Identification of the aldolase responsible for the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from natural sterols in Mycolicibacterium smegmatis

Mycobacterial mutants blocked in ring degradation constructed to achieve C19 synthons production, also accumulate by-products such as C22 intermediates throughout an alternative pathway reducing the production yields and complicating the downstream purification processing of final products. In this work, we have identified the MSMEG_6561 gene, encoding the only aldolase on the chromosome responsible for the transformation of 22-hydroxy-3-oxo-cholest-4-ene-24-carboxyl-CoA (22-OH-BCN-CoA) into the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) precursor (20S)-3-oxopregn-4-ene-20-carboxaldehyde (3-OPA). The deletion of this gene increases the production yield of the C-19 steroidal synthon 4-androstene-3,17-dione (AD) from natural sterols, avoiding the production of 4-HBC as by-product and the drawbacks in the AD purification. The molar yield of AD production using the MS6039-5941-6561 triple mutant strain was checked in flasks and bioreactor improving very significantly compared with the previously described MS6039-5941 strain.