Dissecting the disconnect between circuit activation and dominant adaptive evolution in cytoplasmic phage-assisted continuous evolution (PACE) of an EGFR nanobody.

Introduction: Cytoplasmic phage-assisted continuous evolution (PACE) is widely used due to its low selection pressure and straightforward circuit design. However, the reducing environment limits its applicability to mammalian proteins that rely on disulfide bonds or glycosylation. Methods: To evaluate whether these biochemical constraints inherently hinder cytoplasmic evolution, we confined both the antibody and the target protein to single-domain formats. We examined circuit activation and evolution of a 7D12 nanobody targeting the epidermal growth factor receptor domain III (EGFR DIII). Results: Nanobodies and EGFR DIII expressed in the cytoplasm retained their soluble fractions and pairing activity. These enabled efficient activation of the two-hybrid circuit and robust phage propagation. Despite successful circuit activation, neither non-continuous flow (PANCE) nor PACE generated affinity-enhanced variants under extended drift conditions and graded selection pressures designed to increase mutational diversity. Discussion: Although optimal PACE operating conditions inhibited cheater gene recombination and enabled sustained POI mutagenesis, mutational convergence may be more sensitive to organismal incompatibility than circuit activation. This aligns with the observed decoupling between circuit activation and productive adaptive evolution. Moreover, structural analysis and predicted saturation mutagenesis at the binding interface are consistent with interface accessibility as a plausible constraint. The 7D12 nanobody contains a narrow, protruding CDR3, and most substitutions at the CDR1 -CDR3 interface are neutral or deleterious. Although single-domain mammalian proteins can overcome organismal incompatibility for cytoplasmic PACE circuit activation, their evolvability may be influenced by limitations in the accessibility of beneficial mutations within the binding interface and by their combination with organism-level factors. These results support a multifactorial basis for evolvability in cytoplasmic PACE, involving both interface accessibility and system-level factors.