Tatianna Travieso, Caroline Willis
Faculty Sponsor: Dr. A. Malcolm Campbell
Metabolic engineering can harness metabolic pathways for the production of useful metabolites with applications in the biofuel, pharmaceutical, or industrial chemical industries. We published a metabolic engineering approach called programmed evolution that introduces variation in regulatory elements for the expression of enzymes that control orthogonal metabolic pathways, which employs a riboswitch-based fitness module to transduce the production of a desired metabolite into antibiotic resistance. We validated programmed evolution by optimizing the conversion of caffeine to theophylline by caffeine demethylase, and to avoid several limitations associated with this method, we are developing an in vitro approach to programmed evolution. We designed, constructed, and have begun testing a novel fitness module that responds to the production of theophylline produced by the action of caffeine demethylase produced by cell-free protein synthesis (CFPS). The fitness module encodes a fusion protein composed of three functional units: a Gal4 DNA binding domain (targeting binding sites on a library of DNA regulatory elements for the expression of caffeine demethylase), streptavidin (binding to avidin beads for physical separation of selected DNA regulatory elements), and, in between these two domains, GFP (serving as a biosensor). Using this in vitro selection strategy, we plan to pursue the optimization of CFPS for the discovery of efficient metabolic pathways of interest.