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

(2019) BIOTECHNOLOGY AND BIOENGINEERING. 116(2). p.364-374
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Abstract
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.
Keywords
Escherichia coli, genomic integration, knock-in, PhiC31, serine integrase, ESCHERICHIA-COLI, PHI-C31 INTEGRASE, GENE-EXPRESSION, BUDDING YEAST, OPTIMIZATION, REPLICATION, CHROMOSOME, STABILITY, VIOLACEIN, CLEAVAGE

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Chicago
Snoeck, Nico, Maarten De Mol, Dries Van Herpe, Anke Goormans, Isabelle Maryns, Pieter Coussement, Gert Peters, Joeri Beauprez, Sofie De Maeseneire, and Wim Soetaert. 2019. “Serine Integrase Recombinational Engineering (SIRE) : a Versatile Toolbox for Genome Editing.” Biotechnology and Bioengineering 116 (2): 364–374.
APA
Snoeck, N., De Mol, M., Van Herpe, D., Goormans, A., Maryns, I., Coussement, P., Peters, G., et al. (2019). Serine integrase recombinational engineering (SIRE) : a versatile toolbox for genome editing. BIOTECHNOLOGY AND BIOENGINEERING, 116(2), 364–374.
Vancouver
1.
Snoeck N, De Mol M, Van Herpe D, Goormans A, Maryns I, Coussement P, et al. Serine integrase recombinational engineering (SIRE) : a versatile toolbox for genome editing. BIOTECHNOLOGY AND BIOENGINEERING. 2019;116(2):364–74.
MLA
Snoeck, Nico et al. “Serine Integrase Recombinational Engineering (SIRE) : a Versatile Toolbox for Genome Editing.” BIOTECHNOLOGY AND BIOENGINEERING 116.2 (2019): 364–374. Print.
@article{8581358,
  abstract     = {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.},
  author       = {Snoeck, Nico and De Mol, Maarten and Van Herpe, Dries and Goormans, Anke and Maryns, Isabelle and Coussement, Pieter and Peters, Gert and Beauprez, Joeri and De Maeseneire, Sofie and Soetaert, Wim},
  issn         = {0006-3592},
  journal      = {BIOTECHNOLOGY AND BIOENGINEERING},
  language     = {eng},
  number       = {2},
  pages        = {364--374},
  title        = {Serine integrase recombinational engineering (SIRE) : a versatile toolbox for genome editing},
  url          = {http://dx.doi.org/10.1002/bit.26854},
  volume       = {116},
  year         = {2019},
}

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