Disruption of the oxidative Pentose Phosphate Pathway stimulates high-yield production using resting Corynebacterium glutamicum in the absence of external electron acceptors

Jing Shen, Jun Chen, Christian Solem, Peter Ruhdal Jensen, Jianming Liu*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Identifying and overcoming the limitations preventing efficient high-yield production of chemicals remain to be an important task in metabolic engineering. In an attempt to rewire Corynebacterium glutamicum into producing 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-products formation, we knocked out the malate dehydrogenase and replaced the native E3 subunit of the pyruvate dehydrogenase complex (PDHc) with the one from Escherichia coli, which is only active under aerobic conditions. However, this tampering resulted in a 10 times reduced glycolytic flux as well as a greatly increased NADH/NAD+ ratio. By substituting 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 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 re-channel 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 plaform 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 value.Importance The widely used industrial workhorse C. glutamicum possesses a complex anaerobic metabolism under non-growing 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 is high and our results show that inactivation of the PPP flux in resting cells is a promising strategy when the aim is to use non-growing C. glutamicum cells for producing valuable compounds. Overall, we described the benefits to disrupt the oxidative PPP in non-growing C. glutamicum and provide a feasible approach towards waste valorization.

Original languageEnglish
JournalApplied and Environmental Microbiology
ISSN0099-2240
DOIs
Publication statusAccepted/In press - 2020

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