Two biotechnological approaches to the preparative synthesis of natural dihydrocoumarin

In this work, we describe two different biotechnological processes that provide the natural flavour dihydrocoumarin in preparative scale. Both the presented approaches are based on the en-zyme-mediated reduction of natural coumarin. The first one is a whole-cell process exploiting the reductive activity of the yeast Kluyveromyces marxianus, a Generally Recognized As Safe (GRAS) microorganism that possesses high resistance to the substrate toxicity. Differently, the second is based on the reduction of natural coumarin by nicotinamide adenine dinucleotide phosphate (NADPH) and using the Old Yellow Enzyme reductase OYE2 as catalyst. NADPH is used in catalytic amount since the co-factor regeneration is warranted employing an enzymatic system based on glucose oxidation, in turn catalysed by a further enzyme, namely glucose dehydrogenase (GDH). Both processes compare favourably over the previously reported industrial method as they work with higher coumarin concentration (up to 3 g/L for the enzymatic process) yet allowing the complete conversion of the substrate. Furthermore, the two approaches have significant differences. The microbial reduction is experimentally simple but the isolated dihydrocoumarin yield does not ex-ceed 60%. On the contrary, the enzymatic approach requires the use of two specially prepared re-combinant enzymes, however, it is more efficient, affording the product in 90% of isolated yield.