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.

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.

Expression of heterologous proteins in Aspergillus.

Filamentous fungi, in particular those of the genus Aspergillus have been well exploited for their ability to produce high levels of extracellular proteins in an inexpensive manner. Since many human proteins with the potential to be used therapeutically are secreted and require post-translational modification for biological activity, eukaryotic expression-secretion systems have been targeted for development. Recent developments in DNA-mediated transformation systems have allowed the utilization of Aspergillus as a host for the production of recombinant proteins. Several features such as well-characterized genetics and the availability of many mutants make Aspergillus nidulans the organism of choice for development of expression secretion systems. Recombinant strains contain integrated expression cassettes often in multiple copy, which are mitotically stable. In this review, we discuss the recent progress made in the use of Aspergillus as expression secretion hosts for the production of proteins of therapeutic significance.

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.

The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR.

The regulatory gene, alcR, of Aspergillus nidulans, encodes a protein that induces the expression of the alcA and aldA genes. The alcR gene is inducible, autoregulated, and subject to carbon catabolite repression. We report the complete nucleotide sequence of the alcR gene and its 5' and 3' non-coding regions. In the 5' flanking region of the alcR gene, several repeats and inverted repeats were found, and small sequence similarities were also found with the 5' flanking regions of the alcA and aldA genes. One intron of small size interrupts the open reading frame. The start point of transcription was mapped 50 nucleotides upstream from the putative start codon, and a sequence CAATG was found 5' to the polyadenylation site of the transcript that could play a role in selection of the polyadenylation site. The putative alcR-encoded protein was identified in vivo as an inducible polypeptide of 96 kDa in a transformant carrying multiple copies of the alcR gene.

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.

Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa.

We describe the preparation of an in vitro translation system from a wild-type strain of Neurospora crassa. The system is capable of supporting efficient and faithful translation of native and in vitro transcribed eukaryotic messages. The translation products have minimal background and can be clearly analyzed by SDS-polyacrylamide gel electrophoresis. The method of preparation of the lysate is simple, fast and reproducible. The procedure should be readily applicable to other filamentous fungi.

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.

Cloning and characterization of the aldA gene of Aspergillus nidulans.

We have cloned and sequenced the aldA (encoding aldehyde dehydrogenase) gene of Aspergillus nidulans. The gene contains two introns which are similar in size and structure to other fungal introns. The amino acid sequence of aldehyde dehydrogenase (497 residues) shows a significant level of homology with analogous sequences in other organisms. Comparison of the primary structure of the active sites of the mammalian cytosolic and mitochondrial enzymes shows that the Aspergillus enzyme closely resembles the mammalian mitochondrial enzyme. Analysis of the 5' non-coding region of the aldA gene shows a TATA-like sequence located 90 bp upstream from the initiation codon. Two messenger-RNA start points are located 36 and 42 bp upstream from the start codon.

Comparison of the cis-acting control regions of two coordinately controlled genes involved in ethanol utilization in Aspergillus nidulans.

The alcA and aldA genes of Aspergillus nidulans are regulated in exactly the same manner, being subject to positive control by the product of the alcR gene. We report the complete nucleotide sequence of the alcA gene and its 5' non-coding region, preliminary localization of the region involved in the regulation of alcA expression, and a detailed comparison of this region to the 5' non-coding region of aldA (Pickett et al., 1987). The 5' flanking regions of the genes contain six similar sequence elements. Three of these elements are located upstream from the messenger RNA start points and one is related to a sequence element found in the region responsible for ethanol induction of the yeast ADH2 gene (Beier et al., 1985). The other homologous elements are located within the messenger RNA leader and may be associated with selection of messenger RNA start points. The amino acid sequence of alcohol dehydrogenase I (348 residues) shows a significant level of homology with analogous sequences in other organisms. Gene alcA contains introns which are similar in size and structure to other fungal introns. We discuss the positions of the introns in alcA of A. nidulans with particular reference to the conservation of intron position in and the evolutionary assembly of enzymes which possess NAD-binding domains.

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.

Structure and regulated expression of the SpoC1 gene cluster from Aspergillus nidulans.

We have previously described the organization of a 13.3 kb region of the Aspergillus nidulans genome, designated SpoC1, coding for multiple poly(A)+ RNAs that accumulate in asexual spores but not in somatic cells. We have determined the limits of the SpoC1 gene cluster by investigating the transcriptional features of 53 kb of chromosomal DNA. This segment of the genome codes for at least 19 poly(A)+ RNAs, some of which are transcribed from overlapping regions. The area of developmental regulation is approximately 38 kb in length and is delimited by 1.1-kb direct repeats. With one exception, RNAs transcribed from the central part of the cluster appear late during conidiophore development and accumulate specifically in spores. The exceptional transcript appears earlier during development and accumulates specifically in cells of the conidiophore. In contrast, RNAs encoded at the borders of the cluster occur in both somatic cells and spores. The results indicate that if a chromatin-level control mechanism operates to regulate expression of the SpoC1 gene cluster, as previously suggested by us, additional levels of regulation must also exist.

Alterations in gene expression during heat shock of Achlya ambisexualis.

When exponentially growing cultures of Achlya ambisexualis strain E87 were raised from their normal growth temperature of 30 degrees C to 35 degrees C, the rates of synthesis of a small number of proteins were dramatically increased. The most predominant proteins synthesized in response to heat shock had molecular weights of 70,000 and 78,000, and their increased synthesis was detected as early as 10 min after the shift to 35 degrees C. Changes in the populations of translatable messenger RNAs during heat shock showed that the levels of the mRNA's for all the major induced proteins correlated very closely with the alterations in the in vivo patterns, suggesting a transcriptional level of control of their synthesis. When after a period of heat shock (60 min) the cultures were shifted back to 30 degrees C, recovery of the preshock patterns of protein synthesis was attained after several hours. Different proteins show temporally distinct patterns of recovery. During recovery the levels of translatable mRNA's for the induced proteins also correlated closely with the patterns of in vivo protein synthesis.

RNA and protein synthetic patterns during germination of Polysphondylium pallidum microcysts.

During microcyst germination in the cellular slime mould Polysphondylium pallidum, an immediate rapid increase in the rate of protein synthesis ([3H]leucine incorporation) is observed within 15 min after the initiation of germination. The data, corrected for amino acid pool changes, reveal that the rate of protein synthesis reaches its peak at 1 1/2 h, after which it decreases. A low level of RNA synthesis ([3H]uridine incorporation) is observed after 1 h and this rate increases markedly after 2 h. Analysis of the RNA species shows a low level of synthesis of all ribosomal RNA's which begins between 1 and 2 h and increases after 2 h. The synthesis of a heterogeneously distributed, poly(A)-containing fraction of RNA (presumptive mRNA) is initiated some time after 2 h and the synthesis of a small molecular weight species in the 4-5S region is observed after 3 h. Thus, it seems that Polysphondylium microcysts show sequentially initiated synthesis of RNA during germination.