PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin
PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin
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DOI：
10.1073/pnas.0337542100
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发表时间：
2003-02-18
影响因子：
11.1
通讯作者：
Chater, KF
中科院分区：
文献类型：
--
作者：
Gust, B;Challis, GL;Chater, KF
链霉菌是高G+C革兰氏阳性、产生抗生素的菌丝体土壤细菌。8.7-Mb天蓝色链霉菌基因组先前通过使用Supercos-1克隆的有序文库进行测序。在这里，我们描述了一个有效的程序，通过使用PCR靶向和A-红介导的重组在粘粒克隆中创建精确的基因置换。克隆的链霉菌基因被含有可选择的抗生素抗性和oriT（RK 2）的盒替换，用于通过RP 4介导的属间接合有效转移到链霉菌中。Supercos-1在链霉菌中不复制，但克隆很容易进行双交换重组，从而产生基因替换。抗生素抗性盒的侧翼是用于去除抗生素抗性的酵母FLIP重组酶靶序列和用于产生未标记的非极性突变的oriTRK 2。该技术已被超过20名研究人员成功地用于突变约100个链霉菌基因。作为一个例子，我们描述了它的应用程序中发现的基因参与生产的土臭素，无处不在的气味的土壤。基因Sco 6073（cyc 2）编码具有两个倍半萜合酶结构域的蛋白质，其中只有一个结构域是土臭素生物合成所需的，可能通过由焦磷酸法呢酯的倍半萜合酶产生的germacra-1（10）E，5E-二烯-11-醇中间体。
Streptomycetes are high G+C Gram-positive, anti biotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and A-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLIP recombinase target sequences for removal of the antibiotic resistance and oriTRK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.