Wu Ming-Cheng, Styles Matthew Q, Law Brian J C, Struck Anna-Winona, Nunns Laura, Micklefield Jason
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Microbiology (Reading). 2015 Jul;161(7):1338-47. doi: 10.1099/mic.0.000095. Epub 2015 Apr 15.
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.
来自游动放线菌属菌株ATCC 33076的脂肽类抗生素瑞莫拉宁和由杀真菌链霉菌产生的持久霉素是针对多种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肽结构，由17个氨基酸和一个N端脂肪酸侧链组成，但瑞莫拉宁有一个持久霉素所没有的二甘露糖部分。瑞莫拉宁的甘露糖取代基提高了其水溶性，这在瑞莫拉宁作为艰难梭菌感染潜在治疗药物的开发中很重要。在本研究中，我们确定了瑞莫拉宁生物合成基因簇中ram29编码的假定甘露糖基转移酶的功能。生物信息学分析表明，Ram29是一种整合膜蛋白，具有一个假定的DxD基序，该基序被认为可结合并激活聚异戊二烯磷酸甘露糖供体，以及一个预测可结合瑞莫拉宁苷元受体的胞外C端结构域。将ram29基因克隆到四环素诱导质粒pMS17中，并整合到持久霉素产生菌杀真菌链霉菌的基因组中。在杀真菌链霉菌中诱导ram29表达导致产生单甘露糖基化的持久霉素衍生物，而野生型菌株中不存在这些衍生物。串联质谱分析表明，甘露糖基化发生在持久霉素的Hpg11残基上。除了证实Ram29的功能外，这些发现还证明了较不常见的、与膜相关的、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基多样化。这种策略在未来旨在改善包括抗生素在内的生物活性次生代谢物的物理化学和生物学性质的生物合成工程方法中可能具有重要价值。
