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Published on: April 22, 2016
Novel one-pot ATP regeneration system based on three-enzyme cascade for industrial CTP production
Junzhi Wang1,2,3, Cheng Zheng1,2, Tianyi Zhang1,2
1State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, China.
Objectives:
To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production.
Results:
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-1 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.
Conclusions:
A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.
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