UC Riverside

Methods and approaches for regulating gene expression are central to the success of metabolic engineering. In many cases, an inducible transcription factor or repression system is used to control the expression of one or more target genes. In yeast, a range of such systems are available, but activation of these systems requires a change in culture media, carbon source, or the addition of a costly chemical ligand. Chemically induced dimerization (CID) system provides a mechanism whereby two proteins form a stable heterodimer only in the presence of a small molecule. CID sensors are amenable to engineering, and through the fusion of DNA binding, activator, and repressor domains can be converted from a signaling pathway to a system for inducible gene activation or repression. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. We then adapted sensors as a set of orthogonal synthetic transcription factors that can be used in both gene activation and repression formats and that are inducible by ABA or the agrochemical mandipropamid. We present the optimization of this new gene regulation system; demonstrate use in S. cerevisiae as well as the non-conventional yeasts Kluyveromyces marxianus; and, use the system as a means to control central pyruvate metabolism in K. marxianus by simultaneously activating and repressing pyruvate decarboxylase and pyruvate dehydrogenase. This new synthetic transcription factor system opens a new approach to controlling gene expression that can be broadly used across various yeast species.