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.
Highlights
The variations of the recombination enzymes used, these methods differ in their flexibility of integration site, in copy number and optimal length of integration cassettes, as well as scar-forming or scarless integration of target genes
In case of positive integration, the original DNA-sequence is altered, homology to crRNA disappears and genome cutting is prohibited. Such systems consist of two different components: the CRISPRassociated (Cas) protein and a guide RNA consisting of a trans-activating CRISPR RNA and a programmable CRISPR targeting RNA23
Cells were routinely cultured in Lysogeny Broth (LB) media, recovered in super optimal broth medium supplemented with 20 mM glucose (SOC media) and plated on LB agar
Summary
The variations of the recombination enzymes used, these methods differ in their flexibility of integration site, in copy number and optimal length of integration cassettes, as well as scar-forming or scarless integration of target genes. After selecting the desired mutants, this marker has to be deleted by the action of the recombination enzyme flippase[10] Following this protocol, construction of a genome integrated clone, free from antibiotic resistance marker and helper plasmid, takes at least 2 weeks, whereas FRT-scars still remain in the genome. In case of positive integration, the original DNA-sequence is altered, homology to crRNA disappears and genome cutting is prohibited Such systems consist of two different components: the CRISPRassociated (Cas) protein and a guide RNA consisting of a trans-activating CRISPR RNA (tracrRNA) and a programmable CRISPR targeting RNA (crRNA)[23]. Production strains are equipped with the I-SceI restriction site and the pAIO vector, a ready-to-use strain devoid of antibiotic resistance marker, genetic scares and helper plasmid can be established extremely fast and in just 4 steps
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