Transcriptome Mining of Active Biosynthetic Pathways and Their Associated Products in Streptomyces flaveolus
Transcriptome Mining of Active Biosynthetic Pathways and Their Associated Products in Streptomyces flaveolus
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黄链霉菌活性生物合成途径及其相关产物的转录组挖掘
DOI：
10.1002/anie.201103085
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Liu, Wen
中科院分区：
文献类型：
--
作者：
Qu, Xudong;Lei, Chun;Liu, Wen
天然产物（NPs）是具有令人难以置信的结构多样性的小分子，长期以来一直因其在药物发现和开发中的关键作用而受到赞赏。[1]从生物合成基因簇中产生NP取决于相关基因协调转录为信使RNA（mRNA），这些mRNA翻译为多肽链，以及这些多肽的正确折叠以创建功能性生物合成机制，这些机制合作催化简单前体分子形成复杂结构（图1）。在过去的十年中，许多放线菌基因组的测序揭示了数量惊人的NP生物合成基因簇，其中只有约10%与特征NP有关。[2]虽然生物信息学指导的努力已经成功地将这些NP基因簇中的一些与新的代谢物相关联，[3]其中包括由聚酮酶（PKS），非核糖体肽合成酶（NRPS）和萜烯脱氢酶产生的代谢物，[4]很明显，我们挖掘这些新基因簇以揭示隐藏在其中的化学潜力的能力并没有跟上DNA测序技术的步伐。在后基因组时代，已经开发了针对NP生产的不同阶段的策略，以验证与化学生物合成相关的遗传基础。[3-10]我们使用S。flaveolus DSM 9954作为模型系统[11]，并在此报道了在转录水平上挖掘活性生物合成途径及其相关产物，在转录水平上存在将潜在基因型转化为实际化学型的关键联系。这种有效的策略，它使用基于mRNA的挖掘，有可能被普遍应用，以提高效率的NP发现后，进一步发展，甚至从菌株的基因组序列是未知的。我们首先评估了S.不同发酵培养基中的黄曲霉。在4天发酵中单独评估了我们实验室先前用于产生已知代谢物的6种培养基（参见支持信息中的方法）。通过HPLC分析培养液，结果显示产品特征存在显著差异（参见支持性信息中的图S1）。MediumV，其中S. Flaveolus产生的代谢物种类最多，选择该菌株进行生产的时程分析（见辅助信息中的图S2）。由于在第4天观察到大多数代谢物的最高产量，因此选择该点进行转录组分析。从S. flaveolus菌丝体在第4天转化为互补DNA（cDNA），因此构成条件转录组（参见支持信息中的图S3 a）。探讨S.在这些条件下，通过使用对应于产生不同结构基序的各种生物合成基因的引物，通过PCR筛选所得cDNA（参见支持信息中的方法和图4）。由简并引物产生的PCR产物的长度从280到550 bp变化，并且包括用于组装聚酮化合物碳骨架的模块I型PKS的酮脂酰合酶（KS）结构域、用于激活氨基酸底物的线性NRPS的腺苷酸化（A）结构域、用于在核糖体肽上提供噻唑啉部分的硫肽环化酶（TCDH）、[12 c]（d）NDP-d-葡萄糖-4、6-脱氢酶（NGDH）参与脱氧糖的形成，[12 d]和FAD依赖性卤化酶（FDH）负责卤素原子的掺入。[12e]而tcdh、dgdh和fdh的扩增未能给出..
Natural products (NPs) are small molecules of incredible structural diversity that have long been appreciated for their critical role in drug discovery and development.[1] Production of NPs from biosynthetic gene clusters depends on the coordinated transcription of relevant genes to messenger RNAs (mRNAs), the translation of these mRNAs to polypeptide chains, and the correct folding of these polypeptides to create functional biosynthetic machineries that cooperate to catalyze the formation of complex structures from simple precursor molecules (Figure1). Sequencing of numerous actinomycete genomes during the past decade has revealed a stunning number of NP biosynthetic gene clusters, only about 10% of which have been linked to characterized NPs.[2] While bioinformatics-guided efforts have been successful in correlating some of these NP gene clusters to new metabolites,[3] which include those produced by polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and terpene synthases,[4] it is clear that our ability to mine these new gene clusters to uncover the chemical potential hidden within them has not kept pace with DNA sequencing technology. In the postgenomic era, strategies that target different stages of NP production have been developed to validate the genetic basis that is relevant to chemical biosynthesis.[3–10] We used S. flaveolus DSM 9954 as a model system,[11] and report herein the mining of active biosynthetic pathways and their associated products at the transcriptional level, at which a key linkage of transforming the potential genotype to the practical chemotype exists. This effective strategy, which uses mRNA-based mining, has the potential to be generally applied to increase the efficiency of NP discovery after further development, even from strains for which the genome sequence is unknown. We began by evaluating the chemical profile of S. flaveolus in different fermentation media. Six media, which have been used previously in our laboratory to produce known metabolites (see methods in the Supporting Information), were assessed individually in four-day fermentations. The culture broths were analyzed by HPLC, and showed remarkable differences in product profiles (see FigureS1 in the Supporting Information). MediumV, in which S. flaveolus produced the greatest variety of metabolites, was selected for a time-course analysis of the production (see Figure S2 in the Supporting Information). As the highest yields of most metabolites were observed on day 4, this point was selected for transcriptome analysis. Total RNA isolated from S. flaveolus mycelia on day 4 was converted to complementary DNA (cDNA), and thus constitutes a conditional transcriptome (see Figure S3a in the Supporting Information). To explore the biosynthetic potential of S. flaveolus under these conditions, the resulting cDNAs were screened by PCR by using primers corresponding to a variety of biosynthetic genes that produce diverse structural motifs (see methods and FigureS4 in the Supporting Information). PCR products generated by degenerate primers varied in length from 280 to 550 bps and included those for ketoacylsynthase (KS) domain of modular type I PKS to assemble the polyketide carbon backbone,[12a] adenylation (A) domain of linear NRPS to activate the amino acid substrate,[12b] thiopeptide cyclodehydrase (TCDH) to afford thiazoline moieties on a ribosomal peptide,[12c](d) NDP-d-glucose-4, 6-dehydrase(NGDH) involved in the deoxysugar formation,[12d] and FAD-dependent halogenase (FDH) responsible for incorporation of a halogen atom.[12e] Whereas amplifications of tcdh, dgdh, and fdh failed to give …