Two different strains, Aspergillus awamori TGDTh-4 and A. awamori TGP-3 overexpressing a synthetic gene encoding the plant sweet protein thaumatin, showed an unfolded protein response. To facilitate protein secretion, the chaperone BiPA gene was expressed in A. awamori under control of the strong constitutive promoter of the gpdA gene. A good correlation was observed between the level of the bipA transcript in different strains and the amount of thaumatin secreted. Thaumatin secretion was increased 2-to 2.5-fold in transformants overexpressing the bipA gene compared with the parental strain. Secretion of the homologous proteins ␣-amylase and glucoamylase was not affected by the bipA gene overexpression. The requirement for BiPA for secretion of thaumatin was confirmed by attenuation of the endogenous bipA gene expression with an antisense RNA cassette. The decrease in bipA expression reduced the amount of secreted thaumatin up to 80% without affecting the secretion of the homologous ␣-amylase and glucoamylase proteins. The BiPA protein is, therefore, very important for secretion of some heterologous proteins, such as thaumatin in A. awamori.

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.

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Newcastle Disease Virus (NDV) is an avian enveloped single stranded RNA virus belonging to the family of Paramyxoviridae.The virion consists I11 of a membrane which encloses a helical nucleocapsid and has six major proteins, three belonging t o the ribonucleocapsid and three others associated t o the outer membrane. The lipid envelope contains two transmembrane glycoproteins, the fusion (F) protein and the hemagglutininneuraminidase (HN), and a non-glycosylated matrix (M) protein associated as a peripheral protein to the inner leaflet of the lipid bilayer that is essential in viral budding from the host cell [21. Viral membrane is acquired from the host cell membrane during budding of the virus from the host cell.Several workers I3 and references herein1 have reported the stimulation of influenza virus sialidase (EC 3.2.1.1 8, acylneuraminyl hydrolase) by Ca" but, to our knowledge, this matter has not been investigated in paramyxoviruses such as NDV. Our results indicate that the sialidase activity of the HN protein of Newcastle Disease Virus shows a strong dependence on the ionic composition of the assay mixture, especially the presence of Ca2+ ions, using 2'-(4-methylumbelliferyl~-u-D-N-neuraminic acid as substrate. A specific Ca2+ chelator such as EGTA completely abolishes the sialidase activity at 10 m M concentration (Table 1 .) EGTA seems t o exert its inhibitory effect by means of its chelating capacity since its removal from the sample by exhaustive dialysis restores the original sialidase activity. Table 1. Effect of EGTA on NDV sialidase activity Control 1 m M EGTA