Sakoleva Thaleia, Vesenmaier Florian, Koch Lena, Schunke Jarne E, Novak Kay D, Grobe Sascha, Dörr Mark, Bornscheuer Uwe T, Bayer Thomas
Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.
acib GmbH, Krenngasse 37/2, 8010, Graz, Austria.
Chembiochem. 2025 Jan 2;26(1):e202400712. doi: 10.1002/cbic.202400712. Epub 2024 Nov 6.
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.
酯类是有价值的香气化合物，可由（脂肪族）酮前体通过拜耳-维利格单加氧酶（BVMOs）酶促合成。然而，目前缺少一种用于评估BVMO活性和检测反应产物的基因编码生物传感器系统。在这项工作中，我们在异源宿主大肠杆菌中组装了一个合成酶级联反应——其包含一种酯酶、一种醇脱氢酶和LuxAB。目标酯由BVMO产生，随后被裂解，相应的醇通过人工途径被氧化。最终，荧光素酶LuxAB在体内检测到醛产物，LuxAB是来自发光杆菌属的一种荧光素酶，在醛氧化为相应羧酸盐时会发出生物发光。这种生物传感器系统极大地加速了从聚焦文库中筛选和选择活性BVMO变体的过程，省去了常用的低通量色谱分析。工程酶能够接受线性脂肪族酮，如2-十一烷酮和2-十二烷酮，并表现出改善的酯形成能力。
