Directed evolution of generalist biosensors for single ring aromatics

Biosensors can accelerate the engineering of new biosynthetic pathways. Phloroglucinol is a platform chemical of wide utility that can be produced at limited titers in Escherichia coli. Starting from the TetR family repressor RolR that is responsive to the related compound resorcinol, we were able to employ a combined selection and screen to identify variants that had greatly improved activities with phloroglucinol (EC50 for phloroglucinol of 131 uM, relative to an estimated 42 mM for wild-type RolR). The variants obtained were further screened with a panel of similar single ring aromatics, and several were found to be generalists, consistent with the hypothesis that both natural and directed evolution tend to first create semi-specific pockets prior to further optimization for new function