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

Abstract

The 56th 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 56th 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 56th 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.