Dynamic control over feedback regulatory mechanisms improves NADPH flux and xylitol biosynthesis in engineered E. coli |
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| Affiliation: | 1. Laboratory for Bioinformatics, Graduate School of Systems Biosciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan;2. Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan;1. Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, United States;2. State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, School of Life Sciences, Hubei University, Wuhan, Hubei, 430062, PR China;3. Advanced Biofuels & Bioproducts Process Development Unit, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States;4. Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States;5. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, 94720, United States;6. School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang, 311121, PR China;7. Novo Nordisk Foundation Center for Biosustainability, Technical University Denmark, DK 2970 Horsholm, Denmark;8. Center for Synthetic Biochemistry, Shenzhen Institutes for Advanced Technologies, Shenzhen, Guangdong, 518055, PR China |
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| Abstract: | We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways. |
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| Keywords: | Xylitol NADPH Dynamic metabolic control Metabolic dysregulation |
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