Thaleia Sakoleva, Florian Vesenmaier, Lena Koch, Jarne E. Schunke, Kay D. Novak, Sascha Grobe, Mark Dörr, Uwe T. Bornscheuer, Thomas Bayer

• Esters are valuable aroma compounds and can be produced enzymatically by Baeyer‐Villiger monooxygenases (BVMOs) from (aliphatic) ketone precursors. However, a genetically encoded biosensor system for the assessment of BVMO activity and the detection of reaction products is missing. In this work, we assembled a synthetic enzyme cascade – featuring an esterase, an alcohol dehydrogenase, and LuxAB – in the heterologous host Escherichia coli . Target esters are produced by a BVMO, subsequently cleaved, and the corresponding alcohol oxidized through the artificial pathway. Ultimately, aldehyde products are detected in vivo by LuxAB, a luciferase from Photorhabdus luminescens that emits bioluminescence upon the oxidation of aldehydes to the corresponding carboxylates. This biosensor system greatly accelerated the screening and selection of active BVMO variants from a focused library, omitting commonly used low‐throughput chromatographic analysis. Engineered enzymes accepted linear aliphatic ketones such as 2‐undecanone and 2‐dodecanone and exhibited improved ester formation.