Engineered Bacterial Production of Volatile Methyl Salicylate

The engineering of microbial metabolic pathways over the last two decades has led to numerous examples of cell factories used for the production of small molecules. These molecules have an array of utility in commercial industries and as in situ expressed biomarkers or therapeutics in microbial applications. While most efforts have focused on the production of molecules in the liquid phase, there has been increasing interest in harnessing microbes' inherent ability to generate volatile compounds. Here, we optimized and characterized the production of methyl salicylate, an aromatic compound found mainly in plants, using a common lab strain of E. coli. We utilized genetic components from both microbes and plants to construct the volatile metabolite circuit cassette. In order to maximize production, we explored expression of methyl salicylate precursors, upregulation of expression by increasing ribosomal binding strength and codon optimization of the methyl transferase gene obtained from plant Petunia x hybrida. Last, we validated and quantified the production of methyl salicylate with liquid chromatography or gas chromatography mass spectrometry (LC-MS or GC-MS) and found that the codon optimized strain with precursor supplementation yielded the highest production compared to the other strains. This work characterizes an optimized metabolite producing genetic circuit and sets the stage for creation of an engineered bacteria diagnostic to be used in volatile assays.