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 beta-galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2-mu m 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 beta-galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant beta-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 beta-galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and similar to 2.5 times higher than those previously reported for the beta-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 beta-galactosidase by A. gossypii. Recombinant beta-galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger beta-galactosidase and recombinant beta-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. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:261-268, 2014