Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set
Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set
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DOI：
10.1073/pnas.0803164105
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发表时间：
2008-06-17
影响因子：
11.1
通讯作者：
Yoon, Yeo Joon
中科院分区：
文献类型：
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作者：
Park, Je Won;Hong, Jay Sung Joong;Yoon, Yeo Joon
自从链霉素首次被用作治疗结核病的有效抗生素以来，氨基糖苷类抗生素已被广泛用于治疗各种细菌感染超过60年。然而，氨基糖苷生物合成的途径仍然不清楚，主要是因为难以对产生这类抗生素的放线菌进行遗传操作。庆大霉素属于4,6-二取代氨基糖苷类，含有一个特征核心氨基环醇片段，2-脱氧链胺（2-DOS），最近在刺孢小单孢子虫中发现了其生物合成基因簇，使我们能够解密其生物合成途径。为了确定庆大霉素复合物生物合成途径中第一个假三糖中间体庆大霉素A(2)生成的最小基因集及其功能，我们将来自M. echinospora和其他相关氨基糖苷产生菌株的各种候选基因集引入委内瑞拉链霉菌的非氨基糖苷产生菌株。对2-DOS生物合成基因不同组合的异源表达表明，一个亚群gtmB-gtmA-gacH负责庆大霉素核心氨基环醇部分的生物合成。gtmG与gtmB-gtmA-gacH的表达导致2'- n -乙酰氨基多巴胺的产生，表明gtmG作为糖基转移酶将n -乙酰- d -氨基葡萄糖（GLcNA）添加到2- dos中。gtmM在2′- n -乙酰帕罗玛胺重组委内瑞拉葡萄球菌中的表达产生帕罗玛胺。gtmE在委内瑞拉S. paromamine- production菌株中的表达成功地生成了庆大霉素A(2)，这表明gtmE是另一种将d -木糖附着在paromamine上的糖基转移酶。这些结果代表了阐明假三糖氨基糖苷的完整生物合成途径的体内证据。
Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-D-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches D-Xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.