Cells employ a variety of different sensor biomolecules to dynamically evaluate their environments and trigger appropriate metabolic responses. The ability to program cells with engineered molecules that can sense structural and chemical events is a critical technology for many of the challenges present in biotechnology and medical research. The long term goal of this proposal is to develop platforms for the design of molecular sensors that will be used for the noninvasive, real-time detection of intracellular metabolite concentrations at the population or single cell level and the construction of dynamic control loops for regulating enzyme levels. This proposal focuses on the development of an enabling technology: a universal platform for the design of molecular sensors that can be tailor-made to each pathway or metabolic network of interest and whose properties can be programmed to detect different concentration ranges. The specific objectives are to: (i) generate RNA-based molecular sensors to key metabolites along the benzylisoquinoline alkaloid (BIA) pathway; (ii) engineer Saccharomyces cerev/s/aeto synthesize early BIAs; i) apply the sensors to the noninvasive, real-time detection of key metabolite levels; (iv) expand the sensor design to the dynamic regulation of enzyme levels in response to metabolite accumulation; and (v) solidify a strong base in this field and encourage its evolution by training and educating scientists through integrated research and educational plans. This proposal will incorporate molecular design strategies previously developed in our laboratory for the construction of the proposed RNA-based sensors. Standard molecular biology and biochemistry techniques will be used to generate the transgenic yeast strains. Analytic methods such as high performance liquid chromatography, capillary electrophoresis, and surface plasmon resonance will be used to characterize sensor response and metabolic flux response in engineered cells. The proposed technology has applications in diverse areas of health-related research such as metabolic engineering for drug synthesis, cell-based sensors, localized imaging and detection tools, 'smart' therapeutics, and in vitro diagnostics. The described programmable molecular sensors will have a significant impact in metabolic engineering in that they present a unique and universal platform for the real-time, noninvasive detection of dynamic metabolite concentrations and thereby pathway flux both in cell populations or single cells. This technology will be a powerful research tool for probing and advancing understanding of the dynamic cellular metabolite environment. Due to the flexibility of the sensor platform and the ability of these molecules to 'sense' specific concentrations, these sensors will be powerful research tools in a variety of other applications.
| Michener, Joshua K; Thodey, Kate; Liang, Joe C et al. (2012) Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways. Metab Eng 14:212-22 |
| Hawkins, Kristy M; Smolke, Christina D (2008) Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat Chem Biol 4:564-73 |
