Abstract
A novel approach for the synthesis of vanillin employing a three-step two-enzymatic cascade sequence is reported. Cytochrome P450 monooxygenases are known to catalyse the selective hydroxylation of aromatic compounds, which is one of the most challenging chemical reactions. A set of rationally designed variants of CYP102A1 (P450 BM3) from Bacillus megaterium at the amino acid positions 47, 51, 87, 328 and 437 was screened for conversion of the substrate 3-methylanisole to vanillyl alcohol via the intermediate product 4-methylguaiacol. Furthermore, a vanillyl alcohol oxidase (VAO) variant (F454Y) was selected as an alternative enzyme for the transformation of one of the intermediate compounds via vanillyl alcohol to vanillin. As a proof of concept, the bi-enzymatic three-step cascade conversion of 3-methylanisole to vanillin was successfully evaluated both in vitro and in vivo.
Highlights
Vanillin is one of the most widely used flavouring compounds worldwide
P450 monooxygenases (P450s) catalyse oxidation reactions introducing a single oxygen-atom from molecular oxygen into the substrate with the concomitant reduction of the other oxygen atom to water [9]
We introduce the concept of enzymatic cascade reactions as an effective means to selectively oxidise aromatic compounds for the synthesis of vanillin from the simple aromatic phenylalkane 3-methylanisole (Scheme 1)
Summary
Vanillin is one of the most widely used flavouring compounds worldwide. Besides being a flavour ingredient for food and beverages, it is used as a fragrance ingredient in perfumes and cosmetics [1], as well as an intermediate in the chemical and pharmaceutical industries for the production of antifoaming agents, herbicides and drugs [2]. A variety of biotechnological approaches was developed for the production of vanillin from eugenol, isoeugenol, ferulic acid, lignin, glucose, phenolic stilbene, vanillic acid or aromatic amino acid starting materials from renewable feedstocks [3,4,5]. Aiming at the enzymatic synthesis of vanillin by the selective hydroxylation of an aromatic substrate, cytochrome P450 monooxygenases (P450s) constitute ideal candidates [8]. P450s catalyse oxidation reactions introducing a single oxygen-atom from molecular oxygen into the substrate with the concomitant reduction of the other oxygen atom to water [9]
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