Characterization of Physiological and Transcriptional Properties of Improved Protein Secretion Yeast Strains
The yeast Saccharomyces cerevisiaeis one of the most used organisms for protein production and developing an efficient secretion yeast system is favorable for industrial applications. In our previous study, several improved protein (α-amylase) secretion yeast strains were selected from UV mutagenesis libraries by microfluidic droplet screening and mutations of mutant strains have been identified by whole genome DNA sequencing. These identified mutations can be used as potential targets for strain optimization for protein secretion. However, except enhancement of protein production, the impacts of these mutations on mutant strains remain unknown.
In this work, mutant strains together with wildtype strain are cultured in SD-2×SCAA medium for batch fermentation to characterize their physiological and transcriptional properties. Compared with the wildtype strain, the mutant strains show higher α-amylase production rate, higher maximum specific growth rate on glucose, higher specific glucose uptake rate, higher ethanol production rate, lower yield of biomass from glucose and higher yield of ethanol from glucose. Lower percentage of intracellular α-amylase of mutant strains indicates that less α-amylase is retained by the cell, reflecting efficient secretory pathways of mutant strains.
To further explore the molecular mechanisms in the improved α-amylase secretion mutant strains, genome-wide transcriptional responses at exponential phase of mutant strains and the wildtype strain are analyzed. More genes are significant up- or down-regulation in higher α-amylase production strains, indicating wide global transcriptional adjustments are important to α-amylase production increasing. PHO89is the common down-regulation gene in all mutant strains. Up-regulation of most hexose transporter genes and down-regulation of genes in the glycolysis and TCA cycle are observed in mutant strains. Reporter GO term analysis reveals that GO term related to mitochondrial function, generation of precursor metabolites and energy, cellular respiration, cellular amino acid metabolic process, etc. are down-regulated. In contract, GO term related to ribosome function, cytoplasmic translation, regulation of organelle organization, Golgi vesicle transport, etc. are up-regulated. Genes controlled by transcriptional factors Mss11p, Rox1p, Tup1p, Upc2p and Yox1p are up-regulated, and genes controlled by transcriptional factors Adr1p, Bas1p, Gcr1p, Hap1p, Hap2p/3p/4p/5p CCAAT-binding complex, Opi1p and Sut1p are down-regulated. These results suggest anaerobic behaviors of yeast are favorable for protein production, which is in agreement with other studies from our lab.
The findings in this study help to understand underlying mechanisms of improved protein production strains and will contribute to design yeast cell factories for efficient protein production.