Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis.
Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis.
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DOI：
10.1128/aem.02403-15
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发表时间：
2015-12
影响因子：
4.4
通讯作者：
Smith MC
中科院分区：
文献类型：
--
作者：
Fayed B;Ashford DA;Hashem AM;Amin MA;El Gazayerly ON;Gregory MA;Smith MC
链霉菌属及其近亲中的细菌是具有抗生素活性的次级代谢产物的多产者。这些细菌的基因组测序揭示了潜在的新抗生素途径的丰富来源，其产物从未被观察到。此外，这些新的途径可以提供新的基因，可用于组合生物合成方法，以产生现有抗生素的非天然类似物。在这里，我们探讨使用多个orthopathic整合质粒系统，基于int/attP位点从STG 1，SV 1，和STGBT 1，在异源链霉菌宿主中表达红霉素的聚酮合酶（PKS）。含有三种聚酮合酶基因eryAI、eryAII和eryAIII的链霉菌菌株从三种不同的整合质粒表达，产生糖苷配基中间体6-脱氧腺苷酸B（6-dEB）。构建了另一对整合质粒，其均来源于C31 int/attP基因座，携带用于糖苷配基中间体糖基化的基因盒，具有或不具有合成阿糖胞苷B（EB）所需的剪裁基因eryF。代谢产物的液相色谱-质谱分析表明产生了安哥罗胺基-6-dEB和安哥罗胺基-EB。证明了使用多重整合质粒进行工程表达和组合生物合成的优点。
Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.