A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspergillus awamori

Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin. Transformants with different levels of pdiA mRNA and measured PDIA levels were chosen for examination of the impact that PDIA levels had on thaumatin secretion. The secretion of two native proteins, alpha-amylase and acid phosphatase, was also examined in relation to varying levels of PDIA. Over a range of PDIA levels of 1-8, relative to the native level in strains with just one copy of the pdiA gene, the fraction of alpha-amylase and acid phosphatase in the total secreted protein was unaffected. In contrast, a peak level of thaumatin, about 5-fold higher than in the strain with one copy of pdiA, was found in strains with a relative PDIA level of between two and four. Improved thaumatin production was confirmed in 5-1 fermenters using a strain of A. awamori with six pdiA gene copies, containing 3.2-fold higher levels of PDIA than wild-type strains.     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

Translational control of alpha-amylase gene expression in Aspergillus awamori

Regulation of alpha-amylase gene expression in Aspergillus awamori was studied by analyzing the enzyme activity levels, rate of protein synthesis, and alpha-amylase-specific mRNA levels under various conditions of growth. alpha-Amylase synthesis was sensitive to catabolite repression as glucose repressed its synthesis by about fourfold. The stimulation of alpha-amylase synthesis in the presence of its substrate starch was shown to be due to derepression rather than induction as the enzyme was synthesized at similar rates in both starch and starvation media. Repression and derepression of enzyme synthesis was found to be mediated at the translational level. The cellular levels of alpha-amylase-specific mRNA as measured by an in vitro translation assay system, were almost identical under all conditions of enzyme synthesis. Relative in vivo and in vitro alpha-amylase mRNA template activities suggest that alpha-amylase mRNA is translated much more efficiently during the derepression than under the conditions of repressed synthesis.     

Cloning and heterologous expression of gene encoding A polygalacturonase from Aspergillus awamori

A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.     

Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.     

Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori

In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.     

Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori

The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l(-1) in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP.     

Microencapsulation selection for isolation of yeast mutants with increased secretion of Aspergillus awamori glucoamylase

We have developed a microencapsulation selection method which allows the rapid and quantitative screening of >10(6) yeast cells for enhanced secretion of Aspergillus awamori glucoamylase. The method provides a 400-fold single-pass enrichment for high-secreting mutants, and can be straightforwardly adapted for application to growth-based selection schemes with other microorganisms and enzymes. (c) 1993 John Wiley & Sons, Inc.