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.