Multigram-scale stereoselective synthesis of neurosteroid isomers by gut microbial isolates using plant...

We present a sustainable microbial platform utilizing gut bacteria and a plant-based medium for stereoselective neurosteroid biosynthesis. Through bioinformatics- and structural biology-guided screening of over 3000 bacterial isolates, we identified three gut strains exhibiting distinct stereospecificities: Holdemania filiformis produces isopregnanolone, Clostridium innocuum generates epipregnanolone, and Hungatella effluvii synthesizes pregnanolone. Following heterologous expression in Escherichia coli, we characterized two H. filiformis proteins: steroid 5α-reductase (Hp5αR) and 3β-hydroxysteroid dehydrogenase/reductase (Hf4205). We developed molasses-okara medium (MOM), a plant-derived composite medium combining sugarcane molasses with enzymatically hydrolyzed okara devoid of animal-derived components. In multigram batch whole-cell biotransformation trials using MOM, we achieved over 95% progesterone conversion into target neurosteroid isomers. The inherent stereoselectivity of these whole-cell biotransformations bypasses downstream chiral chromatographic separation, enabling pharmaceutical-grade product recovery through a simple open-column purification. Compared with peptone-yeast extract-glucose media, MOM reduced production costs by 90% and carbon footprint by 95% in whole-cell biotransformation, demonstrating sustainable bioeconomy principles in pharmaceutical biotechnology.