Efficient production of deoxynucleoside-5′-monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system

Abstract

Deoxynucleoside-5′-monophosphates (5′-dNMPs) are the basic components of DNA and are widely used in medicine and as chemical and biochemical reagents. A large amount of effort has been expended to obtain 5′-dNMPs of high quality and at a low cost. However, these procedures are inefficient and inconvenient. In this study, deoxyadenosine-5′-monophosphate (5′-dAMP), 2,6-diaminopurine deoxynucleoside-5′-monophosphate (5′-dDAMP), and deoxycytidine-5′-monophosphate (5′-dCMP) were biosynthesized using recombinant N-deoxyribosyltransferase II (NDT-II), deoxycytidine kinase, and acetate kinase in a one-pot reaction system. The ndt-II gene from Lactobacillus delbrueckii, dck from Bacillus subtilus, and ack from Escherichia coli K12 were overexpressed in E. coli BL21 (DE3). Thymidine was used as the deoxyribose donor; GTP was used as the phosphate donor, and acetyl phosphate was used to regenerate GTP. Under optimized conditions, each 10 mM adenine, 10 mM 2,6-diaminopurine, or 10 mM cytosine were converted into 9.01 mM 5′-dAMP, 8.68 mM 5′-dDAMP, or 6.23 mM 5′-dCMP, respectively. The high yield indicated that this process of biosynthesis of 5′-dAMP, 5′-dDAMP, or 5′-dCMP was efficient and economical, and this one-pot system may also potentially be used for the preparation of other types of 5′-dNMPs.