Computer-directed rational engineering of dioxygenase TcsAB for triclosan biodegradation under cold conditions

The dioxygenase TcsAB is a specific dioxygenase involved in the initial biodegradation of the broad-spectrum antibacterial agent triclosan (TCS). However, it exhibits significantly reduced activity under cold conditions. In this study, a computer-directed approach combining loop engineering and N-terminal truncation was utilized to decrease the thermostability of TcsAB, thereby enhancing its catalytic activity in cold environments. The iterative mutant TcsAB (TcsA^{Y277P/F279P/S311W/A313W}TcsB^{N-terminal truncation}) exhibited a 2.54-fold greater catalytic efficiency than the wild type at 15°C. Molecular dynamics simulations showed that the mutations introduced in the substrate-binding pocket increased its flexibility, leading to enhanced catalytic activity through binding in a more advantageous conformation. This modified dioxygenase was employed as a biological component, and <i>Pseudomonas knackmussii</i> B13 was used as a chassis cell to construct an engineered strain for the efficient degradation of TCS at low temperatures. The objective was to enhance the capacity of TCS bioremediation in natural environments. Insights gained from this study may inform the rational redesign of enzymes related to the robustness of biodegradation of emerging contaminants.IMPORTANCEThe presence of TCS in surface water and wastewater poses a significant risk to aquatic organisms and human health due to its high resistance to degradation. The biodegradation of TCS pollution in the environment through the metabolic processes of microorganisms represents a significant and effective remediation strategy. The dioxygenase TcsAB is the only specific enzyme that has been identified as responsible for the initial biodegradation of TCS. Nevertheless, the enzyme activity responsible for the degradation of TCS was markedly diminished at low temperatures. The actual ambient temperature is frequently lower than the optimum temperature for enzyme reaction, and maintaining the 30°C reaction condition results in high costs and energy consumption for TCS removal. Accordingly, the rational engineering of dioxygenase TcsAB for low-temperature activity will facilitate more efficient and realistic removal of TCS in an aqueous environment.