S69: Design and use of a novel mevalonate-responsive gene regulator for engineering improved mevalonate synthesis in E. coli

Customized metabolite-responsive gene regulators find applications as molecular reporters and as novel gene control tools in synthetic biology and metabolic engineering. In this study the AraC regulatory protein of E. coli was engineered to control gene expression in response to mevalonate, a key intermediate in the synthesis of cholesterol and high-value terpenoid compounds. Residue positions in the AraC binding pocket were randomized and mutants were expressed in E. coli cells containing a pBAD-GFP reporter construct. AraC variants having desired regulatory properties in the presence and absence of mevalonate were isolated via FACS. A unique mevalonate reporter system was subsequently used to screen for improved mevalonate production in E. coli harboring a heterologous mevalonate synthesis operon with randomized ribosomal binding site sequences. Colony screening yielded an RBS mutant resulting in ~10-fold improved mevalonate production.