Design, Evolution, and Characterization of a Xylose Biosensor in Using the XylR/ System with an Expanded Operating Range.

Genetically encoded biosensors are extensively utilized in synthetic biology and metabolic engineering. However, reported xylose biosensors are far too sensitive with a limited operating range to be useful for most sensing applications. In this study, we describe directed evolution of XylR, and construction of biosensors based on XylR and the corresponding operator . The operating range of biosensors containing the mutant XylR was increased by nearly 10-fold comparing with the control. Two individual amino acid mutations (either L73P or N220T) in XylR were sufficient to extend the linear response range to upward of 10 g/L xylose. The evolved biosensors described here are well suited for developing whole-cell biosensors for detecting varying xylose concentrations across an expanded range. As an alternative use of this system, we also demonstrate the utility of XylR and as a xylose inducible system to enable graded gene expression through testing with β-galactosidase gene and the lycopene synthetic pathway. This evolution strategy identified a less-sensitive biosensor for real applications, thus providing new insights into strategies for expanding operating ranges of other biosensors for synthetic biology applications.