No abstract

Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs) are essential components of the yeast protein-trafficking machinery and are required at the majority of membrane fusion events in the cell, where they facilitate SNARE-mediated fusion between the protein transport vesicles, the various membrane-enclosed organelles and, ultimately, the plasma membrane. We have demonstrated an increase in secretory titers for the Talaromyces emersonii Cel7A (Te-Cel7A, a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (Sf-Cel3A, a β-glucosidase) expressed in Saccharomyces cerevisiae through single and co-overexpression of some of the endoplasmic reticulum (ER)-to-Golgi SNAREs (BOS1, BET1, SEC22 and SED5). Overexpression of SED5 yielded the biggest improvements for both of the cellulolytic reporter proteins tested, with maximum increases in extracellular enzyme activity of 22 % for the Sf-Cel3A and 68 % for the Te-Cel7A. Co-overexpression of the ER-to-Golgi SNAREs yielded proportionately smaller increases for the Te-Cel7A (46 %), with the Sf-Cel3A yielding no improvement. Co-overexpression of the most promising exocytic SNARE components identified in literature for secretory enhancement of the cellulolytic proteins tested (SSO1 for Sf-Cel3A and SNC1 for Te-Cel7A) with the most effective ER-to-Golgi SNARE components identified in this study (SED5 for both Sf-Cel3A and Te-Cel7A) yielded variable results, with Sf-Cel3A improved by 131 % and Te-Cel7A yielding no improvement. Improvements were largely independent of gene dosage as all strains only integrated single additional SNARE gene copies, with episomal variance between the most improved strains shown to be insignificant. This study has added further credence to the notion that SNARE proteins fulfil an essential role within a larger cascade of secretory machinery components that could contribute significantly to future improvements to S. cerevisiae as protein production host.

SNAREs (soluble NSF [N-ethylmaleimide-sensitive factor] attachment receptor proteins) are required at the majority of fusion events during intracellular membrane transport and play crucial roles in facilitating protein trafficking between the various membrane-enclosed organelles and the plasma membrane. We demonstrate increases in the secretion of the Talaromyces emersonii Cel7A (a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (a β-glucosidase), through the separate and simultaneous over-expression of different components of the exocytic SNARE complex in Saccharomyces cerevisiae. Over-expression of SNC1 yielded the biggest improvement in Te-Cel7A secretion (71 %), whilst SSO1 over-expression lead to the highest increases in Sf-Cel3A secretion (43.8 %). Simultaneous over-expression of differential combinations of these SNARE components yielded maximal increases of ~52 % and ~49 % for the secretion of Te-Cel7A and Sf-Cel3A, respectively. These increases generally did not cause deleterious growth effects, whilst differential improvement patterns were observed for the two reporter proteins (Sf-Cel3A and Te-Cel7A). Simultaneous over-expression of up to three of these components, in strains secreting the more efficiently expressed Sf-Cel3A, illustrated a slight decrease in osmotic tolerance at elevated NaCl concentrations, as well as a detectable decrease in ethanol tolerance at increased concentrations. This work illustrates the potential of engineering components of the anterograde secretory pathway, particularly its SNARE components, for the improvement of heterologous cellulase secretion.