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1. WO2020108327 - METHOD OF PREPARING TAUROURSODEOXYCHOLIC ACID BY BIOTRANSFORMATION AND APPLICATION THEREOF

Publication Number WO/2020/108327
Publication Date 04.06.2020
International Application No. PCT/CN2019/118856
International Filing Date 15.11.2019
IPC
C12P 33/00 2006.01
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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
33Preparation of steroids
C12N 15/70 2006.01
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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
70Vectors or expression systems specially adapted for E. coli
C12N 15/53 2006.01
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
11DNA or RNA fragments; Modified forms thereof
52Genes encoding for enzymes or proenzymes
53Oxidoreductases (1)
C12N 15/62 2006.01
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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
11DNA or RNA fragments; Modified forms thereof
62DNA sequences coding for fusion proteins
CPC
C07K 2319/00
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
2319Fusion polypeptide
C12N 15/62
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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
11DNA or RNA fragments; Modified forms thereof
62DNA sequences coding for fusion proteins
C12N 15/70
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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
70Vectors or expression systems specially adapted for E. coli
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
C12N 9/0006
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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9Enzymes; Proenzymes; Compositions thereof
0004Oxidoreductases (1.)
0006acting on CH-OH groups as donors (1.1)
C12P 33/00
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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
33Preparation of steroids
Applicants
  • 江苏邦泽生物医药技术股份有限公司 NOVABAND BIOMEDICAL TECHNOLOGY INC. [CN]/[CN]
Inventors
  • 赵志斌 ZHAO, Zhibin
  • 王丹丹 WANG, Dandan
  • 郑祥艳 ZHENG, Xiangyan
  • 李清 LI, Qing
  • 秦松柏 QIN, Songbai
  • 丁峰 DING, Feng
  • 陶京兰 TAO, Jinglan
  • 陈潘海 CHEN, Panhai
  • 曹海兵 CAO, Haibing
Agents
  • 深圳中一联合知识产权代理有限公司 SHENZHEN ZHONGYI UNION INTELLECTUAL PROPERTY AGENCY CO., LTD.
Priority Data
201811446689.529.11.2018CN
Publication Language Chinese (ZH)
Filing Language Chinese (ZH)
Designated States
Title
(EN) METHOD OF PREPARING TAUROURSODEOXYCHOLIC ACID BY BIOTRANSFORMATION AND APPLICATION THEREOF
(FR) PROCÉDÉ DE PRÉPARATION D'ACIDE TAUROURSODÉSOXYCHOLIQUE ET APPLICATION CORRESPONDANTE
(ZH) 生物转化制备牛磺熊去氧胆酸的方法及其应用
Abstract
(EN)
A method of preparing tauroursodeoxycholic acid by biotransformation and an application thereof, comprising genetic codon optimization, engineered bacteria construction, engineered bacteria cultivation, substrate transformation, and product preparation. Tauroursodeoxycholic acid is prepared by transforming a substrate by means of direct fermentation of engineered bacteria, and the substrate is taurochenodeoxycholic acid. The substrate may reach 250 g/L in concentration, the reaction time is short, the transformation rate of the substrate reaches 98% and above, and the obtained product reaches 99% and above in purity; cyclic regeneration of NAD+ in the reaction system helps greatly reduce the usage of coenzyme NAD+, the cost of enzyme catalysis reaction is reduced, and industrial amplification is benefited. Steroid dehydrogenase and coenzyme regeneration enzyme are connected by a flexible polypeptide sequence to construct a fusion protein polymer, binding distances to the substrate and the coenzyme are shorter, transformation reaction is more facilitated, the number of times of fermentations in industrial production is decreased, the process is simplified, and time costs and raw material costs are saved.
(FR)
L'invention concerne également un procédé de préparation d'acide tauroursodésoxycholique par biotransformation et une application correspondante, comprenant une optimisation de codon génétique, une construction de bactéries génétiquement modifiées, une culture de bactéries génétiquement modifiées, une transformation de substrat et une préparation de produit. L'acide tauroursodésoxycholique est préparé par transformation d'un substrat au moyen d'une fermentation directe de bactéries génétiquement modifiées, et le substrat est un acide taurochénodésoxycholique. Le substrat peut atteindre 250 g/L en concentration, le temps de réaction est court, le taux de transformation du substrat atteint 98 % et plus, et le produit obtenu atteint 99 % et plus de pureté; la régénération cyclique de NAD+ dans le système de réaction aide à réduire considérablement l'utilisation de la coenzyme NAD+, le coût de la réaction de catalyse enzymatique est réduit, et l'amplification industrielle en tire avantage. La stéroïde déshydrogénase et l'enzyme de régénération de coenzyme sont reliées par une séquence polypeptidique flexible pour construire un polymère de protéine de fusion, les distances de liaison au substrat et à la coenzyme sont plus courtes, la réaction de transformation est davantage facilitée, le nombre de fois de fermentations dans la production industrielle est réduit, le processus est simplifié, et les coûts en temps et les coûts de matière première sont économisés.
(ZH)
一种生物转化制备牛磺熊去氧胆酸的方法及其应用,包括基因密码子优化、工程菌构建、工程菌培养、底物转化及产物制备;采用工程菌直接发酵转化底物制备牛磺熊去氧胆酸,底物为牛磺鹅去氧胆酸。底物浓度可高达250g/L,反应时间短,对底物的转化率高达98%以上,所获得的产品纯度在99%以上;反应体系中NAD +循环再生,极大降低了辅酶NAD +的使用量,酶催化反应的成本降低,利于工业放大;将类固醇脱氢酶和辅酶再生酶通过柔性多肽序列连接在一起构建成融合蛋白多聚体,与底物和辅酶的结合距离更近,更有利于转化反应的进行,在工业化生产中,减少发酵的次数,简化了工艺,节约时间成本和原料成本。
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