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bioRxiv · 10.1101/2020.07.27.222588

Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineered E. coli

Li, S.·Moreb, E. A.·Ye, Z.·Hennigan, J. N.·Baez Castellanos, D.·Yang, T.·Lynch, M. D.
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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 two-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 16 fold and 100 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 a new NADPH generation pathway, namely 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 enoyl-ACP reductase and glucose-6-phosphate dehydrogenase to broadly enable improved NADPH dependent bioconversions. HighlightsO_LIDecreases in NADPH pools lead to increased NADPH fluxes C_LIO_LIPyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo. C_LIO_LIDynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH flux C_LIO_LIXylitol titers > 200g/L in fed batch fermentations with xylose as a sole feedstock. C_LI

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Li, S., Moreb, E. A., Ye, Z., Hennigan, J. N., Baez Castellanos, D., Yang, T., Lynch, M. D.. 2020-07-27. Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineered E. coli. https://doi.org/10.1101/2020.07.27.222588

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