Wan Jiaying, Ma Nan, Yuan Hua
College of Life Sciences, Shanghai Normal University, Shanghai 200234, China.
Eng Microbiol. 2023 Mar 29;3(3):100085. doi: 10.1016/j.engmic.2023.100085. eCollection 2023 Sep.
Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of uncharacterized natural product (NP) biosynthetic gene clusters (), and this has spurred a renaissance of novel drug discovery. However, the majority of these are often poorly or not at all expressed in their native hosts under laboratory conditions, and thus are regarded as silent/orphan . Currently, connecting silent to their corresponding NPs quickly and on a large scale is particularly challenging because of the lack of universal strategies and enabling technologies. Generally, the heterologous host-based genome mining strategy is believed to be a suitable alternative to the native host-based approach for prioritization of the vast and ever-increasing number of uncharacterized . In the last ten years, a variety of methods have been reported for the direct cloning of of interest, which is the first and rate-limiting step in the heterologous expression strategy. Essentially, each method requires that the following three issues be resolved: 1) how to prepare genomic ; 2) how to digest the bilateral boundaries for release of the target ; and 3) how to assemble the and the capture vector. Here, we summarize recent reports regarding how to directly capture a of interest and briefly discuss the advantages and disadvantages of each method, with an emphasis on the notion that direct cloning is very beneficial for accelerating genome mining research and large-scale drug discovery.
大规模基因组挖掘分析表明，微生物可能蕴藏着大量未表征的天然产物（NP）生物合成基因簇，这推动了新型药物发现的复兴。然而，在实验室条件下，这些基因簇中的大多数在其天然宿主中往往表达不佳或根本不表达，因此被视为沉默/孤儿基因簇。目前，由于缺乏通用策略和支持技术，快速且大规模地将沉默基因簇与其相应的天然产物联系起来极具挑战性。一般来说，基于异源宿主的基因组挖掘策略被认为是一种合适的替代方法，可用于对大量且不断增加的未表征基因簇进行优先级排序，以替代基于天然宿主的方法。在过去十年中，已经报道了多种用于直接克隆感兴趣基因簇的方法，这是异源表达策略中的第一步也是限速步骤。从本质上讲，每种方法都需要解决以下三个问题：1）如何制备基因组DNA；2）如何切割双边边界以释放目标基因簇；3）如何组装基因簇和捕获载体。在此，我们总结了近期关于如何直接捕获感兴趣基因簇的报道，并简要讨论了每种方法的优缺点，重点强调直接克隆对加速基因组挖掘研究和大规模药物发现非常有益的观点。
