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, MceGA, yrbE4AA, yrbE4BA, 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.简单节杆菌（Arthrobacter simplex）在工业中被广泛应用于醋酸可的松（cortisone acetate, CA）的生物转化，但其Δ1-脱氢作用的分子机制仍不明晰。本研究通过比较蛋白质组学分析，鉴定出若干在该反应中发挥潜在作用的蛋白质，这些蛋白主要参与脂质或氨基酸的转运与代谢、能量产生与转换、类固醇降解以及转运相关功能。后续进一步验证了六种蛋白质在该反应中的作用：其中pps、MceGA、yrbE4AA、yrbE4BA与hyp2均表现出正向调控作用，而hyp1则呈现负向调控效果；此外，KsdD5是最优的催化酶。通过在强启动子调控下对多个基因进行联合改造，成功构建得到一株优化菌株，该菌株具备更优异的催化酶活性、底物转运能力与细胞应激耐受性。经生物技术优化后，该菌株的产量峰值与生产效率分别达到初始水平的4.1倍与4.0倍。本研究不仅加深了对Δ1-脱氢作用机制的理解，同时也为高性能类固醇转化菌株的构建提供了有效策略。