Boosting In Vitro Electro-Phosphorylation for Powering Glycosylation of an Antitumor Macrolide

Abstract

Adenosine triphosphate (ATP) serves as the universal energy currency, and its regeneration is crucial for in vitro biotransformation (ivBT). However, translating natural ATP regeneration methods, especially ATP synthase-involved phosphorylation, to sustain energy-intensive ivBT cascades remains challenging. Electrocatalysis provides a promising strategy to generate proton motive force to drive ATP synthase-catalyzed ATP regeneration, denoted as electro-phosphorylation (EP). Here, we reported a boosted EP system enabling the glycosylation of epothilone B (EpoB). Through the knockout of the endogenous atp gene of E. coli and the transformation of an ATP synthase-overexpression plasmid, the distribution density of ATP synthase molecules increased 2.4-fold compared to that of E. coli wild type (WT). By reshaping the ATP synthase-rich membrane into a planar pattern on the proton exchange membrane and integrating it into an electrolytic cell, the optimized EP system accumulated 40.44 ± 5.49 μM ATP, enabling an in vitro multienzymatic cascade to synthesize antitumor macrolides, EpoB glycosides. With maltodextrin as the glycosyl donor, 56.14 ± 3.13 μM EpoB was successfully glycosylated. The present work demonstrated not only a robust ATP regeneration capability but also its practicability in ATP-intensive biosynthesis, such as macrolide glycosylation. The modular platform holds great promise for on-demand energy supply in ivBT and synthetic biology.