Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining.
Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining.
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DOI：
10.1021/acschembio.5b00658
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发表时间：
2015-12-18
影响因子：
4
通讯作者：
Moore BS
中科院分区：
文献类型：
--
作者：
Tang X;Li J;Millán-Aguiñaga N;Zhang JJ;O'Neill EC;Ugalde JA;Jensen PR;Mantovani SM;Moore BS
近期的基因组测序工作使得公共数据库中未表征的或“孤儿”次级代谢生物合成基因簇（BGCs）迅速积累。DNA测序大数据的这种增长给天然产物基因组挖掘的应用领域带来了重大挑战，包括（i）如何对孤儿BGCs的表征进行优先级排序，以及（ii）如何快速将基因与生物合成的小分子联系起来。在此我们表明，通过将编码靶标修饰蛋白的假定抗生素抗性基因与孤儿BGCs相关联，我们在被我们称为靶向基因组挖掘的过程中在通路特异性小分子被揭示之前预测其生物学功能。通过查询86个盐孢菌属细菌基因组的泛基因组中与天然产物BGCs共定位的重复管家基因，我们对一个带有假定脂肪酸合酶抗性基因的孤儿聚酮合酶 - 非核糖体肽合成酶杂合BGC（tlm）进行了优先级排序。我们采用了一种基于转化相关重组的新型合成双链DNA介导的克隆策略，以从基因组DNA中高效捕获tlm和相关的ttm BGCs，并在链霉菌宿主中异源表达它们。我们展示了一组不寻常的硫代四氢呋喃酸天然产物的产生，包括30多年前首次被描述的著名脂肪酸合酶抑制剂硫乳霉素，但在生物合成和自身抗性方面从未在基因水平上进行过研究。这一发现不仅验证了在没有先验知识的情况下发现产生抗生素的基因簇的靶向基因组挖掘策略，而且为研究硫代四氢呋喃酸天然产物生物合成所涉及的新型酶学铺平了道路。
Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or “orphaned” secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs, and (ii) how to rapidly connect genes to biosynthesized small molecules. Here we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes co-localized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and auto-resistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized, but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis.