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

Abstract

The yeast Saccharomyces cerevisiae is a safe microorganism with established industrial-scale culture techniques. Standard laboratory S. cerevisiae strains, such as the representative YPH499, are valuable hosts for producing proteins and chemicals through metabolic engineering. Consequently, there's a high demand for platform strains of S. cerevisiae with enhanced protein production capacity. We have previously established an efficient and straightforward technique for introducing point and structural mutations into yeast via plasmid introduction, leading to the generation of mutant strains with superior phenotypes. In this study, we aimed to develop S. cerevisiae mutants with high protein production capacity using techniques to introduce point and structural mutations. We introduced these mutations into the YPH499/pEUPGGFP strain, which expresses green fluorescent protein (GFP). Since GFP is easily detected by its fluorescence, we selected mutants 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, a carotenoid-producing plasmid was introduced to construct YPH499Mu10G39/pEU20Beta3. YPH499Mu10G39/pEU20Beta3 produced 6.74 mg/g-dry cell carotenoids, 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.