title: Over-expression of native Saccharomyces cerevisiae exocytic SNARE genes increased heterologous cellulase secretion.
abstract: 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.
authors.affiliation: Department of Microbiology, Stellenbosch University, Stellenbosch, 7602, South Africa.
authors.forename: John Henry D
authors.initials: JH
authors.lastname: Van Zyl
authors.forename: Riaan
authors.initials: R
authors.lastname: Den Haan
authors.forename: Willem H
authors.initials: WH
authors.lastname: Van Zyl
mesh_terms: D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D032641:Eurotiales / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D060847:Metabolic Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D002480:Cellulase