Antiterminator-Mediated Unveiling of Cryptic Polythioamides in an Anaerobic Bacterium
Antiterminator-Mediated Unveiling of Cryptic Polythioamides in an Anaerobic Bacterium
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DOI：
10.1002/anie.201108214
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Hertweck, Christian
中科院分区：
文献类型：
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作者：
Behnken, Swantje;Lincke, Thorger;Hertweck, Christian
梭状芽孢杆菌属由一系列高度异质性的专性厌氧微生物组成，这些微生物生活在从土壤到人类肠道的各种生态位中。这些微生物已经得到了广泛的研究，不仅因为它们能够产生有用的溶剂，还因为一些物种产生有害的蛋白质毒素，如肉毒杆菌毒素。[1，2]然而，尽管对梭状芽孢杆菌有大量的知识，直到最近才从这些或任何其他严格的厌氧菌中分离出次生代谢物。然而，对梭状芽孢杆菌基因组序列的挖掘揭示了次生代谢基因的广泛存在，特别是聚酮合成酶和非核糖体多肽合成酶基因。[3，4]我们已经证明，在标准的实验室条件下，编码的途径保持沉默。[4]显然，这种神秘的生物合成基因只有在特定刺激存在的情况下才会被激活。[5，6]这是合理的，因为次生代谢物途径需要大量的ATP结合能量，这在厌氧世界中是一种稀有的商品。我们最近发现，将土壤水提取物添加到纤维素裂解杆菌培养物中，激活了一条原本沉默的代谢途径，导致了一种全新的名为闭硫酰胺的聚硫酰胺(1；方案1)。[7]除了其特殊的结构特征外，闭硫酰胺对多种细菌具有高度的活性，如万古霉素耐药肠球菌(VRE)和甲氧西林耐药金黄色葡萄球菌(MRSA)，并且是第一种来自严格厌氧细菌的抗生素。[7]然而，土壤的可变性质限制了这些微生物的恒定产量，并阻碍了这些微生物的复杂发酵。因此，各种次要的闭硫酰胺同系物逃脱了分离和充分鉴定，生物合成研究也是无与伦比的。在这里，我们提出了一种新的策略，使用一个反终止子基因来触发一个隐藏的生物合成途径，并揭示了七个新的多硫酰胺同系物的结构和抗菌活性。除了揭示结构-活性关系外，我们还使用合成的封闭硫酰胺类似物作为探针，初步了解天然多硫酰胺的生物发生关系。一种已建立的定向诱导次生代谢物生物合成的方法是操纵特定途径的调节基因。[5，8-10]然而，由于闭合硫酰胺生物合成基因位置仍然不明，这种方法是遥不可及的。作为另一种选择，我们专注于可能参与更全面的次生代谢激活的调节元件。[6-11]当分析纤维素裂解杆菌基因组时，我们注意到假定的抗终止子基因nusG的存在。N-利用因子G(NusG)是大肠杆菌中一种重要的蛋白质，可以提高总的转录速度。NusG能够通过促进RNAP的正向易位来减少一些暂停的RNA-聚合酶(RNAP)复合体的占有率。[12]NusG还使RNAP能够阅读转录终止1依赖的位点(图1 A)，特别是在RRN操纵子中，并由于它与核糖体蛋白家族共享的Kyprdes-Onzonis-Woese(KOW)基序的存在而在翻译中发挥作用。[13]提高RNA聚合酶的加工效率有助于有效合成相应的转录本并产生多顺反子mRNA，从而导致蛋白质产量增加。因此，提高nusG的表达率可能潜在地激活纤维素酶C的次生代谢。为了产生高表达抗终止子…的纤维素酶突变体
The genus Clostridium comprises a highly heterogeneous array of obligate anaerobic organisms that inhabit diverse ecological niches, ranging from soil to human intestines. These microorganisms have been extensively studied, not only because of their ability to produce useful solvents, but also because some species produce harmful protein toxins such as botulinum toxin.[1, 2] However, despite the large body of knowledge on clostridia, until recently no secondary metabolites have been isolated from these or any other strict anaerobes. Yet, mining the sequenced genomes of Clostridium species has revealed a widespread occurrence of secondary metabolism genes, in particular polyketide synthase and non-ribosomal peptide synthetase genes.[3, 4] We have shown that the encoded pathways remain silent under standard laboratory conditions.[4] Obviously, such cryptic biosynthesis genes are only activated in the presence of particular stimuli.[5, 6] This is plausible, because secondary metabolite pathways require much ATP-bound energy, a rare commodity in the anaerobic world. We recently discovered that the addition of aqueous soil extract to a C. cellulolyticum culture activates an otherwise silent metabolic pathway, leading to a wholly unprecedented type of polythioamide named closthioamide (1; Scheme 1).[7] Apart from its exceptional structural features, closthioamide is highly active against a variety of bacteria, such as vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), and is the first antibiotic from a strictly anaerobic bacterium.[7] However, the variable nature of soil constrains constant production rates and hampers the complicated fermentation of these organisms. As a result, various minor closthioamide congeners evaded isolation and full characterization, and biosynthetic studies were unapproachable. Herein we present a new strategy using an antiterminator gene to trigger a cryptic biosynthetic pathway and disclose the structures and antibacterial activities of seven novel polythioamide congeners. Besides revealing structure–activity relationships, we used synthetic closthioamide analogues as probes to gain an initial insight into the biogenetic relationships of the natural polythioamides. An established approach for the targeted induction of secondary metabolite biosynthesis is the manipulation of pathway-specific regulatory genes.[5, 8–10] However, as the closthioamide biosynthesis gene locus still remains obscure, this approach was out of reach. As an alternative, we focused on regulatory elements that could be involved in a more global activation of secondary metabolism.[6–11] When analyzing the C. cellulolyticum genome, we noted the presence of a putative antiterminator gene, nusG. N-utilizing factor G (NusG) is an essential protein in E. coli that can increase the overall rate of transcription. NusG has the ability to decrease the occupancy of some of the paused RNA-polymerase (RNAP) complexes by promoting the forward translocation of RNAP.[12] NusG also enables RNAP to read through transcription-terminating 1-dependent sites (Figure 1 A), especially in rrn operons, and plays a role in translation owing to the presence of the Kyprides–Onzonis–Woese (KOW) motif it shares with ribosomal protein families.[13] Increasing the processivity of RNA polymerase aids in the efficient synthesis of the corresponding transcripts and produces polycistronic mRNA, which results in elevated protein production. Thus, increasing the nusG expression rate could potentially activate secondary metabolism in C. cellulolyticum.To generate a C. cellulolyticum mutant overexpressing the antiterminator …