Forest soil metagenome gene cluster involved in antifungal activity expression in Escherichia coli
Forest soil metagenome gene cluster involved in antifungal activity expression in Escherichia coli
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DOI：
10.1128/aem.01911-07
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发表时间：
2008-02-01
影响因子：
4.4
通讯作者：
Lee, Seon-Woo
中科院分区：
文献类型：
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作者：
Chung, Eu Jin;Lim, He Kyoung;Lee, Seon-Woo
我们之前利用两块森林土壤构建了两个 fosmid 文库，总共包含 113,700 个成员。使用酿酒酵母作为目标真菌筛选文库以选择活性抗真菌克隆。来自儒城松树根际土壤文库的一个克隆 pEAF66 显示出酿酒酵母的生长抑制作用。尽管付出了巨大的努力，活性化学物质仍未被分离出来。 pEAF66 的 DNA 序列分析和转座子诱变揭示了 39 个开放阅读框 (ORF)，并表明可能在一个转录单元中的 8 个 ORF 可能直接参与大肠杆菌中抗真菌活性的表达。推导的8个ORF的氨基酸序列与编码II型家族聚酮合酶的核心基因的氨基酸序列相似，例如酰基载体蛋白(ACP)、ACP合酶、转氨酶和ACP还原酶。尽管我们无法从大肠杆菌 KT2440 基因组 DNA 文库中选择类似的活性克隆，但参与抗真菌活性的基因簇在组织上与恶臭假单胞菌 KT2440 的假定抗生素生产位点相似。编码 ATP 结合盒转运蛋白和膜蛋白的 ORF 位于抗真菌基因簇的两端。编码 IclR 家族反应调节因子和 LysR 家族反应调节因子的上游 ORF 参与抗真菌基因表达的正向调节。我们的结果表明宏基因组方法可以作为从不可培养的土壤细菌中寻找新型抗真菌抗生素的替代方法。这是首次报道使用酿酒酵母作为目标真菌从土壤宏基因组中获得抗真菌基因簇。
Using two forest soils, we previously constructed two fosmid libraries containing 113,700 members in total. The libraries were screened to select active antifungal clones using Saccharomyces cerevisiae as a target fungus. One clone from the Yuseong pine tree rhizosphere soil library, pEAF66, showed S. cerevisiae growth inhibition. Despite an intensive effort, active chemicals were not isolated. DNA sequence analysis and transposon mutagenesis of pEAF66 revealed 39 open reading frames (ORFs) and indicated that eight ORFs, probably in one transcriptional unit, might be directly involved in the expression of antifungal activity in Escherichia coli. The deduced amino acid sequences of eight ORFs were similar to those of the core genes encoding type II family polyketide synthases, such as the acyl carrier protein (ACP), ACP synthases, aminotransferase, and ACP reductase. The gene cluster involved in antifungal activity was similar in organization to the putative antibiotic production locus of Pseudomonas putida KT2440, although we could not select a similar active clone from the KT2440 genomic DNA library in E. coli. ORFs encoding ATP binding cassette transporters and membrane proteins were located at both ends of the antifungal gene cluster. Upstream ORFs encoding an IclR family response regulator and a LysR family response regulator were involved in the positive regulation of antifungal gene expression. Our results suggested the metagenomic approach as an alternative to search for novel antifungal antibiotics from unculturable soil bacteria. This is the first report of an antifungal gene cluster obtained from a soil metagenome using S. cerevisiae as a target fungus.