Dataset Information

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

ABSTRACT: Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50-100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.

SUBMITTER: Bubnov DM

PROVIDER: S-EPMC9410887 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

Bubnov Dmitrii M DM Yuzbashev Tigran V TV Khozov Andrey A AA Melkina Olga E OE Vybornaya Tatiana V TV Stan Guy-Bart GB Sineoky Sergey P SP

Nucleic acids research 20220801 15

Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of ...[more]

PMID: 35920321

Similar Datasets

Project description:Lactobacillus casei is a potential cell factory for the production of enzymes and bioactive molecules using episomal plasmids, which suffer from genetic instability. While chromosomal integration strategies can provide genetic stability of recombinant proteins, low expression yields limit their application. To address this problem, we developed a two-step integration strategy in Lb. casei by combination of the LCABL_13040-50-60 recombineering system (comprised of LCABL_1340, LCABL_13050, and LCABL_13060) with the Cre/loxP site-specific recombination system, with an efficiency of ∼3.7 × 103 CFU/µg DNA. A gfp gene was successfully integrated into six selected chromosomal sites, and the relative fluorescence intensities (RFUs) of the resulting integrants varied up to ∼3.7-fold depending on the integrated site, among which the LCABL_07270 site gfp integration showed the highest RFU. However, integrants with gfp gene(s) integrated into the LCABL_07270 site showed various RFUs, ranging from 993 ± 89 to 7,289 ± 564 and corresponding to 1 to 13.68 ± 1.08 copies of gfp gene integration. Moreover, the integrant with 13.68 ± 1.08 copies of the gfp gene had a more stable RFU after 63 generations compared to that of a plasmid-engineered strain. To investigate the feasibility of this system for bioactive molecules with high expression levels, the fimbrial adhesin gene, faeG, from Escherichia coli was tested and successfully integrated into the LCABL_07270 site with 5.51 ± 0.25 copies, and the integrated faeG achieved stable expression. All results demonstrate that this two-step integration system could achieve a high yield of heterologous gene expression by repetitive integration at a targeted chromosomal location in Lb. caseiIMPORTANCE Lactic acid bacteria (LAB), including Lactobacillus casei, have the potential for overexpression of heterologous proteins, such as bioactive molecules and enzymes. However, traditional genetic tools for expression of these proteins show genetic instability or low yields of the desired product. In this study, we provide a procedure for repetitive integration of genes at various chromosomal locations, achieving high-level and stable expression of proteins in Lb. casei without selective pressure. The protocol developed in this study provides an essential reference for chromosomal overexpression of proteins or bioactive molecules in LAB.

Dataset Information

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

Publications

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets