Enzymatic depolymerization of laboratory waste DNA to a dGMP-containing pool and analytical assessment of dGTP in a crude-lysate guanylate kinase system.

BACKGROUND: Deoxynucleoside triphosphates (dNTPs) are essential reagents for molecular biology and synthetic biology, yet their conventional chemical synthesis is costly and environmentally burdensome. Enzymatic routes offer a cleaner alternative but typically start from defined, purified precursors. Recovering nucleotides from laboratory waste DNA would align dNTP supply with circular-biotechnology principles, but this route remains largely unexplored, particularly for guanine nucleotides. This proof-of-concept study tests whether waste DNA can be routed toward deoxyguanosine triphosphate (dGTP) using a simplified crude-lysate enzymatic cascade. METHODS AND RESULTS: Waste DNA was hydrolysed with S1 nuclease into a deoxynucleotide monophosphate pool and subjected to a one-pot phosphorylation cascade combining recombinant Saccharomyces cerevisiae guanylate kinase (GUK1), expressed in Escherichia coli BL21(DE3), with endogenous nucleoside diphosphate kinase activity and a pyruvate kinase/phosphoenolpyruvate ATP-regeneration system. Recombinant GUK1 expression was confirmed by SDS-PAGE. Ion-pair HPLC resolving the monophosphate and triphosphate species showed that S1 hydrolysis reproducibly generated a dGMP-containing nucleotide pool, with the dGMP signal significantly higher in the reaction than in a DNA-free control (~ 1.4-fold; p = 0.023), confirming DNA-dependent depolymerization of the waste-DNA feedstock. High-resolution Q-TOF LC-MS/MS confidently identified dGTP in the reaction through accurate mass ([M - H]⁻ m/z 505.98, C10H16N5O13P3), isotope pattern, acetate adduct, and diagnostic product ions (m/z 158.93, 408.01, 78.96), corresponding to Schymanski confidence level 2. dGTP was present at trace level and was not distinguishable from a DNA-free control by either full-scan LC-MS or ion-pair HPLC, indicating a substantial background of endogenous guanine nucleotides in the crude lysate. An empty-vector cascade control resolved the attribution at the diphosphate level: recombinant GUK1 produced ≈ 2.3-fold more dGDP-the direct product of guanylate kinase-than the empty-vector lysate (n = 3; Welch's t-test, p = 0.007), indicating a specific contribution of the recombinant enzyme above the endogenous background. CONCLUSIONS: A crude-lysate GUK1 cascade acting on waste-DNA-derived substrate yields identifiable dGTP. The product was confirmed structurally but its DNA-dependent formation could not be demonstrated and it was not quantified; the findings therefore establish depolymerization of waste DNA to dGMP and the analytical identity of dGTP as a basis for further development, rather than a quantified conversion process.