A High-Throughput System to Screen For Methyl Halide Transferase Expression and Activity
Maureen Montes, Juan Armas, Lika Chhit, James Khoi Nguyen, Nehemiah Setiawan
Department of Chemistry & Biochemistry
Faculty Supervisor: Raymond Esquerra
As fossil fuel use continues to drive environmental damage and global warming, the need to transition to sustainable alternatives like biofuels has become crucially important. Second generation biofuels, derived from nonfood biomass, help offer a carbon neutral solution that can help serve to transition away from fossil fuels. Methyl halides are of interest to biofuel research as they serve as chemical intermediates in the creation of biofuels. They are naturally produced by marine alga, fungi, and halophytic plants. The enzyme responsible for methyl halide production is methyl halide transferase (MHT). In this study, we will be using a Batis maritima MHT variant due to its relatively high enzymatic activity in using the methyl group from S-adenosyl methionine (SAM) to methylate halides. Naturally occurring MHT is slow growing and has low methyl halide yields, so expression in an industrial organism, such as E.coli bacteria is critical for commercial methyl halide production. In addition we are developing a high-throughput colorimetric system to quantify MHT expression and activity. MHT with a monomeric red fluorescent protein (mCherry) will be used to aid in visualization of expression in vivo creating a novel fusion protein. To measure activity, we will develop a colorimetric assay using 4-(4-nitrobenzyl)pyridine (NBP) to detect methyl-halide production of our enzymes. Our goal of the project is to demonstrate a successful assay that can be made high-throughput and produce results comparable to gas chromatography-mass spectrometer (GC-MS).