Highly active enzymes produced by directed evolution with stability-based selection

Abstract In a directed evolution aimed at improving enzymatic activity, a situation occurs where highly active variants can no longer be obtained from a template protein because the template is already located at a peak (local maximum) in the fitness landscape of activity for the sequence space. To overcome this situation, the template needs to descend the mountain (lose activity) once and climb another higher mountain. However, there is no solid guideline of how the template should go down. Here, we propose a stability index. Previous studies have shown that protein evolution is potentially governed by stability, and that proteins with low activity but high stability are more favorable templates for producing highly active variants. In our earlier works on conventional directed evolution by random mutagenesis of an esterase from Sulfolobus tokodaii, we identified variants with 3-fold higher activity than the wild-type as the highest activity variants. In this work, as a first step, stability-keeping variants were selected by five rounds of random mutagenesis and screening based on halo formation assay using the substrate tributyrin at 70 °C after heat treatment for 30 min at 90 °C. These variants are likely to be scattered at the feet of various mountains in the fitness landscape. Next, these variants were pooled and used as parental proteins for a conventional experiment with activity-based selection, where the activity of variants was assayed using their cell-free extracts on the substrate p-nitrophenyl butyrate at 75 °C. After two rounds of random mutagenesis, we successfully obtained a variant with 9-fold higher activity than the wild-type. These results indicate that the two-step selection by stability and activity enables us more easily to produce markedly activity-improving variants.