Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage.
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 of A. awamori or with the gpdA promoter of Aspergillus nidulans. There was good correlation of tha 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 in A. awamori that result in very high levels of thaumatin production.
Moralejo
F J
FJ
Instituto de Biotecnología INBIOTEC, Parque Científico de León, 24006 León, Spain.
Cardoza
R E
RE
Gutierrez
S
S
Martin
J F
JF
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018628:Gene Dosage; D005822:Genetic Vectors; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D043484:Proprotein Convertases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D013549:Sweetening Agents; D014170:Transformation, Genetic
D000199:Actins; D010940:Plant Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013549:Sweetening Agents; C003427:thaumatin protein, plant; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae