Biosensor-Based Enzyme Engineering Approach Applied To Psicose Biosynthesis

Bioproduction of chemical compounds is of great interest formodern industries, as it reduces their production costs and ecological impact. With the use of synthetic biology, metabolic engineering and enzyme engineering tools, the yield of production can be improved to reachmass production and cost-effectiveness expectations. In this study, we explore the bio-production of D-psicose, also known as D-allulose, a rare non-toxic sugar and a sweetener present in nature in low amounts. D-psicose has interesting properties and seemingly the ability to fight against obesity and type 2 diabetes. We developed a biosensor-based enzyme screening approach as a tool for enzyme selection that we benchmarked with the Clostridium cellulolyticum D-psicose 3-epimerase for the production of D-psicose from D-fructose. For this purpose, we constructed and characterized seven psicose responsive biosensors based on previously uncharacterized transcription factors and either their predicted promoters or an engineered promoter. In order to standardize our system, we created the Universal Biosensor Chassis, a construct with a highlymodular architecture that allows rapid engineering of any transcription factor-based biosensor. Among the seven biosensors, we chose the one displaying themost linear behavior and the highest increase in fluorescence fold change. Next, we generated a library of D-psicose 3-epimerase mutants by error-prone PCR and screened it using the biosensor to select gain of function enzymemutants, thus demonstrating the framework's efficiency.