Tolerance against butanol stress by disrupting succinylglutamate desuccinylase in Escherichia coli

Abstract

Background: The four-carbon alcohol, butanol, is emerging as a promising biofuel and efforts have been undertaken to improve several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of butanol production. Although genomic tools (transcriptomics, proteomics, and metabolomics) have been widely used to investigate the cellular response to butanol stress, the existing knowledge of the molecular mechanisms involved in butanol tolerance is limited, and strain improvement is difficult due to the complexity of the regulatory network. Results: In this study, a butanol-tolerant Escherichia coli was constructed by disrupting gene astE (encoding succinylglutamate desuccinylase) to obtain higher butanol tolerance (increased by 34.6%). To clarify the tolerance mechanism, a metabolome analysis was also performed. As a result, a total of 73 metabolites (11 elevated and 62 decreased) were significantly changed. Most of the downregulated metabolites were mainly involved in the L-arginine degradation pathway, sulfate metabolic pathway, and 2-methylcitrate metabolic pathway. To further analyze the differential gene expression, a transcriptome was created. In total, 311 genes (113 upregulated and 198 downregulated) showed over a twofold difference and were associated with carbohydrate metabolism, energy metabolism, and ABC transporters. The integration of metabolomics and transcriptomics found that acid-activated glutaminase ybaS and the amino acid antiporter gadC were significantly up-regulated, but the levels of L-arginine and glutamate were not significantly increased and decreased. Therefore, the changes of amino acids between strains BW25113 and BW25113-ΔastE were measured by amino acid analysis. The ability of a mutant strain against acid stress was also measured by the growth experiment under various pH conditions in the absence of butanol. Conclusions: Based on the above experiments, it could be concluded that mutant BW25113-ΔastE mainly regulated intracellular pH-homeostasis to adapt to butanol stress, indicating the non-negligible impact of pH on microbial butanol tolerance, broadening our understanding of microbial butanol tolerance and providing a novel strategy for the rational engineering of a more robust butanol-producing host.