Engineering highly functional thermostable proteins using ancestral sequence reconstruction
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- Gillam__Article_1542035960_1
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Commercial biocatalysis requires robust enzymes that can withstand elevated temperatures and long incubations. Ancestral reconstruction has shown that pre-Cambrian enzymes were often much more thermostable than extant forms. Here, we resurrect ancestral enzymes that withstand ~30 °C higher temperatures and ≥100 times longer incubations than their extant forms. This is demonstrated on animal cytochromes P450 that stereo- and regioselectively functionalize unactivated C–H bonds for the synthesis of valuable chemicals, and bacterial ketol-acid reductoisomerases that are used to make butanol-based biofuels. The vertebrate CYP3 P450 ancestor showed a 60T50 of 66 °C and enhanced solvent tolerance compared with the human drug-metabolizing CYP3A4, yet comparable activity towards a similarly broad range of substrates. The ancestral ketol-acid reductoisomerase showed an eight-fold higher specific activity than the cognate Escherichia coli form at 25 °C, which increased 3.5-fold at 50 °C. Thus, thermostable proteins can be devised using sequence data alone from even recent ancestors.
Original language | English |
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Journal | Nature Catalysis |
Volume | 1 |
Issue number | 11 |
Pages (from-to) | 878-888 |
ISSN | 2520-1158 |
DOIs | |
Publication status | Published - 2018 |
Externally published | Yes |
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