Multisite synergistic evolution of oleate hydratase via DCCM and the orthogonal location of distal sites

The evolution of enzymes through multisite combinations is often constrained by the dynamic correlations among residues, resulting in the challenge of achieving additive effects when combining single-site positive mutants. Nevertheless, the incorporation of distal-site residue mutants offers a promising avenue to unlock synergistic interactions, particularly in enzymes with channels that mediate substrate and product transport. While combinatorial mutagenesis typically requires exhaustive screening of extensive mutant libraries to identify superior variants. In this study, a multisite combination strategy using dynamic cross-correlation matrices (DCCM) iterative analysis, coupled with location orthogonal filtration (DCCM/OL) was established based on single-site mutants enhancing enzyme performance by engineering distal residues using the "Structure, SCANEER, and Sequence (3S)" approach. Thirteen beneficial single mutants of oleate hydratase from Staphylococcus aureus (SaOhy), which catalyzes the hydration of linoleic acid, were targeted using the "3S" approach. By employing the DCCM/OL strategy, the number of candidates in the combination mutation library for experimental screening were reduced from 60 to 5. And a triple-site mutant, SaOhy_L151V/I411L/V135A, was ultimately identified, which increased the catalytic efficiencies with linoleic acid and oleic acid by a 4- and 2.3-fold, respectively. The philosophy of multisite mutation based on distal residues using the DCCM/OL strategy effectively achieves an amplification of enzymatic activity with distinct substrates simultaneously. This study offers a promising strategy for the construction of a mutant smart library and multisite combinations to efficiently increase enzymatic performance.