基因簇大片段克隆技术研究进展及挑战

天然产物结构复杂、活性多样，是新药开发的重要来源，对天然产物生物合成途径的研究，有利于探索酶催化的合成机制，促进复杂天然产物的应用。天然产物的生物合成由其对应的基因簇调控，其中大量天然产物生物合成基因簇(biosynthetic gene clusters,BGCs)在野生型菌株中无法表达或表达量低。对这些基因簇的研究，需要进行克隆表达，而如何克隆大片段基因簇并使其表达，从而发现新型天然产物是一个具有挑战性的问题。其中构建基因组文库、转化关联重组(transformation-associated recombination,TAR)、Red/ET重组等是克隆大片段基因簇的重要技术。本文从克隆技术的策略和应用两个方面，总结了这3种克隆技术目前的研究进展，讨论了目前大片段基因簇克隆技术面临的挑战，为研究大片段基因簇提供方法学借鉴。

Research progress and challenges in cloning large gene clusters

Natural products, which feature complex structures and diverse activities, are important sources of new drugs. The research on the biosynthesis pathways of natural products is conducive to exploring the synthesis mechanism catalyzed by enzymes and promoting the application of complex natural products. The corresponding gene clusters regulate the biosynthesis of natural products, and a large number of biosynthetic gene clusters (BGCs) of natural products are not expressed or expressed at low levels in wild-type strains. Thus, these gene clusters are usually cloned before further research on them. It remains challenging to clone and express large gene clusters for the discovery of novel natural products. Construction of genomic library, transformation-associated recombination (TAR), and Red/ET recombination technology are essential techniques for cloning large gene clusters. This study summarizes the research on these three cloning techniques from the aspect of cloning strategy and application. Moreover, we discuss the challenges in cloning large gene clusters, providing a methodological reference for further study.