吸水链霉菌10-22中硫肽类抗生素cyclothiazomycin生物合成机制的研究
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摘要
硫肽类抗生素是一类由微生物生产的重要天然产物,对很多高抗药病原菌都有很强的抑制作用。它们共享一些典型的结构特征,包括含多个半胱氨酸衍生的两个氨基酸残基间的异源环(包括噻唑和噻唑啉)、分子结构中心位置的6元含氮杂环(吡啶或者脱水哌啶环)。多年来,这些不同寻常的氨基酸衍生结构一直吸引着有机化学家们在实验室中尝试通过有机合成的手段来实现,但是生物体本身究竟是如何合成这类产物,却很少有实验数据来描述其生物合成过程。
环噻唑霉素(cyclothiazomycin,CLT)是一种新近分离且已经有立体化学结构鉴定报道的硫肽类抗生素,有肾素抑制剂的应用潜力。它是一种比较特别的硫肽类抗生素,除了具备硫肽类的共有特征外,自身有多处结构较为罕见,包括非2, 3-位噻唑取代的中心吡啶环,噻唑啉和噻唑的数目平分秋色以及一个叔碳上的硫醚键。
本实验室保存的吸水链霉菌应城变种10-22由我国科学家分离得到,能够产生三种农用抗生素的,其中一种多肽类抗生素5102-II的生物合成机制多年来困扰着我们。NRPS思路的失败令我们转而考虑核糖体合成的途径。基于此猜想,利用一对基于核糖体合成途径中特有的环化脱水酶的保守序列设计的兼并引物,我们从10-22的基因组中克隆到了一个0.7kb的目标片段。基于该片段设计的特异引物在10-22基因组文库中钓到了两个阳性柯丝质粒(cosmid)。限制性内切酶消化后的片段序列测序结果证实,其中包含有一小段DNA序列,编码60aa的一个多肽,其中C端18aa的序列和CLT的结构氨基酸序列顺序完全一致。后续的生测实验、QTOF分子量检测分析以及二级质谱碎片分析都证明10-22所产的多肽类抗生素就是CLT。
柯丝质粒测序结果显示整个35kb左右的插入片段中包含了27个ORF。根据ORF的分布情况,我们构建了多个异源表达载体,首次在链霉菌的模式菌株变铅青链霉菌Streptomyces lividans 1326中实现硫肽类抗生素的异源表达,并通过不同ORF的组合定位出了一个完整的环噻唑霉素合成基因簇。这是一个包含了15个ORFs、长度为22.7kb的核苷酸序列,生物信息学分析表明其中唯一一个结构基因cltA编码产生环噻唑霉素前体,前体多肽再经由基因簇中的其他蛋白进行翻译后修饰,最终成熟并释放至胞外。
我们推测整个过程中有9个蛋白参与,其中6个基因,cltBCDEFG,围绕在前体肽基因cltA周围可能成一个大的操纵子,他们负责环噻唑霉素主体结构的合成,包括:CltB负责对半胱氨酸(Cys)残基和其临近的残基之间脱水环化形成分子间的异源噻唑啉;CltC则选择性识别成双的噻唑啉结构而只催化脱氢N端的一个从而形成CLT分子中噻唑啉和噻唑啉平分秋色的局面;2,3位非噻唑环取代的中心吡啶区域的形成则可能包括环化脱水、前导链切除和脱氨基三个过程,CltD由于拥有环化脱水酶和肽酶两个结构域,被认为是负责催化吡啶形成的主力军,并由此推测吡啶形成和前导肽的切除是同时进行的;CltG则很可能是一个脱氨基酶;脱水氨基酸的形成和lantibiotic的情形类似,由两个脱水酶CltE和CltF负责催化形成的。还有两个基因cltM/N,我们认为其编码的产物负责环噻唑霉素最后成环的一个叔碳硫醚键的形成;该硫肽分子完成所有翻译后修饰后,由转运蛋白CltO识别结合成熟产物并将之转运至胞外。
就在2009年初,3个不同的硫肽类抗生素的合成基因簇序列几乎同时在三个不同的实验室得到突破,分析结果相当一致,认为硫肽类抗生素就是一类基因组编码、核糖体合成后经由一系列的后修饰成熟释放的一类抑菌多肽。本研究为这个结论提供了一个新的证据,并首次通过异源表达的手段给出了一个相对简单明晰的完整硫肽合成基因簇,同时也揭示了几个特有的后修饰酶,使得在未来的硫肽合成机制的深入研究以及“非天然”硫肽类化合物的实验室合成上都有一个便利的研究对象和工具。
Thiopeptide antibiotics are an important class of natural products. Although their biosynthesis, especially construction of unusual amino acid residues, attracted organic chemists for many years, few experimental data on the enzymatic polypeptide formation has been obtained.
Cyclothiazomycin (CLT), isolated as a novel and selective inhibitor of human plasma rennin, is a unique bridged macrocyclic thiopeptide, whose stereo structure was recently revealed as containing a dehydroserine, two dehydrothreonine residues, three thiazolines, three thiazoles, and a tri-substituted pyridine. Compared with common thiopeptides, it lacks the characteristic 2- and 3-azole substituent on the central pyridine domain, but instead, possesses an alanine-derived heterocyclic residue with (R)-configuration and a quaternary sulfide to exhibits two macro-cyclic peptide loops.
Streptomyceshygroscopicus var. yingchengensis 10-22 (10-22), isolated by Zhou et al in 1980s’, can produce three antifungal antibiotics, 5102-I, 5102-II and 5102-III. Known as a polypeptide containing Thr, Ser and Cysantibiotic, antibiotic 5102-II can protect against leaf spot of corn (disease caused by Cochliobolus heterostrophus). However, the lack of detailed structural information and the unsuccessful cloning strategy based on non-ribosomal peptide hypothesis have hampered the cloning of its biosynthesis gene cluster for years.
Previously failures raised the possibility that the polypeptide antibiotic 5102-II is synthesized by ribosomal pathway followed by posttranslational modifications. A pair of degenerated primers, designed based on the conserved cyclodehydratase, was used to clone a 700 bp fragment from the genomic DNA of S. hygroscopicus 10-22. Based on this nucleotide sequence, a pair of specific nestle primers was designed to screen the S. hygroscopicus 10-22 genomic library. One of the positive cosmids, 14E6, with a 35 kb insertion, was sequenced (Accession number:FJ472825) and 27 ORFs were predicted in this sequence. Significantly, a 180-nt orf, named as cltA, in the sequence was found to encode a 60-amino acid precursor peptide, of which the C terminal sequence is perfectly consistent with the structural peptide of cyclothiazomycin. Moreover, both the bioassay and Q-TOF analysis idenfied that the polypeptide antiobiotic produced by 10-22 was indeed CLT.
According to the organization of ORFs, a profile of heterologous expression was successfully taken in S. lividans 1326, which is the first time for thiopeptides. Therefore, a minimized gene cluster responsible for the production of CLT has been confirmed to contain 15 ORFs and 9 of them are suggested to be directly involved into the biosynthesis.
Based on the heterologous expression study and the bioinformatic analysis on the minimized intact gene cluster, we proposed an eight-enzyme involved biosynthetic scheme for the posttranslational modifications of cyclothiazomycin. In this scheme, three proteins, CltC, CltD and CltN were suggested to be responsible for the characteristic structural elements: the selectively dehydrogenated thiazolines, tri-substituted central pyridine and tertiary thioether. Importantly, the LP cleavage was deduced to be catalyzed by the C-terminal domain of CltD during the pyridine formation. The other two enzymes encoded by a putative operon, cltMN, were supposed to participate in tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure.
Until early in this year, three groups cloned three different thiopeptides biosynthetases almost at the same time. Here we reported the first cloning and heterlogous expression of the biosynthetic gene cluster of CLT. Remarkably, this rigorous bioinformatic analysis based on the heterologous expression results presented a precise biosynthetic scheme for cyclothiazomycin, which may offer a short-cut access to the biosynthesis of other 'unnatural' products generated by posttranslational modification.
环噻唑霉素(cyclothiazomycin,CLT)是一种新近分离且已经有立体化学结构鉴定报道的硫肽类抗生素,有肾素抑制剂的应用潜力。它是一种比较特别的硫肽类抗生素,除了具备硫肽类的共有特征外,自身有多处结构较为罕见,包括非2, 3-位噻唑取代的中心吡啶环,噻唑啉和噻唑的数目平分秋色以及一个叔碳上的硫醚键。
本实验室保存的吸水链霉菌应城变种10-22由我国科学家分离得到,能够产生三种农用抗生素的,其中一种多肽类抗生素5102-II的生物合成机制多年来困扰着我们。NRPS思路的失败令我们转而考虑核糖体合成的途径。基于此猜想,利用一对基于核糖体合成途径中特有的环化脱水酶的保守序列设计的兼并引物,我们从10-22的基因组中克隆到了一个0.7kb的目标片段。基于该片段设计的特异引物在10-22基因组文库中钓到了两个阳性柯丝质粒(cosmid)。限制性内切酶消化后的片段序列测序结果证实,其中包含有一小段DNA序列,编码60aa的一个多肽,其中C端18aa的序列和CLT的结构氨基酸序列顺序完全一致。后续的生测实验、QTOF分子量检测分析以及二级质谱碎片分析都证明10-22所产的多肽类抗生素就是CLT。
柯丝质粒测序结果显示整个35kb左右的插入片段中包含了27个ORF。根据ORF的分布情况,我们构建了多个异源表达载体,首次在链霉菌的模式菌株变铅青链霉菌Streptomyces lividans 1326中实现硫肽类抗生素的异源表达,并通过不同ORF的组合定位出了一个完整的环噻唑霉素合成基因簇。这是一个包含了15个ORFs、长度为22.7kb的核苷酸序列,生物信息学分析表明其中唯一一个结构基因cltA编码产生环噻唑霉素前体,前体多肽再经由基因簇中的其他蛋白进行翻译后修饰,最终成熟并释放至胞外。
我们推测整个过程中有9个蛋白参与,其中6个基因,cltBCDEFG,围绕在前体肽基因cltA周围可能成一个大的操纵子,他们负责环噻唑霉素主体结构的合成,包括:CltB负责对半胱氨酸(Cys)残基和其临近的残基之间脱水环化形成分子间的异源噻唑啉;CltC则选择性识别成双的噻唑啉结构而只催化脱氢N端的一个从而形成CLT分子中噻唑啉和噻唑啉平分秋色的局面;2,3位非噻唑环取代的中心吡啶区域的形成则可能包括环化脱水、前导链切除和脱氨基三个过程,CltD由于拥有环化脱水酶和肽酶两个结构域,被认为是负责催化吡啶形成的主力军,并由此推测吡啶形成和前导肽的切除是同时进行的;CltG则很可能是一个脱氨基酶;脱水氨基酸的形成和lantibiotic的情形类似,由两个脱水酶CltE和CltF负责催化形成的。还有两个基因cltM/N,我们认为其编码的产物负责环噻唑霉素最后成环的一个叔碳硫醚键的形成;该硫肽分子完成所有翻译后修饰后,由转运蛋白CltO识别结合成熟产物并将之转运至胞外。
就在2009年初,3个不同的硫肽类抗生素的合成基因簇序列几乎同时在三个不同的实验室得到突破,分析结果相当一致,认为硫肽类抗生素就是一类基因组编码、核糖体合成后经由一系列的后修饰成熟释放的一类抑菌多肽。本研究为这个结论提供了一个新的证据,并首次通过异源表达的手段给出了一个相对简单明晰的完整硫肽合成基因簇,同时也揭示了几个特有的后修饰酶,使得在未来的硫肽合成机制的深入研究以及“非天然”硫肽类化合物的实验室合成上都有一个便利的研究对象和工具。
Thiopeptide antibiotics are an important class of natural products. Although their biosynthesis, especially construction of unusual amino acid residues, attracted organic chemists for many years, few experimental data on the enzymatic polypeptide formation has been obtained.
Cyclothiazomycin (CLT), isolated as a novel and selective inhibitor of human plasma rennin, is a unique bridged macrocyclic thiopeptide, whose stereo structure was recently revealed as containing a dehydroserine, two dehydrothreonine residues, three thiazolines, three thiazoles, and a tri-substituted pyridine. Compared with common thiopeptides, it lacks the characteristic 2- and 3-azole substituent on the central pyridine domain, but instead, possesses an alanine-derived heterocyclic residue with (R)-configuration and a quaternary sulfide to exhibits two macro-cyclic peptide loops.
Streptomyceshygroscopicus var. yingchengensis 10-22 (10-22), isolated by Zhou et al in 1980s’, can produce three antifungal antibiotics, 5102-I, 5102-II and 5102-III. Known as a polypeptide containing Thr, Ser and Cysantibiotic, antibiotic 5102-II can protect against leaf spot of corn (disease caused by Cochliobolus heterostrophus). However, the lack of detailed structural information and the unsuccessful cloning strategy based on non-ribosomal peptide hypothesis have hampered the cloning of its biosynthesis gene cluster for years.
Previously failures raised the possibility that the polypeptide antibiotic 5102-II is synthesized by ribosomal pathway followed by posttranslational modifications. A pair of degenerated primers, designed based on the conserved cyclodehydratase, was used to clone a 700 bp fragment from the genomic DNA of S. hygroscopicus 10-22. Based on this nucleotide sequence, a pair of specific nestle primers was designed to screen the S. hygroscopicus 10-22 genomic library. One of the positive cosmids, 14E6, with a 35 kb insertion, was sequenced (Accession number:FJ472825) and 27 ORFs were predicted in this sequence. Significantly, a 180-nt orf, named as cltA, in the sequence was found to encode a 60-amino acid precursor peptide, of which the C terminal sequence is perfectly consistent with the structural peptide of cyclothiazomycin. Moreover, both the bioassay and Q-TOF analysis idenfied that the polypeptide antiobiotic produced by 10-22 was indeed CLT.
According to the organization of ORFs, a profile of heterologous expression was successfully taken in S. lividans 1326, which is the first time for thiopeptides. Therefore, a minimized gene cluster responsible for the production of CLT has been confirmed to contain 15 ORFs and 9 of them are suggested to be directly involved into the biosynthesis.
Based on the heterologous expression study and the bioinformatic analysis on the minimized intact gene cluster, we proposed an eight-enzyme involved biosynthetic scheme for the posttranslational modifications of cyclothiazomycin. In this scheme, three proteins, CltC, CltD and CltN were suggested to be responsible for the characteristic structural elements: the selectively dehydrogenated thiazolines, tri-substituted central pyridine and tertiary thioether. Importantly, the LP cleavage was deduced to be catalyzed by the C-terminal domain of CltD during the pyridine formation. The other two enzymes encoded by a putative operon, cltMN, were supposed to participate in tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure.
Until early in this year, three groups cloned three different thiopeptides biosynthetases almost at the same time. Here we reported the first cloning and heterlogous expression of the biosynthetic gene cluster of CLT. Remarkably, this rigorous bioinformatic analysis based on the heterologous expression results presented a precise biosynthetic scheme for cyclothiazomycin, which may offer a short-cut access to the biosynthesis of other 'unnatural' products generated by posttranslational modification.
引文
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