Despite their intriguing photophysical and photochemical activities, naturally occurring photoenzymes have not yet been repurposed for new-to-nature activities. Here we engineered fatty acid photodecarboxylases to catalyse unnatural photoredox radical C–C bond formation by leveraging the strongly oxidizing excited-state flavoquinone cofactor. Through genome mining, rational engineering and directed evolution, we developed a panel of radical photocyclases to facilitate decarboxylative radical cyclization with excellent chemo-, enantio- and diastereoselectivities. Our high-throughput experimental workflow allowed for the directed evolution of fatty acid photodecarboxylases. An orthogonal set of radical photocyclases was engineered to access all four possible stereoisomers of the stereochemical dyad, affording fully diastereo- and enantiodivergent biotransformations in asymmetric radical biocatalysis. Molecular dynamics simulations show that our evolved radical photocyclases allow near-attack conformations to be easily accessed, enabling chemoselective radical cyclization. The development of stereoselective radical photocyclases provides unnatural C–C-bond-forming activities in natural photoenzyme families, which can be used to tame the stereochemistry of free-radical-mediated reactions.

尽管天然存在的光酶具有令人着迷的光物理和光化学活性，但尚未被重新用于新的自然活动。在这里，我们设计了脂肪酸光脱羧酶，通过利用强氧化性激发态黄酮醌辅助因子来催化非天然光氧化还原自由基 C-C 键的形成。通过基因组挖掘、合理工程和定向进化，我们开发了一组自由基光环化酶，以促进脱羧自由基环化，具有优异的化学选择性、对映选择性和非对映选择性。我们的高通量实验工作流程允许脂肪酸光脱羧酶的定向进化。一组正交的自由基光环化酶被设计用于获得立体化学二元体的所有四种可能的立体异构体，在不对称自由基生物催化中提供完全的非对映和对映异构生物转化。分子动力学模拟表明，我们进化的自由基光环化酶可以轻松获得近攻击构象，从而实现化学选择性自由基环化。立体选择性自由基光环化酶的发展在天然光酶家族中提供了非自然的 C-C 键形成活性，可用于控制自由基介导反应的立体化学。