Engineering a coenzyme-independent dioxygenase for one-step production of vanillin from ferulic acid.

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. IMPORTANCE:The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.