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Biotechnology and Bioengineering

Biotechnology and Bioengineering

IF: 3.5
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Engineering substrate and energy metabolism for living cell production of cytidine-5′-diphosphocholine

Published:30 January 2020 DOI: 10.1002/bit.27291 PMID: 31997310
Yanna Ren, Qi Liu, Haifeng Liu, Xiangshan Zhou, Yuanxing Zhang, Menghao Cai

Abstract

Cytidine-5′-diphosphocholine (CDP-choline) is a widely used neuroprotective drug for multiple indications. In industry, CDP-choline is synthesized by a two-step cell culture/permeabilized cell biotransformation method because substrates often do not enter cells in an efficient manner. This study develops a novel one-step living cell fermentation method for CDP-choline production. For this purpose, the feasibility of Pichia pastoris as a chassis was demonstrated by substrate feeding and CDP-choline production. Overexpression of choline phosphate cytidylyltransferase and choline kinase enhanced the choline transformation pathway and improved the biosynthesis of CDP-choline. Furthermore, co-overexpression of ScHnm1, which is a heterologous choline transporter, highly improved the utilization of choline substrates, despite its easy degradation in cells. This strategy increased CDP-choline titer by 55-folds comparing with the wild-type (WT). Overexpression of cytidine-5′-monophosphate (CMP) kinase and CDP kinase in the CMP transformation pathway showed no positive effects. An increase in the ATP production by citrate stimulation or metabolic pathway modification further improved CDP-choline biosynthesis by 120%. Finally, the orthogonal optimization of key substrates and pH was carried out, and the resulting CDP-choline titer (6.0?g/L) at optimum conditions increased 88 times the original titer in the WT. This study provides a new paradigm for CDP-choline bioproduction by living cells.

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