Biomaterial Database

Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: enzymatic production of CDP-choline from orotic acid (Part II). 1997

T Fujio, and S Teshiba, and A Maruyama

Planning & Development Department/Food, Liquor & Food Division, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.

A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. coli), cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32 degrees C, 21.5 mM (11 g/liter) of CDP-choline was accumulated.

UI	MeSH Term	Description	Entries
D008025	Ligases	A class of enzymes that catalyze the formation of a bond between two substrate molecules, coupled with the hydrolysis of a pyrophosphate bond in ATP or a similar energy donor. (Dorland, 28th ed) EC 6.	Ligase,Synthetases,Synthetase
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009963	Orotic Acid	An intermediate product in PYRIMIDINE synthesis which plays a role in chemical conversions between DIHYDROFOLATE and TETRAHYDROFOLATE.	Potassium Orotate,Sodium Orotate,Zinc Orotate,Acid, Orotic,Orotate, Potassium,Orotate, Sodium,Orotate, Zinc
D010754	Phosphoribosyl Pyrophosphate	The key substance in the biosynthesis of histidine, tryptophan, and purine and pyrimidine nucleotides.	Pyrophosphate, Phosphoribosyl
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D002797	Choline Kinase	An enzyme that is active in the first step of choline phosphoglyceride (lecithin) biosynthesis by catalyzing the phosphorylation of choline to phosphorylcholine in the presence of ATP. Ethanolamine and its methyl and ethyl derivatives can also act as acceptors. EC 2.7.1.32.	ATP Choline Transphosphatase,Choline Phosphokinase,Choline Transphosphatase, ATP,Kinase, Choline,Phosphokinase, Choline,Transphosphatase, ATP Choline
D002798	Diacylglycerol Cholinephosphotransferase	An enzyme that catalyzes the synthesis of phosphatidylcholines from CDPcholine and 1,2-diacylglycerols. EC 2.7.8.2.	Cholinephosphotransferase,Phosphorylcholine-Glyceride Transferase,1-alkyl-2-Acetylglycerol Cholinephosphotransferase,CDP-Choline 1,2-Diglyceride Choline Phosphotransferase,CDP-Choline Cholinephosphotransferase,CDP-Diacylglycerol Synthase,Diacylglycerol-CDP Choline Phosphotransferase,PAF Phosphocholinetransferase,Phosphocholinetransferase,Phosphorylcholineglyceride Transferase,CDP Choline 1,2 Diglyceride Choline Phosphotransferase,CDP Choline Cholinephosphotransferase,CDP Diacylglycerol Synthase,Choline Phosphotransferase, Diacylglycerol-CDP,Cholinephosphotransferase, 1-alkyl-2-Acetylglycerol,Cholinephosphotransferase, CDP-Choline,Cholinephosphotransferase, Diacylglycerol,Diacylglycerol CDP Choline Phosphotransferase,Phosphocholinetransferase, PAF,Phosphorylcholine Glyceride Transferase,Phosphotransferase, Diacylglycerol-CDP Choline,Synthase, CDP-Diacylglycerol,Transferase, Phosphorylcholine-Glyceride,Transferase, Phosphorylcholineglyceride
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D003352	Corynebacterium	A genus of asporogenous bacteria that is widely distributed in nature. Its organisms appear as straight to slightly curved rods and are known to be human and animal parasites and pathogens.
D003566	Cytidine Diphosphate Choline	Donor of choline in biosynthesis of choline-containing phosphoglycerides.	CDP Choline,Citicoline,Cidifos,Citicholine,Cyticholine,Cytidine 5'-Diphosphocholine,5'-Diphosphocholine, Cytidine,Choline, CDP,Choline, Cytidine Diphosphate,Cytidine 5' Diphosphocholine,Diphosphate Choline, Cytidine