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

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. K E Y W O R D SEscherichia coli, genomic integration, knock-in, PhiC31, serine integrase

Heterologous Metabolic Pathways: Strategies for Optimal Expression in Eukaryotic Hosts

“…The viral-mediated gene delivery system is based predominantly on bacteriophage integrases and the corresponding integration sequences: thus, many methods are based on φC31 integrase [ 50 , 51 ], derived from φBT1 [ 52 , 53 ], or comprise several integrases [ 54 ]. Integrase systems can provide means for the efficient integration of large DNA sequences (up to 100 kbp) into specific genomic loci, allowing for iterative or one-step tandem assembly of fragments [ 52 ].…”

“…Вирус-опосредованная система доставки генов основана, в целом, на интегразах бактериофагов и соответствующих последовательностях интеграции. Поэтому во многих методах используют интегразу φC31 [50,51], интегразу φBT1 [52,53] или несколько интеграз [54]. Эти системы могут обеспечить эффективную интеграцию в определенные места генома больших (до 100 т.п.н.)…”

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology

“…Six pairs of nonpalindromic central overlap sequences (TT, CT, GT, CA, CC, and TC) can be used to create orthogonal sites and allows multiple att sites to be used simultaneously (e.g., att P TT will only recombine with att B TT in a specific orientation) [125]. The control of directionality and orthogonality have made serine integrases attractive tools for genome engineering and genetic logic gate design [126,127]. In contrast to CRISPR/Cas-based approaches, site-specific recombination using serine integrases does not rely on endogenous DNA repair pathways, such as NHEJ or HR.…”

“…Although CRISPR/Cas knock-in approaches are able to generate small insertions in a single step, the size of the insertion remains limited by the efficiency of HR. In addition, unlike Cas9, no toxicity has been reported with the usage of serine integrases in several organisms [124,126,128].…”

“…Serine integrases have been used for genome engineering in a variety of organisms, including mice [129], Drosophila melanogaster [130], S. cerevisiae, E. coli [126] and Clostridium ljungdahlii [128]. A recent strategy outlined a portable method to simplify the introduction of multiple genomic insertions using the orthogonal att sites of the PhiC31 serine integrase [126]. Firstly, one or more selective markers flanked by two orthogonal att B sites were integrated into the genome of E. coli as “landing pads”.…”