Aldolase SalA dominates C24 steroidal side-chain-cleavage in the phytosterol degradation from Mycobacterium neoaurum

Understanding the mycobacteria degradation of sterol may facilitate strategies for the directed biotransformation of steroidal drug intermediates. Due to the long and complicated catabolic process, several key steps that involved in the C-C bond cleavage of steroidal side-chain remain unknown. In the present study, an aldolase SalA from Mycobacterium neoaurum was identified that was related to the synthesis of 3-oxo-4-pregnene-20-carboxyl aldehyde and 20-hydroxymethyl pregn-4-ene-3-one (4-HBC). As a member of the thiolase superfamily, the aldolase SalA-SalB is composed of a thiolase-like protein (SalA) and a DUF35 domain (SalB). As a result, the gene salA was determined that had special significance for the synthesis of 4-HBC in sterol metabolism by performing knockout and replenishment. Based on insights provided by the docking model and mutants analysis, the hypothesis of the catalytic mechanism was proposed. Furthermore, the deletion of salA absolutely eliminated the by-product 4-HBC and meanwhile the molar yield of ADD was increased by 10 %. The dominant role of SalA in the C24 steroidal side-chain cleavage as confirmed in this study provided a further clarification on the phytosterol degrading pathway in Mycobacterium neoaurum, which is of significant importance for the directed biotransformation between C19- and C22-steroids.