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 po
werful tools for engineering biomolecules
with useful ne
w functions by directed evolution. Synthetic biology no
w 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 allo
w successive repair of different markers through t
wo 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 gro
wing arsenal of next generation molecular biology tools for synthetic biology.
Keywords:
homologous recombination; directed evolution; mutagenesis; libraries; sexual reproduction; yeast