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Protocols for yTREX/Tn5-based gene cluster expression in Pseudomonas putida.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2019-06-04 , DOI: 10.1111/1751-7915.13402
Robin Weihmann 1 , Andreas Domröse 2 , Thomas Drepper 1 , Karl-Erich Jaeger 1 , Anita Loeschcke 1
Affiliation  

Bacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5‐based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX‐based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine‐1‐carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putidaKT2440 as well as the use of β‐galactosidase‐encoding lacZ as a reporter of production levels.

中文翻译:

在恶臭假单胞菌中基于yTREX / Tn5的基因簇表达的协议。

细菌基因簇代表了次级代谢产物途径的遗传宝库,通常需要在异源宿主中激活才能获得有价值的生物合成产物。我们在这里为yTREX“基因簇移植工具”的应用提供详细的协议:通过酵母重组克隆,可以将感兴趣的基因簇克隆到yTREX载体中,从而实现基因簇向细菌等细菌的牢固结合转移。恶臭假单胞菌,以及随后基于Tn5的转座子插入宿主染色体。取决于基因簇的结构和染色体插入位点,可以从染色体启动子有效地转录各个途径基因,从而使天然产物能够生物合成。我们描述了设计基因簇表达盒,通过酵母重组在yTREX载体中克隆该盒以及随后在恶臭假单胞菌中进行转移和表达的工作流程。作为天然产物的生物合成的基于yTREX移植一个例子,我们提供有关吩嗪-1-羧酸生物合成基因从的克隆和活化细节铜绿假单胞菌恶臭假单胞菌KT2440以及使用β-编码半乳糖苷酶的lacZ作为生产水平的报告物。
更新日期:2019-06-04
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