Novel one-pot ATP regeneration system based on three-enzyme cascade for industrial CTP production
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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.
KeywordsATP 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).
Supplementary Table 1—Strains and Plasmids
Compliance with Ethical Standards
Conflict of interest
All the authors declare no competing financial interest.
- 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
- 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