Protein engineering of toluene monooxygenases for synthesis of hydroxytyrosol

Brouk, Moran; Fishman, Ayelet

doi:10.1016/j.foodchem.2009.02.020

Cited by 32 publications

(54 citation statements)

References 30 publications

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“…The variants were obtained through rational design and directed evolution, as follows. Directed evolution was performed as described previously (6). Generally, by utilizing error-prone PCR, random mutations were generated, thus creating a diverse library of T4MO variants.…”

Section: Resultsmentioning

confidence: 99%

“…Error-prone PCR was performed as described earlier in detail for T4MO (6). Saturation mutagenesis at position I100 was described by Feingersch et al (7).…”

Section: Methodsmentioning

confidence: 99%

“…Analytical methods. The analytical method was the same as we described previously (6). HPLC analysis was performed with an Agilent 1100-series instrument (Agilent Technologies, CA) using an Eclipse XDB-C 18 column (5 m, 4.6 by 150 mm; Agilent Technologies, CA) equipped with a photodiode array detector.…”

Section: Methodsmentioning

confidence: 99%

“…In this work, we combine all three approaches-directed evolution, rational design, and statistical methods-to improve the capacity of toluene 4-monooxygenase (T4MO) to produce an important antioxidant, hydroxytyrosol (6). This phenol, which is naturally present in olives and olive oil, has the highest free radical scavenging capacity and has been shown to be beneficial in preventing various diseases, such as diabetes, atherosclerosis, and cancer (13,19,34).…”

mentioning

confidence: 99%

“…The goal of the present work was to further generate a better T4MO variant for the production of hydroxytyrosol. As the cloning steps associated with producing double and triple mutants of this enzyme are very laborious and time-consuming (e.g., QuikChange mutagenesis cannot be applied due to the large plasmid involved [9 kb], and a three-step PCR is needed for each mutant [5,6]), the integration of the Nov and Wein statistical model was evaluated.…”

mentioning

confidence: 99%

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Improving Biocatalyst Performance by Integrating Statistical Methods into Protein Engineering

Brouk

Nov

Fishman

2010

Appl Environ Microbiol

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Directed evolution and rational design were used to generate active variants of toluene-4-monooxygenase (T4MO) on 2-phenylethanol (PEA), with the aim of producing hydroxytyrosol, a potent antioxidant. Due to the complexity of the enzymatic system-four proteins encoded by six genes-mutagenesis is labor-intensive and time-consuming. Therefore, the statistical model of Nov and Wein (J. Comput. Biol. 12:247-282) was used to reduce the number of variants produced and evaluated in a lab. From an initial data set of 24 variants, with mutations at nine positions, seven double or triple mutants were identified through statistical analysis. The average activity of these mutants was 4.6-fold higher than the average activity of the initial data set. In an attempt to further improve the enzyme activity to obtain PEA hydroxylation, a second round of statistical analysis was performed. Nine variants were considered, with 3, 4, and 5 point mutations. The average activity of the variants obtained in the second statistical round was 1.6-fold higher than in the first round and 7.3-fold higher than that of the initial data set. The best variant discovered, TmoA I100A E214G D285Q, exhibited an initial oxidation rate of 4.4 ؎ 0.3 nmol/min/mg protein, which is 190-fold higher than the rate obtained by the wild type. This rate was also 2.6-fold higher than the activity of the wild type on the natural substrate toluene. By considering only 16 preselected mutants (out of ϳ13,000 possible combinations), a highly active variant was discovered with minimum time and effort.

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Section: Resultsmentioning

confidence: 99%

“…Error-prone PCR was performed as described earlier in detail for T4MO (6). Saturation mutagenesis at position I100 was described by Feingersch et al (7).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Improving Biocatalyst Performance by Integrating Statistical Methods into Protein Engineering

Brouk

Nov

Fishman

2010

Appl Environ Microbiol

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Selective Catalytic Oxidation of CH Bonds with Molecular Oxygen

et al. 2012

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Although catalytic reductions, cross‐couplings, metathesis, and oxidation of CC double bonds are well established, the corresponding catalytic hydroxylations of CH bonds in alkanes, arenes, or benzylic (allylic) positions, particularly with O2, the cheapest, “greenest”, and most abundant oxidant, are severely lacking. Certainly, some promising examples in homogenous and heterogenous catalysis exist, as well as enzymes that can perform catalytic aerobic oxidations on various substrates, but these have never achieved an industrial‐scale, owing to a low space‐time‐yield and poor stability. This review illustrates recent advances in aerobic oxidation catalysis by discussing selected examples, and aims to stimulate further exciting work in this area. Theoretical work on catalyst precursors, resting states, and elementary steps, as well as model reactions complemented by spectroscopic studies provide detailed insight into the molecular mechanisms of oxidation catalyses and pave the way for preparative applications. However, O2 also poses a safety hazard, especially when used for large scale reactions, therefore sophisticated methodologies have been developed to minimize these risks and to allow convenient transfer onto industrial scale.

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Whole‐Cell Carboxylate Reduction for the Synthesis of 3‐Hydroxytyrosol

et al. 2014

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Abstract3‐Hydroxytyrosol (3‐HT) is a phenolic antioxidant that has a number of beneficial effects on human health and is a valuable building block in the synthesis of various pharmaceuticals. Herein, we report a new method for the production of 3‐HT through reduction of 3,4‐dihydroxyphenylacetic acid. The reduction was performed in whole Escherichia coli BL21 (DE3) cells overexpressing carboxylic acid reductase from Nocardia and phosphopantetheinyl transferase from E. coli. An endogenous E. coli aldehyde reducing activity turned out to be highly efficient for further reduction of the aldehyde intermediate to the desired alcohol. The influence of different buffer components, cofactors, and cofactor recycling systems was investigated. A very economic combination of glucose, citrate, and air proved sufficient for recycling of the essential cofactors ATP and NAD(P)H. Selected crucial parameters were then further optimized within a “design of experiments” approach. Finally, first preparative‐scale bioreductions resulted in pure 3‐HT.

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Protein engineering of toluene monooxygenases for synthesis of hydroxytyrosol

Cited by 32 publications

References 30 publications

Improving Biocatalyst Performance by Integrating Statistical Methods into Protein Engineering

Improving Biocatalyst Performance by Integrating Statistical Methods into Protein Engineering

Selective Catalytic Oxidation of CH Bonds with Molecular Oxygen

Whole‐Cell Carboxylate Reduction for the Synthesis of 3‐Hydroxytyrosol

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