Rational design of a novel halotolerant ATP regeneration system for biocatalytic CTP production

BACKGROUND: Acetyl phosphate/acetate kinase system is a cost-saving and efficient ATP regeneration system. However, it suffers from accumulation of salts during catalytic reactions. It inhibits the activity of acetate kinase (ACK), a crucial enzyme of this process, and hence decreases the rate of product accumulation and yield of CTP. RESULTS: A new halotolerant ACK enzyme based ATP regeneration platform was established in this research by applying the concept of rational design to optimize its building blocks. Selection of residues for rational design followed several rules. First, the less conserved residue positions on the protein surface were considered. Second, avoiding significant residue interactions, suchas salt bridges. Third, following the feature of nature occurring halotolerant enzymes, the basic and neutral surface residues were replaced with acidic ones. Our mutants represent high catalytic activities and high IC50 value (inhibit activity by 50%) at high salt concentrations. Also, when the halotolerant ACK was applied to catalyze CTP production, the maximum titer was 213.5 ± 1.6 mM (which is 12.3-fold higher than that of the traditional process), the productivity was 13.3 ± 0.1 mM L⁻¹ h⁻¹, and the molar yield to 5′-cytidylate monophosphate (CMP) and utilization efficiency of energy were 92.4% and 72.7%, respectively. CONCLUSION: This strategy can be applied to modify other enzymes in the industry and gives insight into resolving salt accumulation during biocatalytic processes. In addition, the halotolerant Adenosine Triphosphate (ATP) regeneration system built in this work can be applied to the synthesis of several other high energy compounds.