Genome-Wide Screening to Study Breeding Methods to Improve the Nitrogen Accumulation Ability of Yeast without Gene Recombinant Techniques

Abstract

To remove nitrogen efficiently from high-concentration organic wastewater, we studied breeding methods using Saccharomyces cerevisiae as a model yeast with improved nitrogen accumulation ability. By DNA microarray analysis under various nitrogen concentrations with two nitrogen sources (peptone and L-asparagine), we obtained 295 commonly overexpressed (over 2-fold) genes and 283 commonly underexpressed (under one-half) genes under nitrogen-starvation conditions. We speculated that overexpression or underexpression recombination of some of these genes might enhance nitrogen uptake. Because a complete collection of nonessential gene deletion strains had been created, we investigated the nitrogen accumulation profiles of underexpressed gene deletion strains. From 256 nonessential gene deletion strains, three (URE2, SNO1, and AVT3) were selected. Strain SUD2 (ure2Δ::kanMX4) improved by 1.2-fold total nitrogen per cell (TN/OD₆₆₀) as compared to the parent strain, S288c. Positive selection of methylamine-resistant mutants to obtain URE2 mutants was useful for improving nitrogen accumulation ability without recombinant techniques.