Engineered Biosensors from Dimeric Ligand-Binding Domains

Abstract

Biosensors are important components of many synthetic biology and metabolic engineering applications. Here, we report a second generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. The biosensors, comprising one ligand-binding domain monomer fused to a DNA-binding domain and another fused to a transcriptional activation domain, activate reporter gene expression in response to steroid binding and receptor dimerization. The introduction of a destabilizing mutation to the dimer interface increased biosensor dynamic range by an order of magnitude. Computational redesign of the dimer interface and functional selections were used to create heterodimeric pairs with further improved dynamic range. A heterodimeric biosensor built from the digoxigenin and progesterone ligand-binding domains functioned as a synthetic "AND"-gate, with 20-fold stronger response to the two ligands in combination than to either one alone. We also identified mutations that increase the sensitivity or selectivity of the biosensors to chemically similar ligands. These dimerizing biosensors provide additional flexibility for the construction of logic gates and other applications.

Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae

“… 2019 ) or mutagenesis of the sTF (Jester et al . 2018 ). Moreover, mutagenesis has been reported as a successful method for increasing biosensor specificity (Feng et al .…”

“… 2016 ; Jester et al . 2018 ). Similar approaches may be taken to optimize the dynamic and operational range of the acetic acid biosensor developed here.…”

Strategies for Improving Small-Molecule Biosensors in Bacteria

“…Laboratory studies on a given protein’s activity provide a second source of useful information, with researchers often making targeted mutations to identify important residues or test predictions made using structural models. This strategy has been used in the development of several small-molecule biosensors, including sensors for fentanyl [ 26 ], TNT [ 108 ], and digoxigenin [ 109 ]. Structure and activity information can also be used to identify targeted residues for directed evolution (termed semi-rational design), with prior applications of this approach including improvements to the cationic amino acid biosensor LysG [ 110 ], the choline biosensor BetI [ 111 ], and the tetracycline biosensor TetR [ 112 ].…”

Custom-made transcriptional biosensors for metabolic engineering

“…Upon ligand binding, the two proteins are stable and can homodimerize, resulting in biosensing. This system allows for better range tuning and possible orthogonal biosensing of different ligands [39].…”