Protein engineering of toluene monooxygenases for synthesis of hydroxytyrosol

Hyroxytyrosol (HTyr), an important phenol present in olives, stands out as a compound of high added value due to its exceptional antioxidant, antimicrobial and anticarcinogenic activities. This work describes the synthesis of HTyr via double hydroxylation of 2-phenylethanol (PEA) employing toluene monooxygenases (TMOs) as biocatalysts. Wild-type TMOs were initially evaluated for their ability to oxidise PEA and structurally-related substrates, providing better understanding of the factors responsible for controlling the regiospecificity. Both the length of the alkyl side chain and the presence of the hydroxyl group were found to influence the activity, possibly by interfering with the substrates’ entrance into the active site. Directed evolution of toluene-4-monooxygenase of Pseudomonas mendocina KR1 led to the discovery of variant TmoA S395C with a 15-fold increase in PEA hydroxylation rate. Saturation-mutagenesis at position TmoA I100 resulted in the finding of novel HTyr-producing variants I100A, I100S and I100G.