Effect of gpd box copy numbers in the gpdA promoter of Aspergillus nidulans on its transcription efficiency in Aspergillus niger

In this study, we characterised PgpdA, PgpdA2B, PgpdA3B and PgpdA4B promoters, containing 1-4 copies of gpd box by modifying the gpdA promoter, and constructed pSZHGX-xynB expression vectors, which were introduced into Aspergillus niger CICC2462 through Agrobacterium-mediated transformation. Thus, An (PgpdA-xynB), An (PgpdA2B-xynB), An (PgpdA3B-xynB) and An (PgpdA4B-xynB) homozygous recombinant strains were obtained. The xylanase activity of homozygous recombinant strains was measured. The enzymatic activities of An (PgpdA-xynB), An (PgpdA2B-xynB), An (PgpdA3B-xynB) and An (PgpdA4B-xynB) peaked on the 7th day of fermentation, at 1578.67, 2333.88, 3588.38 and 3183.51 U·mL-1, respectively. SDS-PAGE and qRT-PCR analysis indicated that An (PgpdA3B-xynB), containing three copies of gpd box, demonstrated the highest levels of protein expression and transcription. These results suggested that the PgpdA3B promoter promotes highly efficient transcription and may serve as a strong constitutive promoter for efficient recombinant protein expression. Additionally, a number of constitutive promoters with various transcription efficiencies were identified for the metabolic engineering of A. niger. Accordingly, this study provides a new approach for obtaining promoters with different transcription efficiencies.