State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, China; National Engineering Technique Research Center for Biotechnology, Nanjing, 211816, China.
National Center for Protein Science Shanghai, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai, 201210, China.
J Biotechnol. 2019 Jan 20;290:59-66. doi: 10.1016/j.jbiotec.2018.11.008. Epub 2018 Nov 13.
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.
生物催化已成为生产胞苷二磷酸胆碱（CDP-胆碱）的主要方法，该方法已应用于急性颅脑损伤和脑外科手术后意识障碍的治疗。然而，随着生产的进行，盐会积累并抑制酶活性，最终降低产量和产物积累率。我们的工作通过应用一种经过计算设计的嗜盐胆碱激酶为解决这个问题提供了一个可能的解决方案。该耐盐 CKI（胆碱激酶）是根据一种独特的策略设计的，该策略考虑了不同来源的靶酶中蛋白质表面最易变的残基位置。碱性和中性表面残基被酸性残基取代。这种方法受到天然嗜盐酶特征的启发。工作中的突变体在高盐浓度（超过 1200mM）下表现出更高的催化活性和 IC50（抑制活性 50%）。此外，当在补料分批生产中使用突变体时，与在乙酸盐浓度超过 700mM 时使用野生型 CKI 的过程相比，CDP-胆碱的积累速率提高了一倍。最大浓度为 151±3.2mM，产率为 5.8±0.1mM·L·h，CMP 的摩尔产率和能量利用率分别为 85.3%和 63.5%。我们工作中的计算设计理念也可应用于工业中修饰其他酶，并为缓解工业生产过程中盐积累的影响提供了思路。
