High-frequency oligonucleotide-directed recombination engineering (recombineering) has enabled rapid modification of several prokaryotic genomes to date. Here, we present a method for oligonucleotide-mediated recombineering in the model eukaryote and industrial production host Saccharomyces cerevisiae , which we call yeast oligo-mediated genome engineering (YOGE). Through a combination of overexpression and knockouts of relevant genes and optimization of transformation and oligonucleotide designs, we achieve high gene-modification frequencies at levels that only require screening of dozens of cells. We demonstrate the robustness of our approach in three divergent yeast strains, including those involved in industrial production of biobased chemicals. Furthermore, YOGE can be iteratively executed via cycling to generate genomic libraries up to 10 (5) individuals at each round for diversity generation. YOGE cycling alone or in combination with phenotypic selections or endonuclease-based negative genotypic selections can be used to generate modified alleles easily in yeast populations with high frequencies.

• PMID: 24160921
• DOI full text
• PMC full text
• PubMed

Reference Type

Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

Authors

DiCarlo JE, Conley AJ, Penttilä M, Jäntti J, Wang HH, Church GM

Primary Lit For

Additional Lit For

Review For