Wang Junzhi, Zheng Cheng, Zhang Tianyi, Liu Yingmiao, Cheng Zhuopei, Liu Dong, Ying Hanjie, Niu Huanqing
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.
Biotechnol Lett. 2017 Dec;39(12):1875-1881. doi: 10.1007/s10529-017-2427-x. Epub 2017 Aug 31.
To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production.
We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24 h, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM l respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system.
A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.
开发一种低成本、高效率的新型一锅法多聚磷酸激酶（PPK）系统，用于工业CTP生产中的ATP再生。
我们通过应用三酶级联反应（CMK、NDK和PPK）与基于体外多聚P的ATP再生系统，开发了一种新型一锅法PPK系统。从二十种来源中筛选出PPK，并通过与可溶性蛋白融合表达、构建多顺反子质粒或与分子伴侣GroES/EL共表达使其可溶。通过融合表达、tac-pBAD系统、Rosetta宿主和密码子优化对其他酶的活性进行了优化。24小时后，CDP和CTP的浓度分别达到3.8±0.2和6.9±0.3 mM l，产率约为79%。CTP的摩尔转化率为51%，其产率和转化率比传统系统提高了100%。
开发了一种应用多聚磷酸激酶进行CTP生产的新型一锅法ATP再生系统。
