De Novo Biosynthesis of 22R,23R-diOH-7-Dehydrocampesterol in Yeast for Osmium-Free Synthesis of Brassinolide

22R,23R-Dihydroxylation (22R,23R-diOH) is a critical structural feature of highly active brassinosteroids (BRs), such as brassinolide (BL). This modification can be chemically achieved through Sharpless asymmetric dihydroxylation. However, the reliance on toxic and costly osmium oxide and chiral ligands restricts the commercial viability of highly active BRs. Developing microbial cell factories for the biosynthesis of sterols featuring 22R,23R-diOH using renewable carbon sources will serve as a viable strategy for the sustainable production of highly active BRs. This study presents, for the first time, the microbial production of 22R,23R-diOH-7-dehydrocampesterol (22R,23R-diOH-7-DHCR), a proposed precursor for osmium-free semisynthesis of BL. Initially, an artificial biosynthetic pathway was constructed in yeast by introducing two plant-derived CYP450s, a sterol C-22 hydroxylase and a sterol C-23 hydroxylase, enabling biosynthesis of 22R,23R-diOH-7-DHCR. Subsequently, through the rational design of CYP450s and combinatorial metabolic engineering strategies, including the implementation of a sucrose-triggered GAL cascade system and enhancing the supply of key cofactors NADPH and heme, the production of 22R,23R-diOH-7-DHCR reached 592.14 mg L–1 in a two-stage fed-batch fermentation process using glucose and sucrose as carbon sources. The artificial pathway established in this work provides a foundational step toward the sustainable and osmium-free production of BL.