Recently, Professor Fu Jun's team at the State Key Laboratory of Microbial Technology, Shandong University, in collaboration with associate professor Yin Jia from Hunan Normal University, published an article entitled “Precise genome engineering in Pseudomonas using the phage-encoded homologous recombination and the Cascade-Cas3 system” in Nature Protocols, to describe the development of precise genome engineering technology in Pseudomonas. Professor Fu Jun, Associate Professor Li Ruijuan from Shandong University and Associate Professor Yin Jia from Hunan Normal University are co-corresponding authors. Dr. Zheng Wentao from Shandong University and graduate student Xia Yandong from Hunan Normal University are co-first authors of this paper. Shandong University is the first author institute and the corresponding author institution.

Pseudomonas is a genus of Gram-negative, rod-shaped, and aerobic bacteria, which contains about 200 species. Organisms within this genus are easy to cultivate, diverse and rich in metabolic functions, making them excellent subjects for scientific research. As a highly studied bacterial genus, the number of species for which genomic information is available continues to expand (currently 4,847 genomic sequences in the Pseudomonas genomes database). However, the biological roles of only a few protein-coding regions in Pseudomonas have been experimentally validated, and this low level of open reading frame characterization emphasizes the need for generic and highly efficient genetic manipulation tools to engineer Pseudomonas with precisely defined genotypes.

In recent years, Professor Fu Jun's team developed phage-encoded homologous recombineering systems for Pseudomonas, and the article entitled “Single-stranded DNA-binding protein and exogenous RecBCD inhibitors enhance phage-derived homologous recombination in Pseudomonas” has been published in iScience (2019). Recombineering systems have made a contribution to the genome mining of new natural products from Pseudomonas. The related work has been published in Biotechnology Journal (2021) entitled “Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva”. Then, Fu Jun's team combined a compact Cascade-Cas3 system from P. aeruginosa (PaeCas3c) with a Pseudomonas-specific PEHR system, and the results of the recombineering assay showed that this compact Cascade-Cas3 system can significantly improve PEHR recombineering accuracy. The related work entitled “Cascade-Cas3 facilitates high-accuracy genome engineering in Pseudomonas using phage-encoded homologous recombination” has been published in Engineering Microbiology (2022).

Here, we describe the recent work of the development of precise genome engineering technology in Pseudomonasby combining of phage-encoded homologous recombination and the Cascade-Cas3 system.

Figure 1. Recombineering system optimization and genome editing in a new Pseudomonas strain.

a. Resistance screening, growth curve assessment and transformation optimization are performed in Pseudomonas, including determination of the critical time points for electroporation;b. Screening for the optimal recombination system.

The researchers worked on the key issue of the lack of a universal and efficient genetic manipulation method for Pseudomonas aeruginosa. This PEHR system is based on a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (Rec-TE_Psy) from P. syringaepv. syringae B728a and also contains exogenous elements, including the RecBCD inhibitor (Redγ or Pluγ) or single-stranded DNA-binding protein (SSB), that were added to enhance the PEHR recombineering efficiency. To address the problem of false positives in Pseudomonasediting with the PEHR system, the processive enzyme Cas3 with a minimal Type I-C Cascade based system, was used in combination with PEHR. This study details the construction of PEHR-Cas3 in Pseudomonas aeruginosa based on the phage-encoded homologous recombination (PEHR) system and the PaeCas3c system of the CRISPR-Cas3 system (also known as the compact Cascade-Cas3 system). The pipeline uses standardized gene cassettes combined with the concerted use of SacB counterselection and Cre site-specific recombinase for markerless or seamless genome modification, combined with vectors that possess the selectively replicating template R6K to minimize recombineering background. Compared with general phage recombinases-based editing systems, the PEHR-Cas3 system can effectively improve the screening efficiency of mutants using the cutting ability of Cas3 protein. This study established a methodology for complete genetic manipulation of Pseudomonas species, summarized the process and key tools of recombinant engineering for gene knockout, insertion, and single-base mutation in non-model Pseudomonas species, effectively expanded the library of gene editing tools, and accelerated the process of researching bacterial genomic resources.

This work was supported by the National Key Research and Development Program of China, the National Natural Science Foundation of China, the Shandong Provincial Natural Science Foundation of China, and so on. Shandong University Core Facilities for Life and Environmental Sciences also provided important support.

Link to the article:https://rdcu.be/dkpaf