AbstractAs a next‐generation probiotic, Clostridium tyrobutyricum, which is considered obligate anaerobe, has been extensively considered a promising candidate for future use to promote health benefits. The industrial applications were limited by its weak tolerance to oxygen stress during fermentation. Here, we enhanced the oxidative stress tolerance of C. tyrobutyricum L319 by deleting the peroxide response regulator gene perR using an endogenous CRISPR‐Cas system. The mutant strain exhibited greatly improved growth when exposed to oxygen, which also showed a shortened lag phase compared with the control strain. Consistently, a concomitant increase in butyric acid productivity from 0.08 to 0.13 g/L/h in the presence of oxygen was observed. Decreased intracellular ROS level sand NAD+/NADH ratios were also observed in the mutant strain. Furthermore, transcriptome and binding target analysis revealed that genes encoding transcriptional regulators and proteins related to redox balance were upregulated. Changes in carbohydrate, amino acid and purine metabolisms upon oxygen exposure were also observed. Importantly, phylogenomic analysis indicated that PerR may be a universal engineering target in diverse Clostridium species. These findings will contribute to the development of robust C. tyrobutyricum strains for efficient butyric acid production in the food industry.
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