Suchergebnisse

Employing the homologous DNA recombination apparatus of Saccharomyces cerevisiae as a dynamic engineering tool allows mutant libraries to be constructed in a rapid and efficient manner. Among the plethora of methods based on the yeast's splicing apparatus, site-directed recombination (SDR) is often useful to gather information from mutations discovered in directed evolution experiments. When using SDR, the target gene is divided in segments carrying the selected mutation positions so that the resulting PCR fragments show 50% mutated and 50% wild type residues at the codons of interest. The PCR products are then assembled and cloned into yeast through one-pot transformations with the help of homologous overlapping flanking regions. By screening SDR libraries, the effect of the mutations/reversions at the different positions can be rapidly sorted out in a combinatorial manner. As such, SDR can serve as the `final polishing step´ in a laboratory evolution campaign, revealing beneficial synergies among mutations and/or overriding deleterious mutations. In practice, using SDR it is possible to discern between beneficial and negative epistasis, that is, it should be possible to collect positive synergistic mutations while discarding detrimental substitutions that affect the enzyme's fitness. ; This research was supported by the Spanish Government project BIO2016-79106-R287 Lignolution and the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA.