Optimization, reconstruction and heterologous expression of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri in Escherichia coli and its successive hydroxylation of toluene and benzene

Biological treatment is a cost-effective and environmentally friendly technique for treating toluene. However, this process is limited by the type and quantity of toluene degraders that can be applied to different environments and methods. It is a feasible solution to constructing new genetic engineering organisms for toluene degradation through biotechnology. To facilitate genetic manipulation and improve gene expression, the toluene/o-xylene monooxygenase locus from Pseudomonas stutzeri was reconstructed and artificially synthesized. All six genes were optimized and driven with a monocistronic transcriptional pattern by T7 promoter and terminator to be expressed at a high level. The engineered bacteria can hydroxylate toluene to a mixture of o-, m- and p-cresol, which are then further oxidized to 3- and 4-methylcatechol. Benzene can also be oxidized to phenol, catechol and pyrogallol successively. The negative effects of toluene can be removed by the engineered bacteria via proteomics analysis. Overall, the artificial gene clusters established in this study can be used to construct different toluene and benzene degradation organisms for bioremediation. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1996267 .