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Biotechnology Letters

, Volume 39, Issue 12, pp 1875–1881 | Cite as

Novel one-pot ATP regeneration system based on three-enzyme cascade for industrial CTP production

  • Junzhi Wang
  • Cheng Zheng
  • Tianyi Zhang
  • Yingmiao Liu
  • Zhuopei Cheng
  • Dong Liu
  • Hanjie Ying
  • Huanqing Niu
Original Research Paper

Abstract

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.

Keywords

ATP regeneration system CTP production Fusion expression Molecular chaperone Polyphosphate kinase 

Notes

Acknowledgements

This work was supported by the National High-Tech Research and Development Program of China (863) (2012AA021203), the National Basic Research Program of China (973) (2013CB733602), the Major Research Plan of the National Natural Science Foundation of China (21390204), the National Technology Support Program (2012BAI44G01), the National Natural Science Foundation of China, General Program (2137611), the Program for Changjiang Scholars and Innovative Research Team in university (IRT_14R28), the young investigator grant program of National Natural Science Foundation of China (21506097) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Supporting Information

Supplementary Table 1—Strains and Plasmids

Compliance with Ethical Standards

Conflict of interest

All the authors declare no competing financial interest.

Supplementary material

10529_2017_2427_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 19 kb)

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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical EngineeringNanjing Tech UniversityNanjingChina
  2. 2.National Engineering Technique Research Center for BiotechnologyNanjingChina
  3. 3.Jiangsu National Synergetic Innovation Center for Advanced MaterialsNanjing Tech UniversityNanjingChina

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