iTRAQ-Based Quantitative Proteomic Analysis of Arthrobacter simplex in Response to Cortisone Acetate and Its Mutants with Improved Δ¹‑Dehydrogenation Efficiency

Arthrobacter simplex is extensively used for cortisone acetate (CA) biotransformation in industry, but the Δ¹-dehydrogenation molecular fundamental remains unclear. Herein, the comparative proteome revealed several proteins with the potential role in this reaction, which were mainly involved in lipid or amino acid transport and metabolism, energy production and conversion, steroid degradation, and transporter. The influences of six proteins were further confirmed, where pps, MceG_A, yrbE4A_A, yrbE4B_A, and hyp2 showed positive impacts, while hyp1 exhibited a negative effect. Additionally, KsdD5 behaved as the best catalytic enzyme. By the combined manipulation in multiple genes under the control of a stronger promoter, an optimal strain with better catalytic enzyme activity, substrate transportation, and cell stress tolerance was created. After biotechnology optimization, the production peak and productivity were, respectively, boosted by 4.1- and 4.0-fold relative to the initial level. Our work broadens the understanding of the Δ¹-dehydrogenation mechanism, also providing effective strategies for excellent steroid-transforming strains.