D'Agostino Paul M, Gulder Tobias A M
Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM) , Technical University of Munich , Lichtenbergstraße 4 , 85748 Garching bei München , Germany.
ACS Synth Biol. 2018 Jul 20;7(7):1702-1708. doi: 10.1021/acssynbio.8b00151. Epub 2018 Jul 3.
The need for new pharmacological lead structures, especially against drug resistances, has led to a surge in natural product research and discovery. New biosynthetic gene cluster capturing methods to efficiently clone and heterologously express natural product pathways have thus been developed. Direct pathway cloning (DiPaC) is an emerging synthetic biology strategy that utilizes long-amplification PCR and HiFi DNA assembly for the capture and expression of natural product biosynthetic gene clusters. Here, we have further streamlined DiPaC by reducing cloning time and reagent costs by utilizing T4 DNA polymerase (sequence- and ligation-independent cloning, SLIC) for gene cluster capture. As a proof of principle, the majority of the cyanobacterial hapalosin gene cluster was cloned as a single piece (23 kb PCR product) using this approach, and predicted transcriptional terminators were removed by simultaneous pathway refactoring, leading to successful heterologous expression. The complementation of DiPaC with SLIC depicts a time and cost-efficient method for simple capture and expression of new natural product pathways.
对新型药理学先导结构的需求，尤其是针对耐药性的先导结构，推动了天然产物研究与发现的热潮。因此，已开发出新型生物合成基因簇捕获方法，以高效克隆和异源表达天然产物途径。直接途径克隆（DiPaC）是一种新兴的合成生物学策略，它利用长片段扩增PCR和HiFi DNA组装来捕获和表达天然产物生物合成基因簇。在此，我们通过利用T4 DNA聚合酶（序列和连接无关克隆，SLIC）进行基因簇捕获，减少克隆时间和试剂成本，进一步简化了DiPaC。作为原理验证，使用这种方法将蓝藻哈帕洛辛基因簇的大部分作为一个整体（23 kb PCR产物）进行克隆，并通过同时进行途径重构去除预测的转录终止子，从而实现了成功的异源表达。DiPaC与SLIC的结合描述了一种用于简单捕获和表达新天然产物途径的省时且经济高效的方法。
