Mapping and Redirecting Metabolic Fluxes to Attain Significantly Lessen Oxygen Requirements, Acid Production, and Enhanced Recombinant Culture Productivity

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9622821 Ataai This proposal is on research which addresses one of the bottlenecks in protein production frequently reported for recombinant E. coli, namely overflow production of semi-oxidized sugars and carbohydrates (e.g. acetate) which reduces the total carbon yield and also indicates inefficient metabolic biosynthetic/catabolic fluxes. The specific goals of this research are to: (1) build on the investigators' Bacillus research to experimentally validate (using NMR) the feasibility of optimized flux distribution centered around pyruvate kinase (PYK), (2) transfer the yield-increasing strategy to E. coli by deleting PYK isozymes and analyze via metabolic flux analysis, again coupled with NMR, (3) determine scale-up potential in terms of mutant E. coli concentration, recombinant protein production, and oxygen consumption, and, (4) evaluate whether there is a link between energy-consuming proteolysis and ATP-generation as shown in high glucose feed E. coli cultures. ***