Enzymatic production of pyrimidine nucleotides using Corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: Enzymatic production of CDP-choline from orotic acid and choline chloride .1.

Enzymatic production of cytidine diphosphate choline (CDP-choline) using erotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel, When Corynebacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mM) from erotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently, Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY13505 for the enzymatic reaction using erotic acid as a substrate, CTP was produced at 8.95 g/liter (15.1 mM) after 23 h of this reaction, To produce CDP-choline, two additional enzyme activities were needed, E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CRI gene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system, After 23 h of the reaction using erotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP), ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.