Knockout of glycerol metabolic pathways enables efficient mycolicibacterial phytosterol conversion using glycerol as cosovlent.

Mycobacterial phytosterol conversion naturally occurs within a vegetable oil environment, a process that has been widely adopted in water‒oil two-phase fermentation to produce active pharmaceutical intermediates of steroids. The use of hydroxypropyl-β-CD (HP-β-CD) as a potential replacement for vegetable oils has been explored. However, both approaches encounter challenges, particularly the need for a high concentration of either vegetable oil or HP-β-CD in the fermentation medium, which significantly affects the efficiency of mycobacterial phytosterol conversion as well as the recovery of the resulting products. In this study, we demonstrated that the knockout of glycerol metabolic pathways enables the utilization of glycerol as cosovlent for efficient phytosterol conversion in Mycolicibacterium neoaurum HGMS6, an industrial strain known for its production of 4-androstene-3,17-dione (4-AD). Our bioinformatic analysis revealed two parallel glycerol metabolic pathways in the HGMS6 strain. These pathways are independently initiated by glycerol kinase (GLPK) and glycerol dehydrogenase (ADHC). To investigate their roles, we conducted gene knockout experiments in which both the glycerol kinase gene (glpk) and the glycerol dehydrogenase gene (adhc) were targeted in HGMS6, resulting in the creation of a glycerol metabolism-deficient mutant, denoted HGMS6^{Δadhc/Δglpk}. This double mutant exhibited complete inactivation of mycobacterial glycerol metabolism without any adverse effects on mycobacterial growth when cultured in conventional fermentation medium containing glucose. We then evaluated HGMS6^{Δadhc/Δglpk} in an aqueous phase fermentation medium and determined that fermentation media consisting of 10% glycerol and 0.5% HP-β-CD provided the optimal conversion efficiency. In pilot-scale fermentation experiments, this mutant was highly efficient at converting phytosterols, achieving a remarkable conversion rate of up to 83.8% (mol/mol%). This bioconversion level was on par with that of HGMS6 and HGMS6^{Δadhc/Δglpk} when operating in vegetable oil- or HP-β-CD-containing fermentation media. This study provides a promising strategy for enhancing the efficiency of mycobacterial phytosterol conversion. KEY POINTS: • TheGLPK and ADHC of M. neoaurum were confirmed and examined. • The enzymology of ADHC and GLPK was characterized in vitro. • An adhc/glpk-knockout mutant efficiently converts phytosterols in fermentation media containing 10% (w/v) glycerol and 0.5%(w/v) HP-β-CD.