<p>Stereodivergent
syntheses leading to the different stereoisomers of a product are useful in the
discovery and testing of drugs and agrochemicals. A longstanding challenge in
catalysis, developing sets of stereodivergent catalysts is often solved for enzymes
by screening Nature’s diversity for biocatalysts with complementary
stereoselectivities. Here, Nature’s protein diversity has been leveraged to
develop stereodivergent catalysts for a reaction not known in biology, cyclopropanation
via carbene transfer. By screening diverse native and engineered heme proteins,
we identified globins and serine-ligated cytochromes P450 with promiscuous
activity for cyclopropanation of unactivated alkene substrates. Their activities
and stereoselectivities were enhanced by directed evolution: 1-3 rounds of site-saturation
mutagenesis and screening generated enzymes that catalyze the stereodivergent cyclopropanation
to form each of the four stereoisomers of unactivated alkenes and
electron-deficient alkenes with up to 5,400 total turnovers and 98% enantiomeric
excess. These fully genetically encoded biocatalysts function in whole <i>E. coli</i> cells in mild, aqueous
conditions and provide the first example of enantioselective, intermolecular
iron-catalyzed cyclopropanation of unactivated alkenes via carbene transfer.</p>
Site-saturation Mutagenesis is more Efficient than DNA Shuffling for the Directed Evolution of β-Fucosidase from β-Galactosidase
