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



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−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.


A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.


ATP regeneration system CTP production Fusion expression Molecular chaperone Polyphosphate kinase 



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)


  1. Andexer JN, Richter M (2015) Emerging enzymes for ATP regeneration in biocatalytic processes. ChemBioChem 16:380–386CrossRefPubMedGoogle Scholar
  2. Caschera F, Noireaux V (2015) A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. Metab Eng 27:29–37CrossRefPubMedGoogle Scholar
  3. Chow CK, Palecek SP (2004) Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells. Biotechnol Progr 20:449–456CrossRefGoogle Scholar
  4. Fujio T, Teshiba S, Maruyama A (1997) Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: enzymatic production of CDP-choline from orotic acid (part II). Biosci Biotechnol Biochem 61:960–964CrossRefPubMedGoogle Scholar
  5. Hara KY, Kondo A (2015) ATP regulation in bioproduction. Microb Cell Fact 14:198CrossRefPubMedPubMedCentralGoogle Scholar
  6. Iwamoto S, Motomura K, Shinoda Y, Urata M, Kato J, Takiguchi N, Ohtake H, Hirota R, Kuroda A (2007) Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate. Appl Environ Microbiol 73:5676–5678CrossRefPubMedPubMedCentralGoogle Scholar
  7. Kimura A, Tatsutomi Y, Mizushima N, Tanaka A, Fukuda H (1978) Immobilization of glycolysis system of yeasts and production of cytidine diphosphate choline. Eur J Appl Microbiol Biot 5:13–16CrossRefGoogle Scholar
  8. Noguchi T, Shiba T (1998) Use of Escherichia coli polyphosphate kinase for oligosaccharide synthesis. Biosci Biotechnol Biochem 62:1594–1596CrossRefPubMedGoogle Scholar
  9. Resnick SM, Zehnder AJ (2000) In vitro ATP regeneration from polyphosphate and AMP by polyphosphate: AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A. Appl Environ Microbiol 66:2045–2051CrossRefPubMedPubMedCentralGoogle Scholar
  10. Sato M, Masuda Y, Kirimura K, Kino K (2007) Thermostable ATP regeneration system using polyphosphate kinase from Thermosynechococcus elongatus BP-1 for D-amino acid dipeptide synthesis. J Biosci Bioeng 103:179–184CrossRefPubMedGoogle Scholar
  11. Simon ES, Bednarski MD, Whitesides GM (1988) Synthesis of CMP-NeuAc from N-acetylglucosamine: generation of CTP from CMP using adenylate kinase. J Am Chem Soc 110:7159CrossRefGoogle Scholar
  12. Simon ES, Grabowski S, Whitesides GM (1990) Convenient syntheses of cytidine 5′-triphosphate, guanosine 5′-triphosphate, and uridine 5′-triphosphate and their use in the preparation of UDP-glucose, UDP-glucuronic acid, and GDP-mannose. J Org Chem 55:1834CrossRefGoogle Scholar
  13. Stevenson BJ, Liu JW, Kuchel PW, Ollis DL (2012) Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme. J Biotechnol 157:113–123CrossRefPubMedGoogle Scholar
  14. Sureka K, Sanyal S, Basu J, Kundu M (2009) Polyphosphate kinase 2: a modulator of nucleoside diphosphate kinase activity in mycobacteria. Mol Microbiol 74:1187–1197CrossRefPubMedGoogle Scholar
  15. Thum C, Schneider CZ, Palma MS, Santos DS, Basso LA (2009) The Rv1712 Locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially phosphorylates dCMP. J Bacteriol 191:2884–2887CrossRefPubMedPubMedCentralGoogle Scholar

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

Personalised recommendations