30 results about "Dammarenediol" patented technology

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 invention relates to a biosynthesis method of dammarenediol and provides a producing strain of the dammarenediol. A pichia pastoris engineering strain, which can recombine and express a dammarenediol synthetic enzyme with high efficiency, is constructed through a genetic engineering technical means, a precursor for synthesizing protopanaxadiol, namely the dammarenediol is further synthesized, and a foundation is finally laid for biosynthesis and large-scale industrial production of the protopanaxadiol.

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 discloses an escherichia coli gene engineering strain for synthesizing Dammar enediol and a construction method. The escherichia coli gene engineering strain for synthesizing Dammar enediol contains a squalene synthase gene with 26 amino acid residues at the truncated C end, a 2,3-oxidized squalene synthase gene, a cytochrome-NADPH-reductase 1 gene in arabidopsis with 45 amino acid residues at the truncated C end, and a Dammar enediol synthase gene. The construction method disclosed by the invention is used for successfully constructing the escherichia coli gene engineering strain for synthesizing the Dammar enediol. Compared with other host bacteria, the escherichia coli gene engineering strain disclosed by the invention has the advantages that the way of synthesizing the Dammar enediol by escherichia coli is easy to regulate and control, and metabolic engineering transformation is facilitated, so that a foundation is laid for fermentation and production of the escherichia coli of Dammar cucuribitane saponin.

The invention discloses fusion protein capable of improving dammarenediol conversion efficiency, a construction method and application. The construction steps comprise: excising first 138 bases at 5' end of arabidopsis thaliana cytochrome-NADPH-reductase 1 gene AtCPR1, so as to obtain a sequence shown as SEQ ID NO. 2; (2) removing a termination codon TAA at 3' end of ginseng protopanaxadiol synthase gene PPDS with a sequence shown as SEQ ID NO. 3, and connecting with the base sequence which at 5' end of the sequence shown as SEQ ID NO. 2 and used for coding polypeptide GSTSSGSG, so as to construct a gene member of fusion protein; and (3) connecting the gene member of the fusion protein with saccharomyces cerevisiae cell endogenous promoter and terminator, so as to construct a fusion protein gene expression cassette, and transforming into saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method is capable of improving the conversion rate of dammarenediol to protopanaxadiol.

The invention relates to a protopanoxadiol biosynthesizing method and provides a production bacterial strain of the protopanoxadiol. According to the protopanoxadiol biosynthesizing method, a pichia pastoris engineering strain constructed through a genetic engineering technological means can be used for efficiently co-expressing dammarendiol synthetase and P450 cytochrome synthetase, and further the protopanoxadiol is catalyzed and synthesized. The protopanoxadiol biosynthesizing method lays a foundation for large-scale industrial production of the protopanoxadiol.

The invention discloses a fusion protein capable of increasing the conversion efficiency of dammarendiol, a construction method and an application. The construction method comprises the following steps: (1) excising the front 138 basic groups of the end 5' of the cytochrome-NADPH-reductase 1 gene AtCPR1 in arabidopsis to obtain a sequence shown in SEQ ID NO. 2; (2) removing the termination codon TAA of the end 3' of the synthase gene PPDS of protopanoxadiol in ginseng shown in SEQ ID NO. 3, and connecting with a base sequence of an encoding polypeptide GSTSSG for the end 5' of a sequence shown in SEQ ID NO. 2 to construct a genetic element of the fusion protein; (3) connecting the genetic element of the fusion protein with the internal promoter and the terminator of a saccharomyces cerevisiae cell to construct a genetic expression kit of the fusion protein, and transforming to enter the saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method disclosed by the invention is capable of increasing the conversion efficiency of converting from dammarendiol to protopanoxadiol.

The invention discloses a cultivation method of a novel dammarane-type ginsenoside-containing paddy rice variety and belongs to the field of genetic engineering. The breeding method is characterized in that a full-length cDNA of a gene of a dammarenediol synthetase as a key enzyme of dammarane-type ginsenoside biosynthesis is constructed to double-T-DNA plant expression vectors; the constructed product is subjected to genetic transformation in paddy rice; and through screening cultivation, the novel dammarane-type ginsenoside-containing paddy rice variety is obtained. A test proves that the novel dammarane-type ginsenoside-containing paddy rice variety comprises 0.020% of 20(S)-protopanoxadiol (PPD) and 0.173% of 20(S)-protopanaxatriol (PPT). The cultivation method can be used for cultivation of high-quality ginseng rice varieties containing dammarane-type ginsenoside, can improve a health care value of rice as a main food of a human body, can widen a ginsenoside source, can alleviate the scarcity of a ginsenoside drug source, and has a good application prospect.

The invention discloses a recombinant Yarrowia lipolytica for producing dammarendiol and protopanoxadiol and application. The recombinant Yarrowia lipolytica is constructed by the following steps: introducing an optimized dammarendiol synthase encoding gene DS expression cassette, an optimized protopanoxadiol synthase encoding gene PPDS expression cassette and an optimized encoding gene expressioncassette of cytochrome-NADPH-reductase 1 into Yarrowia lipolytica so as to obtain a recombinant yeast 2, and cutting 1500 nucleotides from the 5'end of a gene HMG1 for encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase to obtain a truncated gene tHMG1 for encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase; and introducing the truncated gene tHMG1 into recombinant yeast 2 to obtainrecombinant yeast 3. Experiments prove that the yield of dammarendiol and protopanoxadiol is increased by acquiring the recombinant Yarrowia lipolytica through a homologous recombination method. Themethod disclosed by the invention provides a basis for artificially synthesizing the dammarendiol and the protopanoxadiol.

The invention provides an American ginseng identification DNA hybridization method. An applied probe applied in the American ginseng identification DNA hybridization method is designed on the basis ofthe locus of Single Nucleotide Polymorphism (SNP) of dammarenediol synthase genes of panax genomes. Experiments of the American ginseng identification DNA hybridization method detects that Chinese ginseng, American ginseng and pseudo-ginseng are high in similarity of dammarenediol synthase genes. For identifying American ginseng more accurately, the American ginseng identification DNA hybridization method designs a number of probe sequences and screens out probe sequences capable of accurately identifying American ginseng.

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 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 a dammarenediol synthase gene of panax japonicus var and applications thereof. The dammarenediol synthase gene of panax japonicus var is prepared by the following steps: carrying out transcriptome sequencing and Denovo splicing on panax japonicus var rhizome by utilizing the second-generation Sole xaHiSeq2000 for the first time; analyzing and finding out a candidate gene for encoding the dammarenediol synthase (DS) in panax japonicus var and performing in vitro cloning, to finally obtain the dammarenediol synthase gene of panax japonicus var, wherein the sequence of the gene is SEQIDNO.1. The gene is genetically transformed into the panax japonicus var through agrobacterium tumefaciens mediated transformation, to obtain transgenic plant with high content of dammarane saponin, thus providing a technical support for the industrial production of the dammarane saponin.

The invention discloses a dammaren diol trimethanol compound, a crystal form A and a preparation method of the dammaren diol trimethanol compound. The X-ray powder diffraction pattern comprises X-raydiffraction peaks as shown at the following 2 theta angles: 6.58 + / -0.2 degrees, 13.35 + / -0.2 degrees, 15.45 + / -0.2 degrees, 20.18 + / -0.2 degrees and 21.83 + / -0.2 degrees. The preparation method disclosed by the invention is safe, simple and convenient, and the prepared dammaren diol trimethanol compound or crystal form A is good in dissolution, high in bioavailability, good in stability, suitablefor drug development and good in marketization prospect.

The invention obtains a hydroxylase mutant SEQ ID Nos:3-4 with the enzyme activity improved by means of directed evolution, the mutant or an expressed microorganism can efficiently catalyze dammarenediol to prepare protopanoxadiol through a hydroxylation reaction, and the hydroxylase mutant has industrial development and application prospects.

The invention belongs to the field of medicines, and relates to application of dammarendiol, in particular to application of dammarendiol in preparation of drugs for preventing and treating autoimmune hepatitis. The research result of the effect of the dammarendiol on mouse liver injury caused by concanavalin A shows that the dammarendiol can obviously reduce the content of transaminase and the liver index of an autoimmune hepatitis model mouse; the research result of the treatment effect of the dammarendiol on rats with autoimmune hepatitis shows that the dammarendiol can obviously improve the proportional imbalance between the level of interleukin and the level of gamma-interferon, obviously increase the level of interleukin 2 and the level of gamma-interferon, and reduce the level of interleukin 4 and the level of interleukin 6; the traditional Chinese medicine composition has a remarkable prevention and treatment effect on autoimmune hepatitis.

The invention obtains a hydroxylase mutant SEQ ID Nos:3-4 with the enzyme activity improved by means of directed evolution, the mutant or an expressed microorganism can efficiently catalyze dammarenediol to prepare protopanoxadiol through a hydroxylation reaction, and the hydroxylase mutant has industrial development and application prospects.

The invention discloses a recombinant Saccharomyces cerevisiae strain and a construction method for producing dammarenediol and protopanaxadiol by using xylose. Through homologous recombination, the promoter of Saccharomyces cerevisiae xylulokinase gene XKS1 is replaced with a promoter Sub P FBA1 , and then introduce xylose reductase XYL1, xylitol dehydrogenase XYL2 expression cassettes, and then increase the activity of transketolase TKL1 and transaldolase TAL1 to obtain recombinant bacterium 1, and then introduce farnesyl diphosphate into recombinant bacterium 1 Farnesyltransferase gene ERG9, squalene monooxygenase gene ERG1 and dammarene diol synthase gene DS were obtained to obtain recombinant bacteria 2, and nicotinamide adenine dinucleotide-hydroxymethyl was introduced into recombinant bacteria 2 Glutaryl-CoA reductase gene NADH-HMGr, farnesyl pyrophosphate synthase ERG20, protopanaxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 were obtained to obtain recombinant bacteria 3, the recombinant brewing wine of the present invention Yeast uses xylose to synthesize dammarenediol and protopanaxadiol.

The invention discloses an Escherichia coli genetic engineering strain for synthesizing dammarenediol and a construction method. The Escherichia coli genetic engineering strain for synthesizing dammarenediol contains a squalene synthase gene with 26 amino acid residues truncated at the C-terminal, 2 , 3‑oxidized squalene synthase gene, cytochrome‑NADPH‑reductase 1 gene and dammarenediol synthase gene in Arabidopsis thaliana truncated by 45 amino acid residues at the C-terminus. The invention successfully constructs the Escherichia coli genetic engineering strain for synthesizing dammarenediol. Compared with other host bacteria, Escherichia coli synthesizes dammarenediol pathway with simple regulation and easy metabolic engineering, which lays the foundation for Escherichia coli fermentation production of dammarane-type saponins.

The probe used in the DNA hybridization method for identifying American ginseng provided by the present invention is based on the single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) site of the dammarenediol synthase (dammarenediol synthase) gene in the Panax genus genome design. The inventor found that the dammarene diol synthase genes of Chinese ginseng, American ginseng, and Panax notoginseng have high similarity in experiments. In order to more accurately identify American ginseng, the inventor designed a variety of probe sequences, and finally screened out the dammarene diol synthase that can be accurately identified. The probe sequence of American ginseng.

The invention belongs to the field of traditional Chinese medicines, and relates to an application of an Erzhi formula compound in preparation of a drug for preventing and treating benign prostatic hyperplasia (BPH). Specifically, the invention relates to an application of a compound in preparation of a medicine selected from the following medicines: (1) a medicine for treating and / or preventing benign prostatic hyperplasia; (2) a medicine for inhibiting proliferation of human prostatic hyperplasia epithelial cells; or (3) a drug for inhibiting PCNA protein expression and / or inhibiting mRNA expression; wherein the compound is selected from dammarendiol, p-hydroxyphenylethanol, ligustrosidic acid, verbascoside, oleoside methyl ester, luteolin, ABiochanin A, echinocystic acid and echinocystic acid-28-O-beta-D-glucose. The Erzhi formula compound or a combination of the Erzhi formula compound is effective in the treatment and / or prevention of benign prostatic hyperplasia.

The invention discloses a fusion protein capable of increasing the conversion efficiency of dammarendiol and a construction method. The construction method comprises the following steps: (1) excising the front 138 basic groups of the end 5' of the cytochrome-NADPH-reductase 1 gene AtCPR1 in arabidopsis to obtain a sequence shown in SEQ ID NO. 2; (2) removing the termination codon TAA of the end 3' of the synthase gene PPDS of protopanoxadiol in ginseng shown in SEQ ID NO. 3, and connecting with a sequence at the end 5' of the sequence shown in SEQ ID NO. 2 to construct a genetic element of the fusion protein; (3) connecting the genetic element of the fusion protein with the internal promoter and the terminator of a saccharomyces cerevisiae cell to construct a genetic expression kit of the fusion protein, and transforming to enter the saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method disclosed by the invention is capable of increasing the conversion efficiency of converting from dammarendiol to protopanoxadiol.

The invention discloses a fusion protein capable of improving the dammarendiol conversion efficiency and a construction method thereof. The construction method comprises the following steps: (1) cutting off the front 138 bases at the 5' end of the cytochrome-NADPH-reductase 1 gene AtCPR1 in arabidopsis to obtain the sequence shown by SEQ ID No.2; (2) removing the termination codon TAA at the 3' end of the protopanoxadiol synthase gene PPDS in ginseng shown by SEQ ID No.3, and connecting the termination codon TAA with the base sequence of the coding polypeptide GGG at the 5' end of the sequence shown by SEQ ID No.2 to construct a gene element of the fusion protein; and (3) connecting the gene element of the fusion protein with the endogenous promoter and terminator of saccharomyces cerevisiae cells to construct a gene expression box of the fusion protein, and converting into the saccharomyces cerevisiae cells for expression. The fusion protein disclosed by the invention can improve the conversion rate of dammarendiol to protopanoxadiol.

The invention discloses fusion protein capable of improving dammarenediol conversion efficiency, a construction method and application. The construction steps comprise: excising first 138 bases at 5' end of arabidopsis thaliana cytochrome-NADPH-reductase 1 gene AtCPR1, so as to obtain a sequence shown as SEQ ID NO. 2; (2) removing a termination codon TAA at 3' end of ginseng protopanaxadiol synthase gene PPDS with a sequence shown as SEQ ID NO. 3, and connecting with the base sequence which at 5' end of the sequence shown as SEQ ID NO. 2 and used for coding polypeptide GSTSSGSG, so as to construct a gene member of fusion protein; and (3) connecting the gene member of the fusion protein with saccharomyces cerevisiae cell endogenous promoter and terminator, so as to construct a fusion protein gene expression cassette, and transforming into saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method is capable of improving the conversion rate of dammarenediol to protopanaxadiol.

The invention discloses a dammarene diol trimethanol compound, crystal form A and a preparation method thereof. The present invention provides a crystal form A of dammarene diol trimethanol, and its X-ray powder diffraction pattern includes the following X-ray diffraction peaks shown at 2θ angles: 6.58°±0.2°, 13.35°±0.2° , 15.45°±0.2° and 20.18°±0.2°, 21.83°±0.2°. The preparation method of the invention is safe and convenient, and the prepared dammarene diol trimethanol or crystal form A has good dissolution, high bioavailability and good stability, is suitable for drug development, and has good market prospects.

The present invention relates to nicotinamide adenine dinucleotide (NADPH)-cytochrome P450 reductase and application thereof. The present invention discloses for the first time the NADPH-cytochrome P450 reductase derived from ginseng, which has good coenzyme properties, can assist cytochrome P450 to exert catalytic activity, and promote the production of protopanaxadiol from the substrate dammarenediol. The invention also discloses a polynucleotide encoding the NADPH-cytochrome P450 reductase, an expression vector and a host cell for expressing the NADPH-cytochrome P450 reductase, and a method for producing protopanaxadiol. The application of ginseng-derived NADPH‑cytochrome P450 reductase can significantly increase the production efficiency of protopanaxadiol, thereby increasing the yield of ginsenoside biosynthesis.

The invention discloses a fusion protein capable of increasing the conversion efficiency of dammarendiol, a construction method and an application. The construction method comprises the following steps: (1) excising the front 138 basic groups of the end 5' of the cytochrome-NADPH-reductase 1 gene AtCPR1 in arabidopsis to obtain a sequence shown in SEQ ID NO. 2; (2) removing the termination codon TAA of the end 3' of the synthase gene PPDS of protopanoxadiol in ginseng shown in SEQ ID NO. 3, and connecting with a base sequence of an encoding polypeptide GSTSSG for the end 5' of a sequence shown in SEQ ID NO. 2 to construct a genetic element of the fusion protein; (3) connecting the genetic element of the fusion protein with the internal promoter and the terminator of a saccharomyces cerevisiae cell to construct a genetic expression kit of the fusion protein, and transforming to enter the saccharomyces cerevisiae cell for expression. The fusion protein constructed by the method disclosed by the invention is capable of increasing the conversion efficiency of converting from dammarendiol to protopanoxadiol.

The invention discloses a genetically engineered bacterium for producing protopanaxadiol, which expresses squalene synthase gene, 2,3-oxide squalene synthase gene, dammarene synthase gene and dammarene synthase gene in Escherichia coli. Enzyme gene, protopanaxadiol synthase gene and nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase gene engineering bacteria. The biosynthesis of protopanaxadiol by using the genetically engineered bacteria has the advantages of short fermentation time, simple culture medium, meeting the requirements of modern industry, being beneficial to popularization and application, and the like.

The invention discloses a cultivation method of a novel dammarane-type ginsenoside-containing paddy rice variety and belongs to the field of genetic engineering. The breeding method is characterized in that a full-length cDNA of a gene of a dammarenediol synthetase as a key enzyme of dammarane-type ginsenoside biosynthesis is constructed to double-T-DNA plant expression vectors; the constructed product is subjected to genetic transformation in paddy rice; and through screening cultivation, the novel dammarane-type ginsenoside-containing paddy rice variety is obtained. A test proves that the novel dammarane-type ginsenoside-containing paddy rice variety comprises 0.020% of 20(S)-protopanoxadiol (PPD) and 0.173% of 20(S)-protopanaxatriol (PPT). The cultivation method can be used for cultivation of high-quality ginseng rice varieties containing dammarane-type ginsenoside, can improve a health care value of rice as a main food of a human body, can widen a ginsenoside source, can alleviate the scarcity of a ginsenoside drug source, and has a good application prospect.