Biosensors provide valuable parts for synthetic biology; however, few platforms exist that are malleable to a variety of ligands, are portable between species, or can be used in simple in-vitro diagnostics. Many tools exist for domesticating and engineering non-conventional microorganisms, but only a few biosensing systems have been developed for use in such species. In our previous work, we demonstrated that the plant PP2C hormone sensing system (PYR1/HAB1) can be reprogrammed to respond to new ligands1, can be ported into Saccharomyces cerevisiae and Arabidopsis thaliana2, and can be used in ELISA-like assays. Here, we port this system into non-model hosts, greatly expand the classes of ligands that can bind to PYR1 mutants, and use the PYR1/HAB1 system for dynamic regulation to optimize biochemical production3. We have screened both computationally generated mutants as well as DNA shuffle libraries and discovered PYR1 biosensors for a wide array of terpenes. From all 20 identified terpenes one or two enzymatic steps away from geranyl pyrophosphate, we obtained 29 unique sensors for 4 of the screened chemicals. In the thermotolerant, fast-growing yeast Kluyveromyces marxianus, we optimized sensor component expression for >40-fold response fluorescent output, used growth-coupled terpene sensors to detect intracellular terpene production, and used a dual activation/repression biosensor system as dynamic regulators for cell growth/production to enhance ethyl acetate production. We have shown that the portable and adaptable PYR1 biosensors serve as a potent tool in synthetic biology.
