A Procedure to Design One-Pot Multi-enzyme System for Industrial CDP-Choline Production.

Fermentation and chemical methods for industrial cytidine diphosphate choline (CDP-choline) catalytic production both suffer from several disadvantages such as relatively low efficiency and productivity. To overcome these problems, we applied the concept of synthetic biology to develop a new one-pot multi-enzyme system to produce CDP-choline from orotic acid. Enzymes from different sources were selected and optimized as building blocks of the system, and parameters such as oxygen supply were also optimized. This system shows a titer of 37.6 ± 1.1 mM and a reaction rate of 1.6 mM L-1 h-1, both increase 66 % from traditional processes. It also has an efficiency of energy of 25.4%, improves 2-folds. This new one-pot CDP-choline-producing system has a potential for industrial use, and the procedure to design one-pot multi-enzyme system can be applied to build other one-pot system producing energy-rich compounds.

Efficient synthesis of cytidine diphosphate choline (CDP-choline) and its analogs.

An efficient P(V)-N activation approach for the synthesis of cytidine diphosphate choline (CDP-choline) and related ribo- and deoxyribonucleotide analogs has been established.

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.

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.

[Preparation, properties and therapeutic potential of cytidine diphosphocholine incorporated into liposomes]. / Preparazione, proprietà e potenzialità terapeutiche della citidin-difosfocolina associata a liposomi.

Cytidine diphosphocholine (CDC) was incorporated into unilamellar egg lecithin liposomes with a yield of 23% and subsequently administered to rats by intracardiac injection. Unlike free CDC, 0.2% of which was absorbed by the brain, 21% of the CDC-liposomes was absorbed by the brain, while their relative distribution among brain phospholipids was similar. The changes in blood concentration and brain content of CDC, with and without liposomes, were measured at different times after administration. The CDC-liposome association significantly increased, unlike CDC alone, the levels of dopamine catabolites in the striatum of rats treated with haloperidol (0.15 mg/Kg/die) after 5 days of intravenous administration (20 mg/Kg/die).

Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates.

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 degrees C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP X G1P and G6P were synthesized from glucose and inorganic phosphate at 70 degrees C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.

Studies on enzyme-substrate interactions of cholinephosphotransferase from rat liver.

In order to elucidate the reaction mechanism and the substrate-binding sites, CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), prepared from rat liver microsomal fraction, has been subjected to kinetic analysis and substrate specificity studies. Kinetic evidence supports the hypothesis of a Bi-Bi sequential mechanism, involving a direct nucleophilic attack of diacylglycerol on CDPcholine during the reaction. To investigate the substrate requirements for recognition and catalysis, several CDPcholine analogs, modified in the nitrogen base or in the sugar or in the pyrophosphate bridge, have been synthesized, characterized and assayed as substrates and/or inhibitors of the reaction. The amino group on the pyrimidine ring, the 2'-alcoholic function of the ribose moiety as well as the pyrophosphate bridge have been identified as critical sites for enzyme-substrates interactions.

[Transport of cytidinediphosphocholine in different biological models. I - Synthesis and characterization of (5 3H, Met 14 C) cytidinediphosphocholine]. / Trasporto della citidindifosfocolina in differenti modelli biologici. I - Sintesi e caratterizzazione della (5 3H, Met 14C) citidindifosfocolina.

In order to investigate the transport of CDP-choline across the biological membranes we have synthesized the double labeled CDP-choline. The (5 3H, methyl 14C) CDP-choline is synthesized by a crossed condensation of (5 3H) CMP and methyl 14C) p-choline using DCC as condensing agent. The product of the reaction is identified by chromatographics and spectroscopics methods.