Overexpression and lack of degradation of thaumatin in an aspergillopepsinA-defective mutant of Aspergillus awamori containing an insertion in the pepA gene

A gene encoding the sweet-tasting protein thaumatin (tha) with optimized co don usage was expressed in Aspergillus awamori. Mutants of A. awamori with reduced proteolytic activity were isolated. One of these mutants, named lpr -66, contained an insertion of about 200 bp in the pepA gene, resulting in an inactive aspergillopepsin A. In vitro thaumatin degradation tests confir med that culture broths of mutant lpr66 showed only a small thaumatin-degra ding activity. A. awamori lpr66 has been used as host strain for thaumatin expression cassettes containing the tha gene under the control of either th e cahB (cephalosporin acetylhydrolase) promoter of Acremonium chrysogenum o r the gdhA (glutamate dehydrogenase) promoter of Aspergillus awamori. Resid ual proteolytic activities were repressed by using a mixture of glucose and sucrose as carbon sources and L-asparagine as nitrogen source. Degradation of thaumatin by acidic proteases was prevented by maintaining the pH value at 6.2 in the fermenter. Expression of cassettes containing the gdhA promo ter was optimal in ammonium sulfate as nitrogen source, whereas transforman ts expressing the tha gene from the cahB promoter yielded higher thaumatin levels using L-asparagine as nitrogen source. Under optimal fermentation co nditions, yields of 105 mg thaumatin/l were obtained, thus making this ferm entation a process of industrial interest.