Biocatalytic Synthesis of dNTPs from Bacterial Biomass

Abstract Deoxyribonucleoside triphosphates (dNTPs) are crucial reagents widely utilized in nucleic acid amplification. However, current synthesis methods are often plagued by environmental pollution for chemical synthesis and the high costs of phosphate donors for enzymatic synthesis. To address these challenges, this study developed a fully enzymatic method that utilizes inexpensive polyphosphate as the phosphate donor to recover deoxyribonucleoside monophosphates (dNMPs) from waste biomass and further synthesize all four dNTPs. Initially, the dNMPs were recovered from discarded Escherichia coli cell paste via genomic DNA extraction and enzymatic hydrolysis. Secondly, two enzymatic catalysis systems were developed including the engineered MrPPK2 for purine substrates achieving 69% conversion rate and a tailored two-enzyme cascade comprising dTMP-K, dCMP-K, and SmcPPK2 for pyrimidine substrates achieving 64% and 60% conversion rates. Finally, the produced dNTPs demonstrated functional performance comparable to that of commercial counterparts in PCR amplification across multiple genomic templates. This study provides an environmentally friendly enzymatic synthesis route for dNTPs with greater promise in cost control.