Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation.

Autor: Seco EM; Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Darwin 3, Campus UAM Cantoblanco, Madrid, 28049, Spain., Fernández LÁ; Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Darwin 3, Campus UAM Cantoblanco, Madrid, 28049, Spain.
Jazyk: angličtina
Zdroj: Microbial biotechnology [Microb Biotechnol] 2022 May; Vol. 15 (5), pp. 1374-1391. Date of Electronic Publication: 2021 Nov 09.
DOI: 10.1111/1751-7915.13967
Abstrakt: The probiotic strain Escherichia coli Nissle 1917 (EcN) is a common bacterial chassis in synthetic biology developments for therapeutic applications given its long track record of safe administration in humans. Chromosomal integration of the genes of interest (GOIs) in the engineered bacterium offers significant advantages in genetic stability and to control gene dose, but common methodologies relying on the transformation of EcN are inefficient. In this work, we implement in EcN the use of bacterial conjugation in combination with markerless genome engineering to efficiently insert multiple GOIs at different loci of EcN chromosome, leaving no antibiotic resistance genes, vector sequences or scars in the modified bacterium. The resolution of cointegrants that leads to markerless insertion of the GOIs requires expression of I-SceI endonuclease and its efficiency is enhanced by λ Red proteins. We show the potential of this strategy by integrating different genes encoding fluorescent and bioluminescent reporters (i.e. GFP, mKate2, luxCDABE) both individually and sequentially. We also demonstrate its application for gene deletions in EcN (ΔflhDC) and to replace the endogenous regulation of chromosomal locus (i.e. flhDC) by heterologous regulatory elements (e.g. tetR-Ptet) in order to have an ectopic control of gene expression in EcN with an external inducer to alter bacterial behaviour (e.g. flagellar motility). Whole-genome sequencing confirmed the introduction of the designed modifications without off-target alterations in the genome. This straightforward approach accelerates the generation of multiple modifications in EcN chromosome for the generation of living bacterial therapeutics.
(© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)
Databáze: MEDLINE
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