Construction of high protein-producing mutant yeast strains via point and structural mutageneses and their use for carotenoid production

Yeast Saccharomyces cerevisiae is a safe microorganism with established industrial-scale culture techniques, making it a useful host for protein and chemical production through metabolic engineering. Therefore, S. cerevisiae platform strains with high protein production are needed. In this study, we aimed to develop mutant S. cerevisiae strains with high protein production using techniques to introduce point and structural mutations. Point and structural mutations were introduced into the YPH499/pEUPGGFP strain, which expresses green fluorescent protein (GFP) in S. cerevisiae YPH499, and mutant strains were selected based on their fluorescence intensity. Consequently, YPH499/pEUPGGFP/Mu10G39, with a GFP fluorescence intensity 2.5-fold higher than that of the parent strain, was successfully obtained. Then, GFP expression plasmid was removed from the mutant and a carotenoid-producing plasmid was introduced to construct YPH499Mu10G39/pEU20Beta3. YPH499Mu10G39/pEU20Beta3 produced 6.74 mg/g-dry cell carotenoids after 72 h of culture, which was 2.9-fold higher than that produced by the parent strain. Transcriptome analysis suggested that YPH499Mu10G39 exhibited improved energy production, amino acid precursor supply, ribosome function, and stress tolerance, which may have contributed to its high protein production. In conclusion, by introducing point and structural mutations, we successfully developed the platform strain, YPH499Mu10G39, which is useful for the high production of various proteins. In the future, various proteins and useful chemicals can be produced through metabolic engineering using YPH499Mu10G39 as a platform strain.