High-level living cell production of cytidine-5'-diphosphocholine in metabolically engineered yeast

Abstract

Industrial production of neuroprotective drug CDP-choline is accomplished via permeabilized or lysed cell biotransformation because of the inefficient penetration of substrates into intact cells. We previously proposed a novel one-step living cell method for CDP-choline production by engineered yeast, but obtained low titer and molar yield. This study develops a high-production strain with improved molar yield by metabolic engineering strategies. The selective markers previously integrated into host cell were recovered for facilitating genetic modification, which however resulted a strain with improved CDP-choline titer and molar yield to CMP. Knockout of 5'-NT or CDA in CMP sinking pathway but not APY in CTP sinking pathway further improved CDP-choline titer and molar yield to CMP. However, overexpression of seven enzymes in CTP synthetic pathway showed no positive functions. Finally, optimization of CMP and choline phosphate levels for the optimized recombinant strains achieved a high-level CDP-choline of ~30 g/L, which was enhanced by 400% compared to the previous work. Also, the molar yield of CDP-choline to CMP increased from 40% to 84.7%. The titer and molar yield are comparable to the reported permeabilized or lysed cell based biotransformation methods. It represents a novel and competitive paradigm for the potential industrial production of CDP-choline.

Keywords: Cytidine-5′-diphosphocholine; Living cell production; Metabolic engineering; Pichia pastoris.