Thaumatin Production in Aspergillus awamori by Use of Expression Cassettes with Strong Fungal Promoters and High Gene Dosage

ABSTRACT Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain. The best expression results were obtained with the gdhA promoter ofA. awamori or with the gpdA promoter ofAspergillus nidulans. There was good correlation oftha gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems inA. awamori that result in very high levels of thaumatin production.

Sequence analysis and transformation by the catabolic 3-dehydroquinase (QUTE) gene from Aspergillus nidulans

The induction of catabolic 3-dehydroquinase by quinic acid in Aspergillus nidulans has been shown to involve transcriptional control and yields a single major 0.8 kb mRNA. The nucleotide sequence of the catabolic 3-dehydroquinase QUTE gene has been determined and contains a single uninterrupted open reading frame of 462 bases encoding a 16,505 Da protein of 153 residues. Comparison with the corresponding QA2 gene of Neurospora crassa reveals the absence of 75 nucleotides encoding 25 amino acids from the centre of the QUTE gene of A. nidulans and the presence of 21 additional nucleotides at its 3′ end. There is no nucleotide or amino acid homology between these two elements. A 16 bp inverted repeat (5′ GGCAGAGCGTTCTGCC) shows similarity to such repeats found in other fungal promoters. The functional integrity of the QUTE gene was demonstrated by the transformation of a qutE mutant strain which regains growth on quinic acid as sole carbon source. Four of the twelve transformed strains examined contained vector sequences integrated at the qutE locus, and these strains all exhibited normal regulation of 3-dehydroquinase even when 16 copies of the QUTE gene were present.