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Synthetic protein scaffolds provide modular control over metabolic flux


Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/l) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.

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Figure 1: Employing metazoan machinery for modular control over pathway flux.
Figure 2: Heterologous protein-protein interaction domain/ligands provide direct control over enzyme stoichiometry of a synthetic complex.
Figure 3: Synthetic scaffolds built from modular protein-protein interaction domains provide modular control over metabolic pathway flux.
Figure 4: Enhancement of mevalonate production is scaffold-dependent.
Figure 5: Improved efficiency from pathway scaffolding allows higher titers to be achieved with faster growth of the production host.
Figure 6: Improvement of glucaric acid titers by scaffolding the bottleneck step enzymes Ino1 and MIOX.


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We thank A. Arkin, J. Dietrich, E. Dueber, L. Katz, and W. Whitaker for comments and discussion during the preparation of the manuscript. We also thank members of the Dueber and Keasling labs for experimental help and discussions. This work was supported by funding from UC Berkeley QB3 Institute (J.E.D.), National Science Foundation (NSF) Synthetic Biology Engineering Research Center grant no. EEC-0540879 (J.E.D., J.D.K, K.L.J.P., T.S.M.), NSF grant no. CBET-0756801 (J.E.D.), the Bill and Melinda Gates Foundation (J.D.K), Joint BioEnergy Institute (J.D.K.), the Office of Naval Research Young Investigator Program grant no. N000140510656 (K.L.J.P. and T.S.M.).

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Authors and Affiliations



J.E.D. conceived the project, designed all experiments and wrote the manuscript. J.E.D. and G.C.W. co-performed the experiments, and G.C.W. edited the manuscript. G.R.M. constructed and performed preliminary experiments used as a foundation for experiments included in this paper. T.S.M. contributed an experimental role for glucaric acid pathway experiments and edited the manuscript. C.J.P. contributed an experimental role for mass spectrometry experiments. A.V.U. contributed a supportive role in performing experiments for Supplementary Information Materials. K.L.J.P. contributed in development of the glucaric acid pathway and edited the manuscript. J.D.K. contributed general advice, especially with the mevalonate biosynthesis pathway, resource support and critical advice for manuscript preparation.

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Correspondence to John E Dueber.

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Jay Keasling has a financial interest in Amyris and LS9.

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Dueber, J., Wu, G., Malmirchegini, G. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27, 753–759 (2009).

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