High-level expression of Aspergillus niger b-galactosidase in Ashbya gossypii.
Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the b-galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2-mm plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of bgalactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant b-galactosidase in A. gossypii, leading to 2- and 8-fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active b-galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and 2.5 times higher than those previously reported for the b-galactosidase-high producing S. cerevisiae NCYC869-A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5-fold increase in the secretion of active b-galactosidase by A. gossypii. Recombinant b-galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger b-galactosidase and recombinant b-galactosidase produced by yeast. 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.
Frederico
F
Magalhes
Tatiana Q
TQ
Aguiar
Carla
C
Oliveira
Lucília
L
Domingues
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D005947:Glucose