New biocatalysts for one pot multistep enzymatic synthesis of pyrimidine nucleoside diphosphates from readily available reagents

•Corynebacterium ammoniagenes ATCC 19350 was used as biocatalyst for UMP synthesis.•NDP including ribo, deoxyribo and arabinonucleosides were obtained by S. cerevisiae.•UDP was prepared by one-pot biotransformation from uracil, uridine or orotic acid.•TDP was obtained by two consecutive one-pot biotransformations starting from glucose.

Since the preparation of nucleoside 5′-diphosphates by classical methodologies is complex, multistep enzymatic systems were explored to synthesize pyrimidine nucleoside 5′-diphosphates starting from readily available reagents. Different strategies were combined to prepare uridine- and thymidine 5′-diphosphates as ribo- and deoxyribonucleoside models, respectively. For uridine 5′-diphosphate synthesis, conversions between 38 and 66% were achieved, using a simple methodology that involves commercial yeast extract as biocatalyst and biocatalytically in situ prepared uridine 5′-monophosphate. Corynebacterium ammoniagenes ATCC 19350 was used for the first time as biocatalyst to synthesize uridine 5′-monophosphate from uracil and orotic acid while Raoultella planticola was the selected biocatalyst for uridine 5′-monophosphate synthesis from uridine. The overall performances of all the tested approaches were similar but the use of uracil leads to a more suitable and cheaper process. Alternatively, for thymidine 5′-diphosphate synthesis two consecutive one pot multistep enzyme systems were assayed. In the first biotransformation, 2′-deoxyribose 5-phosphate was formed from glucose by Erwinia carotovora whole cells followed by the action of phosphopentomutase and thymidine phosphorylase affording thymidine in 85% conversion relative to 2′-deoxyribose 5-phosphate. Finally, in the second one pot reaction, the nucleoside was converted to thymidine 5′-diphosphate by the combined action of Escherichia coli BL21 pET22b-phoRp and Saccharomyces cerevisiae.

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