Journal
ACS SYNTHETIC BIOLOGY
Volume 1, Issue 12, Pages 602-609Publisher
AMER CHEMICAL SOC
DOI: 10.1021/sb3000904
Keywords
homologous recombination; directed evolution; mutagenesis; libraries; sexual reproduction; yeast
Categories
Funding
- NIH [R01GM62867, R01GM096064]
- NSF [CHE0957569]
- National Institute of General Medical Sciences [F32GM089031]
Ask authors/readers for more resources
Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available