Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching bei München, Germany.
Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching bei München, Germany.
Metab Eng. 2018 May;47:334-345. doi: 10.1016/j.ymben.2018.03.010. Epub 2018 Mar 13.
Specialized metabolites from bacteria are an important source of inspiration for drug development. The genes required for the biosynthesis of such metabolites in bacteria are usually organized in so-called biosynthetic gene clusters (BGCs). Using modern bioinformatic tools, the wealth of genomic data can be scanned for such BGCs and the expected products can often structurally be predicted in silico. This facilitates the directed discovery of putatively novel bacterial metabolites. However, the production of these molecules often requires genetic manipulation of the BGC for activation or the expression of the pathway in a heterologous host. The latter necessitates the transplantation of the BGC into a suitable expression system. To achieve this goal, powerful cloning strategies based on in vivo homologous recombination have recently been developed. This includes LCHR and LLHR in E. coli as well as TAR cloning in yeast. Here, we present Direct Pathway Cloning (DiPaC) as an efficient complementary BGC capturing strategy that relies on long-amplicon PCR and in vitro DNA assembly. This straightforward approach facilitates full pathway assembly, BGC refactoring and direct transfer into any vector backbone in vitro. The broad applicability and efficiency of DiPaC is demonstrated by the discovery of a new phenazine from Serratia fonticola, the first heterologous production of anabaenopeptins from Nostoc punctiforme and the transfer of the native erythromycin BGC from Saccharopolyspora erythraea into Streptomyces. Due to its simplicity, we envisage DiPaC to become an essential method for BGC cloning and metabolic pathways construction with significant applications in metabolic engineering, synthetic biology and biotechnology.
细菌的次生代谢产物是药物开发的重要灵感来源。细菌中这些代谢物生物合成所需的基因通常组织在所谓的生物合成基因簇（BGC）中。利用现代生物信息学工具，可以扫描基因组数据中的 BGC，并在计算机上对预期的产物进行结构预测。这有助于有针对性地发现潜在的新型细菌代谢产物。然而，这些分子的产生通常需要对 BGC 进行遗传操作以激活或在异源宿主中表达途径。后者需要将 BGC 移植到合适的表达系统中。为了实现这一目标，最近开发了基于体内同源重组的强大克隆策略。这包括大肠杆菌中的 LCHR 和 LLHR 以及酵母中的 TAR 克隆。在这里，我们提出了直接途径克隆（DiPaC）作为一种有效的 BGC 捕获策略，该策略依赖于长扩增子 PCR 和体外 DNA 组装。这种简单的方法促进了完整途径的组装、BGC 的重构以及在体外直接转移到任何载体骨架中。DiPaC 的广泛适用性和效率通过从 Serratia fonticola 中发现一种新的吩嗪、从 Nostoc punctiforme 中首次异源生产 anabaenopeptins 以及将天然红霉素 BGC 从 Saccharopolyspora erythraea 转移到 Streptomyces 中得到了证明。由于其简单性，我们设想 DiPaC 将成为 BGC 克隆和代谢途径构建的重要方法，在代谢工程、合成生物学和生物技术中有重要的应用。
