Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Abstract

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 ␣-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 ␣-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous ␣-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-␣-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. KEYWORDS protein secretion, mitochondria, ERES, ROS, SEC16T he yeast Saccharomyces cerevisiae is a widely used cell factory for the production of recombinant proteins (1). The advantages of S. cerevisiae are fast growth, easy cultivation, and ability to perform posttranslational modifications and secrete proteins to the extracellular medium, which facilitates the purification of the protein (2, 3). However, S. cerevisiae naturally secretes only a few proteins, such as aspartyl protease, invertase, and ␣-factor pheromone (4, 5); therefore, it has evolved a secretory pathway with a relatively low capacity. Many different strategies have been used to engineer this pathway with the objective of improving heterologous protein production (6-8). When expressing a protein that has a secretory signal peptide, the protein is cotranslationally translocated into the endoplasmic reticulum (ER) lumen, where disulfide bond formation occurs with the initial glycosylation (9). The secretion of recombinant proteins is affected by their signal peptide sequences (10). A synthetic leader sequence increases human insulin precursor production, whereas the use of the ␣-factor leader sequence increased ␣-amylase production (7). Excessive mannose glycosylation of recombinant proteins can reduce the efficiency of protein secretion, and it was recently found that

COPII gets in shape: Lessons derived from morphological aspects of early secretion

“…Sec16 expression levels control overall secretion in yeast and determine the size and function of ERES and Golgi in Trypanosoma brucei. Sec16A levels and its dynamic interactions with ERES link cell proliferation with secretion, to control cell growth and survival in mammals, and its alternative splicing controls the overall efficiency of COPII‐mediated traffic . In mammalian cells, cargo availability regulates COPII assembly and vesicle formation at ERES .…”

Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production

“…Although previous studies suggested various strategies for improving protein production at different levels including transcription, translation, posttranslational processes, etc. (12)(13)(14)(15)(16)(17), they rarely focused on processes associated with intracellular retention of the protein in the secretory pathway.…”

RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae

“…(C) The amylase production of strains with fine-tuned expression of PDI1 and TDA3 was quantified in strains with various copies (number) of PDI1 expression (P) and TDA3 interference (T) cassettes integrated. (D) Reassessment of amylase yield from strains with fine-tuned expression and selected strains from a recombineered PDI1-TDA3rc library (13,46,47). The integration sites and integrated segments from the PDI1-TDA3rc library strain with the highest amylase production were characterized.…”