From the Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), and Department of Pharmaceutical Biotechnology, Saarland University, Campus C2 3, Saarbrücken 66123, Germany.
Naicons Scrl, Via G. Fantoli, 16/15, Milan 20138, Italy.
J Biol Chem. 2010 Oct 22;285(43):32710-32719. doi: 10.1074/jbc.M110.146803. Epub 2010 Aug 14.
The GE81112 tetrapeptides (1-3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.
GE81112 四肽（1-3）代表了一类结构独特的抗生素，作为原核生物蛋白质合成的特异性抑制剂。在这里，我们报道了从链霉菌 L-49973 中克隆和测序的 GE81112 生物合成基因簇，并开发了用于链霉菌 L-49973 的遗传操作系统。GE81112 四肽抗生素的生物合成基因簇（getA-N）位于包含 29 个开放阅读框（开放阅读框）的 61.7-kb 区域内，其中 14 个被分配到生物合成基因簇。序列分析表明，GE81112 簇由六个非核糖体肽合酶（NRPS）基因组成，这些基因编码不完整的二结构域 NRPS 模块和一个单独的游离 NRPS 结构域，以及编码其他生物合成和修饰蛋白的基因。通过使 NRPS 基因 getE 失活导致 GE81112 生产完全缺失突变体，从而证实了克隆基因簇在 GE81112 生物合成中的参与。此外，我们通过在大肠杆菌中的表达和体外酶促测定来表征途径中的 NRPS A 结构域。从综合化学和生物合成方法中确定了 GE81112 中大多数手性中心的先前未知的立体化学。总的来说，这些发现使我们能够提出 GE81112 生物合成的合理模型。这些结果进一步为通过遗传工程开发这些有前途的抗生素化合物的新衍生物开辟了道路。
