Kirk J. Hayenga

Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori.

We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.

Primary structure of Mucor miehei aspartyl protease: evidence for a zymogen intermediate

The gene encoding the aspartyl protease of the filamentous fungus Mucor miehei has been cloned in Escherichia coli and the DNA sequenced. The deduced primary translation product contains an N-terminal region of 69 amino acid (aa) residues not present in the mature protein. By analogy to the evolutionarily related mammalian gastric aspartyl proteases it is inferred that the primary secreted product is a zymogen containing a 47-aa propeptide. This propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium. To study the effects of modifications of the protease structure on its maturation by enzyme-engineering methods, an efficient expression system was sought. In E. coli, transcription of the preproenzyme coding sequence from a bacterial promoter results primarily in the accumulation of unsecreted, enzymatically inactive polypeptides, immunologically related to the authentic protease. In Aspergillus nidulans expression of the cloned gene, probably from its own promoter, results in the secretion into the culture medium of polypeptides which, compared to the authentic protease, are similar in specific activity, but differ in the character of their asparagine-linked oligosaccharides.

Aspergillus Niger var. Awamori as a Host for the Expression of Heterologous Genes

Among the diversity of cellular systems that have been developed for the expression of heterologous gene products, certain species of filamentous fungi possess features which make them exceptionally attractive for this purpose. These include (a) the ability to produce high levels (>25 grams per liter) of secreted protein in submerged culture, (b) a long history of safe use in the production of enzymes, antibiotics, and biochemicals which are used for human consumption, and (c) established fermentation processes which are inexpensive by comparison with animal cell culture processes done on a similar scale. These attributes have prompted several biotechnology companies to explore the use of filamentous fungi as hosts for the expression and secretion of foreign proteins. Some of the heterologous gene products which have been made using fungal expression systems are shown in Table 1. Compared to highly refined expression systems such as Escherichia coli or Saccharomyces cerevisiae, the evolution of filamentous fungi as hosts has barely begun, and many fundamental aspects of cell biology and biochemistry in fungi have not been studied. Fortunately, many of the molecular details and principles which have been elucidated in yeast and in mammalian cell systems appear to be applicable to the study of heterologous gene expression and protein secretion in filamentous fungi as well.

Isolation and characterization of the Aspergillus oryzae gene encoding aspergillopepsin O

We have cloned and determined the nucleotide sequence of a genomic DNA segment from Aspergillus oryzae which contains pepO, the gene encoding the aspartic proteinase, aspergillopepsin O (PEPO). The organization of pepO is strikingly similar to that of pepA from A. niger var. awamori (previously called A. awamori) in that both are composed of four exons and three introns with virtually identical lengths, and the positions of the introns are exactly conserved. From the deduced amino acid (aa) sequence, it appears that PEPO, like other fungal aspartic proteinases, is synthesized as a zymogen containing a putative N-terminal prepro-region of 77 aa followed by a mature protein of 327 aa. Southern blotting experiments suggest that a single copy of pepO exists in the A. oryzae genome.