Site saturation mutagenesis of ribosomal protein L42 at 56th residue and application as a consecutive selection marker for cycloheximide resistance in yeast

Abstract

The 56^th residue of ribosomal protein L42 (Rpl42) determines the sensitivity of yeast cells to the antibiotic cycloheximide (CYH). In this study, we identified the relationship between the 56^th residue of Rpl42 and the function of the ribosome by site saturation mutagenesis. The resulting 20 RPL42 mutants harbouring one of 20 amino acids at the 56^th residue were classified into five groups: sensitive to CYH (RPL42aP); weak resistance (RPL42aA, RPL42aM, RPL42aC, RPL42aN, RPL42aD, RPL42aS, and RPL42aT), moderate resistance (RPL42aL, RPL42aI, RPL42aV, RPL42aG, and RPL42aH), and strong resistance (RPL42aQ, RPL42aE, RPL42aR, and RPL42aK) to CYH; and non-functional (RPL42aF, RPL42aY, and RPL42aW). Three RPL42a mutants from each group, RPL42aA, RPL42aL, and RPL42aQ, were used as CYH-resistant selection marker genes for the sequential transformation of CYH-sensitive yeast. A series of RPL42 mutants conferring different levels of resistance to CYH should be useful for the dose-dependent multiple selection of prototrophic industrial yeasts.