OxyR regulates the oxidative stress response in Zymomonas mobilis during oxic growth and anoxic biofuel fermentation

The bacterium Zymomonas mobilis is widely studied for its potential as an industrial biofuel producer. Anoxic fermentation by Z. mobilis in lignocellulosic hydrolysate can generate bioethanol from renewable plant biomass. In this study, we deleted a gene from the Z. mobilis genome encoding a homolog of OxyR, a transcription factor that activates an oxidative stress response in bacteria to reduce reactive oxygen species (ROS). Deletion of this transcription factor inhibited growth of Z. mobilis in oxic, but not anoxic, conditions in laboratory media. A ROS probe revealed that the oxyR response is required to reduce intracellular ROS during oxic growth. Importantly for biofuel production, the absence of oxyR inhibited growth and delayed ethanol production during anoxic hydrolysate fermentation. To determine the source of oxidative stress in hydrolysate, we grew{Delta} oxyR in a synthetic hydrolysate containing known inhibitors found in hydrolysate. There was no growth defect in{Delta} oxyR in the synthetic hydrolysate, indicating that known inhibitory compounds are not the source of anoxic oxidative stress. We determined that Ammonia-Fiber Expansion (AFEX) switchgrass hydrolysate contains significant peroxide concentrations. Addition of catalase to hydrolysate improves growth of both{Delta} oxyR and wild-type Z. mobilis in hydrolysate. This study uncovers an important source of stress to Z. mobilis during biofuel fermentation. ImportanceFermentation of non-food biomass is a promising avenue for sustainable production of fuels and chemicals, but several challenges currently limit applicability of this technology. One major hurdle is that when biomass is deconstructed into a fermentable form, many byproducts are generated that inhibit microbial fermentation. Here, we investigated how a fermentative bacterium, Zymomonas mobilis, experiences oxidative stress during anoxic biomass fermentation, and identified genes important in this response. These findings provide a better understanding of the stresses faced by Z. mobilis during biofuel production. Fully understanding the effects of hydrolysate on biofuel-producing microbes is crucial for optimizing production and making carbon-neutral fuel a reality.