title: Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis.
abstract: Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli's highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold-increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Bujara
authors.forename: Michael
authors.initials: M
authors.lastname: Schümperli
authors.forename: René
authors.initials: R
authors.lastname: Pellaux
authors.forename: Matthias
authors.initials: M
authors.lastname: Heinemann
authors.forename: Sven
authors.initials: S
authors.lastname: Panke
mesh_terms: D004099:Dihydroxyacetone Phosphate / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006019:Glycolysis; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D013997:Time Factors
chemical_list: D004099:Dihydroxyacetone Phosphate