Recombinant saccharomyces cerevisiae for heterologous synthesis of ginsenoside F2 and construction method of recombinant saccharomyces cerevisiae
A technology for recombining Saccharomyces cerevisiae and ginsenosides, applied in the biological field, can solve problems such as unreported
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Embodiment 1
[0042] Example 1 Construction Method of the First Recombinant Saccharomyces cerevisiae (Recombinant Bacteria 1) for Heterologous Synthesis of Ginsenoside F2
[0043] Glycosyltransferase GTK1 is derived from Bacillus subtilis, and glycosyltransferase UGT1 is derived from Ginseng, both of which were codon-optimized by Saccharomyces cerevisiae from Wuhan Jinkairui Bioengineering Co., Ltd. to obtain the optimized glycosyltransferase The GTK1 gene is shown in SEQ ID NO.1; the optimized glycosyltransferase UGT1 gene is shown in SEQ ID NO.2; synthesized by chemical synthesis, the gene shown in SEQ ID NO.1 and SEQ ID NO.2 After the indicated gene is fused, it is introduced into Saccharomyces cerevisiae producing protopanaxadiol PPD to obtain the first recombinant Saccharomyces cerevisiae that synthesizes ginsenoside F2 heterologously, referred to as recombinant strain 1.
[0044] (1) GTK1 and UGT1 fusion protein construction
[0045] The stop codon of the optimized glycosyltransferas...
Embodiment 2
[0050] Example 2 Construction method of the second recombinant Saccharomyces cerevisiae strain (abbreviated as recombinant bacterium 2) for heterologous synthesis of ginsenoside F2
[0051] Knockout the diacylglycerol acyltransferase DGA1 gene of recombinant bacterium 1 and overexpress the diacylglycerol kinase DGK1 gene to obtain recombinant bacterium 2, the nucleotide sequence of the diacylglycerol acyltransferase DGA1 gene is shown in SEQ ID No.3 ; The nucleotide sequence of the diacylglycerol kinase DGK1 is shown in SEQ ID NO.4.
[0052] (1) Diacylglycerol kinase DGK1 gene expression module construction
[0053] Using the Saccharomyces cerevisiae ATCC208352 genome as a template,
[0054] Using DGA1-F (SEQ ID NO.22)
[0055] PCR amplification of the DGA1L fragment with TEF1-DGA1-R (SEQ ID NO.23), and PCR amplification of the P TEF1 Fragment, using TEF1-DGK1-F (SEQ ID NO.26) and CYC1-DGK1-R (SEQ ID NO.27) to PCR amplify the DGK1 fragment, using DGK1-CYC1-F (SEQ ID NO.28) ...
Embodiment 3
[0057] Example 3 Construction Method of the Third Recombinant Saccharomyces cerevisiae (Recombinant Bacteria 3) for Heterologous Synthesis of Ginsenoside F2
[0058] The gene of β-glucose hydrolase EGH1 was knocked out in recombinant bacterium 2, and the genes of phosphoglucomutase PGM1 and UDP glucose pyrophosphorylase were overexpressed to obtain recombinant bacterium 3.
[0059] The nucleotide sequence of the β-glucohydrolase EGH1 is shown in SEQ ID NO.5;
[0060] The nucleotide sequence of the phosphoglucomutase PGM1 is shown in SEQ ID NO.6;
[0061] The nucleotide sequence of the UDP glucose pyrophosphorylase UGP1 is shown in SEQ ID NO.7;
[0062] (1) Construction of expression modules of phosphoglucomutase PGM1 and UDP glucose pyrophosphorylase UGP1
[0063] Using the Saccharomyces cerevisiae ATCC208352 genome as a template, use EGH1L-F (SEQ ID NO.34) and PGK1-EGH1L-R (SEQ ID NO.35) to PCR amplify the EGH1L fragment, and use EGH1-PGK1-F (SEQ ID NO.36 ) and PGM1-PGK1-R...
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