De Novo Synthesis And Optimization Of Campesterol And Pregnenolone In Yarrowia Lipolytica

Campesterol and pregnenolone are important precursor and intermediate to heterologous synthetic steroid drugs.This study planed to introduce heterologous campesterol and pregnenolone synthesis pathways in Yarrowia lipolytica,and carry out a variety of optimization strategies,including screening heterologous protein source,knocking out competitive pathway,optimizing acetyl-CoA supplement and distribution and truncating or redirecting enzyme,hoping to achieve high-level expression of campesterol and pregnenolone in Yarrowia lipolytica.Heterologous 7-dehydrocholesterol reductase(DHCR7)should be introduced in Yarrowia lipolytica for campesterol synthesis.Here,6 DHCR7 souces were chosen and screened,finding that the best source was zebrafish(Danio rerio).Through bioinformatics analysis,D409 E,G408P and L404F(or L404M)were realized the three key mutations to affect the enzyme activity,such as D409 E mutation in DHCR7 from Xenopus laevis reduced campesterol production by 86%.A campesterol overproduction strain SyBE_Yl02060056 was obtained by knocking out ERG5 for ergosterol synthesis and introducing DHCR7 from Danio rerio,and this strain only accumulated campesterol as sterol,which was 1.38-fold than the highest yield that has been reported.Then,this strain was chosen as a chassis to characterize the regulation of 13 acetyl-CoA accumulation or distribution related genes to campesterol production under glucose or sunflower seed oil as carbon source.It was concluded that when oil as carbon source,three key metabolic regulation targets including HMGR,ACL and POX2 were identified,and campesterol production was increased by 1.32-fold,1.22-fold and 1.19-fold via overexpressing these genes,respectively.Finally,the highest campesterol titer of 941.5 mg/L was realized in 5-L bioreactor via fermentation process optimization,which is now known to be the the highest yield.Campesterol was catalized by the P450 scc system(including CYP11A1,Ad R and Ad X)for pregnenolone synthesis in Yarrowia lipolytica.36 combinations about different P450 scc sources were chosen and screened,for the first time pregnenolone was de novo synthesis in Yarrowia lipolytica,and CYP11A1 from Sus scrofa with AdR and AdX from Bos taurus was the best combination.To explore the influence factors of pregnenolone synthesis,including substrate specificity,campesterol existent form,contact of enzyme with precursor and enzyme activity,ect,it found that boththe spatial isolation of enzyme with precursor and enzyme activity insufficience reduced pregnenolone production significantly.By applying a strong promoter and redirecting CYP11A1 from Sus scrofa to lipid particles' membrane,pregnenolone yield of 3.1±0.5 mg/L was reached,which increased 13-fold over original recombination strain.A variety of strategies were used to optimize the campesterol and pregnenolone synthesis in Yarrowia lipolytica in this study,it would offer many effective ways to improve other terpenes accumulation in Yarrowia lipolytica,and provide reference for high-yield terpene in other microorganisms.Also,campesterol and pregnenolone overproduction strains here would be apply for further biosynthesis of subsequent steroid drugs.