Transforming a Historical Chemical Synthetic Route for Vanillin Starting from Renewable Eugenol to a Cell‐Free Bi‐Enzymatic Cascade

Vanillin is one of the most important aroma compounds, naturally occurring in vanilla pods. Many routes to access natural vanillin from various renewables have been investigated, including a natural five-step microbial transformation of eugenol to vanillin. Readily available eugenol was also the starting material for a chemical two-step sequence to vanillin employed in the 19^th century. Here we show that a two-step sequence can also be realized using biocatalysts only and run it in one-pot simultaneously. This was achieved by isomerizing the C=C double bond of eugenol by oxidation to coniferyl alcohol followed by oxidative C=C cleavage catalyzed by newly identified enzymes. Thus, two oxidative steps catalyzed by two different biocatalysts - one containing flavin and the other a non-heme iron(II) cofactor - were successfully run simultaneously just requiring molecular oxygen as oxidant for each step. Using natural eugenol sources, e. g. clove oil, vanillin was obtained with 91 % product formation. This study shows that natural pathways like the microbial transformation of eugenol to vanillin involving five steps can be shortened, hereto just two simultaneous steps, by exploiting and combining the repertoire of promiscuous enzymatic activities present in different organisms leading to new-to-nature cascades.