I will present two projects from my laboratory, whose aim is to create biochemical technologies to improve global health and environmental quality.
First, I will discuss a series of chemical biology and biophysical advances we have made toward understanding and improving CRISPR- and recombinase-based molecular detection for infectious disease monitoring. First, we discover that recombinase-based DNA amplification could be governed by liquid-liquid phase separation, where the condensate formation enhances the nucleic acid amplification process. Through volumetric and high-resolution imaging, we identify the recombinase component of the reaction as the key regulator orchestrating distinct core-shell arrangements of proteins within multiphase condensates, creating ideal microenvironments for amplification. Second, we use protein engineering techniques including unnatural amino acid incorporation with genetic code expansion to dissect the principles behind the multiphasic organization of recombinase-anchored condensates. This further leads to identification of recombinase mutants with distinct phase separation propensities and improved reaction efficiency. Third, such improved recombinase-based amplification is coupled to CRISPR-based detection and applied by our team to clinical and environmental detection of neglected fatal tropical disease pathogens, whose monitoring and diagnosis are challenging yet critically important for timely treatment.
I will then discuss our discovery of MG8, a mesophilic polyethylene terephthalate (PET) hydrolase from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different salinity and temperature conditions, outperforming several naturally occuring and engineered hydrolases in degrading PET. To expand functionalities of MG8, we employ a mechanism-based trap via genetic encoding of 2,3-deaminopropionic acid (DAP) to replace the catalytic serine residue of MG8, thereby converting a PET-degrading enzyme into a covalent binder of PET. We show that MG8(DAP) can be used to attach protein cargos to PET as well as other polyester plastics and can serve as a modular platform for bio-functionalization of PET and polyesters. Analyses of the crystal structure of MG8 reveal features which allow the enzyme to function well at mesophilic temperatures and identify hotspots for further enzyme engineering using a high-throughput mass spectrometry approach. An engineered MG8 (eMG8) is coupled to cell-based upcycling, where PET degradation products are biochemically converted into new chemicals through engineered metabolic pathways.
