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 (Part I).
Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Cornybacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mM) from orotic 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 KY12505 for the enzymatic reaction using orotic 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 CKI 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 orotic 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.
Fujio
T
T
Planning & Development Department/Food, Liquor & Food Division, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.
Maruyama
A
A
D019731:Carbon-Nitrogen Ligases; D002794:Choline / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D003566:Cytidine Diphosphate Choline / Q000138:chemical synthesis*; D003570:Cytidine Triphosphate / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D008025:Ligases / Q000096:biosynthesis; D009963:Orotic Acid / Q000737:chemistry / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011742:Pyrimidine Nucleotides / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014530:Uridine Diphosphate / Q000096:biosynthesis; D014542:Uridine Monophosphate / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014544:Uridine Triphosphate / Q000096:biosynthesis
D011742:Pyrimidine Nucleotides; D003566:Cytidine Diphosphate Choline; D014530:Uridine Diphosphate; D009963:Orotic Acid; D003570:Cytidine Triphosphate; D010754:Phosphoribosyl Pyrophosphate; D014542:Uridine Monophosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D002794:Choline; D014544:Uridine Triphosphate