Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Abstract

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae , and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10 4 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

Synthetic Biology Triggers New Era of Antibiotics Development

“…More importantly, the 30 kb myxochromide S biosynthetic gene cluster, and even ~60 kb biosynthetic gene cluster for epothilones, were ef fi ciently chromosomal integrated into Myxococcus xanthus and Pseudomonas chromosomes by transposition (Wenzel et al 2005 ) , which would be dif fi cult using electroporation due to the large size of DNA. Harnessing endonuclease-stimulated homologous recombination, Wingler et al developed an user-friendly and highyielding tool called Reiterative Recombination, for multigene pathway assembly in vivo with seamless and robustness stitch (Wingler and Cornish 2011 ) . As the expanding of twenty-fi rst-century genetic manipulation toolkit and advance in genome synthesis, the design of excellent cell chassis will be easier.…”

“…Generally, the related coding genes clustered together on the genome in modularity, and each catalytic domain has certain substrate or product fl exibility, which facilitates interchange of domains between different sources or pathways to generate a large natural product derived compound library for antibiotics screening. Other important technological progress, such as DNA synthesis (Bugl et al 2007 ) , DNA manipulation technology for pathway/whole genome assembly (Isaacs et al 2011 ;Shao and Zhao 2009 ;Wingler and Cornish 2011 ;Zhang et al 1998 ) , and in silico microbial system simulation Price et al 2003 ;Weber et al 2009 ;Yadav et al 2009 ) , has led to encouraging achievements in production of fi ne chemicals (Keasling 2010 ) , bio-fuels (Clomburg and Gonzalez 2010 ) and pharmaceuticals (Medema et al 2011 ;Neumann and Neumann-Staubitz 2010 ) . Moreover, the yield and productivity often could increase hundreds fold after multiple cycles of strains improvement.…”

Design-based re-engineering of biosynthetic gene clusters: plug-and-play in practice

“…Alternatively, there are methods for iterative DNA assembly into the genomes of Bacillus subtilis [46,47] and Saccharomyces cerevisiae [48 ]. These are the methods of choice for creation of libraries of one pathway, but not for assembly of many different pathways at once.…”

Advanced DNA assembly technologies in drug discovery

“…The system is commercialized by Life Technologies under the name of GeneArt Ò HighOrder Genetic Assembly System. Finally, the recombinogenic properties of yeast have been harnessed by the 'reiterative recombination' method [32]. The process is based on the expression of homing endonucleases that induce recombination at specific sites.…”