Abstract
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 ∼13,000 possible combinations), a highly active variant was discovered with minimum time and effort.