Nguyen Kien T, Kau David, Gu Jian-Qiao, Brian Paul, Wrigley Stephen K, Baltz Richard H, Miao Vivian
Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, MA 02421, USA.
Mol Microbiol. 2006 Sep;61(5):1294-307. doi: 10.1111/j.1365-2958.2006.05305.x.
In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.
在许多肽类抗生素中，修饰氨基酸对生物活性至关重要。氨基酸3 - 甲基 - 谷氨酸（3mGlu）仅在三种环脂肽抗生素中被发现：达托霉素以及由玫瑰孢链霉菌产生的A21978C家族、由天蓝色链霉菌产生的钙依赖性抗生素和由弗氏链霉菌产生的A54145。我们研究了参与A21978C生物合成的非核糖体肽合成酶基因以及下游基因dptG、dptH、dptI和dptJ，预计它们分别编码一种功能未知的保守蛋白、一种硫酯酶、一种甲基转移酶（MTase）和一种色氨酸2,3 - 双加氧酶。缺失dptGHIJ会降低总体脂肽产量，并导致产生一系列含有Glu12而非3mGlu12的新型A21978C类似物。仅用dptI或其天蓝色链霉菌同源物glmT进行互补，可恢复突变体中含3mGlu化合物的生物合成。与A21978C相比，含Glu12的衍生物对金黄色葡萄球菌的活性较低。进一步的遗传分析表明，dptGHIJ基因座的成员协同促进了A21978C的最佳产量；缺失dptH、dptI或dptJ基因会显著降低产量，而从强ermEp*启动子表达dptIJ或dptGHIJ会大幅增加脂肽产量。结果表明这些基因在达托霉素的生物合成中起重要作用，并且dptI编码一种Glu甲基转移酶。
