Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17
Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17
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DOI：
10.1128/aac.03512-14
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发表时间：
2014-08
影响因子：
4.9
通讯作者：
M. Spohn;Norbert Kirchner;A. Kulik;Angelika Jochim;F. Wolf;P. Muenzer;O. Borst;H. Gross;W. Wohlleben;E. Stegmann
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文献类型：
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作者：
M. Spohn;Norbert Kirchner;A. Kulik;Angelika Jochim;F. Wolf;P. Muenzer;O. Borst;H. Gross;W. Wohlleben;E. Stegmann
摘要 过去几十年来抗生素耐药病原菌的出现是迫切需要新型抗菌剂的原因之一。发现新的抗感染化合物的策略是评估次级代谢产物生产者的遗传能力和激活隐秘基因簇（基因组挖掘）。无枝酸菌属（Amycolatopsis）是一种以其合成医学上重要产品的潜力而闻名的属。然而，日本无枝酸菌在标准实验室条件下不产生抗生素。与大多数无枝酸菌株不同，日本无枝酸菌可以通过不同的方法进行遗传处理。为了激活可能的沉默糖肽簇，我们将编码巴希霉素生物合成转录激活剂的基因，即来自无枝霉 (bbrAba) 的 bbr 基因引入日本刺孢菌中。这导致产生抗生素活性化合物。对日本刺参进行全基因组测序后，通过基因组挖掘鉴定出 29 个隐秘基因簇。这些基因簇之一是推定的糖肽生物合成基因簇。利用生物信息学工具，利霉素（syn. ristocetin）是一种 III 型糖肽，具有抗菌活性，用于诊断血管性血友病和 Bernard-Soulier 综合征，被推断为该基因簇的可能产物。通过高效液相色谱和质谱 (HPLC-MS)、串联质谱 (MS/MS) 和核磁共振 (NMR) 光谱进行的化学分析证实了重组日本曲霉/pRM4-bbrAba 合成利霉素 A 的计算机预测。
ABSTRACT The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is Amycolatopsis. However, Amycolatopsis japonicum does not produce an antibiotic under standard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina (bbrAba), into A. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of A. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum/pRM4-bbrAba synthesizes ristomycin A.