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

Author:

Asin-Garcia Enrique12ORCID,Garcia-Morales Luis1,Bartholet Tessa1,Liang Zhuobin34,Isaacs Farren J345ORCID,Martins dos Santos Vitor A P126ORCID

Affiliation:

1. Laboratory of Systems and Synthetic Biology, Wageningen University & Research , Wageningen 6708 WE, The Netherlands

2. Bioprocess Engineering Group, Wageningen University & Research , Wageningen  6700  AA , The Netherlands

3. Department of Molecular, Cellular, and Developmental Biology, Yale University , New Haven , CT  06520 , USA

4. Systems Biology Institute, Yale University , West Haven , CT  06516 , USA

5. Department of Biomedical Engineering, Yale University , New Haven , CT  06520 , USA

6. LifeGlimmer GmbH , Berlin  12163 , Germany

Abstract

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.  

Funder

Dutch Research Council

European Union Horizon

US Department of Energy, Office of Science, Office of Biological Environmental Research

Publisher

Oxford University Press (OUP)

Subject

Genetics

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