State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China.
National Engineering Technique Research Center for Biotechnology, Nanjing 211816, China.
Biotechnol J. 2018 Jul;13(7):e1700577. doi: 10.1002/biot.201700577. Epub 2018 Mar 12.
Salt accumulation often impedes cytidine diphosphate choline (CDP-choline) in vitro biosynthetic process. In this work a halotolerant in vitro enzymatic system is developed to solve this problem. It applies a halotolerant choline-phosphate cytidylyltransferase (CCT) obtained from rational design instructed by a unique strategy, which refers to one of the features of naturally occurring halophilic enzymes. By increasing acidic residues on protein surface where is most variable with respect to amino acid in the sequence alignment with other CCT, the mutants are obtained. The mutants represent higher catalytic activities and IC50 values (inhibit activity by 50%) at high-salt concentrations. Furthermore, when the halotolerant CCT is applied to in vitro one-pot biosynthesis of CDP-choline, the maximum titer and productivity are 161 ± 3.5 mM and 6.2 ± 0.1 mM L h , respectively. When acetate concentration increases, it still keeps relatively high reaction rate and is 2.2-fold higher than process using wild-type CCT (3.87 mM L h comparing with 1.74 mM L h ). This halotolerant system has great potential for industrial use, and the rational design concept can be applied to modify other enzymes, addressing the salt accumulation problem in in vitro systems, and gives insight into resolving by-product inhibition during reaction.
盐积累通常会阻碍胞苷二磷酸胆碱（CDP-胆碱）的体外生物合成过程。在这项工作中，开发了一种耐盐体外酶系统来解决这个问题。它应用了一种耐盐胆碱磷酸胞苷转移酶（CCT），该酶是通过一种独特的策略（即自然发生的嗜盐酶的特征之一）进行合理设计获得的。通过增加蛋白质表面上的酸性残基，这些残基在与其他 CCT 的序列比对中最易发生变化，从而获得了突变体。突变体在高盐浓度下表现出更高的催化活性和 IC50 值（抑制活性 50%）。此外，当耐盐 CCT 应用于 CDP-胆碱的体外一锅法生物合成时，最大浓度和产率分别为 161±3.5 mM 和 6.2±0.1 mM·L-1·h-1。当乙酸盐浓度增加时，它仍然保持相对较高的反应速率，比使用野生型 CCT 的过程高 2.2 倍（3.87 mM·L-1·h-1 比 1.74 mM·L-1·h-1）。这个耐盐系统具有很大的工业应用潜力，并且合理的设计概念可以应用于修饰其他酶，解决体外系统中的盐积累问题，并深入了解反应过程中的副产物抑制问题。
