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

LEWKOWICS, E; MEDICI, ROSARIO; GARAYCOCHEA, J; VALINO, A; CANEPA, GASPAR; BOUVIER, LEON; PEREIRA, CA; IRIBARREN, ADOLFO

Barcelona, Spain

Congreso; 14th European Congress on Biotechnology; 2009

Natural and modified nucleoside-5-monophosphates (NMPs) arevaluable compounds widely used in biochemical studies, as activepharmacological components or as flavour enhancers in the foodindustry. NMPs are usually obtained by microbial fermentationor by isolation from hydrolysates of nucleic acids. Methods forthe production of modified NMPs are based either on chemical oron enzymatic phosphorylation of the corresponding nucleosides.Chemical phosphorylation involves polluting multi-step reactionsand varied yields depending on the method employed and theinitial compound; therefore, enzymatic phosphorylation offers aprofitable option.Non-specific acid phosphatases (NSAPs) are a group of secretedenzymes that are able to hydrolyse a broad range of organic phosphoestersand present optimal activity at acidic to neutral pHvalues. These enzymes are widely distributed among enteric bacteria,and in addition to their intrinsic phosphatase activity, some ofthem exhibit phosphotransferase activity. NSAPs were applied tothe phosphorylation of several compounds using different organicand inorganic phosphate donors.With the aim to prepare NMPs, some biocatalysts with nucleosidephosphotransferase activity belonging to the Enterobacter,Klebsiella, Citrobacter and Serratia genera had been selected fromour bacterial strain collection. Natural and modified nucleosideswere tested and NMPs were prepared regioselectively using sodiumpyrophosphate as phosphate donor.To increase the productivity of the process, we developed twogenetically modified strains of Escherichia coli which overexpressedNSAPs of Enterobacter aerogenes and Klebsiella planticola. These newrecombinant microorganisms (E. coli BL21/petEapho and E. coliBL21/petKppho) showed higher activity than the correspondingwild type strains. Reductions in the reaction times from 4 hoursto 15 min and from 24 hours to 40 min in case of inosine andfludarabine, respectively, were obtained.A biocatalyzed process to synthesize nucleosides employingthe combined action of phosphopentomutase and nucleosidephosphorylases was explored by our group, recently. Ribose-5-phosphate (R5P) and 2-deoxyribose-5-phosphate (DR5P), thestarting materials for this strategy, are usually prepared by chemicalmethods. As biocatalyzed alternative, these compounds wereobtained using E. coli BL21/petEapho and BL21/petKppho. Again,an important improvement in reaction times was observed, since10 min instead of 24 hours was necessary to prepare DR5P with E.coli BL21/petEapho.