Empirical testing of how changing regulatory module membership affects module function within central metabolism

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In teamwork a group uses coordinated directions to maximize their success. This concept is also thought to be at the core of how metabolism functions in life. A team of genes/enzymes are organized by directions from a transcription factor to function as a cohesive unit. Understanding how biological modules function is essential for all efforts to either engineer or improve organisms for nearly any purpose. However, this concept has not actually been tested. This project uses genome engineering approaches to cause individual genes to lose their ability to function as a gene and test how the module functions. This effort trains graduate students and postdoctoral fellows to integrate synthetic biology engineering with genomics and computational data analysis. The project provides direct research experience to first-generation and transfer undergraduate researchers to broaden the future pool of researchers.This project tests a foundational assumption of biology, that the members of a network are coordinately regulated to optimize the trait being produced by the network and to maximize efficiency. However, it is not clear that all of the genes in a holoenzyme or metabolic pathway need to be coordinated or that there is some threshold at which genes can be dropped from a regulatory module without functional consequence. This project uses genome engineering to directly test this theory by manipulating promoters to remove genes and pathways from specific regulons. The effect of these manipulations is measured with a wide array of phenotyping platforms to test consequences at all levels of a trait from transcript to metabolite to whole organism. These results allow a direct empirical test of how module membership at the holoenzyme, pathway or super-pathway level influences those modules' function. The project directly measures how many members of a regulon must be coordinated to create the proper trait response. These results contribute to the development of new theories and concepts to improve the understanding of how organisms function.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.