Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis.
Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis.
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DOI：
10.1093/nar/gkad009
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发表时间：
2023-02-22
影响因子：
14.9
通讯作者：
Challis, Gregory L.
中科院分区：
文献类型：
--
作者：
Huang, Chuan;Zabala, Daniel;de los Santos, Emmanuel L. C.;Song, Lijiang;Corre, Christophe;Alkhalaf, Lona M.;Challis, Gregory L.
DNA测序技术和生物信息学的进步揭示了微生物产生结构复杂的特殊代谢物的巨大潜力，这些代谢物在医学和农业中具有多种用途。然而，这些分子通常需要结构修饰以优化它们的应用，这可能很难使用合成化学。生物工程提供了一种补充的方法，结构修改，但往往受到遗传的棘手性，并需要一个彻底的了解生物合成基因的功能。在异源宿主中表达专门的代谢物生物合成基因簇（BGC）可以克服这些问题。然而，目前的BGC克隆和操作方法效率低下，缺乏保真度，并且可能非常昂贵。在这里，我们报告了一个基于酵母的平台，利用转化相关重组（TAR）的高效捕获和BGC的并行操作。作为一个概念的证明，我们克隆，异源表达和遗传分析BGC的结构相关的非核糖体肽eponemycin和TMC-86 A，澄清这些重要的蛋白酶体抑制剂的生物合成中剩余的模糊性。我们的研究结果表明，eponemycin BGC还指导TMC-86 A的生产，并揭示了启动这两种代谢产物组装的对比机制。此外，我们的数据揭示了4，5-脱氢-L-亮氨酸（dhL）的生物合成和掺入机制，这是一种不寻常的非蛋白质氨基酸，可掺入TMC-86 A和eponemycin中。
Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin.
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