Discovery and mechanistic characterization of a probiotic‐origin 3β‐ OH ‐Δ ^5‐6 ‐cholesterol‐5β‐reductase directly converting cholesterol to coprostanol

Cholesterol serves as a fundamental molecule in various structural and biochemical pathways; however, high cholesterol levels are linked to cardiovascular diseases. Some selected strains of Lactobacilli are known for modulating cholesterol levels. However, the molecular mechanism underlying cholesterol transformation by lactobacilli has remained elusive. This study describes the discovery and function of a microbial 3β‐OH‐Δ ^5‐6 ‐cholesterol‐5β‐reductase (5βChR) from Limosilactobacillus fermentum NKN51, which directly converts cholesterol to coprostanol, thereby unraveling this longstanding mystery. Protein engineering of the reductase enzyme identified the cholesterol and NADPH interacting amino acid residues, detailing the catalytic mechanism of 5βChR. Phylogenetic analyses highlight the prevalence of 5βChRs in gut commensal lactobacilli, which share a common evolutionary origin with plant 5β reductases. Meta‐analysis of microbiomes from healthy individuals underscores the importance of 5βChR homologs, while a cohort study demonstrates an inverse association between 5βChR abundance and diabetes. The discovery of the 5βChR enzyme and its molecular mechanism in cholesterol metabolism paves the way for a better understanding of the gut‐associated microbiome and the design of practical applications to ameliorate dyslipidemia.