Aureobasidin A (AbA) is a potent and unique yeast antibiotic that kills S. cerevisiae at low concentrations (Takesako et. al. 1993). The drug is a cyclic multipeptide (Figure 1) that acts by inhibiting inositol phosphorylceramide synthase, an essential yeast enzyme. A mutant enzyme, encoded by the AUR1-C gene, confers resistance to AbA and can be used as a highly effective selectable marker that requires little to no optimization. Our optimized Matchmaker Gold Yeast Two-Hybrid System leverages this by including a reporter strain containing a stably-integrated AUR1-C gene, allowing for simple and rapid colony screening in yeast two-hybrid (Y2H) libraries using AbA selection.
Figure 1. Structure of Aureobasidin A. Aureobasidin A (AbA; MW 1,100) is a cyclic depsipeptide antibiotic isolated from the fungus Aureobasidium pullulans R106. AbA inhibits the product of the yeast AUR1 gene (inositol phosphorylceramide synthase) and is toxic to S. cerevisiae at low concentrations (0.1 µg/ml). The gene product of a dominant mutant allele, AUR1-C, confers resistance to AbA and its expression can be used as a selectable marker.
Low background with AbA screening
AbA selection virtually eliminates the high number of background colonies that often plague low-stringency primary screens that rely on nutritional markers alone (e.g., HIS3). Because AbA actually kills sensitive cells, rather than merely retarding their growth, AbA-based selection greatly favors the growth and identification of true positive clones. In general practice, a high percentage of clones that emerge from low-stringency primary screens using AbA selection are subsequently verified on high-stringency secondary screens that select for all four Matchmaker Gold reporters (AUR1-C, HIS3, ADE2, and MEL1).
AbA: ampicillin for yeast
Many researchers have yearned for a yeast selection system similar to those used for E. coli or mammalian cells. In fact, AbA is used for yeast in a similar way as ampicillin and kanamycin are used for cloning in E. coli, or as G418 is used for selection of stably-transfected mammalian cell clones. AbA resistance is far easier to use in Y2H library screening than auxotrophic reporters, which often require additional optimization steps to achieve selective growth conditions.
