Sustainable supply of wilforic acid C via decompartmentalization of peroxisomal acetyl-CoA and systematic engineering in yeast

Abstract

Wilforic acid C is the key precursor in the biosynthesis of celastrol. The microbial production of triterpenoids, including wilforic acid C, is often hindered by limited availability of the necessary precursor due to the compartmentalization of acetyl-CoA metabolism. In this study, we designed a decompartmentalization strategy to enhance wilforic acid C biosynthesis in Saccharomyces cerevisiae by redirecting peroxisomal acetyl-CoA to the cytosol. It was achieved by manipulating the glyoxylate cycle and introducing Aspergillus nidulans ATP-citrate lyase (AnACL), which enabled an efficient supply of cytosolic acetyl-CoA. Optimization of the synthetic pathway and semi-rational design of ThCYP712K1 proteins further boosted production. Additionally, lipid droplet expansion and NADPH regeneration modules were integrated to improve overall metabolic flux. The resulting engineered strain LAC168 produced 263.55 mg L⁻¹ wilforic acid C in shake-flask culture, and reached 584.78 mg L⁻¹ in a 10 L bioreactor. This study offers a generalizable strategy for cytosolic acetyl-CoA supply and acetyl-CoA-derived chemicals production and represents the highest reported titer of wilforic acid C to date.