An Enzyme Cascade Synthesis of Vanillin

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.

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward aromatic hydroxylation are shown (red spheres). Mutated positions in the heme domain (residues 1–471) and in the reductase domain (472–1049) are listed below, with refs: 1 (), 11 ( − ), 23 (), 42 (), 47 ( , , , , − ,, , , − , − , , , , − , , , − , , − ), 51 ( , , , , , , , − , , , , , , , , , − ,, , ), 58 ( − ), 64 ( , , , , , − , ), 68 ( , ), 72 ( , ), 73 (), 74 ( , , , , , , , …”

“… All positions of the P450 BM3 heme domain that have been mutated and show improvement toward aromatic hydroxylation are shown (red spheres). Mutated positions in the heme domain (residues 1–471) and in the reductase domain (472–1049) are listed below, with refs: 1 ( 338 ), 11 ( 338 − 340 ), 23 ( 338 ), 42 ( 338 ), 47 ( 5 , 23 , 25 , 158 , 163 − 165 , 167 , 170 , 173 , 175 − 177 , 182 − 184 , 186 , 187 , 189 , 193 − 195 , 200 , 202 , 338 − 347 , 384 , 349 − 354 ), 51 ( 5 , 23 , 25 , 158 , 165 , 167 , 173 , 175 − 177 , 182 , 184 , 186 , 187 , 189 , 193 , 194 , 202 , 342 − 344 , 346 , 355 , 356 ), 58 ( 357 − 359 ), 64 ( 173 , 183 , 200 ...…”

The beauty of biocatalysis: sustainable synthesis of ingredients in cosmetics

“…In an alternative route, a P450 BM3 variant converted 3-methylanisole to vanillyl alcohol and subsequent oxidation using vanillyl alcohol oxidase variant F454Y from Penicillium simplicissimum produced vanillin, but only 1% after 12 h in the cascade in vivo . 369…”

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

“…BM3 variants have also been shown to target a range of benzene derivatives. The production of vanillin from 3-methylanisole was achieved in a whole-cell E. coli system via a three-step cascade using the BM3 variant A328L combined with mutated vanillyl alcohol oxidase, albeit at a low conversion of 11.7% and a yield of 1.1% [ 86 ]. WT BM3 and the selected variants also selectively hydroxylate the methyl-substituted benzene derivatives, pseudocumene and mesitylene, to the phenolic building blocks used in the α-tocopherol synthesis via quinolone intermediates.…”