Biocatalysis has become the main approach to produce cytidine diphosphate choline (CDP-choline)， which has been applied for treatment of acute craniocerebral injury and consciousness after brain surgery. However， salt accumulates with the production and inhibits enzyme activity， and eventually reduces yield and product accumulation rate. Our work provided a possible solution to this problem by applying a computational designed halophilic choline kinase. The halotolerant CKI (choline kinase) was designed following a unique strategy considering the most variable residue positions on the protein surface among target enzymes from different sources. The basic and neutral surface residues were replaced with aci... More
Biocatalysis has become the main approach to produce cytidine diphosphate choline (CDP-choline)， which has been applied for treatment of acute craniocerebral injury and consciousness after brain surgery. However， salt accumulates with the production and inhibits enzyme activity， and eventually reduces yield and product accumulation rate. Our work provided a possible solution to this problem by applying a computational designed halophilic choline kinase. The halotolerant CKI (choline kinase) was designed following a unique strategy considering the most variable residue positions on the protein surface among target enzymes from different sources. The basic and neutral surface residues were replaced with acidic ones. This approach was enlightened by features of natural halophilic enzymes. Mutants in the work represented higher catalytic activities and IC50 (inhibit activity by 50%) at high salt concentrations (over 1200 mM). Furthermore， when the mutant was used in fed-batch production， the CDP-choline accumulation rate doubled comparing with process using wild-type CKI at acetate concentration of over 700 mM. The maximum titer was 151 ± 3.2 mM， the productivity was 5.8 ± 0.1 mM·L h， and molar yield to CMP and utilization efficiency of energy were 85.3 and 63.5%. The idea of computational design in our work can also be applied to modify other enzymes in industry， and sheds light on alleviating effect of salt accumulation during industrial manufacturing process.
