Optimizing cytochrome P450 activity for quillaic acid biosynthesis in Saccharomyces cerevisiae

QS-21, a saponin extract from the Chilean tree Quillaja saponaria, is gaining popularity as a potent vaccine adjuvant, but its production is constrained due to its low natural abundance and complex chemical structure of the triterpenoid saponins, which hinder large-scale production through plant extraction or chemical synthesis. Microbial biosynthesis presents a promising alternative, with Saccharomyces cerevisiae emerging as a desirable host for triterpenoid production. A key step toward microbial QS-21 synthesis is the efficient biosynthesis of its aglycone core, the triterpenoid quillaic acid. However, its efficient production remains limited by challenges of cytochrome P450 enzyme (CYP450s) activity, including cofactor availability and electron transfer efficiency. To address this limitation, we applied a multi-faceted metabolic engineering strategy to optimise CYP450 activity, including CYP450 expression and cytochrome P450 reductase (CPR) selection. Additionally, aligning CYP450 expression with the ethanol phase, enhanced the metabolic flux toward quillaic acid synthesis, leading to an 85-fold increase in titre. Together, these strategies led to a quillaic acid titre of 385 {+/-} 14 mg/L in flask fermentation. Fed-batch bioreactor fermentations increased quillaic acid titre to 471 {+/-} 20 mg/L and significantly increased the selectivity for QA from 32.6% to 65.1% of the total triterpenoids produced. These findings demonstrate the effectiveness of enhancing CYP450 activity through targeted strategies and reveal potential bottlenecks in CYP450 expression, providing valuable insights for future optimization of triterpenoid production in yeast.