Production of cytidine 5'-diphosphorylcholine with high utilization of ATP by whole cells of Saccharomyces cerevisiae.

Cytidine 5'-diphosphorylcholine (CDP-choline) was produced using a high efficiency ATP regeneration system and the Kennedy pathway in whole cells of Saccharomyces cerevisiae As 2.398. Out of eight variables, KH(2)PO(4), glycerol and (NH(4))(2)SO(4) were considered to be the most significant factors by response surface methodology including a Plackett-Burman design, path of steepest accent and central composite design. The optimum levels of the three variables were 20.13g/L KH(2)PO(4), 12.35g/L glycerol and 0.49g/L (NH(4))(2)SO(4), respectively. Energy utilization efficiency increased from 10.59% to 16.72% and choline chloride conversion yields increased from 12.35% to 42.78%. A high efficiency ATP regeneration system improves CDP-choline production.

Effect of NH4+ and glycerol on cytidine 5'-diphosphocholine synthesis in Saccharomyces cerevisiae.

Both stimulation of ammonium ion on the glycolytic flux and regulation by glycerol of enzymes in Kennedy pathway for cytidine diphosphate choline production in S. cerevisiae were studied. The conventional transformation course featured four stages. Firstly, CMP and choline chloride were phosphorylated and CDP-choline was formed rapidly; secondly, the rate of CDP-choline formation declined and CMP was not detected in the mixture; thirdly, CMP was released and the CDP-choline concentration reached a peak; Fourthly, the compound concentrations did not practically changes eventually. Using the central composite design, the concentration, yield, and utilization efficiency of energy reached 24.7 mmol/L, 82.3% and 10.6%, with 30 mmol/L of ammonium ion and 1% (V/V) of glycerol, respectively. Ammonium ion not only strengthened the glycolytic pathway, but also coordinated the reaction rate between the glycolytic pathway and the Kennedy pathway. Glycerol alleviated the activity decrease of the key enzymes in the mixture.