Steroidal compounds, characterized by a dihydrophenanthrene carbon skeleton, represent the second-largest class of drugs with their production being dependent on pregnenolone as a crucial precursor.The biosynthesis of pregnenolone depends on the cytochrome P450 scc side-chain cleavage system, which consists of the monooxygenase CYP11A1 and electron transport chains AdX and AdR.Unlike chemical synthesis, de novo microbial synthesis offers a more cost-effective alternative, despite of it involving complex synthetic and regulatory pathways.In this study, we utilised CRISPR/Cas9 to integrate codon-optimized P450 scc side chain degradation cascade into the genome of a Saccharomyces cerevisiae strain that accumulate Campesterol;a substrate for production of pregnenolone.This was through utilisation of a group allocation pair strategy employed to screen CYP11A1, AdX and AdR from different sources.Ultimately, the most efficient cytochrome P450 scc system comprised CYP11A1 from Lynx rufus and AdX/AdR from Yarrowia lipolytica.Next, we screened eight strong Saccharomyces cerevisiae promoters to drive CYP11A1 expression.When TEF1p was used as the promoter, the highest pregnenolone yield reached 12.28 mg/L.Further optimization by enhancing the upstream sterol synthesis pathway increased the fermentation yield to 38.75 mg/L.Finally, fed-batch fermentation in a 5 L bioreactor achieved a pregnenolone production of 116.43 mg/L.This study not only demonstrates the effectiveness of the group allocation strategy for optimizing the cytochrome P450 scc system but also establishes a versatile platform for steroid production.
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