Ancestral Sequence Reconstruction Enhances Gene Mining Efforts for Industrial Ene Reductases by Expanding Enzyme Panels with Thermostable Catalysts

Ancestral sequence reconstruction (ASR) has been used to produce enzymes that exhibit enhanced stability, activity, and promiscuity compared to wild type (WT) enzymes. In this work, we compiled a panel of 57 closely related wild type ene reductases and 56 counterpart ancestral enzymes and statistically compared their biocatalytic properties. Enzyme activity, selectivity, thermostability, and expression level distributions were used to compare their performance. ASR and WT enzymes exhibited comparable activity and overexpression levels, but ASR enzymes displayed enhanced average thermostability (by 9 degrees C) compared to their wild-type counterparts. Our results demonstrate that ASR-derived ene reductase enzymes may be used as a complement to wild-type enzymes and may provide a superior source of biocatalysts for alkene reduction for industrial applications.