Elucidating the mechanisms of styrene tolerance in Gluconobacter oxydans using adaptive laboratory evolution

Styrene-containing wastewater, primarily originating from petrochemical industries, has become a significant environmental concern due to its increasingly detriment to fresh water. Styrene biodegradations in aquatic environments via bioelectrocatalysis and immobilized Gluconobacter oxydans (G. oxydans) offers a promising solution; however, it is influenced by the inhibitory and toxicity impact of styrene concentration. Here, adaptive laboratory evolution was utilized to generate G. oxydans mutants with enhanced tolerance to styrene. The evolution experiments were performed at low concentration to simulate typical wastewater conditions. Physiological and biochemical characteristics shows that styrene not only induces cell apoptosis, impeding the normal growth of cells and ATP supply, but also damages the function of cell membrane and cell motility. Nonetheless, phenotypic heterogeneity and the increased biofilm formation ability are main factors responsible for the high styrene tolerance. Comparative transcriptomic analysis and whole genome sequencing indicate that the genes required for styrene tolerance was traced back to the mutations in flagella protein FlgE, as well as the upregulation in transcription factors MarR and HipB. The stimulatory effect of FlgE on the evolved strain primarily manifests in cell motility, chemotaxis, and biofilm formation. MarR and HipB positively regulated the observed tolerance phenotypes, and MarR plays a more significant role than HipB in regulating styrene tolerance in G. oxydans. Besides conferring tolerance to styrene, MarR overexpression was also shown to significantly enhance resistance against other solvents. Overall, this study discerns the potential mechanisms underlying styrene tolerance and highlights that regulating the expression abundance of MarR is a key mechanism for improving styrene tolerance, providing valuable guidance for the practical application of G. oxydans in styrene-containing wastewater treatment.