Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method of recombinant saccharomyces cerevisiae

A technology of Saccharomyces cerevisiae and ginsenoside, applied in the biological field

Active Publication Date: 2022-07-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no research on the production of terpenoid natural products, especially triterpene saponins, by modifying glycerol metabolism

Method used

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  • Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method of recombinant saccharomyces cerevisiae
  • Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method of recombinant saccharomyces cerevisiae
  • Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method of recombinant saccharomyces cerevisiae

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1 The construction method of recombinant Saccharomyces cerevisiae 1 (recombinant bacteria 1)

[0045] The glycosyltransferase UGT1 gene expression cassette, the PGM2 gene expression cassette of glucose phosphate mutase and the UDP glucose pyrophosphorylase UGP1 gene expression cassette were introduced into Saccharomyces cerevisiae W3a to obtain recombinant bacteria 1.

[0046] The glycosyltransferase UGT1 is derived from ginseng (Ginseng), and after codon optimization of Saccharomyces cerevisiae by Wuhan Jinkarui Bioengineering Co., Ltd., the nucleic acid sequence of the glycosyltransferase UGT1 gene is shown in SEQ ID NO.1; the The nucleotide sequence of the phosphoglucomutase PGM2 gene is derived from Saccharomyces cerevisiae and is shown in SEQ ID NO.2; the nucleotide sequence of the UDP glucose pyrophosphorylase UGP1 gene is derived from Saccharomyces cerevisiae and is shown in SEQ ID NO.3 shown.

[0047] (1) Construction of expression cassette

[0048]...

Embodiment 2

[0055] Embodiment 2 The construction method of recombinant Saccharomyces cerevisiae 2 (recombinant bacteria 2)

[0056] The glycerol transporter CjFPS1 gene expression cassette, the glycerol dehydrogenase OpGDH gene expression cassette and the dihydroxyacetone (DHA) kinase DAK1 gene expression cassette were introduced into the recombinant bacteria 1 to obtain the recombinant bacteria 2.

[0057] The glycerol transporter CjFPS1 is derived from Cyberlindnera jadinii, and after codon optimization of Saccharomyces cerevisiae by Wuhan Jinkarui Bioengineering Co., Ltd., the nucleic acid sequence of the glycerol transporter CjFPS1 gene is shown in SEQ ID NO.4; the glycerol dehydrogenase OpGDH It is derived from Ogataeapara polymorpha, and after codon optimization of Saccharomyces cerevisiae by Wuhan Jinkarui Bioengineering Co., Ltd., the nucleic acid sequence of the OpGDH gene of glycerol dehydrogenase is shown in SEQ ID NO.5; the source of the dihydroxyacetone (DHA) kinase DAK1 In S...

Embodiment 3

[0064] Embodiment 3 The construction method of recombinant Saccharomyces cerevisiae 3 (recombinant bacteria 3)

[0065] Introducing the 3-hydroxy-3-methylglutaryl-CoA reductase HMGr gene expression cassette into recombinant bacteria 2 to obtain recombinant bacteria 3;

[0066] The 3-hydroxy-3-methylglutaryl-CoA reductase HMGr is derived from Silicibacter pomeroyi, and after codon optimization of Saccharomyces cerevisiae by Wuhan Jinkarui Bioengineering Co., Ltd., 3-hydroxy-3-methylglutaryl is obtained The nucleic acid sequence of the coenzyme A reductase HMGr gene is shown in SEQ ID NO.7.

[0067] (1) Construction of expression cassette

[0068] As shown in Table 3, using the genome of Saccharomyces cerevisiae ATCC208352 as a template, the MET17L fragment was amplified by PCR using MET17L-F (SEQ ID NO.50) and Bleo-MET17L-R (SEQ ID NO.51); the pSH65 plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) as a template, using MET17L-Bleo-F (SEQ ID NO.52) and TDH3-Bleo-...

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Abstract

The invention discloses recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and a construction method thereof, and the construction method comprises the following steps: (1) introducing a glycosyl transferase UGT1 gene expression cassette, a glucose phosphate mutase PGM2 gene expression cassette and a UDP glucose pyrophosphorylase UGP1 gene expression cassette into saccharomyces cerevisiae capable of synthesizing protopanoxadiol to obtain recombinant bacteria 1; (2) introducing a glycerol transport protein CjFPS1 gene expression cassette, a glycerol dehydrogenase OpGDH gene expression cassette and a dihydroxyacetone kinase DAK1 gene expression cassette into the recombinant bacterium 1 to obtain a recombinant bacterium 2; (3) introducing a 3-hydroxy-3-methylglutaryl coenzyme A reductase HMGr gene expression cassette into the recombinant bacterium 2 to obtain a recombinant bacterium 3; experiments prove that the yields of the ginsenoside CK of the recombinant bacteria 1, 2 and 3 are 109.84 mg / L, 152.12 mg / L and 330.94 mg / L in sequence.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Saccharomyces cerevisiae for metabolizing glycerol to produce ginsenoside CK and a construction method and application. Background technique [0002] Ginsenoside CK (abbreviated as CK) belongs to the protopanaxadiol type saponin and has biological properties such as anti-cancer, anti-allergy, anti-aging, and anti-diabetes. The content of CK in ginseng is extremely low. According to reports, it is mainly obtained by hydrolysis of the main ginsenosides Rb1, Rd, Rg2 and other ginsenosides through mild acid hydrolysis and alkali treatment, microbial transformation and enzymatic transformation, and the unit cost is still high. In recent years, research in synthetic biology has continued to develop, enabling microorganisms to synthesize compounds of animal and plant origin. Although some researchers have constructed strains that can produce ginsenoside CK, the yield still ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/54C12N15/61C12N15/53C12N15/31C12N1/19C12P33/20C12R1/865
CPCC12N15/81C12N9/1051C12N9/90C12N9/1241C12N9/0006C12N9/1205C07K14/39C12P33/20C12Y204/01017C12Y504/02002C12Y207/07009C12Y101/01006C12Y207/01029
Inventor 卢文玉张传波田锦平刘瑞霞
Owner TIANJIN UNIV
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