Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry CV4 7AL, UK.
Nucleic Acids Res. 2023 Feb 22;51(3):1488-1499. doi: 10.1093/nar/gkad009.
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.
DNA 测序技术和生物信息学的进步揭示了微生物在医学和农业领域产生结构复杂的特殊代谢物的巨大潜力，这些代谢物具有多种用途。然而，这些分子通常需要进行结构修饰，以优化其应用，而使用合成化学方法通常很难实现。生物工程提供了一种结构修饰的互补方法，但通常受到遗传复杂性的阻碍，并且需要对生物合成基因功能有透彻的了解。在异源宿主中表达特殊代谢物生物合成基因簇（BGCs）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，该平台利用转化相关重组（TAR）来高效捕获和并行操作 BGCs。作为概念验证，我们克隆、异源表达并对结构相关的非核糖体肽 eponemycin 和 TMC-86A 的 BGC 进行了遗传分析，澄清了这些重要蛋白酶体抑制剂生物合成中仍然存在的一些模糊性。我们的结果表明，eponemycin BGC 还指导 TMC-86A 的产生，并揭示了这两种代谢物组装的不同机制。此外，我们的数据阐明了 4,5-脱氢-l-亮氨酸（dhL）的生物合成和掺入机制，dhL 是一种罕见的非蛋白氨基酸，掺入到 TMC-86A 和 eponemycin 中。
