase fusion protein gene CYP52M1-AtCPR1 into the recombinant fungi (1).

The invention discloses a recombinant yarrowia lipolytica strain for producing protopanoxadiol by utilizing xylose and a construction method and application of the recombinant yarrowia lipolytica strain. Yarrowia lipolytica ATCC 201249 is used as an original strain, optimized xylose reductase XYL1, xylitol dehydrogenase XYL2 and an overexpressed endogenous xylulokinase XKS expression cassette areintroduced, and a recombinant strain 1 is obtained; a dammarendiol synthase gene DS, a protopanoxadiol synthase gene PPDS and a cytochrome P450 reductase gene ATR1 are introduced into the recombinantstrain 1 to obtain a recombinant strain 2; a gene tHMG1 for encoding nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase, a gene erg9 for squalene synthase and an erg20 for encoding farnesene pyrophosphate synthase in a truncated mevalonic acid pathway are introduced into the recombinant strain 2 to obtain a recombinant strain 3; and the recombinant yarrowia lipolytica isused for producing the protopanoxadiol by using the xylose, and the shake flask yield reaches 80.88 mg / L.

The invention discloses recombinant yarrowia lipolytica for heterogenous synthesis of betulinic acid and a construction method of the recombinant yarrowia lipolytica. The construction method comprisesthe following steps: by using a homologous recombination method, introducing an optimized lupeol synthase encoding gene opAtLUP1, an optimized cytochrome P450 enzyme encoding gene opCYP716A12 and anoptimized cytochrome P450 reductase 1 encoding gene opAtCPR1 into yarrowia lipolytica so as to obtain a recombinant bacterium 1; and introducing a shortcut 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding gene tHMG1, a squalene synthase encoding gene Erg9 and the optimized lupeol synthase encoding gene opAtLUP1 into the recombinant bacterium 1, so as to obtain a recombinant bacterium 2. Experiments show that the recombinant yarrowia lipolytica which is constructed by using the method disclosed by the invention and applied to heterogenous synthesis of betulinic acid has a high yield ofbetulinic acid.

The invention discloses recombinant saccharomyces cerevisiae for producing levopimaric diene and levopimaric acid and a construction method. The method comprises the following steps: introducing a modified L-pimaric diene synthase encoding gene T delta LPS, an optimized cytochrome P450 enzyme encoding gene CYP720B1 and an optimized cytochrome P450 reductase encoding gene TcCPR into saccharomyces cerevisiae to obtain a recombinant bacterium 1; introducing a 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 into the recombinant bacterium 1 to obtain a recombinant bacterium 2; introducing a farnesyl pyrophosphate synthase encoding gene Erg20 into the recombinant bacterium 2 to obtain a recombinant bacterium 3; and replacing a wild squalene synthase promoter in the recombinant bacterium 3 with a Met3 promoter to obtain a recombinant bacterium 4; experiments prove that the fermentation of the recombinant bacteria disclosed by the invention can improve the yields of the levopimaric diene and the levopimaric acid; and a foundation is laid for artificial cell synthesis of the levopimaric diene and the levopimaric acid.