Cell-free screening of peptide tags for droplet-like assembly formation of glucose dehydrogenase

Glucose dehydrogenases (GDHs) are oxidoreductases that catalyze the oxidation of d-glucose to glucono-δ-lactone and are widely used in industrial applications such as biosensors, bioelectrodes, and biocatalytic cofactor regeneration. In particular, NAD(P)⁺-dependent GDHs are frequently employed as NAD(P)H-regenerating enzymes, with extensive efforts devoted to improving their robustness. BmGDH_M6, an engineered variant of the Bacillus megaterium GDH with enhanced chemical and organic solvent tolerance, was developed as a potent cofactor-regeneration enzyme. In parallel, enzyme condensation via liquid-liquid phase separation has attracted increasing attention as a potential mechanism for organizing enzyme-catalyzed reactions, and short peptides capable of promoting condensate formation. However, efficient screening and evaluation of such peptide tags using conventional cell-based expression systems have remained challenging. This study established a peptide tag screening strategy using the PURE system, a reconstituted cell-free protein synthesis platform, and applied it to BmGDH_M6 as a model enzyme. A small library of metabolic enzymes transiently assembling (META) body-forming signal (METAfos) tags and intrinsically disordered region (IDR)-derived peptide tags was evaluated with respect to expression, solubility, and assembly behavior within a defined in vitro environment. From this library, a short peptide tag, K7G3, 10-amino-acids-long, was identified that conferred droplet-like assembly-forming properties on BmGDH_M6 under specified crowding conditions. These results demonstrated that the PURE system provided a rapid and controllable platform for screening peptide tags and down-selection of candidates that modulate enzyme assembly behavior in vitro.