Optimizing archaeal enzyme expression in the yeast Saccharomyces cerevisiae

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 low biomass yields. To circumvent these problems we attempted to create a high-yield archaeal enzyme expression system using the yeast Saccharomyces cerevisiae. We utilized the tetrameric β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus as our model protein.; We engineered the β-glucosidase to be secreted into the culture medium and have demonstrated the β-glucosidase's secretion and activity. Through gene copy optimization we obtained a transformant capable of secreting approximately 12 mg/L in batch culture, a 3-fold improvement over single gene overexpression. All transformants retained large intracellular fractions of inactive β-glucosidase, indicative of an intracellular bottleneck. By using immunofluorescence, confocal microscopy, and cell fractionation by sucrose density centrifugation the bottleneck was found to reside in the endoplasmic reticulum (ER).; Expression at moderately elevated temperatures (between 30–40°C) dramatically increased beta-glucosidase yields (>400% per cell) and practically eliminated the ER bottleneck. Investigation of β-glucosidase secretion and the cellular heat shock response revealed that increased expression temperature improved β-glucosidase folding and secretion yields, a result we believe is directly related to the natural characteristics of hyperthermophilic proteins at low (∼30°C) temperatures.; As a secondary method to improve β-glucosidase secretion yields we investigated the role of ER-resident proteins BiP and PDI. Overexpression of a single BiP and PDI gene increased β-glucosidase secretion a modest 64%. In an attempt to optimize the effects of BiP and PDI we created β-glucosidase expressing strains with variable BiP and PDI levels. Strains with up to 3-fold higher β-glucosidase levels than the control were isolated. Examination of a subset of improved strains established that increased BiP levels decreased β-glucosidase secretion improvements while increased PDI levels increased β-glucosidase improvements, an interesting result since β-glucosidase contains only one cysteme per monomer. We postulate that PDI may be acting as a unique chaperone or possibly forming transient, mixed disulfide bonds with β-glucosidase during folding.