Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii.
To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-beta-D: -lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.
Ribeiro
Orquídea
O
IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Wiebe
Marilyn
M
Ilmén
Marja
M
Domingues
Lucília
L
Penttilä
Merja
M
D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D021381:Protein Transport; D004718:Saccharomycetales / Q000235:genetics* / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology*
D005656:Fungal Proteins; C056374:endoglucanase 1; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase