Protocols for RecET‐based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

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.

Unraveling Plant Natural Chemical Diversity for Drug Discovery Purposes

“…Microbial hosts are generally considered more amenable than plants to fermentation process (Atanasov et al, 2015). Among them, classical work horses like E. coli or S. cerevisiae, or newcomers like Bacillus subtilis and Pseudomonas putidaare (Nikel et al, 2014;Loeschcke and Thies, 2015;Choi and Lee, 2020) can be cited. Another interesting example is the recent high-cell-density fermentation strategies developed for heterologous production in Pichia pastoris (W.-C. .…”

Industrial biotechnology of Pseudomonas putida: advances and prospects

“…Thus, one copy of the Flp recognition target (FRT) site will always remain after excision, limiting the repeated use of these procedures (Nikel and de Lorenzo 2013b). Subsequently, efficient genome editing methods that do not leave selection markers nor foreign DNA sequences, such as CRISPR/Cas9 technologies (Aparicio et al 2018;Kim et al 2020;Pham et al 2020;Wirth et al 2020;Zhou et al 2020), DNA recombineering (Aparicio et al 2020a;Aparicio et al 2020b;Choi et al 2018;Choi and Lee 2020), and homologous recombination-based DNA editing (Galvão and de Lorenzo 2005;Graf and Altenbuchner 2011;Martínez-García and de Lorenzo 2011), have been developed and applied. The most widespread homologous recombination-based technique for genome engineering in P. putida involves two rounds of recombination.…”

“…Since many of the biosynthetic gene clusters required for the production of natural products are larger than 6 kb (Loeschcke and Thies 2015), sophisticated tools are needed for chromosomal integration. One such tool is the recent developed RecET-based markerless recombineering system for P. putida (Choi and Lee 2020). The site-specific integration of a 7.4-kb violacein cluster could be shown (Choi et al 2018).…”

Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics

“…In some cases, the expression of λ red recombineering genes was proven to be toxic, as shown for the enterobacteriaceae Pantoea ananatis (Katashkina et al, 2009), requiring the use of mutant strains to express λ red genes (Katashkina et al, 2009). Adaptation of Rac recombineering was proven successful in case of Zymomonas mobilis (Wu et al, 2017), Acinetobacter baumannii (Tucker et al, 2019), Corynebacterium glutamicum (Huang et al, 2017) and Pseudomonas putida (Choi and Lee, 2020) among others.…”