It is particularly important to mention here that H79 was chosen as the input FuncLib in order to try to enhance and replace the interaction of D-Lac with H79 Unfortunately all mutations at the H79 an

It is crucial to note that the selection of H79 as the input FuncLib was aimed at enhancing and replacing the interaction of D-Lac with this residue. Regrettably, all mutations at the H79 and N146 sites resulted in the deactivation of D-Lac. Based on sequence conservation analysis and combinatorial mutational activity assays, it appears that these two residues may have catalytic or binding effects, making it challenging to identify alternative residues. Furthermore, the T213, Q219, and I309 mutations all had unfavorable synergistic mutagenic effects with F308G. Nonetheless, certain mutations in N96 and A271 exhibited a strong synergistic mutagenic effect with F308G (Figure 3G). Thus far, FuncLib has been successfully utilized to enhance the activity or functional diversity of enzymes 41-45. Nevertheless, even if the sequence space of FuncLib or the MD simulations enabled us to select superior combinations to the WT (including combinatorial mutations in H79, N146, T213, Q219, and I309), direct synthetic gene expression of multiple combinatorial mutations may allow the more superior mutants to escape the local optimal space. After preliminary screening by MD simulations and appropriate disaggregation of combinatorial mutations, the application scenarios and potential of FuncLib can be further expanded and improved