Discovery of β-hydroxybutyrate binding proteins and their engineering as enhanced biological recognition elements for continuous biosensing

β-hydroxybutyrate (BHB) is a key physiological metabolite and biomarker, serving as a primary energy source for various cells and tissues during low glucose availability. There is significant academic and clinical interest in continuous BHB biosensing, with applications in the study and management of metabolic disorders. However, continuous monitoring of BHB remains challenging due to a lack of suitable biological recognition elements. Through this work, we discovered insights at the intersection of bacterial metabolism and biosensing technology through the identification of BHB binding proteins (BHBBPs), a new group of periplasmic solute-binding proteins (SBPs) that specifically interact with BHB. After recombinant production of BHBBPs, the specificity and affinity of each protein were thoroughly evaluated through an extensive analytical pipeline, including differential scanning fluorimetry (thermal shift assay) and tryptophan fluorescence spectroscopy (TFS). Binding data obtained from TFS revealed wild-type dissociation constants in the nano- to low micromolar range and suggested D-BHB, the natural enantiomer of BHB and human metabolite, as the preferred ligand for most tested proteins, including the proteins “Tt.2” from Thermus thermophilus, “EDC24” from Paucimonas lemoignei, and “NovoS” from Noviherbaspirillum sedimenti. These findings were corroborated further via isothermal calorimetry. Overall, 7 unique wild-type proteins were identified and biochemically characterized. Consequently, there is strong evidence for the classification of over 4000 putative proteins without previously-annotated function as hydroxyalkanoate binding proteins. These SBPs may facilitate cellular translocation of BHB through the membrane of certain prokaryotic organisms. Given the physiological range of BHB spans the micro- to millimolar range, it was necessary to adjust the binding constants of wild-type BHBBPs to improve their suitability for BHB biosensing. Structural analyses facilitated by machine learning based structural prediction, AlphaFold2, along with molecular docking and dynamics simulations, were used to identify key residues in the binding pocket and model the conformational dynamics of substrate binding/unbinding. Targeted point mutations in conserved polar residues of several BHBBPs reduced the binding affinity of the wild-type BHBBPs EDC24 and NovoS by more than 50-fold. Thus, BHBBPs were engineered through site-directed mutagenesis to achieve a collective affinity range covering nearly 4 orders of magnitude, from nanomolar to millimolar recognition of BHB. Leveraging the versatility of SBP-based biosensing, these receptors and their broad affinity range could facilitate the development of effective bioanalytical tools for BHB detection across diverse physiological environments.