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© 2018. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli λ phage recombinase β – an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recβ homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus‐wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Recβ functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads – and beyond.

Details

Title
A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities
Author
Ricaurte, Deirdre E 1 ; Esteban Martínez‐García 1 ; Nyerges, Ákos 2 ; Pál, Csaba 2 ; de Lorenzo, Víctor 1 ; Aparicio, Tomás 1 

 Systems Biology Program, Centro Nacional de Biotecnología (CNB‐CSIC), Madrid, Spain 
 Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary 
Pages
176-188
Section
Research Articles
Publication year
2018
Publication date
Jan 2018
Publisher
John Wiley & Sons, Inc.
e-ISSN
17517915
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2299781512
Copyright
© 2018. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.