Engineered biosynthesis of enduracidin lipoglycopeptide antibiotics using the ramoplanin mannosyltransferase Ram29.
Engineered biosynthesis of enduracidin lipoglycopeptide antibiotics using the ramoplanin mannosyltransferase Ram29.
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DOI：
10.1099/mic.0.000095
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发表时间：
2015-07
期刊：
影响因子：
--
通讯作者：
Micklefield J
中科院分区：
文献类型：
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作者：
Wu MC;Styles MQ;Law BJ;Struck AW;Nunns L;Micklefield J
来自游动放线菌ATCC 33076的脂肽雷莫拉宁和由杀真菌链霉菌产生的雷莫拉定是针对许多耐药革兰氏阳性病原体的有效抗生素。虽然这两种抗生素具有相似的环状肽结构，包含17个氨基酸和N-末端脂肪酸侧链，但雷莫拉宁具有雷莫拉定缺乏的二甘露糖部分。雷莫拉宁的甘露糖基取代基增强了水溶性，这在雷莫拉宁作为艰难梭菌感染的潜在治疗药物的开发中是重要的。在这项研究中，我们已经确定了假定的甘露糖基转移酶编码ram 29从雷莫拉宁生物合成基因簇的功能。生物信息学显示，Ram 29是一个完整的膜蛋白与推定的DxD基序，建议结合，并激活，聚异戊二烯磷酸甘露糖供体和胞质外的C-末端结构域，预测结合雷莫拉宁糖苷配基受体。将ram 29基因克隆到四环素诱导型质粒pMS 17中，并整合到产黄曲霉素的S.杀真菌的。诱导ram 29在S.杀真菌剂导致产生单甘露糖基化的双孢菌素衍生物，其不存在于WT菌株中。串联质谱分析表明，甘露糖基化发生在Hpg 11残基的阿那西定。除了证实Ram 29的功能外，这些发现还证明了不太常见的膜相关的聚异戊二烯磷酸糖依赖性糖基转移酶如何用于天然产物的糖多样化。这样的策略可能是有价值的，在未来的生物合成工程方法，旨在改善包括抗生素在内的生物活性次生代谢产物的物理化学和生物学特性。
The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.