Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations |
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| Authors: | Keith E.J. Tyo Curt R. Fischer Fritz Simeon Gregory Stephanopoulos |
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| Affiliation: | 3. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;4. Center for Cancer Research, Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129;5. Department of Medicine, Division of Hematology-Oncology, Massachusetts General Hospital, Boston, Massachusetts 02115;6. Department of Life Sciences, University of Coimbra, 3004-517 Coimbra, Portugal;1. Masdar Institute of Science and Technology, Water and Environmental Engineering Program, P.O. Box 54224, Abu Dhabi, United Arab Emirates;2. Massachusetts Institute of Technology, Department of Chemical Engineering, 77 Massachusetts Avenue, Cambridge, MA 02139, USA;1. AnoxKaldnes, Veolia Water Technologies, Klosterängsvägen 11A, 226 47 Lund, Sweden;2. Dept. of Biotechnology and Biosystems, Veolia Recherche et Innovation (VERI) – Centre de Recherche de Maisons Laffitte, Chemin de la Digue – BP 76, 78603 Maisons-Laffitte, France;1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States;2. Whitehead Institute for Biomedical Research, W.M. Keck Imaging Facility, 9 Cambridge Center, Room 447, Cambridge, MA 02142, United States;1. Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China;2. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States;3. School of Life Sciences, Tsinghua University, Beijing 100084, China |
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| Abstract: | Poly-3-hydroxybutyrate (PHB) titers in Escherichia coli have benefited from 10+ years of metabolic engineering. In the majority of studies, PHB content, expressed as percent PHB (dry cell weight), is increased, although this increase can be explained by decreases in growth rate or increases in PHB flux. In this study, growth rate and PHB flux were quantified directly in response to systematic manipulation of (1) gene expression in the product-forming pathway and (2) growth rates in a nitrogen-limited chemostat. Gene expression manipulation revealed acetoacetyl-CoA reductase (phaB) limits flux to PHB, although overexpression of the entire pathway pushed the flux even higher. These increases in PHB flux are accompanied by decreases in growth rate, which can be explained by carbon diversion, rather than toxic effects of the PHB pathway. In chemostats, PHB flux was insensitive to growth rate. These results imply that PHB flux is primarily controlled by the expression levels of the product forming pathway and not by the availability of precursors. These results confirm prior in vitro measurements and metabolic models and show expression level is a major affecter of PHB flux. |
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