Talens Assisted Multiplex Engineering for Accelerated Genome Evolution in Saccharomyces Cerevisiae

Genome engineering is an essential tool of constructing a genotype to achieve a desired phenotype, a challenge whose difficulty is influenced by the selectivity and scale of genomic alteration required. Here we report TALENs Assisted Multiplex Engineering (TAME) as a generally applicable genome engineering method for editing the genome and accelerating the genome evolution in yeast. Through the iterative cycles of TAME induced mutants coupled with efficient screening by fluorescence reporter, TAME can continuously and rapidly improve simple target trait by accumulating multiple beneficial genetic modifications in evolving yeast genome. In order to assess its efficiency further, we then show the application of TAME for improved ethanol tolerance, a key and complicated trait for microbial production of fuels. Five rounds of iterative TAME screening led to acting synergistically to confer the engineered yeast strain with substantially improved ethanol tolerance. Therefore, TAME is a broadly applicable platform for accelerated evolution for desired traits in eukaryotes.