Respiration is essential for aerobic growth of Zymomonas mobilis ZM4.

Abstract

Zymomonas mobilis is an alpha-proteobacterium that is a promising platform for industrial scale production of biofuels due to its efficient ethanol fermentation and low biomass generation. Z. mobilis is aerotolerant and encodes a complete respiratory electron transport chain, but the benefit of respiration for growth in oxic conditions has never been confirmed, despite decades of research. Growth and ethanol production of wild-type Z. mobilis is poor in oxic conditions indicating that it does not benefit from oxidative phosphorylation. Additionally, in previous studies, aerobic growth improved significantly when respiratory genes were disrupted (ndh) or acquired point mutations (cydA and cydB), even if respiration was significantly reduced by these changes. Here, we obtained clean deletions of respiratory genes ndh and cydAB, individually and in combination, and showed, for the first time, that deletion of cydAB completely inhibited O2 respiration and dramatically reduced growth in oxic conditions. Both respiration and aerobic growth were restored by expressing a heterologous, water-forming NADH oxidase, noxE. Oxygen can have many negative effects, including formation of reactive oxygen species (ROS) or directly inactivating oxygen sensitive enzymes. Our results suggest that the effect of molecular oxygen on enzymes had a greater negative impact on Z. mobilis than formation of ROS. This result shows that the main role of the electron transport chain in Z. mobilis is reducing the intracellular concentration of molecular oxygen, helping to explain why it is beneficial for Z. mobilis to use electron transport chain complexes that have little capacity to contribute to oxidative phosphorylation. IMPORTANCE A key to producing next-generation biofuels is to engineer microbes that efficiently convert non-food materials into drop-in fuels, and to engineer microbes effectively, we must understand their metabolism thoroughly. Zymomonas mobilis is a bacterium that is a promising candidate biofuel producer, but its metabolism remains poorly understood, especially its metabolism when exposed to oxygen. Although Z. mobilis respires with oxygen, its aerobic growth is poor, and disruption of genes related to respiration counterintuitively improves aerobic growth. This unusual result has sparked decades of research and debate regarding the function of respiration in Z. mobilis. Here, we used a new set of mutants to determine that respiration is essential for aerobic growth and likely protects the cells from damage caused by oxygen. We conclude that the respiratory pathway of Z. mobilis should not be deleted from chassis strains for industrial production because this would yield a strain that is intolerant of oxygen, which is more difficult to manage in industrial settings.