Based on our previous work on a geometry-conservative redox-isomerization, the commercial alcohol dehydrogenase P2-G03 from the Codexis KRED enzyme kit was quickly identified as suitable tool for the intended ring-contraction methodology, combining excellent stereoselectivity in the ketoreduction with the complete inability to engage in subsequent hemiacetal dehydrogenations. In a first screening to identify biocatalysts that would be capable to selectively oxidize the furanose intermediates, model substrate rac-1a was initially reduced by means of P2-G03 together with glucose dehydrogenase and glucose in order to render the reduction fully irreversible. Under these conditions, 1a was quickly consumed, without the observation of any of the corresponding lactone products. The reactions were run in a pH 7.5 phosphate buffer to suppress acid-induced dehydration (i.e. retro-Achmatowicz reactions). Supplementing the reaction mixture with an excess of acetone as oxidant and a second alcohol dehydrogenase, in selected cases, clean conversion of the presumptive pyranose-furanose mixture to the desired butenolide 7a was observed. Out of a set of 26 commercial biocatalysts, seven ADHs showed significant amounts of lactone products, and most of them actually featured high selectivities for the fivemembered products. Since the enzymes KRED101, KRED130 and NADH101 themselves are not capable to recycle their nicotinamide cofactor, additional P2-G03 was provided to serve this purpose and translate the acetone additive into an oxidative environment. Particularly P3-H12 and evo030 stood out with excellent butenolide selectivities and conversions >95% (Scheme 3a). The differences in selectivities (e.g. 3:1 for P2-B02 vs 30:1 for P3-H12) indicate that the pyranose-furanose interconversion is reversible and that the selectivity in product formation is at least partially governed by catalyst selectivity according to the Curtin-Hammett principle (see also Supplementary Table ). Interestingly, as opposed to the in vivo experiments, the undesired aromatization was not detected under any circumstances. With these promising results in hand, we next aimed for a true cascade catalysis without distinct reduction and oxidation periods, a more challenging approach with seemingly contradictory demands on redox balances of the two individual biocatalysts. Even though the two ADHs may directly interact via oxidized or reduced cofactors, the necessity of a moderately reductive environment, to avoid immediate pyranose dehydrogenation to the d-lactones, prompted us to incorporate a redox mediator into the design (Scheme 3b). Most alcohol dehydrogenases can typically convert acetone to isopropanol, and vice versa, via consumption of nicotinamide cofactors. We envisaged that isopropanol as additive would thus enable a quick reduction of the pyranone in order to force the formation or the furanose-pyranose mixture. A second dehydrogenase with affinity for the furanose substrate could subsequently employ the resulting acetone to in-situ generate its oxidized NAD(P) + and irreversibly transform the hemiacetal to the desired butenolide. To our very delight, at 1% isopropanol (v/v) four of the previously identified dehydrogenation biocatalysts paired in a constructive manner with the Scheme 3 Selection of biocatalysts and redox mediators for the single-step ring-contracting redox isomerization of pyranone 1a to butenolide 7a.