In silico directed evolution of humanized peptide transporters via computational epistatic rescue.

The human intestinal peptide transporter 1 (SLC15A1, PepT1) is a critical determinant of oral drug bioavailability, yet its thermodynamic characterization and structural tractability remain challenging. While bacterial orthologues like the Escherichia coli DtpA transporter offer highly stable structural surrogates, significant cross-species variations in the binding microenvironment limit their pharmacological fidelity. Traditional structure-based engineering aimed at engrafting the human pharmacophore onto bacterial scaffolds is frequently hindered by combinatorial explosion and severe thermodynamic frustration, as non-native side-chains disrupt co-evolved local packing. To bypass the empirical bottlenecks of in vitro directed evolution, we present a fully in silico co-evolutionary pipeline leveraging deep contextual protein language models (PLMs). By mapping 19 pharmacologically critical human PepT1 residues onto the DtpA sequence, we utilized the 650-million parameter ESM-2 model to conduct zero-shot mutational profiling. We identified 14 primary humanizing mutations to which the model assigned very low likelihoods (below 10 - 5). To resolve these energetic conflicts, we deployed an automated epistatic rescue algorithm to sweep the flanking microenvironmental topologies. This computationally efficient heuristic successfully identified localized, non-native compensatory mutations for all high-risk targets without relying on traditional molecular dynamics. Most notably, the highly deleterious Q41R and R305F substitutions were buffered by Y38D and I304K secondary mutations, yielding 5,215-fold and 11,614-fold increases in the model likelihood assigned to the primary substitution, respectively; these are changes in sequence likelihood and not calculated free energies. Binding dynamics were assessed via docking calculations and all-atom membranous molecular dynamics simulations. This deep learning-driven framework computationally rationalizes the engraftment of the human binding pocket, reducing the potential experimental screening space and yielding a humanized surrogate that requires experimental validation of folding, binding and transport before use in pharmacological screening.