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Applications of glycosyltransferase and related materials thereof in construction of engineering bacteria for producing ginsenoside Rb1 and Rg1

A technology of ginsenosides and biomaterials, applied in transferase, genetic engineering, application, etc., can solve the problems of complex biosynthesis of glycosylated saponins and no relevant glycosyltransferase genes found

Active Publication Date: 2019-11-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, relevant progress has been made in the research on the biosynthetic pathway of ginsenosides, including some key enzymes in the synthetic pathway of ginsenosides, such as squalene synthase (Squalene synthase, which catalyzes the isoprenoid pathway towards sterols and triterpene saponins). SS), squalene epoxidase (Squaleneepoxidase, SE) that catalyzes the generation of 2,3-oxidized squalene, dammarene diol synthase (DS) that catalyzes the generation of dammarene diol, and responsible for hydroxylation Cytochrome P450 enzymes, etc., but the biosynthesis of various glycosylated saponins in Panax notoginseng is relatively complicated, and has not yet been fully explored
In the study of the effect of calcium ions on the biosynthesis of Panax notoginseng and ginsenoside Rb1, Zhong Jianjiang et al. found that the crude enzyme solution extracted from Panax notoginseng plant tissue cells had the activity of catalyzing the production of ginsenoside Rd from ginsenoside Rb1, but no related glycosyltransferases were found. Genes (Yue CJ et al., Biotechnol Bioeng, 2005, 89(4):444-452)

Method used

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  • Applications of glycosyltransferase and related materials thereof in construction of engineering bacteria for producing ginsenoside Rb1 and Rg1
  • Applications of glycosyltransferase and related materials thereof in construction of engineering bacteria for producing ginsenoside Rb1 and Rg1
  • Applications of glycosyltransferase and related materials thereof in construction of engineering bacteria for producing ginsenoside Rb1 and Rg1

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Embodiment 1

[0069] Example 1. Application of glycosyltransferases Pn3-32, Pn1-31, Pn3-29 and Pn3-31 and their coding genes in the preparation of ginsenoside Rb1 and ginsenoside Rg1

[0070] (1) Construction of recombinant plasmids

[0071] 1. Construction of genetic elements

[0072] 1. Acquisition of Panax notoginseng cDNA

[0073] 1) Extraction of total RNA: Panax notoginseng (from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, the public can obtain from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) leaves 200mg, grind with liquid nitrogen and extract total RNA by CTAB method : Add 1ml 2×CTAB extract to a 1.5ml centrifuge tube, preheat at 65°C, add 20μl 2-ME; add a small amount of powder (about 50mg), mix well, keep warm at 65°C for 10 minutes, shake well 5 times; 4°C , centrifuge at 12000rpm for 10min, remove the supernatant and extract with an equal volume of chloroform / isoamyl alcohol; centrifuge at 12000rpm for 10min at 4°...

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Abstract

The invention discloses applications of glycosyltransferase and related materials thereof in the construction of engineering bacteria for producing ginsenoside Rb1 and Rg1. A glycosyltransferase genePn3-32 which can catalyze ginsenoside Rd to generate ginsenoside Rb1 can be successfully identified through a synthetic biological method; and the gene can simultaneously catalyze ginsenoside F1 to generate ginsenoside Rg1 and construct recombinant yeast producing the ginsenoside Rb1 and the ginsenoside Rg1. Through experiments, the constructed recombinant yeast producing the ginsenoside Rb1 and the ginsenoside Rg1 can simultaneously generate the ginsenoside Rb1 and the ginsenoside Rg1. Pn1-31, Pn-3-29, Pn3-31 and Pn3-32 glycosyltransferase genes in medicinal plant radix notoginseng are firstly utilized to continuously catalyze protopanaxadiol and protopanaxatriol to synthetize the ginsenoside Rb1, the ginsenoside Rg1 and corresponding intermediates, so that novel cases can be provided formicrobial strains to produce natural products.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of glycosyltransferase and related materials in constructing engineering bacteria producing ginsenosides Rb1 and Rg1. Background technique [0002] Ginsenoside Rb1 (Ginsenoside Rb1), Ginsenoside Rd (Ginsenoside Rd) and Ginsenoside Rg1 (Ginsenoside Rg1) are all dammarane-type triterpene saponins, mainly distributed in Araliaceae plants such as ginseng, Panax notoginseng, American ginseng, etc. Among them, it has a variety of pharmacological activities and is a chemical substance of great medicinal value. Among them, ginsenoside Rb1 has improved regulatory effects on the central nervous system, cardiovascular system, immune system and anti-tumor. Ginsenoside Rd has significant biological activity in cardiovascular system, immune system, renal function protection and anti-tumor effect. Ginsenoside Rg1 has a natural anti-oxidation effect, affects the nervous ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P33/20C12N15/81C12N1/19C12R1/865
CPCC12N9/1048C12P33/20C12P33/005C12N15/81
Inventor 张学礼戴住波王金鹤
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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