Efficient Microbial Synthesis of Key C21 Steroid 16-Dehydroprogesterone from Diosgenin by Engineered <i>Mycolicibacterium</i> sp. HK-90

C21 steroids represent essential precursors for the industrial synthesis of corticosteroids, yet their conventional manufacturing processes are hampered by suboptimal efficiency and substantial environmental burdens. In this study, we established a biocatalytic route to synthesize the pivotal C21 steroid precursor 16-dehydroprogesterone (16-DPG) from diosgenin, employing an engineered strain, Mycolicibacterium sp. HK-90. Key metabolic engineering strategies included (i) targeted knockout of kstD and kshA genes to disrupt the steroid core degradation pathway, generating the chassis strain mHust-ΔkstD-ΔkshA. This modification prevented diosgenin degradation while enabling its conversion to C19 steroid 4-androstenedione (4-AD) via C21 steroid intermediates. (ii) Identification and characterization of a Baeyer–Villiger monooxygenase (BV-2539) as the key enzyme mediating C21-to-C19 steroid conversion during diosgenin catabolism. Subsequent inactivation of BV-2539 in mHust-ΔkstD-ΔkshA completely abolished 4-AD, thereby redirecting diosgenin catabolism toward the accumulation of 16-DPG. (iii) Discovery and knockout of a transcriptional repressor (IclR-2535) significantly constrained the production yield. The resulting engineered strain, mHust-C21, efficiently synthesized 33.80 g/L 16-DPG (89.7% molar yield) from 50 g/L diosgenin under optimized cultivation conditions. The synthesis pathway achieves a yield far exceeding those of conventional processes while eliminating toxic Cr(VI) oxidants and streamlining production steps. This work offers a novel strategy for the sustainable manufacturing of 16-DPG.