Overexpression and lack of degradation of thaumatin in an aspergillopepsin A-defective mutant of Aspergillus awamori containing an insertion in the pep  A gene

Abstract

A gene encoding the sweet-tasting protein thaumatin (tha) with optimized codon usage was expressed in Aspergillus awamori. Mutants of A. awamori with reduced proteolytic activity were isolated. One of these mutants, named lpr66, contained an insertion of about 200 bp in the pepA gene, resulting in an inactive aspergillopepsin A. In vitro thaumatin degradation tests confirmed that culture broths of mutant lpr66 showed only a small thaumatin-degrading activity. A. awamori lpr66 has been used as host strain for thaumatin expression cassettes containing the tha gene under the control of either the cahB (cephalosporin acetylhydrolase) promoter of Acremonium chrysogenum or the gdhA (glutamate dehydrogenase) promoter of Aspergillus awamori. Residual 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 fermentor. Expression of cassettes containing the gdhA promoter was optimal in ammonium sulfate as nitrogen source, whereas transformants expressing the tha gene from the cahB promoter yielded higher thaumatin levels using L-asparagine as nitrogen source. Under optimal fermentation conditions, yields of 105 mg thaumatin/l were obtained, thus making this fermentation a process of industrial interest.

High-yield Secretion of the Recombinant Sweet-Tasting Protein Thaumatin I

“…Taken together, the maintenance of the hydrophobic environment at the N-terminal region of the thaumatin molecule curred at this site; this may be due to the deficiency of Kex2 protease. These results are similar to the previous report by Moralejo et al (1999) who used A. awamori as a host.…”

“…In some studies, acceptable production yield was achieved using artificial genes with optimized codon usage encoding thaumatin II (Weickmann et al, 2004, Daniell et al, 2000, but the recombinant protein was obtained as insoluble inclusion bodies, thereby requiring a renaturation procedure using a reduced/oxidized glutathione system to obtain a soluble, correctly folded, and active thaumatin II. Another expression system using Aspergillus awamori has been attempted to satisfy the expression yield of recombinant thaumatin, but three forms of recombinant thaumatin that differ in one amino acid at the N-terminus were obtained, resulting in a non-homogeneous product (Moralejo et al, 1999, Lombraña et al, 2004.…”

Monitoring Global Gene Expression of Proteases and Improvement of Human Lysozyme Production in thenptBGene Disruptant ofAspergillus oryzae

“…8) In Aspergillus awamori, deletion of pepB greatly increased thaumatin production in combination with a pepA defective mutant. 9,10) In A. oryzae, the anti-sense RNA technique repressed serin-type carboxypeptidases and increased human lysozyme (HLY) production. 11) In addition, we have employed systematic gene disruption of five protease genes (pepA, pepE, alpA, tppA, and palB), and double disruption of pepE and tppA genes successfully increased HLY production in A. oryzae.…”

Silencing of the Aspergillopepsin B ( pepB ) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production

“…Proteolytic degradation is considered one of the major problems during protein production in Aspergillus species (3,7,29,37). Proteolytic degradation affects mainly heterologous proteins and can be explained, in part, by the presence in these proteins of recognition sites for proteases (e.g., the KEX2 system) in greater numbers than in the homologous proteins (9).…”

“…Thaumatin degradation by aspergillopepsin A has been determined, and the inactivation of this protease resulted in a significant increase of extracellular thaumatin (29). Even when an aspergillopepsin A-deficient mutant, lpr66, was used as the host strain (29), some thaumatin degradation still occurred, mainly at late stages of fermentation, indicating the presence of other proteases different from aspergillopepsin A in culture broths of this strain.…”