Laccase is a multicopper-containing enzyme that catalyzes the oxidation of phenolic compounds. lcc1 cDNA coding for a secretory laccase of Pycnoporus coccineus was expressed under the maltose inducible amyB promoter in Aspergillus oryzae and under the galactose inducible GAL10 promoter in Saccharomyces cerevisiae. Laccase activities, which were undetectable in the absence of copper, were observed by increasing copper concentrations in the media for both systems. The amounts of secreted laccase protein but not lcc1 mRNA increased in proportion to copper concentrations in A. oryzae. The extracellular activities of native A. oryzae amylase and recombinant RNase-T1 expressed from the same amyB promoter in A. oryzae were constant regardless of copper concentrations. Our results indicate that a high copper concentration is required for the production of active laccase in heterologous hosts and that the copper is required for a post-transcriptional process.

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form.