期刊
ACS SYNTHETIC BIOLOGY
卷 2, 期 12, 页码 741-749出版社
AMER CHEMICAL SOC
DOI: 10.1021/sb400117c
关键词
yeast; genome engineering; recombineering; MAGE; Saccharomyces cerevisiae; oligonucleotide transformation
资金
- Department of Energy [DE-FG02-02ER63445]
- Synthetic Biology Engineering Research Center from the National Science Foundation [SA5283-11210]
- National Institutes of Health [1DP5OD009172-01]
- Academy of Finland [260957]
- Academy of Finland (AKA) [260957, 260957] Funding Source: Academy of Finland (AKA)
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 105 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.
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