Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida.,Microbial Biotechnology

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Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2019-02-14 , DOI: 10.1111/1751-7915.13374
Kyeong Rok Choi ₁ , Sang Yup Lee _{1,

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Affiliation

Pseudomonas putida has emerged as a promising host for the production of chemicals and materials thanks to its metabolic versatility and cellular robustness. In particular, P. putida KT2440 has been officially classified as a generally recognized as safe (GRAS) strain, which makes it suitable for the production of compounds that humans directly consume, including secondary metabolites of high importance. Although various tools and strategies have been developed to facilitate metabolic engineering of P. putida, modification of large genes/clusters essential for heterologous expression of natural products with large biosynthetic gene clusters (BGCs) has not been straightforward. Recently, we reported a RecET‐based markerless recombineering system for engineering P. putida and demonstrated deletion of multiple regions as large as 101.7 kb throughout the chromosome by single rounds of recombineering. In addition, development of a donor plasmid system allowed successful markerless integration of heterologous BGCs to P. putida chromosome using the recombineering system with examples of – but not limited to – integrating multiple heterologous BGCs as large as 7.4 kb to the chromosome of P. putida KT2440. In response to the increasing interest in our markerless recombineering system, here we provide detailed protocols for markerless gene knockout and integration for the genome engineering of P. putida and related species of high industrial importance.

中文翻译：

基于 RecET 的无标记基因敲除和整合方案以在恶臭假单胞菌中表达异源生物合成基因簇。

恶臭假单胞菌因其代谢多功能性和细胞稳健性而成为生产化学品和材料的有前途的宿主。特别是恶臭假单胞菌KT2440 已被正式归类为公认安全 (GRAS) 菌株，这使其适用于生产人类直接食用的化合物，包括高度重要的次级代谢物。尽管已经开发了各种工具和策略来促进恶臭假单胞菌的代谢工程，但对具有大型生物合成基因簇 (BGC) 的天然产物异源表达所必需的大基因/簇的修饰并不简单。最近，我们报道了一种基于 RecET 的无标记重组工程系统恶臭假单胞菌，并通过单轮重组证明了整个染色体上多达 101.7 kb 的多个区域的缺失。此外，供体质粒系统的开发允许使用重组系统成功地将异源 BGC 无标记整合到恶臭假单胞菌染色体上，例如（但不限于）将多个大至 7.4 kb 的异源 BGCs 整合到恶臭假单胞菌染色体上KT2440。为了响应对我们的无标记重组系统日益增长的兴趣，我们在此为恶臭假单胞菌和具有高度工业重要性的相关物种的基因组工程的无标记基因敲除和整合提供了详细的协议。

更新日期：2019-02-14

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