Novel cell factory for the production of 24-epi-ergosterol, an un-natural semi-synthetic precursor for the production of brassinolide in Yarrowia lipolytica.

Brassinolide (BL) is the most bioactive plant growth regulator among Brassinosteroids (BRs), belonging to the sixth class of plant hormones. However, its low natural abundance limits large-scale agricultural applications. An unnatural sterol, 24-epi-ergosterol, was proposed as a semi-synthetic precursor for economic production of BL. Here, we constructed a synthetic pathway for 24-epi-ergosterol in Yarrowia lipolytica, which has abundant acetyl-CoA content and a hydrophobic intracellular environment. Initially, we introduced a mutant plant-derived Δ²⁴⁽²⁸⁾ sterol reductase (Dwf1) into Y. lipolytica to enable 24-epi-ergosterol production. The production of 24-epi-ergosterol was subsequently enhanced by regulating sterol homeostasis, optimizing transcriptional regulators, and overexpressing key pathway genes. Next, the accumulation of 24-epi-ergosterol was further improved by increasing acetyl-CoA levels and adjusting lipid metabolism. Finally, the 24-epi-ergosterol production reached 1626.85 mg/L after optimizing the fermentation conditions and performing a fed-batch culture in a 2 L fermenter. This study represents the first successful de novo synthesis of 24-epi-ergosterol in Y. lipolytica, offering a novel approach for the industrial production of BL.