Optimizing Mutant F308G Activity Through Combinatorial Mutagenesis and MD Simulation

The FuncLib module within the CSFp strategy was employed to enhance the activity of mutant F308G. Nine residues in close proximity to the substrate within the binding pocket were selected for diversification (Figure 3A). A sequence space for D-Lac was generated, yielding 1000 variants (Table S2), of which only the top 50 with the most favorable energy were considered (Figure 3B). Each of these 50 variants underwent a 30 ns MD simulation, and ΔMM/GBSA was calculated relative to WT (Figure 3C). Subsequently, 10 variants with favorable ΔMM/GBSA values were selected for gene synthesis and validation to explore the diversity of multipoint combinatorial mutations while minimizing screening intensity (Figure 3D). However, the results indicated that half of the 10 combination mutants were completely inactivated, and the optimal mutant activity exhibited only a 0.4-fold increase relative to WT, significantly lower than that observed in F308G (Figure 3E). These findings suggest that the epistasis effect of mutations negatively impacts the outcomes obtained using FuncLib. To mitigate the adverse effects of detrimental mutations and prevent the loss of promising mutants from the sequence space, each residue of all combinatorial mutants in Figure 3D was mutated in combination with F308G, resulting in a total of 13 double mutants (Figure 3F). While five of the 13 double mutants lost activity entirely, encouragingly, mutants N96S/F308G, A271N/F308G, and A271D/F308G exhibited a 13-16-fold increase in activity compared to WT. Furthermore, N96S and A271N/D were mutated in combination with F308G, leading to the mutants N96S/A271D/F308G and N96S/A271N/F308G, which demonstrated a 9- and 17-fold increase in activity, respectively (Figure 3G).