Enzymatic Ways To Synthesize Deoxynucleotides And Their Analogs Coupled With A GTP Regeneration System

Deoxynucleoside-5’-monophosphates (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 dNMPs of high quality and at a low cost. However, these procedures are inefficient and inconvenient. In this study, deoxyadenosine-5’-monophosphate (dAMP),2,6-diaminopurine-deoxynucleoside-5’-monophosphate (dDAMP),2’-Chloro-deoxynucleoside-5’-monophosphate (CldAMP) and deoxycytidine-5’-monophosphate (dCMP) were biosynthesized using recombinant N-deoxyribosyltransferase II (NDT-II), nucleoside phosphorylase (NPase), deoxycytidine kinase (dCKase) and acetate kinase (ACKase) in a one-pot reaction system. The ndl-II gene from Lactobacillus delbrueckii, dck from Bacillus sublilus and deoD, deoA, ack from E. coli K12were 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, each10mM adenine,10mM2,6-diaminopurine2.5mM2’-Chloro-adenine or10mM cytosine was converted into9.01mM dAMP,8.68mM dDAMP1.83CldAMP or6.23mM dCMP, respectively. Moreover, this pathway also shows the ability to transfer adonosine, cytidine, cytarabine and vidarabine into adenosine-monophosphate (AMP), cytidine-monophosphate (CMP), Cytarabine-Monophosphate (Arc-AMP) vidarabine-monophosphate (Ara-AMP). respectively. The high yield indicated that this one-pot system was efficient and economical, and suitable be used in industry.