Ribose-Binding Protein Mutants With Improved Interaction Towards the Non-natural Ligand 1,3-Cyclohexanediol.

Bioreporters consist of genetically modified living organisms that respond to the presence of target chemical compounds by production of an easily measurable signal. The central element in a bioreporter is a sensory protein or aptamer, which, upon ligand binding, modifies expression of the reporter signal protein. A variety of naturally occurring or modified versions of sensory elements has been exploited, but it has proven to be challenging to generate elements that recognize non-natural ligands. Bacterial periplasmic binding proteins have been proposed as a general scaffold to design receptor proteins for non-natural ligands, but despite various efforts, with only limited success. Here, we show how combinations of randomized mutagenesis and reporter screening improved the performance of a set of mutants in the ribose binding protein (RbsB) of , which had been designed based on computational simulations to bind the non-natural ligand 1,3-cyclohexanediol (13CHD). Randomized mutant libraries were constructed that used the initially designed mutants as scaffolds, which were cloned in an appropriate bioreporter system and screened for improved induction of the GFPmut2 reporter fluorescence in presence of 1,3-cyclohexanediol. Multiple rounds of library screening, sorting, renewed mutagenesis and screening resulted in 4.5-fold improvement of the response to 1,3-cyclohexanediol and a lower detection limit of 0.25 mM. All observed mutations except one were located outside the direct ligand-binding pocket, suggesting they were compensatory and helping protein folding or functional behavior other than interaction with the ligand. Our results thus demonstrate that combinations of ligand-binding-pocket redesign and randomized mutagenesis can indeed lead to the selection and recovery of periplasmic-binding protein mutants with non-natural compound recognition. However, current lack of understanding of the intermolecular movement and ligand-binding in periplasmic binding proteins such as RbsB are limiting the rational production of further and better sensory mutants.