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

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Microbial Biotechnology

Background:

• Chromosomal integration of biosynthetic pathways is crucial for stable, low-variability production of high-value chemicals by industrial microbial strains.
• Current methods for introducing multiple transcription units into microbial genomes are often laborious and exhibit variable efficiency, particularly with large or complex constructs.

Purpose of the Study:

• To develop a novel, efficient, and versatile methodology for chromosomal integration of large biosynthetic gene clusters in microbial hosts.
• To demonstrate the utility of this new toolbox for applications beyond simple integration, including gene deletion and in vivo assembly.

Main Methods:

• Development of serine integrase recombinational engineering (SIRE), combining recombinase-mediated cassette exchange (RMCE) with PhiC31 integrase orthogonal att sites.
• Application of SIRE in Escherichia coli for the integration of a 10.3 kb biosynthetic gene cluster.
• Demonstration of SIRE's capabilities for multitargeting, operon deletion, and in vivo assembly.

Main Results:

• SIRE achieved 100% efficiency for the integration step of a 10.3 kb gene cluster into the E. coli genome without requiring selection markers.
• The SIRE toolbox proved versatile, enabling large fragment integration, multitargeting, operon deletion, and in vivo assembly.
• The serine integrase PhiC31 was successfully applied in Saccharomyces cerevisiae as a marker recovery tool, highlighting the method's portability.

Conclusions:

• SIRE is a powerful and versatile toolbox for engineering microbial genomes, facilitating the stable integration of large DNA fragments.
• This methodology offers significant advantages over existing techniques for developing industrial microbial strains for biotechnological applications.
• The portability of the serine integrase PhiC31 suggests broader applicability of the SIRE approach across different microbial systems.