Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.     

Purification, properties and primary structure of thioredoxin from Aspergillus nidulans.

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (M(r) = 11,564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%).     

应用实时荧光PCR技术检测构巢曲霉的初步研究

目的 根据构巢曲霉（Aspergillus nidulans）3-磷酸甘油醛脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase,GAPDH）基因特异位点设计并合成Taqman探针及引物,建立构巢曲霉实时荧光 PCR检测方法。方法 应用lasergene7.1软件对构巢曲霉与13种常见曲霉主要包括黑曲霉（A.niger）、烟曲霉（A.fumigatus）、杂色曲霉（A.versicolor）、土曲霉（A.terrus）、黄曲霉（A. flavus）、温特曲霉（A.wentii）、寄生曲霉（A. parasiticus）、泡盛曲霉（A.awamori）、米曲霉（A. oryzae ）、棒曲霉（A.cavatus）、赤曲霉（A.ruber ）、亮白曲霉（A.ochraceus）及赭曲霉（A.ochraceus）GAPDH基因序列比对分析,在特异位点设计引物和探针,建立构巢曲霉实时荧光 PCR检测方法,并对该方法进行特异性及敏感性分析。结果 用曲霉属22种41株不同曲霉及其他属的12株病原真菌验证实验表明,所建立的荧光PCR方法特异性强;检测灵敏度可达4.03×10－12μg/ml的模板DNA。 结论 应用实时荧光PCR技术能够有效检测构巢曲霉,该方法具有特异、灵敏、快速等特点,可在实际工作中应用。     

Apparent mRNA instability in Aspergillus nidulans and Aspergillus terreus of a heterologous cDNA encoding the major capsid antigen of Rotavirus

Two expression plasmids designed to produce the rotaviral VP6 protein in Aspergillus nidulans and Aspergillus terreus have been constructed. In one of these plasmids the inducible A. terreus Gla1 glucoamylase gene promoter and Gla1 signal sequence are fused to the VP6 cDNA to enable induction and extracellular secretion of the final protein product; in the other, the strong, constitutive A. nidulans gpdA gene promoter has been employed. A. nidulans and A. terreus transformants containing intact copies of these plasmids have been obtained but neither intra- nor extra-cellular VP6 protein was detectable. Northern analysis indicated specific degradation of the VP6 mRNA. This lack of VP6 mRNA stability may be related to fundamental differences between the general structure of Aspergillus mRNA and that of rotavirus, including codon usage and AU/GC ratio.     

Analysis of two Aspergillus nidulans genes encoding extracellular proteases

Characterization of prtADelta mutants, generated by gene disruption, showed that the prtA gene is responsible for the majority of extracellular protease activity secreted by Aspergillus nidulans at both neutral and acid pH. The prtA delta mutation was used to map the prtA gene to chromosome V. Though aspartic protease activity has never been reported in A. nidulans and the prtADelta mutants appear to lack detectable acid protease activity, a gene (prtB) encoding a putative aspartic protease was isolated from this species. Comparison of the deduced amino acid sequence of PrtB to the sequence of other aspergillopepsins suggests that the putative prtB gene product contains an eight-amino-acid deletion prior to the second active site Asp residue of the protease. RT-PCR experiments showed that the prtB gene is expressed, albeit at a low level.     

Cloning of the riboB locus of Aspergillus nidulans

We have complemented the riboB2 mutation of Aspergillus nidulans by transformation with a plasmid library of wild-type (wt) sequences. We have isolated, by marker rescue from a riboB+ transformant, a plasmid that complements riboB2 efficiently. From this plasmid we have subcloned an A. nidulans sequence that complements riboB2 efficiently and that integrates by homologous recombination at a site closely linked to the riboB locus. We conclude that this sequence contains the wt riboB+ allele.     

Intracellular and extracellular production of proteins in Aspergillus under the control of expression signals of the highly expressed Aspergillus nidulans gpdA gene.

The expression in Aspergillus is described of genes, coding for intracellular and extracellular proteins controlled by the promoter region of the constitutively and efficiently expressed glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) of Aspergillus nidulans. Both the homologous gpdA and the heterologous Escherichia coli beta-galactosidase (lacZ) and beta-glucuronidase (uidA) genes could be expressed intracellularly at levels as high as 10-25% of total soluble protein. Efficient extracellular production of A. niger glucoamylase could be achieved with a fusion-gene containing the region of the glucoamylase gene coding for the mature protein preceded by a synthetic fungal signal sequence. Extracellular production of a heterologous protein, E. coli beta-glucuronidase, with such a fusion was much less efficient. Only very low levels of beta-glucuronidase were detected in the culture fluid, whereas considerable enzyme activity was detected in the mycelium.     

Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.     

Unique regulatory mechanism for D-galactose utilization in Aspergillus nidulans

This study describes two novel regulators, GalX and GalR, that control d-galactose utilization in Aspergillus nidulans. This system is unique for A. nidulans since no GalR homologs were found in other ascomycetes. GalR shares significant sequence identity with the arabinanolytic and xylanolytic regulators AraR and XlnR, but GalX is more distantly related.     

Transformation of Aspergillus aculeatus using the drug resistance gene of Aspergillus oryzae and the pyrG gene of Aspergillus nidulans

Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5'-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.     

Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.     

acon-3, the Neurospora crassa ortholog of the developmental modifier, medA, complements the conidiation defect of the Aspergillus nidulans mutant

Aspergillus nidulans and Neurospora crassa are ascomycetes that produce asexual spores through morphologically distinct processes. MedA, a protein with unknown function, is required for normal asexual and sexual development in A. nidulans. We determined that the N. crassa ortholog of medA is acon-3, a gene required for early conidiophore development and female fertility. To test hypotheses about the evolutionary origins of asexual development in distinct fungal lineages it is important to understand the degree of conservation of developmental regulators. The amino acid sequences of A. nidulans MedA and N. crassa ACON-3 shared 37% identity and 51% similarity. acon-3 is induced at late time points of conidiation. In contrast, medA is constitutively expressed and MedA protein localizes to nuclei in all tissue types. Nonetheless, expression of acon-3 using its native promoter complemented the conidiation defects of the A. nidulans ΔmedA and medA15 mutants. We conclude that the biochemical activity of the medA orthologs is conserved for conidiation.     

The Aspergillus niger acuA and acuB genes correspond to the facA and facB genes in Aspergillus nidulans

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2% propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutants lacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu+ phenotype with a clone of ACS (facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified by cross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB is analogous to the A. nidulans facB regulatory gene.