A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.

Microbial Biotechnology
Deirdre E RicaurteTomás Aparicio

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 ran...Continue Reading

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Citations

May 12, 2018·Biotechnology and Bioengineering·Emily FreedCarrie A Eckert
May 10, 2019·Biotechnology Journal·Lorena Fernández-CabezónPablo I Nikel
Oct 11, 2019·Environmental Microbiology·Tomas AparicioVíctor de Lorenzo
May 30, 2020·Proceedings of the National Academy of Sciences of the United States of America·Timothy M WannierGeorge M Church
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Jun 6, 2020·ACS Synthetic Biology·Carlos Piñero-LambeaMaria Lluch-Senar

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Methods Mentioned

BETA
genetic modification
PCR

Software Mentioned

MAGE
Rec2
BlastP

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