Construction Of Cell Factories For High Production Of Bioactive Ginsenoside Compound K In Saccharomyces Cerevisiae

The protopanaxadiol type ginsenoside Compound K(CK)is a medicinal ingredient in precious medicinal plants such as ginseng.It has shown strong anti-inflammatory,anti-diabetic,anti-cancer and other biological activities in vitro cell experiments and animal experiments.At present,these compounds are mainly obtained by separating and extracting from plants or hydrolyzing other saponins.However,due to the shortage of ginseng resources and the complex separation process,the wide application of ginsenoside CK is seriously limited.Using synthetic biology technology to transform Saccharomyces cerevisiae cell factory to produce natural products can not only avoid all kinds of thorny problems caused by chemical synthesis and separation,but also control the supply of raw materials and reduce production costs.In this study,Saccharomyces cerevisiae BY4742 was used as the starting strain to obtain a group of chassis strains YSBYT30 suitable for ginsenoside synthesis through the enhancement of mevalonate pathway and 2,3-oxidosqualene pathway.Then,the engineering strain AP was obtained by heterologous expression of damarenedialcohol II synthase gene and protopanaxadiol synthase gene from ginseng.The intermediates including squalene and dammarenediol II(DD)were accumulated in large quantities,and the yield and conversion efficiency of protopanaxadiol(PPD)were low.Lipid droplets are a kind of special organelles in yeast cells that have the ability to temporarily store intracellular hydrophobic substances.Therefore,the lipid droplets of AP strain were extracted and analyzed.The results showed that most of DD and squalene were stored in lipid droplets.Meanwhile,fluorescence colocalization analysis showed that DDS was mainly distributed in the endoplasmic reticulum and lipid droplets,and PPDS was mainly distributed in the endoplasmic reticulum.Therefore,there is a problem of space isolation between the enzyme and substrate in AP engineering bacteria,which affects the catalytic efficiency of the enzyme and results in the accumulation of precursor.Subsequently,for the first time,lipid droplets membrane protein Pln1 p was used to localize PPDS and DDS to solve the spatial isolation between the enzyme and substrate,and the successful locale localization of the enzyme was confirmed by fluorescence co-localization analysis.The fermentation results showed that the compartment localization of DDS did not improve the DD metabolic flux,but the successful compartment localization of PPDs increased the PPD conversion rate to 86.43%,which was 2.58 times of the control strain PTA.The effects of the size and number of lipid droplets on the yield and conversion of PPD were further explored.The conversion and yield of PPD under different lipid droplet forms were evaluated by enhancing several key genes of tag biosynthesis,increasing the number of lipid droplets in yeast and knocking out SEI1 gene to form super lipid droplets.The glycosyltransferases Synpn3-29 from Panax notoginseng were introduced into the chassis strain with high PPD conversion rate,and the engineering strains of ginsenoside CK was obtained by continuously enhancing the expression of the module.The engineering strains of ginsenoside CK was fermented in a 5 L high-density fermentor by fed batch fermentation,and the high-density production of CK reached 4879 mg/L,which is the highest yield of ginsenosides reported so far.