Overexpression of an archaeal protein in yeast: secretion bottleneck at the ER.
Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging. Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. To determine whether a eukaryotic host, S. cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase's secretion and activity. We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a transformant capable of secreting approximately 10 mg/L in batch culture. All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck. Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck. Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association. Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields.
Smith
Jason D
JD
Department of Chemical Engineering, University of Delaware, Newark 19716, USA. robinson@che.ude1.edu
Robinson
Anne Skaja
AS
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics; D011786:Quality Control; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013696:Temperature; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
D001617:beta-Glucosidase