Construction of a binary vector for the expression of the Aspergillus niger McoD laccase gene in the industrial filamentous fungus Aspergillus oryzae

The filamentous fungus Aspergillus oryzae, also named as the koji mold, is a preferred host for enzyme production due to its prominent secretion ability into the culture medium. Fungal laccases are widely used in different industrial processes, especially in removing environmental pollutants. Up till now, little was known about the roles of some laccase genes from the black mold Aspergillus niger. The McoD laccase gene from A. niger includes three exons interrupted by two short introns. The respective coding sequence of the gene is computationally predicted to encode a secreted laccase of 563 amino acids. In this study, a binary vector carrying the A. nigerMcoD gene was successfully constructed for heterologous expression in the edible fungus A. oryzae. This vector was transformed into A. oryzae using the Agrobacterium tumefaciens-mediated transformation method. The transformation efficiency was relatively high in two different auxotrophic A. oryzae strains, including RIB40∆pyrG and VS1∆pyrG. All the tested transformants possess in their genomes the construct for expression of the McoD laccase gene under control of the strong A. oryzae amyB promoter. The selected transformants were examined for the laccase activity using the ABTS substrate. The results showed that in comparison to the wild-type fungal strains, the transgenic strains could oxidise ABTS to form the sea green colour, which can be seen directly on the agar plate.