The video shows the setup and analysis of a chemoenzymatic reaction with the AmbDH3 domain on the semipreparative scale. The synthesis of the enzyme precursor as well as a typical procedure for the production of the catalyst are described. The last two decades have seen a tremendous progress in the understanding of secondary metabolite biosynthesis on the genetic, the enzymatic as well as on the structural level. Such biosynthetic pathways contain a plethora of enzymes that have the potential to become useful biocatalysts. In accordance with their natural role, they would be particularly attractive for an application in natural product synthesis. The ambruticins are a group of polyketide natural products with attractive antifungal activity profile and a low toxicity in mammal. Their biosynthesis contains a couple of enzymes that act via unprecedented mechanisms and which are attractive for an application in the chemoenzymatic synthesis of natural products. The unusual cyclase domain AmbDH3 performs an oxa-Michael addition leading to the formation of one of the tetrahydropyran rings of the ambruticins. As this structural motif is present in many natural products, we are evaluating the potential of this domain to be used as a flexible biocatalytic tool for preparative scale application. The video shows the setup and analysis of a chemoenzymatic reaction with the AmbDH3 domain on the semipreparative scale. The synthesis of the enzyme precursor as well as a typical procedure for the production of the catalyst are described.