Synthesis of nucleoside and pentose monophosphates by acid phosphatase over-producing whole cells

LEWKOWICZ, ELIZABETH S; MEDICI, ROSARIO; GARAYCOECHEA, JUAN; VALINO, ANA; CANEPA, GASPAR; BOUVIER, LEÓN ALBERTO; PEREIRA, CLAUDIO; IRIBARREN, ADOLFO

Barcelona

Congreso; 14 European Congress on Biotechnology; 2009

Natural and modified nucleoside-5-monophosphates (NMPs) are valuable compounds widely used in biochemical studies, as active pharmacological components or as flavour enhancers in the food industry. NMPs are usually obtained by microbial fermentation or by isolation from hydrolysates of nucleic acids. Methods for the production of modified NMPs are based either on chemical or on enzymatic phosphorylation of the corresponding nucleosides. Chemical phosphorylation involves polluting multi-step reactions and varied yields depending on the method employed and the initial compound; therefore, enzymatic phosphorylation offers a profitable option. Non-specific acid phosphatases (NSAPs) are a group of secreted enzymes that are able to hydrolyse a broad range of organic phosphoesters and present optimal activity at acidic to neutral pH values. These enzymes are widely distributed among enteric bacteria, and in addition to their intrinsic phosphatase activity, some of them exhibit phosphotransferase activity. NSAPs were applied to the phosphorylation of several compounds using different organic and inorganic phosphate donors. With the aim to prepare NMPs, some biocatalysts with nucleoside phosphotransferase activity belonging to the Enterobacter, Klebsiella, Citrobacter and Serratia genera had been selected from our bacterial strain collection. Natural and modified nucleosides were tested and NMPs were prepared regioselectively using sodium pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of Enterobacter aerogenes and Klebsiella planticola. These new recombinant microorganisms (E. coli BL21/petEapho and E. coli BL21/petKppho) showed higher activity than the corresponding wild type strains. Reductions in the reaction times from 4 hours to 15 min and from 24 hours to 40 min in case of inosine and fludarabine, respectively, were obtained. A biocatalyzed process to synthesize nucleosides employing the combined action of phosphopentomutase and nucleoside phosphorylases was explored by our group, recently. Ribose-5-phosphate (R5P) and 2 -deoxyribose-5-phosphate (DR5P), the starting materials for this strategy, are usually prepared by chemical methods. As biocatalyzed alternative, these compounds were obtained using E. coli BL21/petEapho and BL21/petKppho. Again, an important improvement in reaction times was observed, since 10 min instead of 24 hours was necessary to prepare DR5P with E.coli BL21/petEapho.