Enzyme Engineering and Cofactor Recycling Enable Efficient Biosynthesis of Neuroactive 3α-OH-5β-H Steroids with C3 Glycosylation

Steroids represent a class of bioactive molecules with diverse physiological functions. Notably, 5β-dihydrosteroids and 3α-OH-5β-H steroids exhibit neuroactive properties and uterine contraction inhibitory effects. AKR1D1 is the sole human enzyme that catalyzes both 3-keto-Δ 4 steroid 5β-reduction and bile acid biosynthesis. A point mutation at position 120 (E120H) in AKR1D1 abolished its 5β-reductase activity while conferring hydroxysteroid dehydrogenase (HSD) activity. This study establishes an efficient multienzyme cascade for the biosynthesis of 3α-OH-5β-H steroids and their C3-glucosylated derivatives from 3β-OH-Δ 5 precursors. This was achieved via rational engineering of AKR1D1 and E120H to alleviate active-site steric hindrance at Y132/W230, which dramatically enhanced catalytic efficiency─showcasing a 7.8-fold (M8) and 20.7-fold (M16) increase in k cat / M m, yielding 94% and 62% in key reductions, respectively. Subsequent glucosylation using a glucosyltransferase (YjiC)/Sucrose synthase (SuSy) system proceeded in 93% yield. Our integrated approach, combining enzyme modification, cofactor recycling, and cascade design, offers a powerful strategy for complex steroid synthesis.