A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases

Background: Oxygenases catalyze the hydroxylation of a wide variety of orga nic substrates, An ability to alter oxygenase substrate specificities and i mprove their activities and stabilities using recombinant DNA techniques wo uld expand their use in processes such as chemical synthesis and bioremedia tion. Discovery and directed evolution of oxygenases require efficient scre ens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples. Results: Horseradish peroxidase (HRP) couples the phenolic products of hydr oxylation of aromatic substrates to generate colored and/or fluorescent com pounds that are easily detected spectroscopically in high-throughput screen ing. Coexpression of the coupling enzyme with a functional mono- or dioxyge nase creates a pathway for the conversion of aromatic substrates into fluor escent compounds in vivo, We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450(cam) by fluorescence digital imaging, Colors generated by the HRP coupling reactio n are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecif icities. Conclusions: The coupled oxygenase-peroxidase reaction system is well suite d for screening oxygenase libraries to identify mutants with desired featur es, including higher activity or stability and altered reaction specificity , This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for o ptimizing oxygenase reaction conditions.