AUTHOR=Maeda Tomoya , Koch-Koerfges Abigail , Bott Michael 

TITLE=Relevance of NADH Dehydrogenase and Alternative Two-Enzyme Systems for Growth of Corynebacterium glutamicum With Glucose, Lactate, and Acetate

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=8

YEAR=2021

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.621213

DOI=10.3389/fbioe.2020.621213

ISSN=2296-4185

ABSTRACT=<p>The oxidation of NADH with the concomitant reduction of a quinone is a crucial step in the metabolism of respiring cells. In this study, we analyzed the relevance of three different NADH oxidation systems in the actinobacterial model organism <italic>Corynebacterium glutamicum</italic> by characterizing defined mutants lacking the non-proton-pumping NADH dehydrogenase Ndh (Δ<italic>ndh</italic>) and/or one of the alternative NADH-oxidizing enzymes, <sc>L</sc>-lactate dehydrogenase LdhA (Δ<italic>ldhA</italic>) and malate dehydrogenase Mdh (Δ<italic>mdh</italic>). Together with the menaquinone-dependent <sc>L</sc>-lactate dehydrogenase LldD and malate:quinone oxidoreductase Mqo, the LdhA-LldD and Mdh-Mqo couples can functionally replace Ndh activity. In glucose minimal medium the Δ<italic>ndh</italic> mutant, but not the Δ<italic>ldhA</italic> and Δ<italic>mdh</italic> strains, showed reduced growth and a lowered NAD<sup>+</sup>/NADH ratio, in line with Ndh being the major enzyme for NADH oxidation. Growth of the double mutants Δ<italic>ndh</italic>Δ<italic>mdh</italic> and Δ<italic>ndh</italic>Δ<italic>ldhA</italic>, but not of strain Δ<italic>mdh</italic>Δ<italic>ldhA</italic>, in glucose medium was stronger impaired than that of the Δ<italic>ndh</italic> mutant, supporting an active role of the alternative Mdh-Mqo and LdhA-LldD systems in NADH oxidation and menaquinone reduction. In <sc>L</sc>-lactate minimal medium the Δ<italic>ndh</italic> mutant grew better than the wild type, probably due to a higher activity of the menaquinone-dependent <sc>L</sc>-lactate dehydrogenase LldD. The Δ<italic>ndh</italic>Δ<italic>mdh</italic> mutant failed to grow in <sc>L</sc>-lactate medium and acetate medium. Growth with <sc>L</sc>-lactate could be restored by additional deletion of <italic>sugR</italic>, suggesting that <italic>ldhA</italic> repression by the transcriptional regulator SugR prevented growth on <sc>L</sc>-lactate medium. Attempts to construct a Δ<italic>ndh</italic>Δ<italic>mdh</italic>Δ<italic>ldhA</italic> triple mutant were not successful, suggesting that Ndh, Mdh and LdhA cannot be replaced by other NADH-oxidizing enzymes in <italic>C. glutamicum</italic>.</p>