Identification and Analysis of the Balhimycin Biosynthetic Gene Cluster and Its Use for Manipulating Glycopeptide Biosynthesis in Amycolatopsis mediterranei DSM5908

Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genes bgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

本研究通过反向克隆技术（reverse-cloning approach），从糖肽类抗生素巴利霉素（balhimycin）的产生菌地中海拟无枝菌酸菌（Amycolatopsis mediterranei）DSM5908中，克隆得到7个完整基因与1个不完整基因，并完成了相关表征。基于糖基转移酶（glycosyltransferase）的序列信息设计寡核苷酸（oligonucleotides）引物，扩增得到一段900 bp的糖基转移酶基因片段，以此为探针筛选得到长度为9882 bp的DNA片段。在所鉴定得到的开放阅读框（open reading frames）中，3个基因（oxyA至C）与细胞色素P450单加氧酶（cytochrome P450 monooxygenases）序列具有显著同源性，1个基因（bhaA）与卤化酶（halogenase）具有序列同源性，而bgtfA至C基因则属于糖基转移酶家族。通过基因失活实验敲除加氧酶与糖基转移酶编码基因以消除其功能，构建了巴利霉素生物合成突变株。敲除加氧酶基因后得到的突变株SP1-1无法合成具有抗菌活性的化合物。通过高效液相色谱-质谱联用法（high-performance liquid chromatography–mass spectrometry）、肽段片段分析与氨基酸组成分析对产物进行结构解析，结果表明上述加氧酶参与了该非常规七肽的芳香侧链之间的偶联过程。通过敲除糖基转移酶基因bgtfB构建的突变株HD1，可产生至少4种未经糖基化修饰但仍保留抗菌活性的化合物。