Lori J. Wilson

Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.

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.

Use of Aspergillus overproducing mutants, cured for intergrated plasmid, to overproduce heterologous proteins

Aspergillus niger var. awamori was previously transformed with a vector designed to express a fused glucoamylase-prochymosin gene and bearing the Neurospora crassa pyr4 gene as a selectable marker. Mutant strains that overproduced the glucoamylase-prochymosin fusion protein were derived from one of the transformants. Despite the fact that the expression vector was integrated into the genome of these strains it was possible to obtain strains from which the vector sequences had been removed. This was performed by selection against the pyr4 gene present on the expression vector using 5-fluoroorotic acid. The cured strains were retransformed in order to investigate production of heterologous proteins using other expression vectors. In addition to the glucoamylase-prochymosin fusion protein, the mutant Aspergillus strains also over-produced Rhizomucor miehei aspartic proteinase but not preprochymosin produced as a non-fusion protein. The ability to select for loss of integrated plasmid from Aspergillus transformants may prove to be important for a variety of applications.

Cloning, characterization, and expression of two α-amylase genes from Aspergillus niger var. awamori

SummaryUsing synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an α-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two α-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (≥98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.

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.

Transformation ofAspergillus awamori andA. niger by electroporation

A method is described which allows transformation ofAspergillus awamori andA. niger mediated by electroporation. This procedure gave transformation frequencies similar to those obtained with polyethylene glycol. ForA. niger no differences were observed between the two procedures with respect to the number of integrated plasmid copies or the frequency of homologous integration.

The oliC3 gene of Aspergillus niger: isolation, sequence and use as a selectable marker for transformation

SummaryThe oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycinresistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5′ untranslated sequences with those from other fungi.

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.

Structural Changes Leading to Increased Enzymatic Activity in an Engineered Variant of Bacillus Lentus Subtilisin

Much of the recent effort of subtilisin protein engineering has centered on the subtilisin from Bacillus lentus. This enzyme has higher alkaline performance than either subtilisin BPN’ from Bacillus amyloliquefaciens or subtilisin Carlsberg from Bacillus licheniformis. While the amino acid sequence of B. lentus subtilisin differs at 106 positions from subtilisin BPN’, including six deleted residues at positions 37a, 58, and 161 to 164, the three-dimensional structures of these subtilisins are very similar and it is possible to draw direct correlations between them.