Engineering the cytochrome P450 to enhance parthenolide production in Saccharomyces cerevisiae

Parthenolide is confirmed to be an important component of the anticancer drug-ACT001. However, parthenolide biosynthesis in <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>) was greatly hindered by the low conversion rate of its precursor, costunolide. In this study, the Position Specific Scoring Matrix (PSSM) was used to analyze the sequence evolutionary information of parthenolide synthase from <i>Tanacetum parthenium</i> (TpPTS), and a series of mutants were designed and validated. Notably, when the mutant of TpPTS-Y22G was introduced in <i>S. cerevisiae</i>, the parthenolide titer increased by 110 % compared to that of the TpPTS wild-type. Considering TpPTS as an endoplasmic reticulum-localized cytochrome P450 and the importance of heme supply, endoplasmic-associated molecular chaperone HRD1 (hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1) and heme biosynthesis gene HEM2 (aminolevulinate dehydratase) were overexpressed in <i>S. cerevisiae</i> to improve TpPTS expression and catalytic activity. As a result, a titer of 27.08 mg/L parthenolide was achieved in a shake flask, which was further increased by 209 %. Additionally, the conversion rate of costunolide to parthenolide increased from 20.4 % to 51.8 % compared to the initial strain yYTQ001. Eventually, a parthenolide titer of 99.71 mg/L was achieved in a 5-L bioreactor. Our research provides effective strategies and valuable references for engineering rate-limiting cytochrome P450 enzymes to improve sesquiterpenes production in <i>S. cerevisiae</i>.