Guo Yuan, Lu Bo, Tang Hongchi, Bi Dewu, Zhang Zhikai, Lin Lihua, Pang Hao
Guangxi Academy of Sciences Nanning 530007 China
Guangxi University Nanning 530004 China
RSC Adv. 2019 Apr 15;9(21):11683-11695. doi: 10.1039/c8ra09711a. eCollection 2019 Apr 12.
: 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 -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. : In this study, a butanol-tolerant was constructed by disrupting gene (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 and the amino acid antiporter 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. : 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.
四碳醇丁醇正成为一种有前景的生物燃料，人们已努力改进多种微生物宿主以用于丁醇生产。然而，大多数生物对丁醇的耐受性非常低（高达2%（v/v）），这限制了丁醇生产的经济可行性。尽管基因组工具（转录组学、蛋白质组学和代谢组学）已被广泛用于研究细胞对丁醇胁迫的反应，但关于丁醇耐受性所涉及的分子机制的现有知识有限，并且由于调控网络的复杂性，菌株改良很困难。
在本研究中，通过破坏astE基因（编码琥珀酰谷氨酸去琥珀酰化酶）构建了一株丁醇耐受性菌株，以获得更高的丁醇耐受性（提高了34.6%）。为阐明耐受机制，还进行了代谢组分析。结果，总共73种代谢物（11种升高和62种降低）发生了显著变化。大多数下调的代谢物主要参与L-精氨酸降解途径、硫酸盐代谢途径和2-甲基柠檬酸代谢途径。为进一步分析差异基因表达，构建了转录组。总共311个基因（113个上调和198个下调）显示出两倍以上的差异，并且与碳水化合物代谢、能量代谢和ABC转运蛋白有关。代谢组学和转录组学的整合发现，酸激活的谷氨酰胺酶和氨基酸反向转运蛋白显著上调，但L-精氨酸和谷氨酸的水平没有显著升高和降低。因此，通过氨基酸分析测量了菌株BW25113和BW25113-ΔastE之间氨基酸的变化。还通过在无丁醇的各种pH条件下的生长实验测量了突变菌株对酸胁迫的能力。
基于上述实验，可以得出结论，突变体BW25113-ΔastE主要通过调节细胞内pH稳态来适应丁醇胁迫，这表明pH对微生物丁醇耐受性有不可忽视的影响，拓宽了我们对微生物丁醇耐受性的理解，并为合理改造更强大的丁醇生产宿主提供了新策略。
