Thaumatin production in Aspergillus awamori by use of expression cassetteswith strong fungal promoters and high gene dosage

Four expression cassettes containing strong fungal promoters, a signal sequ ence for protein translocation, a KEX protease cleavage site, and a synthet ic gene (tha) encoding the sweet protein thaumatin II were used to overexpr ess this protein in. Aspergillus awamori lpr66, a PepA protease-deficient s train. The best expression results were obtained with the gdhA promoter of A. awamori or with the gpd4 promoter of Aspergillus nidulans, There was goo d correlation of tha tha gene dosage, transcript levels, and thaumatin secr etion. The thaumatin gene was expressed as a transcript of the expected siz e in each construction (1,9 or 1.4 kb), and the transcript levels and thaum atin 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 transf ormant was purified to homogeneity, giving one major component and two mino r components, In all cases, cleavage of the fused protein occurred at the K EX recognition sequence. This work provides new expression systems in A. aw amori that result in very high levels of thaumatin production.