Identification of Enoyl-CoA Hydratase EchA19 in Mycolicibacterium neoaurum Involved in the First β-Oxidation Pathway of Phytosterols for Key Steroidal Intermediate Synthesis.

Microbial transformation has enabled phytosterols as readily available and bio-renewable starting materials for the industrial synthesis of steroidal active pharmaceutical ingredients (APIs). Editing the phytosterol side chain would create various steroidal compounds with a specific C17-side chain, which will greatly facilitate the synthesis of steroidal APIs. Precise cleavage of the phytosterol side chain requires identification of the key enzymes and the reaction pathways of phytosterol side chain metabolism. In this study, a hydratase EchA19 was identified in Mycolicibacterium neoaurum NRRL B-3805, a strain which was engineered by traditional mutation and screening or genetic manipulation, generating recombinant strains for the industrial-scale production of androstenedione (AD), androstadienedione (ADD), and 9α-hydroxy-androstenedione (9α-OH-AD) from phytosterols. It was found that EchA19 is the key hydratase affecting the first β-oxidation pathway of phytosterol side chain metabolism. The previously proposed carboxylation at the C28 position might occur after the cleavage of the C24 branched alkyl side chain, rather than after the dehydrogenation reaction. This study has provided us with new insights and a deeper understanding of the metabolic pathways of phytosterol side chain, and laid a foundation for synthesizing valuable steroid drug intermediates from phytosterols through metabolic regulation by precisely editing the side chain.