Serine integrase recombinational engineering (SIRE): A versatile toolbox for genome editing.

Autor: Snoeck N; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium., De Mol ML; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium., Van Herpe D; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium., Goormans A; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium., Maryns I; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium., Coussement P; Inbiose NV, Ghent, Belgium., Peters G; Inbiose NV, Ghent, Belgium., Beauprez J; Inbiose NV, Ghent, Belgium., De Maeseneire SL; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium., Soetaert W; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Department Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2019 Feb; Vol. 116 (2), pp. 364-374. Date of Electronic Publication: 2018 Nov 15.
DOI: 10.1002/bit.26854
Abstrakt: Chromosomal integration of biosynthetic pathways for the biotechnological production of high-value chemicals is a necessity to develop industrial strains with a high long-term stability and a low production variability. However, the introduction of multiple transcription units into the microbial genome remains a difficult task. Despite recent advances, current methodologies are either laborious or efficiencies highly fluctuate depending on the length and the type of the construct. Here we present serine integrase recombinational engineering (SIRE), a novel methodology which combines the ease of recombinase-mediated cassette exchange (RMCE) with the selectivity of orthogonal att sites of the PhiC31 integrase. As a proof of concept, this toolbox is developed for Escherichia coli. Using SIRE we were able to introduce a 10.3 kb biosynthetic gene cluster on different locations throughout the genome with an efficiency of 100% for the integrating step and without the need for selection markers on the knock-in cassette. Next to integrating large fragments, the option for multitargeting, for deleting operons, as well as for performing in vivo assemblies further expand and proof the versatility of the SIRE toolbox for E. coli. Finally, the serine integrase PhiC31 was also applied in the yeast Saccharomyces cerevisiae as a marker recovery tool, indicating the potential and portability of this toolbox.
(© 2018 Wiley Periodicals, Inc.)
Databáze: MEDLINE
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