Abstract
Summary 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.
Highlights
Cellular robustness and versatile metabolism of Pseudomonas putida have characterized this Gram-negative soil bacterium as an attractive workhorse of metabolic engineering for the bio-based production of chemicals and materials (Nikel et al, 2016; Nikel and de Lorenzo, 2018)
Integration of heterologous biosynthetic gene clusters (BGCs) to P. putida chromosome for the production of heterologous secondary metabolites has relied on time-consuming homologous recombination with selection markers (Wenzel et al, 2005; Gross et al, 2006; Cao et al, 2012; Gong et al, 2016) and unpredictable transposonmediated random insertion (Glandorf et al, 2001; Chai et al, 2012; Loeschcke et al, 2013; Domrose et al, 2015, 2017), requiring the development of rapid and reliable integration system for programmed introduction of heterologous BGCs to P. putida chromosome
To prepare donor plasmids for gene knockout, directly go to step vi. iv Inoculate cells harbouring a correctly constructed adaptor plasmid into 5 ml of LB medium supplemented with 10 lg mlÀ1 tetracycline, incubate at 37°C with rotary shaking at 200 rpm and isolate the adaptor plasmid to clone genes of interest to be integrated to P. putida chromosome
Summary
Cellular robustness and versatile metabolism of Pseudomonas putida have characterized this Gram-negative soil bacterium as an attractive workhorse of metabolic engineering for the bio-based production of chemicals and materials (Nikel et al, 2016; Nikel and de Lorenzo, 2018). Ii Assemble the four amplified products (i.e. two HAs, (MCS-)lox71-tetA(C)-lox66 cassette, and sacB-ori cassette) to construct the donor plasmid for gene knockout or adaptor plasmid for the cloning genes of interest (Fig. 1B).
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