Commercial enzymatic processes require robust catalysts able to withstand elevated temperatures and long incubations, conditions under which most native enzymes perform poorly. Incremental increases in thermostability can be achieved by repeated rounds of mutagenesis and screening, but general strategies are needed for designing highly thermostable enzymes a priori. Here we show that enzymes can be created that can withstand temperatures ~ 30 °C higher and incubations ≥ 100 times longer than extant forms in a single step using ancestral reconstruction. We exemplify the approach with the first ancestral resurrections of two unrelated enzyme families: cytochrome P450 monooxygenases, that stereo-and regioselectively functionalize un-activated C-H bonds in pharmaceutical, flavour, fragrance and other fine chemical syntheses; and ketol acid reductoisomerases, used to make butanol-based biofuels. This shows thermostability can be designed into proteins using sequence data alone, potentially enhancing the economic feasibility of any process or product requiring a highly stable protein.
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