Old yellow enzyme-catalyzed desymmetrizing desaturation

Flavin-dependent old yellow enzymes (OYEs) have been widely employed as biocatalysts for the stereoselective anti -hydrogenations of activated C C bonds through a “Ping-Pong” bi-bi mechanism. Over the past decade, studies have expanded their reactivity to new-to-nature radical transformations by exploiting their catalytic promiscuity and adaptability to protein engineering. Recently, our group repurposed OYEs to enable asymmetric carbonyl desaturation by inverting their native activity, addressing key limitations of traditional desaturation approaches—such as harsh reaction conditions and inadequate stereochemical control. By directed evolution of an OYE from Geobacillus kaustophilus ( Gk OYE), we achieved desymmetrizing desaturation of diverse cyclohexanones to form the corresponding cyclohexenones featuring a remote quaternary stereogenic center under mild conditions with excellent stereoselectivity. This chapter provides a comprehensive description of the development of Gk OYE-based desymmetrizing desaturation system, including high-throughput enzyme engineering, protein expression, analytical and scalable reaction set-ups, offering a roadmap for adapting OYEs to challenging desaturative transformations.