Enzymatic production of deoxyguanosine triphosphate from waste DNA by a recombinant guanylate kinase expressed in Escherichia coli BL21(DE3)

The ability to overcome industrial scalability and environmental sustainability constraints largely depends on the use of recombinant enzymes with high catalytic efficiency that can be produced at low cost. Such enzymes enable the valorization of biological waste or low-value biomass, forming the basis of biocatalytic production processes that enhance resource efficiency and align with circular bioeconomy principles. In this context, deoxyguanosine triphosphate (dGTP), a fundamental building block of DNA synthesis, represents a particularly valuable target molecule due to its essential role in GC-rich DNA synthesis, high demand in molecular biology applications, and the economic and environmental drawbacks associated with its conventional chemical synthesis. In this study, a novel enzymatic process based on a “waste-to-value” approach was developed to convert laboratory-derived genomic and waste DNA into dGTP. DNA was enzymatically fragmented using S1 nuclease and a Benzonase/Exonuclease III combination to generate nucleotide mixtures enriched in dGMP. These intermediates were subsequently phosphorylated using Escherichia coli BL21(DE3) cell lysates overexpressing GUK1. Owing to the endogenous kinase activities present in ZYM5052 autoinduced lysates, dGMP was efficiently converted into dGTP without the requirement for externally added nucleoside diphosphate kinases. The phosphorylation steps were supported by an ATP regeneration system comprising phosphoenolpyruvate and pyruvate kinase. dGTP production was confirmed and quantified by HPLC analysis, revealing a characteristic retention time of 5–7 minutes. A five-point external calibration curve (0.5–2.0 µM) exhibited excellent linearity ( R² = 0.9999). The comparative analysis of dGTP production revealed that using laboratory-derived waste DNA as a substrate for biocatalytic reaction resulted in a highly efficient process, producing yielding 1.416 ± 0.025 µM of dGTP, which significantly surpassed the yield from purified genomic DNA (1.155 ± 0.034 µM; p  = 0.0004). The maximum biocatalytic output was achieved through a nuclease cocktail treatment (Exonuclease III/Benzonase), resulting in a yield of 1.719 ± 0.178 µM, representing a significant increase over standard S1 nuclease digestion ( p  = 0.0057). These findings confirm that utilizing crude biological waste can effectively replace expensive purified substrates for sustainable nucleotide biosynthesis. In conclusion, this study demonstrates that waste DNA can be transformed into high-value nucleotides through an environmentally friendly, efficient, and scalable biocatalytic approach compatible with circular bioeconomy frameworks.