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1. WO2020107548 - HIGHLY EFFICIENT METHOD FOR CATALYZING SYNTHESIS OF SANGUINARINE AND CHELERYTHRINE

Publication Number WO/2020/107548
Publication Date 04.06.2020
International Application No. PCT/CN2018/121630
International Filing Date 18.12.2018
IPC
C12P 17/18 2006.01
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PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
17Preparation of heterocyclic carbon compounds with only O, N, S, Se, or Te as ring hetero atoms
18containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
C12N 15/53 2006.01
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09Recombinant DNA-technology
11DNA or RNA fragments; Modified forms thereof
52Genes encoding for enzymes or proenzymes
53Oxidoreductases (1)
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15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
80for fungi
81for yeasts
C12N 1/19 2006.01
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1Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
14Fungi ; Culture media therefor
16Yeasts; Culture media therefor
19modified by introduction of foreign genetic material
C12R 1/865 2006.01
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RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C-C12Q75
1Microorganisms
645Fungi
85Saccharomyces
865Saccharomyces cerevisiae
CPC
C12N 15/81
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15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
80for fungi
81for yeasts
C12N 2800/22
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2800Nucleic acids vectors
22Vectors comprising a coding region that has been codon optimised for expression in a respective host
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9Enzymes; Proenzymes; Compositions thereof
0004Oxidoreductases (1.)
0012acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
0026acting on CH-NH groups of donors (1.5)
0032with oxygen as acceptor (1.5.3)
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9Enzymes; Proenzymes; Compositions thereof
0004Oxidoreductases (1.)
0071acting on paired donors with incorporation of molecular oxygen (1.14)
0073with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
C12N 9/0081
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9Enzymes; Proenzymes; Compositions thereof
0004Oxidoreductases (1.)
0071acting on paired donors with incorporation of molecular oxygen (1.14)
0077with a reduced iron-sulfur protein as one donor (1.14.15)
0081Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
C12P 17/18
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
17Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
18containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
Applicants
  • 湖南美可达生物资源股份有限公司 MICOLTA BIORESOURCE INC., LTD. [CN]/[CN]
Inventors
  • 黄鹏 HUANG, Peng
  • 曾建国 ZENG, Jianguo
Agents
  • 长沙楚为知识产权代理事务所(普通合伙) CHANGSHA CHUWEI INTELLECTUAL PROPERTY AGENCY (GENERAL PARTNERSHIP)
Priority Data
201811423227.127.11.2018CN
Publication Language Chinese (ZH)
Filing Language Chinese (ZH)
Designated States
Title
(EN) HIGHLY EFFICIENT METHOD FOR CATALYZING SYNTHESIS OF SANGUINARINE AND CHELERYTHRINE
(FR) PROCÉDÉ HAUTEMENT EFFICACE POUR CATALYSER LA SYNTHÈSE DE LA SANGUINARINE ET DE LA CHÉLÉRYTHRINE
(ZH) 高效酶催化合成血根碱与白屈菜红碱的方法
Abstract
(EN)
A highly efficient method for catalyzing the synthesis of sanguinarine and chelerythrine, comprising: from the known protopine-6-hydroxylase gene, dihydrobenzophenanthridine oxidase gene and cytochrome P450 reductase gene, by means of heterologous expression and result comparison and analysis, screening out an optimal gene having high expression efficiency, then performing codon optimization on the selected optimal gene; constructing the optimal gene sequence on an expression vector, then transferring same into yeast engineering bacteria for transformation so as to obtain recombinant yeast engineering strains; finally, feeding the recombinant yeast engineering bacteria by using a leaf raw material liquid precursor of Macleaya cordata for fermentation, so as to obtain a product. The invention improves the enzyme catalytic efficiency of sanguinarine and chelerythrine in various aspects such as gene level and fermentation process; the leaf raw material liquid, which is not a traditional medicinal part of Macleaya cordata, is directly used for fermentation with the engineering bacteria, and protopine and allocryptine having high alkaloid content in leaves are converted into high-value sanguinarine and chelerythrine to achieve the comprehensive utilization of Macleaya cordata resources.
(FR)
La présente invention porte sur un procédé hautement efficace pour catalyser la synthèse de la sanguinarine et de la chélérythrine comprenant : le criblage d'un gène optimal ayant une efficacité d'expression élevée, à partir du gène de protopine-6-hydroxylase connu, du gène de dihydrobenzophénanthridine oxydase et du gène de la cytochrome P450 réductase, au moyen d'une expression hétérologue, d'une comparaison et d'une analyse de résultat, puis la réalisation d'une optimisation de codon sur le gène optimal sélectionné; la construction de la séquence de gène optimale sur un vecteur d'expression, puis son transfert dans des bactéries génétiquement modifiées de levure pour une transformation de façon à obtenir des souches génétiquement modifiées de levure recombinée; enfin, l'alimentation des bactéries génétiquement modifiées de levure récombinée à l'aide d'un précurseur liquide de matière première de feuille de Macleaya cordata pour la fermentation, de façon à obtenir un produit. L'invention améliore l'efficacité catalytique enzymatique de la sanguinarine et de la chélérythrine sous divers aspects tels que le niveau de gène et le processus de fermentation; le liquide de matière première de feuille, qui n'est pas une partie médicinale traditionnelle de Macleaya, cordata est directement utilisé pour la fermentation avec les bactéries génétiquement modifiées, et la protopine et l'allocryptine ayant une teneur en alcaloïdes élevée dans les feuilles sont converties en sanguinarine et chélérythrine à valeur élevée pour obtenir l'utilisation complète de ressources de Macleaya cordata.
(ZH)
一种高效酶催化合成血根碱和白屈菜红碱的方法,其分别从已知的普罗托品-6-羟基化酶基因、二氢苯并菲啶氧化酶基因及细胞色素P450还原酶基因中通过异源表达和结果比对分析,筛选出表达效率高的最优基因,然后对选出的最优基因进行密码子优化;再将优化基因序列构建到表达载体上,之后转入酵母工程菌中进行转化获得重组酵母工程菌株;最后用博落回的叶片原料液前体饲喂重组酵母工程菌进行发酵,即得。本发明从基因水平、发酵工艺等多方面上提高血根碱和白屈菜红碱的酶催化效率,利用博落回的非传统药用部位叶片原料液直接与工程菌进行发酵,将叶片中生物碱含量高的原阿片碱和别隐品碱转化成高价值的血根碱和白屈菜红碱,以实现博落回资源的综合利用。
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