Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species

Abstract

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications.

Design and construction towards a pan-microbial toolkit

“…The high diversity of promoter variants found in the donor underscores the robustness of our library assembly and delivery protocols. The number of transconjugant colony-forming units (CFU) obtained varied over several orders of magnitude between recipient species, likely due to variation in the conjugation efficiency, from a maximum of 2.1 x 10 7 CFU for E. coli to 20 CFU for H. alkaliphila ( Supplementary Figure 12A ). As expected, we found the number of promoters detected by NGS in each recipient transconjugant library was positively correlated with the number of transconjugant CFU obtained ( Supplementary Figure 12B ), highlighting the importance of efficient DNA delivery methods for library screening.…”

“…One promising avenue to achieving this is the development of widely-accessible resources that provide: (1) highly diverse, combinatorial libraries of genetic parts compatible with a broad range of microbes, and (2) validated high-throughput screening methods to rapidly identify functional genetic parts in target microbes (Figure 1). This design can dramatically simplify the process of identifying functional genetic parts in non-model microbes, as exemplified in several recent studies [1][2][3][4][5][6][7][8] .…”