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Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis


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.

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Figure 1: Setup for MS-based quantitative real-time analysis.
Figure 2: Quantitative metabolic real-time analysis of in vitro multienzyme network dynamics for a glycolytic network engineered for the production of DHAP with integrated cofactor regeneration.
Figure 3: Identification of rate-limiting steps for DHAP production in the upper part of E. coli's engineered in vitro glycolysis (on the basis of CFX of W3110 Δamn tpiA:Kn).
Figure 4: Operon construction and optimization of in vivo expression levels for improved in vitro DHAP production in a cell-free system obtained from W3110 Δamn tpiAkn provided with plasmids as indicated.


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We would like to thank N. Zamboni, U. Sauer, M. Oldiges and C. Wandrey for help with MS analyses. This work was supported by the EU-FP6 projects EUROBIOSYN and NANOMOT.

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M.B. did the experiments and analyzed the data and wrote the manuscript with S.P., who also supervised the work. R.P. and M.S. helped to construct the setup, and M.H. supervised part of the work.

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Correspondence to Sven Panke.

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The authors declare no competing financial interests.

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Supplementary Methods, Supplementary Figures 1–11 and Supplementary Tables 1–5 (PDF 2301 kb)

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Bujara, M., Schümperli, M., Pellaux, R. et al. Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis. Nat Chem Biol 7, 271–277 (2011).

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