Wang Zhe-Chong, Stegall Hayden, Miyazawa Takeshi, Keatinge-Clay Adrian T
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, 78712, USA.
Microb Cell Fact. 2025 May 27;24(1):125. doi: 10.1186/s12934-025-02741-w.
Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.
We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.
Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.
试图通过改造模块化聚酮合酶（PKSs）来生成天然产物类似物的工程师在对大片段DNA进行基因组编辑时面临重大挑战。
我们描述了一种CRISPR-Cas9系统，该系统用于对委内瑞拉链霉菌ATCC 15439中的PKS进行重新编程，该PKS有助于生物合成大环内酯类抗生素苦霉素。我们首先通过生成缺乏巨型合酶基因pikAI-pikAIV或整个苦霉素生物合成基因簇但在互补时产生苦霉素的菌株来证明其精确编辑能力。然后，我们用它将苦霉素合酶中的4.4 kb模块替换为其他合酶的模块，以产生两种活性与苦霉素相似的新大环内酯类抗生素。
我们的基因编辑工具能够有效替换链霉菌中广泛且重复的DNA区域。
