@article{MTMT:3390047,
	title = {Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.},
	url = {https://m2.mtmt.hu/api/publication/3390047},
	author = {Nyerges, Ákos and Csörgő, Bálint and Draskovits, Gábor and Kintses, Bálint and Szili, Petra and Ferenc, Györgyi and Révész, Tamás and Ari, Eszter and Nagy, István and Bálint, Balázs and Vásárhelyi, Bálint Márk and Bihari, Péter and Számel, Mónika and Balogh, Dávid and Papp, Henrietta and Kalapis, Dorottya and Papp, Balázs and Pál, Csaba},
	doi = {10.1073/pnas.1801646115},
	journal-iso = {P NATL ACAD SCI USA},
	journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
	volume = {115},
	unique-id = {3390047},
	issn = {0027-8424},
	abstract = {Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology.},
	keywords = {ESCHERICHIA-COLI; DRUG-RESISTANCE; URINARY-TRACT-INFECTIONS; QUINOLONE RESISTANCE; ANTIMICROBIAL RESISTANCE; Synthetic biology; RANDOM MUTAGENESIS; Homologous recombination; DIRECTED EVOLUTION; GENE MODIFICATION; COLI DIHYDROFOLATE-REDUCTASE; STRANDED-DNA OLIGONUCLEOTIDES; multiplex automated genome engineering; high-throughput mutagenesis},
	year = {2018},
	eissn = {1091-6490},
	pages = {E5726-E5735},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X; Csörgő, Bálint/0000-0003-0397-6845; Ferenc, Györgyi/0000-0002-3456-319X; Ari, Eszter/0000-0001-7774-1067; Vásárhelyi, Bálint Márk/0000-0003-1782-8691; Papp, Henrietta/0000-0003-3887-5657}
}

@article{MTMT:3315234,
	title = {A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.},
	url = {https://m2.mtmt.hu/api/publication/3315234},
	author = {Ricaurte, DE and Martinez-Garcia, E and Nyerges, Ákos and Pál, Csaba and de Lorenzo, V and Aparicio, T},
	doi = {10.1111/1751-7915.12846},
	journal-iso = {MICROB BIOTECHNOL},
	journal = {MICROBIAL BIOTECHNOLOGY},
	volume = {11},
	unique-id = {3315234},
	issn = {1751-7907},
	abstract = {Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli lambda phage recombinase beta - 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 Recbeta 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 Recbeta 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.},
	year = {2018},
	eissn = {1751-7915},
	pages = {176-188},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X}
}

@article{MTMT:3038670,
	title = {A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species},
	url = {https://m2.mtmt.hu/api/publication/3038670},
	author = {Nyerges, Ákos and Csörgő, Bálint and Nagy, István and Bálint, Balázs and Bihari, Péter and Lázár, Viktória and Apjok, Gábor and Umenhoffer, Kinga and Bogos, Balázs and Pósfai, György and Pál, Csaba},
	doi = {10.1073/pnas.1520040113},
	journal-iso = {P NATL ACAD SCI USA},
	journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
	volume = {113},
	unique-id = {3038670},
	issn = {0027-8424},
	abstract = {Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.},
	keywords = {EXPRESSION; IN-VIVO; RESISTANCE; GENES; ESCHERICHIA-COLI; EVOLUTION; DNA-REPAIR; Synthetic biology; MUTL; REPAIR MUTANTS; methyl-directed mismatch repair; off-target effects; recombineering; genome engineering},
	year = {2016},
	eissn = {1091-6490},
	pages = {2502-2507},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X; Csörgő, Bálint/0000-0003-0397-6845}
}