Isolation of Butanol- and Isobutanol-Tolerant Bacteria and Physiological Characterization of Their Butanol Tolerance
Isolation of Butanol- and Isobutanol-Tolerant Bacteria and Physiological Characterization of Their Butanol Tolerance
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DOI：
10.1128/aem.02900-13
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发表时间：
2013-11-01
影响因子：
4.4
通讯作者：
Kamagata, Yoichi
中科院分区：
文献类型：
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作者：
Kanno, Manabu;Katayama, Taiki;Kamagata, Yoichi
尽管它们作为生物燃料生产平台的重要性，但到目前为止，只有非常有限数量的丁醇耐受性细菌被发现。在这里，我们从各种环境样本中广泛探索了丁醇和异丁醇耐受性细菌。共分离出耐受性大于2.0% （vol/vol）丁醇和异丁醇的好氧和厌氧细菌16株。16S rRNA基因测序结果表明，分离菌株在系统发育上分布于厚壁菌门和放线菌门至少9个属：Bacillus、Lysinibacillus、Rummeliibacillus、Brevibacillus、Coprothermobacter、Caloribacterium、Enterococcus、hydrogenanaerobobacterium和cellulosimicroum。其中10个分离株在系统发育上与先前鉴定的丁醇耐受性细菌不同。两株相对较高的丁醇耐受性菌株CM4A（需氧菌）和GK12（专性厌氧菌）进一步进行了表征。在丁醇暴露下，两种菌株的膜脂肪酸组成发生了变化，CM4A和GK12分别表现出饱和脂肪酸和环丙烷脂肪酸（CFAs）以及长链脂肪酸的增加，这可能有助于维持膜的流动性。从菌株CM4A中克隆出编码cfa合成酶的基因（cfa），并在大肠杆菌中表达。与没有cfa基因的大肠杆菌相比，重组大肠杆菌对丁醇和异丁醇的耐受性相对较高，表明cfa基因可以赋予其溶剂耐受性。连续传代使菌株GK12暴露于丁醇中，甚至提高了其生长速度，这表明尚不清楚的机制也可能有助于其耐溶剂性。综上所述，结果表明，环境中存在多种能够在2.0%丁醇中生长的耐丁醇和异丁醇细菌，它们具有各种策略来保持结构完整性，以抵抗有害溶剂。
Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol-and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus, Lysinibacillus, Rummeliibacillus, Brevibacillus, Coprothermobacter, Caloribacterium, Enterococcus, Hydrogenoanaerobacterium, and Cellulosimicrobium, within the phyla Firmicutes and Actinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli. The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol-and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.