Enhancing phytosterol tolerance and Repeated-Batch androstenedione production in Mycolicibacterium by modulating...

Global protein lysine acylation, driven by intracellular acyl-coenzyme A (acyl-CoA) accumulation during phytosterol catabolism, has emerged as a potential regulatory mechanism in steroid biotransformation, yet its role in androstenedione (AD) production by Mycolicibacterium remains unexplored. Here, we identified and functionally characterized two antagonistic enzymes in Mycobacterium sp. LZ2 (Msp): MpKat, a GNAT-family acyltransferase catalyzing lysine succinylation, and MpSir, an NAD⁺-dependent Sirtuin-family deacylase. Genome-wide prediction indicated that 21.56% of lysine residues in the Msp proteome are potential acylation sites, underscoring the broad regulatory impact of this modification. Targeted genetic manipulation revealed that MpSir overexpression increased AD yield by 13.28% (to 83.24%), and MpKat knockout improved yield by 8.86%, while MpKat overexpression decreased yield by 8.69%. Reducing global acylation levels alleviated oxidative stress, elevated NAD⁺/NADH ratios, enhanced phytosterol tolerance, and improved cell viability. In repeated-batch fermentation, the MpSir-overexpressing strain achieved an average AD yield of 76.5% with a 51% reduction in fermentation time compared to the wild type. This work demonstrates for the first time that modulation of protein acylation via the MpKat/MpSir regulatory axis is a viable and effective strategy to enhance steroid bioconversion in mycobacteria, offering a new dimension for metabolic engineering beyond conventional pathway optimization.