Design And Construction Of 7-dehydrocholesterol Overproducing Saccharomyces Cerevisiae

7-Dehydrocholesterol（7-DHC）is the direct precursor of vitamin D₃.Its derivatives 25-hydroxy-VD₃ and 1,25-dihydroxy-VD₃ have extremely high medical value.In the previous research,our laboratory successfully achieved the biosynthesis of 7-DHC using Saccharomyces cerevisiae as chassis,but there are still problems such as low 7-DHC production and excessive accumulation of intermediate metabolites.This study will improve the supply of precursors,block metabolic branches,screen and regulate rate-limiting exogenous enzyme,regulate liposomes and phospholipid metabolism,and rearrange the organelle locations of enzymes in the synthesis pathway to optimize the chassis and pathway and achieve higher 7-DHC production.Aiming at solving the problems of insufficient precursors for 7-DHC synthesis and weak catalytic activity of DHCR24,this study overexpressed the endogenous genes ERG10,ERG13,t HMG1,ERG12,ERG8,ERG19,IDI1 and ERG20 in the mevalonate pathway to enhance the supply of precursors and knocked out the ergosterol synthesizing genes ERG5 and ERG6 to block the metabolic branch.For the only exogenous enzyme DHCR24 in the 7-DHC synthesis pathway,11 species sources were screened.It was found that gallus-derived DHCR24 obtained the highest 7-DHC production.Then,through adjusting its copy number,promoter,genome integration site and expression mode,7-DHC output at shake-flask increased from 36.1 mg/L（the starting strain Sy BE＿Sc01250050）to 169.1 mg/L（generating strain Sy BE＿Sc0125XJ06）.For the interference of yeast endogenous sterol and lipid metabolism on the 7-DHC synthesis pathway,this study tried to perturb the lipid metabolism of the strains.By overexpressing or knocking out genes related to phospholipid metabolism,it was found that 7-DHC production increased 48.9%,103.0%and 20.6%respectively by knocking out of genes NEM1,OSH2 and FLD1.7-DHC production of the NEM1knockout strain at the level of the 5-L fermentor reached 1.07 g/L,which is the highest7-DHC production that has been reported so far.Aiming at solving the problem of organelle isolation of the enzymes in the 7-DHC synthesis pathway,this study tried to regulate the enzymes in post-squalene pathway spatially to balance the metabolic flow.The enzymes originally located in the endoplasmic reticulum（ER）in the post-squalene pathway are divided into three modules,ERG2-ERG3-DHCR24,ERG1-ERG11-ERG24,and ERG25-ERG26-ERG27,which are overexpressed in the ER or relocated in the lipid droplets（LDs）.The results showed that when ERG2-ERG3-DHCR24 and ERG25-ERG26-ERG27 relocalized on LDs and ERG1-ERG11-ERG24 overexpressed at ER at the same time,the shake-flask level of 7-DHC production was increased by 92.1%compared with the control strain Sy BE＿Sc0125XJ06,reaching 360.6 mg/L,which is the highest 7-DHC output on shake-flask level that has been reported so far.This study increased the production of 7-DHC to a large extent and provides a certain reference for the subsequent research on the synthesis of sterols in biological cells.