Enzymatic depolymerization of laboratory waste DNA to a dGMP- containing pool and analytical assessment of dGTP in a crude-lysate guanylate kinase system
cilt.53, sa.1503, ss.2-7, 2026 (Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 53 Sayı: 1503
- Basım Tarihi: 2026
- Doi Numarası: 10.1007/s11033-02612693-y
- Derginin Tarandığı İndeksler: Scopus
- Sayfa Sayıları: ss.2-7
- İstanbul Üniversitesi Adresli: Evet
Özet
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 alterna-
tive 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 sub-
jected 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 find-
ings 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.