PATHWAY ANALYSIS, ENGINEERING, AND PHYSIOLOGICAL CONSIDERATIONS FOR REDIRECTING CENTRAL METABOLISM

The rate and yield of producing a metabolite is ultimately limited by the ability to channel metabolic fluxes from central metabolism to the desired biosynthesis pathway. Redirection of central metabolism thus is essential to high-efficiency production of biochemicals. This task begins with pathway analysis, which considers only the stoichiometry o f the reaction networks but not the regulatory mechanisms. An approach extended from convex analysis is used to determine the basic reaction modes, which allows the determination of optimal and suboptimal flux distributions, yield, and the dispensable sets of reactions. Genes res ponsible for reactions in the same dispensable set can be deleted simu ltaneously. This analysis serves as an initial guideline for pathway e ngineering. Using this analysis, we successfully constructed an Escher ichia coli strain that can chan nel the metabolic flow from carbohydra te to the aromatic pathway with theoretical yield. This analysis also predicts a novel cycle involving phosphoenolpyruvate (PEP) carboxykina se (Pck) and the glyoxylate shunt, which can substitute the tricarboxy lic acid cycle with only slightly less efficiency. However, the full c ycle could not be confirmed in vivo, possibly because of the regulator y mechanism not considered in the pathway analysis. In addition to the kinetic regulation, we have obtained evidence suggesting that central metabolites are involved in specific regulons in E. coli. Overexpress ion of PEP-forming enzymes (phosphoenolpyruvate synthase [Pps] and Pck ) stimulates the glucose consumption rate, represses the heat shock re sponse, and negatively regulates the Ntr regulon. These results sugges t that some glycolytic intermediates may serve as a signal in the regu lation of the phosphotransferase system, heat shock response, and nitr ogen regulation. However, the role of central metabolites in these reg ulations has not been determined conclusively. (C) 1996 John Wiley & S ons, Inc.