Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans

Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores. These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores. The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis. We have isolated the hymA mutant, in which conidiophore development is affected at the metula stage. In the mutant metulae do not differentiate properly but come to resemble hyphae (hym?=?hypha-like metulae). In this paper we have analyzed the corresponding gene. It encodes a highly expressed 44?kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man. We constructed hym deletion strains of Saccharomyces cerevisiae and of A. nidulans and found that the gene is essential in S. cerevisiae but is dispensable in the filamentous fungus. A cellular function for the Hym protein has not yet been defined in any organism. To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A.?nidulans and the C-terminal half of the mouse homologue MO25. This hybrid protein could fully substitute for HymA function in A. nidulans. In addition, the mouse protein itself partially rescued the hymA mutation in the fungus. HymA is thus highly conserved in evolution and probably serves similar functions. The fact that hymA is required for conidiophore development in A. nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis.     

An aureobasidin A resistance gene isolated from Aspergillus is a homolog of yeast AUR1, a gene responsible for inositol phosphorylceramide (IPC) synthase activity

The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity. We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1. A single point mutation in the aurA gene of A. nidulans confers a high level of resistance to aureobasidin A. The A. nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A. fumigatus, A. niger, and A. oryzae. The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A. fumigatus showed 87% identity to that of A. nidulans. The AurA proteins of A. nidulans and A. fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S. cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts. These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi. Received: 27 August 1998 / Accepted: 19 November 1998     

Cloning and characterisation of the sagA gene of Aspergillus nidulans: a gene which affects sensitivity to DNA-damaging agents

Mutations within the sagA gene of Aspergillus nidulans cause sensitisation to DNA-damaging chemicals but have no effect upon spontaneous or damage-induced mutation frequency. The sagA gene was cloned on a 19-kb cosmid-derived fragment by functional complementation of a sagA1 sagC3 double mutant; subsequently, a fragment of the gene was also isolated on a 3.9-kb genomic subclone. Initial sequencing of a small section of the 19-kb fragment allowed the design of primers that were subsequently used in RTPCR experiments to show that this DNA is transcribed. A 277-bp fragment derived from the transcribed region was used to screen an A. nidulans cDNA library, resulting in the isolation of a 1.4-kb partial cDNA clone which had sequence overlap with the genomic sagA fragment. This partial cDNA was incomplete but appeared to contain the whole coding region of sagA. The sagA1 mutant was shown to possess two mutations; a G-T transversion and a+1 frameshift due to insertion of a T, causing disruption to the C-terminal region of the SagA protein. Translation of the sagA cDNA predicts a protein of 378 amino acids, which has homology to the Saccharomyces cerevisiae End3 protein and also to certain mammalian proteins capable of causing cell transformation. Received: 1 August 1998 / Accepted: 9 November 1998     

Overexpression of fIbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brIA and premature initiation of development

Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores. We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A. nidulans conidiophore development. flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures. The requirement for fIbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development. The wild-type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A. nidulans asexual life cycle. Overexpression of fIbA using an Inducible promoter resulted in misscheduled expression of brlA in vegetative ceils and caused hyphal tips to differentiate into spore-producing structures. Sequence analysis of a nearly full-length fIbA cDNA clone showed that fibA is predicted to encode a 717-amino-acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein. SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination. We propose that fIbA plays a related role in a signalling pathway for Aspergillus conidiophore development.     

Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans

A gene for a synthetic protein-based polymer, G-(VPGVG)₁₁₉-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study represents the first attempt to express a synthetic gene (with no natural analog) in a fungus. Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

FigA,a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae,Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca²⁺ rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.     

The uvsC and uvsE genes of Aspergillus nidulans are not required for the mutagenic repair of UV damage

The uvsC gene of Aspergillus nidulans is a homolog of the RAD51 gene of Saccharomyces cerevisiae. However, with respect to its effects on UV mutagenesis, it differs from the yeast gene, since it seems to be required for UV mutagenesis; however, this conclusion is based only on data from resting conidia. To further clarify the functional role of the uvsC gene, we tested the UV mutability of strains bearing a uvsC mutation in resting as well as in germinating conidia, by the p-fluoro-phenyl-alanine resistance test. We also evaluated the mutability of the uvsE mutant which belongs to the same epistatic group. Our results show that the uvsC and uvsE genes do not have a significant role in the mutagenic UV-repair pathway. Received: 20 January 1998 / Accepted: 22 April 1998     

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form. Received: 19 March 1997 / Received revision: 19 May 1997 / Accepted: 1 June 1997     

Repression of the expression of genes encoding xylanolytic enzymes in Aspergillusoryzae by introduction of multiple copies of the xynF1 promoter

A xylanase gene, xynF1, was cloned and characterized from a shoyu koji mould Aspergillus oryzae KBN616. The xynF1 gene was found to be comprised of 1484 bp with ten introns. The deduced amino acid sequence encodes a protein consisting of 327 amino acids (35,402 Da) which is very similar to the fungal family F xylanases such as Aspergillus nidulans XlnC, Aspergillus kawachii XynA and Penicillium chrysogenum XylP. The intron/exon organization of xynF1 is very similar to that of the fungal family F xylanase genes. Plasmid pXPR64, which contains 64 copies of the xynF1 promoter region (PxynF1) in the same direction, was constructed and introduced into A. oryzae. This led to reduced expression of both xylanase and β-xylosidase genes in the transformants. Received: 18 May 1998 / Received revision: 7 July 1998 / Accepted: 9 July 1998     

Improved ethanol production by glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae

The anaerobic performance of gpd1Δ and gpd2Δ mutants of Saccharomyces cerevisiae was characterized and compared to that of a wild-type strain under well-controlled conditions by using a high-performance bioreactor. There was a 40% reduction in glycerol level in the gpd2Δ mutant compared to the wild-type. Also the gpd1Δ mutant showed a slight decrease in glycerol formation but to a much lesser degree. As a consequence, ethanol formation in the gpd2Δ mutant was elevated by 13%. In terms of growth, the gpd1Δ mutant and the wild-type were indistinguishable. The gpd2Δ mutant, on the other hand, displayed an extended lag phase as well as a reduced growth rate under the exponential phase. Even though glycerol-3-phosphate dehydrogenase 2 (GPD2) is the important enzyme under anaerobic conditions it can, at least in part, be substituted by GPD1. This was indicated by the higher expression level of GPD1 in the gpd2Δ mutant compared to the wild type. These results also show that the cells are able to cope and maintain redox balance under anaerobic conditions even if glycerol formation is substantially reduced, as observed in the gpd2Δ mutant. One obvious way of solving the redox problem would be to make a biomass containing less protein, since most of the excess NADH originates from amino acid biosynthesis. However, the gpd2Δ mutant did not show any decrease in the protein content of the biomass. Received: 16 February 1998 / Received revision: 16 March 1998 / Accepted: 1 June 1998     

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase. Received: 16 June 1998 / Received last revision: 21 August 1998 / Accepted: 3 September 1998     

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

Characterisation of a starch-hydrolysing enzyme of Aspergillus niger

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 °C and glucose, maltose and maltodextrins at 70 °C as primary products, suggested significant applications for the enzyme in starch-processing industries. Received: 29 October 1998 / Received revision: 11 January 1999 / Accepted: 19 January 1999     

Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1α, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-α protein consisted of 460 amino acids possessing high identity to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A. oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 °C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C. Received: 28 November 1997 / Received revision: 24 February 1998 / Accepted: 6 March 1998     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

Cloning and characterization of an endo-β-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938

We describe the identification and expression cloning of two novel enzymes, a β-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-β-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0–6.0, and a temperature activity optimum around 40 °C. Both enzymes show only low sequence identity to other known enzymes. Received: 6 August 1998 / Received revision: 29 October 1998 / Accepted: 30 October 1998     

Phosphorylation of the pheromone-responsive Gβ protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function

The pheromone-responsive G_β subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310–346, ste4Δ310–346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho⁻) alleles of STE4: ste4-T320 A/S335A and ste4-T322 A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4Δ310–346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive G_α of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310–346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1. Received: 14 February 1998 / Accepted: 28 February 1998     

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M _r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression. Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998