EAGER: Systematic Approach to Improve Probability of Metabolic Engineering Success

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The field of metabolic engineering continues to excel at producing specialty and commodity chemicals from simple, renewable feedstocks, but focus on the field's successes in literature and media neglect the larger picture: many metabolic engineering efforts produce industrially irrelevant titers in the early stages of research and sometimes throughout the duration of the project. This EAGER award will address the question of whether there are fundamental steps that can be taken early in metabolic engineering projects to maximize their success rates. The PI will address his hypothesis that the regulation of the amount of cofactors (small molecules that enable certain enzymes to function) is central to the ability to control metabolic pathways in cells in such a way as to enable the production of useful biochemicals for biomanufacturing purposes. In addition, the PI will train graduate and undergraduate students in interdisciplinary research and expose high school students to cutting edge research in this field.Technical description: The investigator will apply a broadly applicable, systematic approach to improve the success rate of metabolic engineering endeavors by performing a rigorous case study of the production of a complex biochemical in E. coli. The PI hypothesizes that the dynamic regulation of cofactor biosynthesis is key to metabolic engineering success, and will use the CRISPR interference (CRISPRi) system to achieve dynamic cofactor expression control in order to test his hypothesis. He will examine the fate of accumulating cofactor and cellular response via pulse-chase metabolic analysis and quantification with LC-MS/MS. Cellular response to cofactor accumulation will be monitored using comparative RNA-seq (transcriptomic) and iTRAQ (proteomic) analysis between wild-type and engineered strains. The PI will then use results from this case study to begin to generalize rules concerning co-factor regulation and impacts on metabolic engineering success.