Author(s): Magalhães, Frederico ; Dinis, Cláudia ; Aguiar, Tatiana Quinta ; Oliveira, Carla Cristina Marques de ; Domingues, Lucília
Date: 2013
Persistent ID: http://hdl.handle.net/1822/32867
Origin: RepositóriUM - Universidade do Minho
Subject(s): Ashbya gossypii; Aspergillus niger ß-galactosidase; High recombinant ß-galactosidase secretion levels; A. gossypii GPD and TEF promoters; Saccharomyces cerevisiae PGK1 and ADH1 promoters
Ashbya gossypii is a filamentous Saccharomycete that has been recently considered as a host for the expression of recombinant proteins. However, the expression levels obtained were low, even though similar to those observed in Saccharomyces cerevisiae for the same proteins. Here, to further assess the potential of this fungus as a recombinant protein producer, the -galactosidase from Aspergillus niger was successfully expressed and secreted by the A. gossypii ATCC10895 strain from 2-micron plasmids carrying the native signal sequence, under the regulation of four different promoters: A. gossypii TEF and GPD promoters, and S. cerevisiae ADH1 and PGK1 promoters. The native TEF promoter revealed to be the best promoter for the expression of recombinant -galactosidase in A. gossypii, leading to 2 and 7 times more extracellular activity than the GPD promoter and the heterologous promoters, respectively. Furthermore, the levels of recombinant - galactosidase activity secreted by A. gossypii were up to 37 times higher than those secreted by the S. cerevisiae CEN.PK 113-7D strain transformed with the same plasmids. In addition, A. gossypii expressed 2.5 times more extracellular -galactosidase activity than the previously reported A. niger -galactosidase producing S. cerevisiae NCYC869-A3/pVK1.1 strain. Partial characterization of the recombinant -galactosidase secreted by A. gossypii revealed that this enzyme is extensively glycosylated, as the recombinant -galactosidase expressed in yeast and the native A. niger -galactosidase. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus.
