title: A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species.
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.
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary;
authors.forename: Ákos
authors.initials: Á
authors.lastname: Nyerges
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary; csorgo.balint@brc.mta.hu pal.csaba@brc.mta.hu.
authors.forename: Bálint
authors.initials: B
authors.lastname: Csörgő
authors.affiliation: Symbiosis and Functional Genomics Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary; SeqOmics Biotechnology Ltd., Mórahalom H-6782, Hungary.
authors.forename: István
authors.initials: I
authors.lastname: Nagy
authors.affiliation: SeqOmics Biotechnology Ltd., Mórahalom H-6782, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Bálint
authors.affiliation: SeqOmics Biotechnology Ltd., Mórahalom H-6782, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Bihari
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary;
authors.forename: Viktória
authors.initials: V
authors.lastname: Lázár
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary;
authors.forename: Gábor
authors.initials: G
authors.lastname: Apjok
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary;
authors.forename: Kinga
authors.initials: K
authors.lastname: Umenhoffer
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary;
authors.forename: Balázs
authors.initials: B
authors.lastname: Bogos
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary;
authors.forename: György
authors.initials: G
authors.lastname: Pósfai
authors.affiliation: Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary; csorgo.balint@brc.mta.hu pal.csaba@brc.mta.hu.
authors.forename: Csaba
authors.initials: C
authors.lastname: Pál
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics; D005818:Genetic Engineering; D016680:Genome, Bacterial; D009154:Mutation
keywords: genome engineering; methyl-directed mismatch repair; off-target effects; recombineering; synthetic biology