Tuning the Specificity of the Recombinant Multicomponent Toluene o -Xylene Monooxygenase from Pseudomonas sp. Strain OX1 for the Biosynthesis of Tyrosol from 2-Phenylethanol

Abstract

Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.

Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics

“…Positions 101 and 103 were chosen for mutagenesis based on earlier results with ToMO (Cafaro et al 2005;Notomista et al 2009;Notomista et al 2011;, T4MO (McClay et al 2005;Tao et al 2005b;Tao et al 2004a), and TpMO (Fishman et al 2005), which indicated that these positions are important for regiospecific oxidation of aromatics. Position 214 was selected based on our earlier results with ToMO, which indicated that this position is a gate residue C catechol, HQ hydroquinone, R resorcinol, NP nitrophenol, N naphthol, ND not detected, SD standard deviation a Expressed in E. coli TG1 whole cells and induced with 1 mM IPTG during mid-exponential phase b The initial formation rates of dihydroxybenzenes were determined via HPLC from 0.5 mM phenol c The overall degradation rates as indicated by the production of chloride were determined over 18 h with 67 μM TCE and important for enhanced aromatic oxidation and chlorinated ethene degradation .…”

“…2). The importance of position 103 as an active residue in ToMO, T4MO, and TpMO has also been reported (Cafaro et al 2005;Fishman et al 2005;McClay et al 2005;Mitchell et al 2002;Notomista et al 2011;Tao et al 2005b;Tao et al 2004a). Through site-directed mutagenesis, ToMO variant E103G was identified with enhanced para hydroxylation of toluene (85 % p-cresol vs 45 % for wild type) and o-xylene (99 % 3,4-dimethyl phenol vs 80 % for wild type) (Cafaro et al 2005).…”

“…Through site-directed mutagenesis, ToMO variant E103G was identified with enhanced para hydroxylation of toluene (85 % p-cresol vs 45 % for wild type) and o-xylene (99 % 3,4-dimethyl phenol vs 80 % for wild type) (Cafaro et al 2005). In a different study, same variant ToMO E103G was shown to increase the efficiency of ToMO for the bioconversion of 2-phenylethanol to p-tyrosol (Notomista et al 2011). Using saturation mutagenesis, TpMO variant G103S was obtained with 3-fold enhancement in m-nitrophenol formation from nitrobenzene (96 % vs 34 % by wild-type TpMO) (Fishman et al 2005).…”

“…These wild-type TMOs and their variants formed by rational and random mutagenesis have been shown to have potential for green chemistry and bioremediation applications (Fishman et al 2006). Some of the compounds that can be produced using the TMO family members are catechol (intermediates for the synthesis of agrochemicals and pharmaceuticals) (Vardar and Wood 2004), 2-naphthol (50,000 t/year market) (Tao et al 2005a;Tao et al 2005b), 4-nitrocatechol (inhibitor of nitric oxide synthase) , indirubin (anti-cancer compound) , tyrosol (nutraceutical for the prevention of cancer and cardiovascular diseases) (Notomista et al 2011), and chiral sulfoxides (functionalized substances in pharmaceuticals and chiral auxiliaries in organic synthesis) (Feingersch et al 2008). For bioremediation, they can degrade mixtures of chlorinated aliphatics (health-threatening pollutants), including trichloroethylene (TCE) (Canada et al 2002;Ryoo et al 2000;Shim et al 2001;.…”

The role of substrate binding pocket residues phenylalanine 176 and phenylalanine 196 on Pseudomonas sp. OX1 toluene o‐xylene monooxygenase activity and regiospecificity

“…The biocatalyst toluene‐ o ‐xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 (Cafaro et al, ; Radice et al, ) belongs to a remarkable family of bacterial multicomponent monooxygenases (BMM; Notomista et al, ) and has been shown to have a great potential for biotechnological and environmental applications (Notomista et al, ; Vardar and Wood, ; Vardar et al, ,).…”