A High-Efficiency Geranylation Platform Enabled by Rational Tuning of a Single-Residue-Coded Chemo- and Regioselective Switch in Aromatic Prenyltransferases

Geranylation is an efficient post-modification strategy for enhancing or introducing new properties to molecules; however, achieving both high efficiency and precision remains challenging. Aromatic prenyltransferases (PTs) provide a mild and sustainable approach, yet their practical application is often limited by the trade-off between broad substrate scope and low selectivity, and successfully balancing these properties is rare. Here, using the pharmaceutically relevant cannabigerol framework as a model, we identify a single-residue switch in a prenyltransferase from Aspergillus terreus (AtaPT) that governs both chemo- and regioselectivity. Rational tuning of this switch preserves broad substrate scope while enhancing selectivity, enabling the selective formation of each of the three possible mono-geranylated products in the model reaction. Structural analysis reveals that this residue modulates the selectivity and activity through coupled steric and electrostatic effects. Notably, transplantation of this site into three homologous PTs similarly reprogrammed selectivity, establishing a conserved and generalizable control mechanism. Leveraging these insights, we achieved selective geranylation across 36 aromatic substrates, affording 41 products. Integration with an efficient geranyl donor regeneration system enabled preparative-scale synthesis, reaching titers up to 324 mg/L. This work defines a unifying mechanism for selectivity control in PTs and provides a high-efficiency platform for geranylation.