Multigram-scale stereoselective synthesis of neurosteroid isomers by gut microbial isolates using plant biomass-derived medium

Neurosteroids are vital therapeutics for mood disorders, with FDA-approved allopregnanolone (Zulresso) for postpartum depression and zuranolone for major depressive disorder representing breakthrough treatments. However, current production methods rely on costly animal-derived sources or non-stereoselective chemical synthesis that require extensive chiral purification steps. Here, we present a sustainable microbial platform utilizing gut bacteria and a completely plant-based medium for stereoselective neurosteroid biosynthesis. Through bioinformatics- and structural biology-guided screening of more than 3000 bacterial isolates, we identified three anaerobic gut strains exhibiting distinct stereospecificities: Holdemania filiformis produces isopregnanolone (3{beta}-hydroxy-5-pregnan-20-one), Clostridium innocuum generates epipregnanolone (3{beta}-hydroxy-5{beta}-pregnan-20-one), and Hungatella effluvii synthesizes pregnanolone (3-hydroxy-5{beta}-pregnan-20-one). We developed Molasses-Okara Medium (MOM), a fully 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 >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 to using peptone-yeast-glucose media for whole-cell biotransformation, MOM reduced production costs by 90% and carbon footprint by 95% that embodies sustainable bioeconomy principles in pharmaceutical biotechnology. Technology Readiness BoxWe argue that this gut microbiota-derived neurosteroid bioproduction technology has reached a Technology Readiness Level (TRL) of 4, having been validated in laboratory environments with the demonstrated multigram-scale synthesis of high-purity neurosteroids. The platform integrates stereoselective bacterial isolates (Holdemania filiformis, Clostridium innocuum, and Hungatella effluvii) with a sustainable plant-based fermentation medium (molasses-okara medium), achieving >90% progesterone conversion efficiency, >99.9% stereochemical purity, and the successful production of 0.7-0.9 g of neurosteroids per gram of progesterone across multiple 1 L fed-batch fermentations. Compared with conventional chemical synthesis approaches that require expensive chiral catalysts and multi-step purification, this microbial platform offers inherent stereoselectivity while eliminating animal-derived media components. Despite these advantages, several challenges remain for industrial implementation, including scale-up validation beyond laboratory conditions, optimization of anaerobic bioprocess control at pilot scale, and ensuring consistent performance under variable industrial feedstock conditions. Addressing these issues will require pilot-scale demonstration (10-50 L bioreactors), process robustness validation, and supply chain development for plant-based feedstocks. Regulatory pathway development will also be essential for pharmaceutical applications, particularly establishing precedents for gut microbiota-derived therapeutic compounds under existing cGMP frameworks HighlightsO_LIIdentification of gut bacteria for stereoselective synthesis of neurosteroid isomers (isopregnanolone, epipregnanolone, pregnanolone) with >99% chiral purity C_LIO_LISustainable plant biomass-based medium replacing animal-derived components for whole-cell progesterone biotransformation C_LIO_LIMulti-gram scale production of progestogenic neurosteroids and one-step-open-column purification bypassing chiral chromatographic separation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/671209v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@11147b0org.highwire.dtl.DTLVardef@1799d64org.highwire.dtl.DTLVardef@14c33deorg.highwire.dtl.DTLVardef@1e14e21_HPS_FORMAT_FIGEXP M_FIG C_FIG