David Ivor Gwynne

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.

A phosphate-repressible acid phosphatase gene from Aspergillus niger: its cloning, sequencing and transcriptional analysis

The cloning and sequencing of an Aspergillus niger gene encoding a secreted form of phosphate-repressible acid phosphatase by complementation of a pacA (phosphate-repressible acid phosphatase) mutant of Aspergillus nidulans is described. The gene contains two introns, 201 and 265 nt in length, and codes for a 1.6-kb transcript. Both phosphate concentration and pH of the growth medium affect the level of expression of the gene in A. niger. Similar regulation is observed in A. nidulans transformants. A putative signal peptide, resembling known signal sequences of yeast, is identified.

Cloning of a new bidirectionally selectable marker for Aspergillus strains

Mutants that lack adenosine triphosphate sulfurylase (ATPsase; EC 2.7.7.4) are unable to use sulfate as sole source of sulfur and are also resistant to selenate. These mutants, denoted sC-, are readily obtained from any strain of Aspergillus niger or Aspergillus nidulans by the strong selection for selenate resistance. We have cloned the gene encoding ATPsase from A. nidulans by complementation of an sC mutant strain of A. nidulans with a gene library and show that plasmids containing this gene transform both A. niger and A. nidulans sC- strains, restoring their ability to grow on sulfate as sole sulfur source. The fact that strong selection for either sC+ or sC- can be applied provides a simple way of delivering genetically engineered constructs to any strain of A. niger including strains of industrial importance. In addition, this system is useful for gene replacements and other genomic DNA manipulations in Aspergillus species.

Heterologous protein secretion directed by a repressible acid phosphatase system of Aspergillus niger

A new expression-secretion system of Aspergillus niger which directs the secretion of heterologous proteins is described. The promoter and signal peptide-encoding region of the phosphate-repressible aphA gene of A. niger, when fused to the coding region of the human interferon alpha 2 (hIFN alpha 2)-encoding gene (hIFN alpha 2), drives the expression of this gene and the secretion of the hIFN alpha 2 protein. Synthesis of hIFN alpha 2 in either A. niger or A. nidulans transformants carrying these constructs was regulated by inorganic phosphate (Pi) present in the medium, so that derepression of heterologous protein expression can be attained by lowering Pi concentration.

Cloning and molecular analysis of the ornithine carbamoyl transferase gene of Aspergillus niger

We have cloned the gene encoding ornithine carbamoyl transferase (OCTase) from Aspergillus niger. The structure and complete nucleotide sequence of this gene have been determined. The gene encodes an mRNA of 1.3 kb. The transcription unit contains an open reading frame of 1110 nucleotides (nt) which shows strong homology to the OCTase of Aspergillus nidulans along most of its length. The N terminus, which shows little or no homology to other OCTases, is highly basic and is probably involved in mitochondrial targeting.

Characterization of an Aspergillus nidulans genomic DNA fragment conferring phosphate-non-repressible acid-phosphatase activity

A clone from an Aspergillus nidulans library was identified by its ability to confer enhanced staining for acid phosphatase (APase) activity upon phosphatase-deficient A. nidulans mutants. This APase activity is not repressed by high phosphate concentrations in the medium. The 2.9-kb nucleotide sequence in the region of the clone responsible for the effect reveals two potential protein-coding genes with a common N terminus. One corresponds to an open reading frame (ORF) with no introns, encoding 330 amino acids (aa). The other, shorter gene encoding 113 or 117 aa has the first 65 or 69 codons in common with the long ORF; then, after a single 165-nt intron with a fungal consensus lariat sequence and splice junctions, there are a further 48 codons in a different reading frame. Both correspond in sense direction, and the shorter gene in length, with the only detectable transcript in this region, but both differ from all known APase sequences. The possible identity of these ORFs with the pacG gene is discussed.