Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes

Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.     

Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans.

Calcium and calmodulin have been widely implicated in the control of cell proliferation. We have created a strain of the genetically tractable filamentous fungus, Aspergillus nidulans, that is conditional for calmodulin expression. This was accomplished by replacing the unique endogenous calmodulin gene with one regulated by the inducible alcohol dehydrogenase (alcA) gene promoter by homologous recombination. This strain cannot grow when the cells are incubated in medium containing a carbon source that represses the alcA promoter. Characterization of the arrested cells shows that 83% are blocked in the G2 phase of the cell cycle. The block is due to very low levels of calmodulin and is fully reversible upon changing to medium that contains an inducer of the alcA promoter. The rate of cell proliferation in this strain is dependent upon both the intracellular calmodulin and extracellular Ca2+ concentrations. Raising the calmodulin concentration by inducing the alcA promoter not only causes the cells to enter the proliferative cycle more quickly and to grow faster, but also decreases the concentration of extracellular Ca2+ required to support growth by 10-fold, as compared with cells grown in noninducing medium. Thus both the intracellular calmodulin and extracellular Ca2+ concentrations are important and interactive factors in regulating the nuclear division cycle of Aspergillus nidulans.     

Regulation of alcR, the positive regulatory gene of the ethanol utilization regulon of Aspergillus nidulans

The alcR positive control gene is necessary for the expression of both alcA (coding for alcohol dehydrogenase ADH I), and aldA (coding for aldehyde dehydrogenase, AldDH) in Aspergillus nidulans. Using a cloned alcR probe and Northern blots analysis we show that: (1) alcR itself is inducible; (2) alcR inducibility depends on the expression of the alcR gene itself; and (3) alcR is subject to carbon catabolite repression and its expression is controlled by the negatively acting creA wide specificity gene. The repression of alcR is sufficient to explain the carbon catabolite repression of ADH I and AldDH.     

Identification of Developmental Regulatory Genes in Aspergillus Nidulans by Overexpression

Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [ alcA (p) ] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA (p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA (p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA (p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA (p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA (p) :: brlA induction. Sequence analyses of the DNA fragments under alcA (p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA (p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.     

An inducible expression system for the production of human lactoferrin in Aspergillus nidulans.

The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter. Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.     

Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulans.

The ethanol utilization pathway in Aspergillus nidulans is a model system, which has been thoroughly elucidated at the biochemical, genetic and molecular levels. Three main elements are involved: (a) high level expression of the positively autoregulated activator AlcR; (b) the strong promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA); and (c) powerful activation of AlcR by the physiological inducer, acetaldehyde, produced from growth substrates such as ethanol and l-threonine. We have previously characterized the chemical features of direct inducers of the alc regulon. These studies allowed us to predict which type of carbonyl compounds might induce the system. In this study we have determined that catabolism of different amino acids, such as L-valine, L-isoleucine, L-arginine and L-proline, produces aldehydes that are either not accumulated or fail to induce the alc system. On the other hand, catabolism of D-galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes. We show that the formation of a direct inducer from carboxylic esters does not depend on alcA-encoded alcohol dehydrogenase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physiological aldehyde inducer.     

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.     

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus.

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.     

Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans.

Aspergillus niger grows poorly on acetamide as a nitrogen or carbon source and lacks sequences detectably homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans. We have taken advantage of these observations to develop a transformation system for A. niger using the amdS gene as a dominant heterologous marker for selecting transformants on the basis of acetamide utilization. Transformants varied in their ability to grow on amide media and the number of integrated copies of the amdS plasmid ranged from 1 or 2 to greater than 100. Southern analysis of transformants revealed that the multiple copies were integrated into the chromosome in tandem arrays. This result indicates that transformation of A. niger is more similar to mammalian cells than to yeast. Analysis of enzyme activity levels and RNA levels showed that most of the copies of amdS were expressed. Mitotic stabilities of transformants were found to be high. A transformant containing greater than 100 copies of the amdS gene was impaired in omega-amino acid utilization, a result that has also been found in A. nidulans. Since, in A. nidulans, omega-amino acids induce acetamidase via a characterizied regulatory gene (amdR/intA) this observation implies that titration of an analogous A. niger regulatory gene product by multiple amdS copies has occurred. Additional evidence suggested that the amdS gene is regulated in A. niger. It has also been shown that an unselected plasmid can be co-transformed with the amdS plasmid into A. niger.     

The Aspergillus niger acuA and acuB genes correspond to the facA and facB genes in Aspergillus nidulans

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2% propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutants lacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu+ phenotype with a clone of ACS (facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified by cross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB is analogous to the A. nidulans facB regulatory gene.