ABSTRACT In a previous study, system level analysis of adaptively evolved yeast mutants showing improved galactose utilization revealed relevant mutations. The governing mutations were suggested to be in the Ras/PKA signaling pathway and ergosterol metabolism. Here, site-directed mutants having one of the mutations RAS2 Lys77 , RAS2 Tyr112 , and ERG5 Pro370 were constructed and evaluated. The mutants were also combined with overexpression of PGM2 , earlier proved as a beneficial target for galactose utilization. The constructed strains were analyzed for their gross phenotype, transcriptome and targeted metabolites, and the results were compared to those obtained from reference strains and the evolved strains. The RAS2 Lys77 mutation resulted in the highest specific galactose uptake rate among all of the strains with an increased maximum specific growth rate on galactose. The RAS2 Tyr112 mutation also improved the specific galactose uptake rate and also resulted in many transcriptional changes, including ergosterol metabolism. The ERG5 Pro370 mutation only showed a small improvement, but when it was combined with PGM2 overexpression, the phenotype was almost the same as that of the evolved mutants. Combination of the RAS2 mutations with PGM2 overexpression also led to a complete recovery of the adaptive phenotype in galactose utilization. Recovery of the gross phenotype by the reconstructed mutants was achieved with much fewer changes in the genome and transcriptome than for the evolved mutants. Our study demonstrates how the identification of specific mutations by systems biology can direct new metabolic engineering strategies for improving galactose utilization by yeast.