ABSTRACT
We have characterized growth and protein processing characteristics of Aspergillus niger strains carrying a disrupted allele of the previously cloned and characterized kexB gene [Appl. Environ. Microbiol. 66 (2000) 363] encoding a furin-type endoprotease. Deletion of the single-copy gene confirms it to be non-essential but disruptant strains exhibit a morphologically distinct phenotype characterized by hyperbranching. Processing of homologous pro-proteins and fusion proteins comprised of a heterologous protein fused down-stream of glucoamylase and separated at the fusion junction by an endoproteolytic cleavage site was compared in wildtype and mutant strains of A. niger. We show that maturation of the native glucoamylase requires KexB, whereas maturation of aspergillopepsin does not. The processing of fusion proteins carrying Lys-Arg requires KexB, although alternative endoproteases are capable of cleaving protein fusions at sites adjacent to Lys-Arg.
Subject(s)
Aspergillus niger/genetics , Aspergillus niger/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Protein Processing, Post-Translational/physiology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Furin/genetics , Furin/metabolism , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Fungal/physiology , Transcriptional Activation/physiologyABSTRACT
The gene coding for benzoate-para-hydroxylase (bphA) of Aspergillus niger was cloned using differential hybridisation techniques and complementation of mutants deficient in this enzyme activity. The nucleotide sequence of the gene was determined, the presence of two introns was shown and the transcription start and termination sites were determined. The structure of the mRNA upstream from the long open reading frame (ORF) is unusual. It contains two small, overlapping ORFs whose function is unknown. Comparison of the deduced amino acid sequence of the protein with the sequences present in the databanks, indicated a significant similarity of BPH to the superfamily of cytochrome P450 enzymes. Further analysis revealed that this protein is a member of a new P450 gene family designated P450LIII. The gene is designated CYP53. To increase the BPH activity of A. niger, multiple copies of the bphA gene were introduced into the genome of a recipient strain by transformation. Although increased intracellular levels of the BPH protein could be detected, the BPH enzyme activity was decreased, suggesting titration of another essential component.
Subject(s)
Aspergillus niger/genetics , Cytochrome P-450 Enzyme System/genetics , Multigene Family , Oxygenases/genetics , Amino Acid Sequence , Aspergillus niger/enzymology , Base Sequence , Blotting, Western , Cloning, Molecular , Gene Expression Regulation, Fungal , Genes, Fungal , Genetic Complementation Test , Introns , Molecular Sequence Data , Mutation , Oxygenases/metabolism , Restriction Mapping , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
The nucleotide sequence of the Aspergillus niger tryptophan C (trpC) gene was determined. Northern hybridization and S1-mapping experiments showed the presence of a 2.6 kb trpC poly(A)+ RNA with two very short (5 and 6 nucleotides) noncoding 5'-regions. Comparison of the predicted amino acid sequence with that of trp gene proteins of pro- and eukaryotic organisms revealed three functional domains (G, C, F) in the A. niger TrpC protein which catalyse the glutamine amidotransferase reaction (GAT), the indole-3-glycerol phosphate synthase reaction (IGPS) and the N-(5'-phosphoribosyl) anthranilate isomerase reaction (PRAI), respectively. These domains are highly conserved and bordered by short areas showing less homology. Within the F domain of the trpC gene in A. niger, A. nidulans and Neurospora crassa, a region encoding 30 amino acids was found which is absent in the analogous genes of Saccharomyces cerevisiae and prokaryotic organisms. This region has features of a mutated in-phase intron.
