Moderate expression of SEC16 increases protein secretion by Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous beta-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased beta-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous beta-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-beta-glucosidase, indicating that this mechanism is of general relevance. IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae. This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future

Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

Abstract The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of α-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpression strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-α-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast

Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of alpha-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpression strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-alpha-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast

Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1.

The wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) yellow (stripe) rust resistance genes Yr15, YrG303, and YrH52 were discovered in natural populations from different geographic locations. They all localize to chromosome 1B but were thought to be non-allelic based on differences in resistance response. We recently cloned Yr15 as a Wheat Tandem Kinase 1 (WTK1) and show here that these three resistance loci co-segregate in fine-mapping populations and share an identical full-length genomic sequence of functional Wtk1. Independent ethyl methanesulfonate (EMS)-mutagenized susceptible yrG303 and yrH52 lines carried single nucleotide mutations in Wtk1 that disrupted function. A comparison of the mutations for yr15, yrG303, and yrH52 mutants showed that while key conserved residues were intact, other conserved regions in critical kinase subdomains were frequently affected. Thus, we concluded that Yr15-, YrG303-, and YrH52-mediated resistances to yellow rust are encoded by a single locus, Wtk1. Introgression of Wtk1 into multiple genetic backgrounds resulted in variable phenotypic responses, confirming that Wtk1-mediated resistance is part of a complex immune response network. WEW natural populations subjected to natural selection and adaptation have potential to serve as a good source for evolutionary studies of different traits and multifaceted gene networks

Efficient protein production by yeast requires global tuning of metabolism

The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion