| Organic-solvent-tolerance of microorganism is very important for various industrial fields such as biocatalysis and biotransformation, renewable biofuels production and environment remediation. Therefore, the understanding of the organic solvent tolerant mechanisms of microorganism is of great importance to enhancing the solvent-tolerance of microorganisms and their industrial applications.Escherichia coli is an important model strain due to its clear genetic background and the simple gene operation. In this study, DNA microarrays were employed to assess the transcriptome profile of a n-butanol tolerant strain JM109/pHACMB8 harboring σ70 mutant B8. Compared with the wild-type strain JM109/pHACMWT, 329 differentially expressed genes(197 up-regulated and 132 down-regulated)(p<0.05; FC≥2) were identified. We first classified the genes expression profiles and analyzed the genes function by cluster of orthologous groups(COG). The functional groups related to butanol tolerance are involved in energy production and conversion, signal transduction mechanisms, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, cell motility etc. The specific metabolic pathways of differentially expressed genes were analyzed by KEGG database. These genes are mainly involved in glyoxylate and dicarboxylate metabolism, pyruvate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis and ABC transports etc.Based on the analysis of DNA microarray, 15 up-regulated genes and 14 down-regulated genes were studied by gene overexpression and gene knockout method. The results showed that glcF, gcl, yhaR, ybbQ, tdcE overexpression strains and △yibT, △yghW, △ybjC knockout strains exhibited higher n-butanol tolerance. Among them, the membrane fatty acid compositions and surface hydrophobicity of △yibT and △ygh W were measured. It was noticed that the proportion of unsaturated fatty acid(UFA) in total fatty acids, especially palmitoleic acid(C16:1) and oleic acid(C18:1), were increased by 20%~30% in both knockouts. And the surface hydrophobicity of △ygh W and △yibT was also increased compared with the control. Additionally, we identified a membrane protein(YbjC) by GFP fusion analysis, and noticed that the intracellular butanol content of the ybjC over-expression strain was 25% higher than that of the control. Moreover, we constructed overexpression strains of glc cluster. The glcA overexpression strain could barely grow, while glc cluster co-expression strains grew better than the control. Finally, the organic acid content of glcF, glcG and gcl over-expression strains were measured in this study. The results showed that pyruvate concentrations in cell cultures of both gcl and glcF overexpressed strains were 4.66 and 1.63 fold of the control, and butanol stress would further increase the content of pyruvate. Conclusions in this study are as follows:(1) The changes of fatty acid composition and hydrophobic property of cell membrane may be contributed to the enhanced butanol tolerance of △yghW and △yibT.(2) Gene(ybjC) encoding a membrane protein may be related to its role in butanol transportation.(3) glcA gene is regulated by other genes in gcl cluster.(4) The up-regulation of gcl and glcF genes are favorable for the cell growth by replenishing pyruvate, and butanol stress would further increase the content of pyruvate, this may play an important role in cell energy production.In this study, the organic solvent tolerant mechanisms of several genes in E. coli were investigated. Our results could provide theoretical basis for engineering the solvent tolerance of microorganisms. |
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