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

Abstract

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.