Dynamic Metabolic Control Improves NADPH Flux and Xylitol Biosynthesis in Engineered E. coli

Metabolic engineering has broad applications in the biosynthesis of a wide variety of products, including biofuels, pharmaceuticals, and food chemicals. Xylitol is a sugar alcohol with a primary use as a sweetener and is produced at ~125,000 tons annually. Compared with the chemical synthesis method, biosynthetic production of xylitol has the potential to decrease costs and be more environmentally friendly. Xylitol can be synthesized from xylose via an NADPH dependent reductase. In our work, we reported improved NADPH flux and xylitol biosynthesis in engineered E. coli using 2-stage dynamic metabolic control, where products are made in a metabolically productive phosphate depleted stationary phase. The implementation of this approach relies on the combined use of controlled proteolysis and gene silencing, using degron tags and CRISPR interference, 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.

Keywords: Xylitol, NADPH, Metabolic engineering, Biosynthesis, Dynamic metabolic control

Reference:

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