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Identifying and overcoming the limitations preventing efficient high-yield production of chemicals remain important tasks in metabolic engineering. In an attempt to rewire Corynebacterium glutamicum to produce ethanol, we attained a low yield (63% of the theoretical) when using resting cells on glucose, and large amounts of succinate and acetate were formed. To prevent the by-product formation, we knocked out the malate dehydrogenase and replaced the native E3 subunit of the pyruvate dehydrogenase complex (PDHc) with that from Escherichia coli , which is active only under aerobic conditions. However, this tampering resulted in a 10-times-reduced glycolytic flux as well as a greatly increased NADH/NAD + ratio. When we replaced glucose with fructose, we found that the glycolytic flux was greatly enhanced, which led us to speculate whether the source of reducing power could be the pentose phosphate pathway (PPP) that is bypassed when fructose is metabolized. Indeed, after shutting down the PPP by deleting the zwf gene, encoding glucose-6-phosphate dehydrogenase, the ethanol yield on glucose increased significantly, to 92% of the theoretical. Based on that, we managed to rechannel the metabolism of C. glutamicum into d-lactate with high yield, 98%, which is the highest that has been reported. It is further demonstrated that the PPP-inactivated platform strain can offer high-yield production of valuable chemicals using lactose contained in dairy waste as feedstock, which paves a promising way for potentially turning dairy waste into a valuable product. IMPORTANCE The widely used industrial workhorse C. glutamicum possesses a complex anaerobic metabolism under nongrowing conditions, and we demonstrate here that the PPP in resting C. glutamicum is a source of reducing power that can interfere with otherwise redox-balanced metabolic pathways and reduce yields of desired products. By harnessing this physiological insight, we employed the PPP-inactivated platform strains to produce ethanol, d-lactate, and alanine using the dairy waste whey permeate as the feedstock. The production yield was high, and our results show that inactivation of the PPP flux in resting cells is a promising strategy when the aim is to use nongrowing C. glutamicum cells for producing valuable compounds. Overall, we describe the benefits of disrupting the oxidative PPP in nongrowing C. glutamicum and provide a feasible approach toward waste valorization.

applied and environmental microbiology

Disruption of the Oxidative Pentose Phosphate Pathway Stimulates High-Yield Production Using Resting Corynebacterium glutamicum in the Absence of External Electron Acceptors