New high-throughput screening technologies to improve cannabinoid production in yeast.

SUMMARY Natural products derived from plants have played crucial roles in the treatments of many diseases throughout historyHoweverscarce natural sources and complex chemical synthesis have limited research and development and increased costs on these productsAn example of this is the non psychoactive cannabinoids found in cannabis plantsThere are overlow abundant cannabinoids that are known to be safe and have beneficial effects ranging from bone growth to neuroprotectionYet agricultural production of these cannabinoids is not environmentally or economically sustainableThustheir therapeutic potentials are unfortunately limited by their availabilities for research and commercial productionAlternative biosynthetic production routes in Scerevisiae are being investigated to increase accessibility to these high potential therapeuticsFurthermassive libraries of yeast mutants are being generated for the purpose of identifying cellular pathways that enhance cannabinoid biosynthesisHowevera major bottleneck in this biosynthetic approach is the lack of high throughput screening technology that can screen these massive libraries efficientlyCurrent analytically methods are low throughputexpensivecumbersomeand complexAnother problem is that these methods do not enable individual mutant cells to be screened for key metabolite productionwhich is widely recognized as a limiting problem in metabolic engineeringSingle cell isolation techniques have been explored as high throughput means to quickly identify and separate metabolically and physiologically favorably mutants for further strain evolutionThereforefluorescent sensors that can inform on the intracellular concentrations of valuable metabolites in living cells and enable high throughput cell sorting would be highly useful tools for any industrial production of natural productsIn this applicationwe will use our fluorescent aptamer technology to develop genetically encoded sensors that can active fluorescence upon binding to target cannabinoid metabolitesThese sensors can then be expressed in yeast to monitor cannabinoid production levels in real time and used in flow cytometry based screening applicationsTaken togetherthe experiments in this application provide the foundation for a novel approach that can be widely used to improve industrial production of natural products by allowing culture conditions or genetically modified organisms with improved production characteristics to be rapidly identified PROJECT NARRATIVE There are overcannabinoids produced in cannabis plants at low abundance that are known to be safe and have a wide range of proven medical benefitsBiosynthetic production approaches are currently being explored to increase natural cannabinoids productionbut existing product screening methods cannot keep up with recent genomic advances in metabolic engineeringThuswe propose to develop a HTS platform that will enable researchers to rapidly identify genetically modified cells that exhibit improved biosynthesis of non psychoactive cannabinoids and improve industrial production of natural products