Gene Assembly and Combinatorial Libraries in S. cerevisiae via Reiterative Recombination

While mutagenesis of single genes is now common practice in molecular biology, engineering multiple target genes still requires complex cloning techniques and thus is limited to expert laboratories. Here, we describe “Reiterative Recombination,” a user-friendly DNA assembly technique in Saccharomyces cere visiae for the integration of an indefinite number of DNA fragments sequentially into the yeast genome. The high efficiency of chromosomal integration can further be utilized for the assembly of large combinatorial libraries for metabolic engineering.