Abstracts

Our group interests are in the broad areas of enzyme catalysis, enzyme engineering, systems biocatalysis, metabolic engineering and synthetic biology. This talk will highlight our contributions on creation of new luciferins and luciferases using enzyme engineering and biocatalysis approach. The first topic is the development of Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from Vibrio campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the non-engineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells. The second topic is the use of rational and computer-assisted protein engineering approach to enhance Fluc stability. We successfully generated two thermostable variants of Fluc with superior biochemical properties than the wild-type enzyme. These variants could be expressed in HEK293T and exhibited greater light signals than that of the native enzyme. For the last topic, we will discuss enzymatic cascades employing flavin-dependent dehalogenase or monooxygenases to catatalyze one-pot reactions to synthesize various new luciferins. We have developed Luminescence-related Method for Specific detection (LUMOS) which can be used for detection of pesticides and metabolites of pesticides in food, environmental, and biological samples called. LUMOS technology provides high sensitivity of detection in a range of ppt levels with the accuracy comparable to the gold standard methods of using HPLC-MS. We have used LUMOS technology in local communities in northern Thailand as a surveillance tool to detect pesticide contamination in food and environmental samples.