Improving the production of 22‐hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one in Mycolicibacterium smegmatis

Abstract

Abstract The 22‐hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (4‐HBC) is a C22 steroid synthon of pharmaceutical interest that can be produced as a lateral end‐product of the catabolism of natural sterols (e.g., cholesterol or phytosterols). This work studies the role of an aldehyde dehydrogenase coded by the MSMEG_6563 gene of Mycolicibacterium smegmatis, named msRed, in 4‐HBC production. This gene is located contiguously to the MSMEG_6561 encoding the aldolase msSal which catalyses the retroaldol elimination of acetyl‐CoA of the metabolite intermediate 22‐hydroxy‐3‐oxo‐cholest‐4‐ene‐24‐carboxyl‐CoA to deliver 3‐oxo‐4‐pregnene‐20‐carboxyl aldehyde (3‐OPA). We have demonstrated that msRed reduces 3‐OPA to 4‐HBC. Moreover, the role of msOpccR reductase encoded by MSMEG_1623 was also explored confirming that it also performs the reduction of 3‐OPA into 4‐HBC, but less efficiently than msRed. To obtain a M. smegmatis 4‐HBC producer strain we deleted MSMEG_5903 (hsd4A) gene in strain MS6039‐5941 (ΔkshB1, ΔkstD1) that produces 4‐androstene‐3,17‐dione (AD) from natural sterols (cholesterol or phytosterols). The triple MS6039‐5941‐5903 mutant was able to produce 9 g/L of 4‐HBC from 14 g/L of phytosterols in 2 L bioreactor, showing a productivity of 0.140 g/L h−1. To improve the metabolic flux of sterols towards the production of 4‐HBC we have cloned and overexpressed the msSal and msRed enzymes in the MS6039‐5941‐5903 mutant rendering a production titter of 12.7 g/L with a productivity of 0.185 g/L h−1, and demonstrating that the new recombinant strain has a great potential for its industrial application.