Construction Of An Engineered Yeast Strain Producing Ginsenoside Rh2

Panax ginseng, belonging to Araliaceae, is a traditional Chinese medicine. Ginsenosides are the main bioactive constituents of ginseng, can be extracted from the roots, stems, and leaves of Panax ginseng, and are highly medicine-valuable. At present the yield of ginsenosides cannot meet their enormous market demand. The synthetic biology had provided a new strategy for the development of nature products and it might be an effective method to improve the yield of ginsenoside. Previous studies have shown that ginsenoside Rh2 is derivied from 2,3-oxidosqualene. Firstly, 2,3-oxidosqualene is converted into dammarenediol with the catalysis of dammarenediol synthase; then dammarenediol is converted into protopanaxadiol under the hydroxylation of protopanaxadiol synthase at C-12 position; finally uridine diphosphate(UDP)-dependent glycosyltransferase catalyzes the transfer of a glucose moiety from UDP-glucose to C3 hydroxyl group of protopanaxadiol to generate ginsenoside Rh2. Saccharomyces cerevisiae has the capacity of 2,3-oxidosqualene biosynthesis, which is the precursor for synthesis of ginsenoside Rh2. In this study, an engineered ginsenoside-Rh2-producing Saccharomyces cerevisiae was constructed, which can heterologously express the above-mentioned three enzymes employed in ginsenoside Rh2 biosynthesis.The key enzymes protopanaxadiol synthase and UDP-dependent glycosyltrans-ferase were encoded by the genes D12 H and UGT-D-3OH-1 respectively. In this study these two genes were amplified by PCR and subcloned into the expression plasmid p ESC-URA to give a recombinant coexpression plasmid p ESC-D12H-UGT. The recombinant plasmid p ESC-D12H-UGT was transformed into Saccharomyces cerevisiae YPH501 with another plasmid p AUR123- DS, and the engineered yeast was named as YPDDG. SDS-polyacrylamide gel electrophoresis was used to verify expression of the three genes after being induced by galactose. The result of SDS-PAGE for YPDDG showed the protein bands of dammarenediol synthase, protopanaxadiol synthase and UDP-dependent glycosyltransferase; while the control showed no band at according sites. HPLC was used to verify the production of ginsenoside Rh2. The main contents of this study are shown as follows:(1) Total RNA was isolated from panax ginseng hairy roots cultured for 21 d, from which c DNA was obtained. According to the sequences in Genebank, primers for D12 H and UGT-D-3OH-1 were designed by Primer 5.0. Then both genes were cloned by RT-PCR.(2) The two key genes D12 H and UGT-D-3OH-1 were ligated into the cloning vector p MD18-T to construct plasmids p T-D12 H and p T-UGT. Sequencing and enzyme cleavage were performed for verification.(3) The p T-D12 H was digested by Bam H I and Kpn I; while the expression plasmid p ESC-URA was linearized by the same restriction enzymes. Then D12 H gene from p T-D12 H was subcloned into p ESC-URA using T4 DNA Ligase to get p ESC-D12 H. By the same method, UGT-D-3OH-1 gene from p T-UGT was subcloned into p ESC-D12 H to construct p ESC-D12H-UGT. The p ESC-D12H-UGT was then transformed into Saccharomyces cerevisiae with another plasmid p AUR123-DS. Transformants were screened by two different screening medium. PCR and sequencing were performed to verify the successful construction. The resulted engineered yeast was named as YPDDG.(4) Expression of dammarenediol synthase, protopanaxadiol synthase and UDP-dependent glycosyltransferase from YPDDG was verified by SDS-polyacryl-amide gel electrophoresis. The result showed that the genes D12 H, DS and UGT-D-3OH-1 could be expressed in YPDDG.(5) HPLC was used to verify the expression of ginsenoside Rh2 and to assay the expression level. The result was 3.74×10-2mg/L.(6) Growth condition optimization for YPDDG was preformed. The optimal growth conditions of YPDDG were 30℃, p H 7.5, 220 rpm, and content of medium 80 m L. Under this culture conditions, the content of ginsenoside Rh2 in YPDDG was 4.13×10-2 mg/L.