Abstract
The current biocatalytic method of industrial Cytidine triphosphate (CTP) production suffers from reaction rate loss. It is caused by gradually increasing acetate salt concentration, which inhibits enzyme activities and decreases the final yield. This work gave a possible solution to this problem through computational aided design of CMP kinase (CMPK), an enzyme in the CTP production system, to increase its stability in solution with high acetate salt concentration. Enlightened by the features of natural halophilic enzymes, the basic and neutral surface residues were replaced with acidic amino acids. This protein design strategy effectively increased the activity of CMPK in the working condition (acetate concentration over 1200 mM). The halotolerant CMPK was applied in fed-batch production of CTP. The maximum titer was 201.4 ± 1.6 mM, and the productivity was 12.6 mM L−1 h−1, increased 26.4% and 27.8% from the process using wild-type CMPK, respectively.
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The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
References
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 55:1834–1841. https://doi.org/10.1021/jo00293a030
Lee SG, Kim BG (2006) Recombinant escheichia coli-catalyzed production of cytidine 5′-triphosphate from cytidine 5′-monophosphate. J Ind Eng Chem 12:757–761
Adibhatla RM, Hatcher JF, Dempsey RJ (2002) Citicoline: neuroprotective mechanisms in cerebral ischemia. J Neurochem 80:12–23. https://doi.org/10.1046/j.0022-3042.2001.00697.x
Warden AC, Williams M, Peat TS, Seabrook SA, Newman J, Dojchinov G, Haritos VS (2015) Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst. Nat Commun 6:1–10. https://doi.org/10.1038/ncomms10278
Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA (2004) PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res 32:665–667. https://doi.org/10.1093/nar/gkh381
Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, Onufriev A, Simmerling C, Wang B, Woods RJ (2005) The Amber biomolecular simulation programs. J Comput Chem 26:1668–1688. https://doi.org/10.1002/jcc.20290
Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, Ben-Tal N (2016) An improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res 44(2016):W344–W350. https://doi.org/10.1093/NAR/GKW408
Madern D, Zaccai G (2004) Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein. Biochimie 86:295–303. https://doi.org/10.1016/j.biochi.2004.04.004
Raghunathan G, Sokalingam S, Soundrarajan N, Madan B, Munussami G, Lee SG (2013) Modulation of protein stability and aggregation properties by surface charge engineering. Mol Biosyst 9:2379–2389. https://doi.org/10.1039/c3mb70068b
Gribenko AV, Patel MM, Liu J, McCallum SA, Wang C, Makhatadze GI (2009) Rational stabilization of enzymes by computational redesign of surface charge-charge interactions. Proc Natl Acad Sci USA 106:2601–2606. https://doi.org/10.1073/pnas.0808220106
Zheng C, Li Z, Yang H, Zhang T, Niu H, Liu D, Wang J, Ying H (2019) Computation-aided rational design of a halophilic choline kinase for cytidine diphosphate choline production in high-salt condition. J Biotechnol 290:59–66. https://doi.org/10.1016/j.jbiotec.2018.11.008
Pasricha S (2020) Research article research article. Arch Anesthesiol Crit Care 4:527–534
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).
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Wen, Q., Zhang, J., Miao, R. et al. Computational aided design of a halotolerant CMP kinase for enzymatic synthesis of cytidine triphosphate. Bioprocess Biosyst Eng 46, 499–505 (2023). https://doi.org/10.1007/s00449-022-02827-4
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DOI: https://doi.org/10.1007/s00449-022-02827-4