iTRAQ-Based Quantitative Proteomic Analysis of Arthrobacter simplex in Response to Cortisone Acetate and Its Mutants with Improved A1-Dehydrogenation Efficiency

Arthrobacter simplex is extensively used for cortisone acetate (CA) biotransformation in industry, but the A1 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 A1-dehydrogenation mechanism, also providing effective strategies for excellent steroid-transforming strains.

Automated summary

Through comparative proteome analysis, several proteins involved in A1 dehydrogenation were identified, including those related to lipid or amino acid transport and metabolism, energy production and conversion, steroid degradation, and transporter proteins. The impact of six proteins on the reaction was confirmed, with pps, MceGA, yrbE4AA, yrbE4BA, and hyp2 showing positive effects and hyp1 exhibiting a negative effect. Additionally, KsdD5 was identified as the most effective catalytic enzyme. Through gene manipulation and the use of a stronger promoter, an optimum strain with improved enzyme activity, substrate transportation, and cell stress tolerance was created. Biotechnology optimization resulted in a 4.1-fold increase in production peak and productivity compared to the initial level. Our findings enhance understanding of the A1 dehydrogenation mechanism and provide effective strategies for developing excellent steroid-transforming strains.