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首页> 外文期刊>Applied and Environmental Microbiology >Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis
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Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

机译：用于工程化红霉素基因簇在链霉菌属种中表达的多重整合质粒。和组合生物合成

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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 ?BT1, 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 ?C31 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.
机译：链霉菌属及其近亲属中的细菌是具有抗生素活性的次生代谢产物的多产者。这些细菌的基因组测序已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。而且，这些新途径可以提供可用于组合生物合成方法的新基因，以产生现有抗生素的非天然类似物。我们在这里探索基于噬菌体TG1，SV1和ΔBT1的int / attP位点的多个直系同源整合质粒系统的使用，以在异源链霉菌宿主中表达红霉素的聚酮化合物合酶（PKS）。含有从三个不同整合质粒表达的三个聚酮化合物合酶基因eryAI，eryAII和eryAIII的链霉菌菌株产生糖苷配基中间体6-脱氧赤藓醇B（6-dEB）。还构建了另一对整合质粒，均来自ΔC31int / attP基因座，携带一个基因盒，用于糖苷配基中间体的糖基化，带有或不带有合成赤藓醇内酯B（EB）所需的定制基因eryF。 。代谢物的液相色谱-质谱分析表明产生了Angolosaminyl-6-dEB和Angolosaminyl-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势。

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