Fast and antibiotic free genome integration into Escherichia coli chromosome

Abstract

Genome-based Escherichia coli expression systems are superior to conventional plasmid-based systems as the metabolic load triggered by recombinant compounds is significantly reduced. The efficiency of T7-based transcription compensates for low gene dosage (single copy) and facilitates high product formation rates. While common Gene Bridges’ λ-red mediated recombination technique for site directed integration of genes into the host genome is very efficient, selection for positive clones is based on antibiotic resistance markers and removal thereof is often time consuming. For the generation of industrial production strains, flexibility in terms of integration site is not required, yet time from gene design to a stable clone is a quite relevant parameter. In this study, we developed a fast, efficient and antibiotic-free integration method for E. coli as production strain. We combined the λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. In a first step, λ-red proteins are performing genome integration of a linear, antibiotic marker-free integration cassette. The engineered host strain carries the I-SceI restriction sequence at the attTn7 site, where the integration event happens. After homologous recombination and integration at the target site, site-specific genome cleavage by endonuclease I-SceI is induced, thereby killing all cells still containing an intact I-SceI site. In case of positive recombination events, the genomic I-SceI site is deleted and cleavage is no longer possible. Since plasmids are designed to contain another I-SceI restriction site they are destroyed by self-cleavage, a procedure replacing the time-consuming plasmid curing. The new plasmid-based “All-In-One” genome integration method facilitates significantly accelerated generation of genome-integrated production strains in 4 steps.

A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli

“…Transcription was stopped by the synthetic tZenit terminator element [ 76 ]. Expression cassettes were either integrated into the host genome of BL21(DE3) (New England Biolabs, USA) and HMS174(DE3) (Merck Millipore, Germany) via a previously described method [ 77 ] or cloned into a pET30a plasmid backbone containing a cer [ 78 ] sequence (pET30a cer ) with subsequent transformation into the production hosts. Amino acid sequences of all signal sequences and peptides are listed in the supplementary material (Table S1).…”

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield

“…The co-translational dsbA SS was used to mitigate cytosolic fluorescence. The dsbA SS -sfGFP construct was amplified from an in-house pET30a plasmid according to the same procedure as the Fab’s and integrated into the genome of BL21­(DE3) …”

“…The dsbA SS -sfGFP construct was amplified from an in-house pET30a plasmid according to the same procedure as the Fab's 41 and integrated into the genome of BL21(DE3). 93 Media and Cultivation Conditions. Cell Banks.…”

Massive integration of large gene libraries in the chromosome of Escherichia coli

“…attTn7 , attB ) (Landy, 2015 ; Peters, 2019 ). Engineering these sites at various positions of the chromosome also enables integration at multiple loci (Egger et al., 2020 ). Another option is the use of transposon‐encoded CRISPR‐Cas systems, which utilize designed crRNA molecules to guide the insertion of the exogenous DNA into a custom target site in the genome (Vo et al., 2020 ).…”