Isolation and characterization of the nuclease O gene (nucO) from Aspergillus oryzae

Nuclease O in the mycelia of Aspergillus oryzae has been purified 55-fold by successive steps of chromatography from the filtrate of the autolyzate. The molecular mass of nuclease O was 32 kDa, as estimated by SDS polyacrylamide-gel electrophoresis. The nuclease O gene (nucO) encoding this enzyme was cloned and sequenced. The open reading frame is interrupted by four introns with conserved splice sites and contains 328 amino-acid residues of the mature enzyme. A. nidulans transformants obtained by introduction of the cloned nucO gene produced 2.5-times as much nuclease O as the wild-type strain, showing that the cloned DNA fragment encodes nuclease O. Received: 6 March / 15 May 1996     

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

Summary Wa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.The sequence reported in this paper has been submitted to the EMBL/GenBank through DDBJ (accession no, X52525).     

Cloning of an Aspergillus niger invertase gene by expression in Trichoderma reesei

The filamentous fungus Aspergillus niger produces two glycosylated forms of the sucrose-hydrolysing enzyme, invertase. In contrast, some Trichoderma species lack invertase and are unable to utilise sucrose as a sole carbon source. Using an A. niger genomic library constructed in a cosmid vector containing the ura5 gene of Podospora anserina as a selectable marker, and the T. reesei ura5^- strain as a sucrose-minus recipient strain, an A. niger invertase gene (suc1) has been cloned by a sib selection procedure. PAGE and enzyme analysis confirmed that transformants had acquired invertase activity. The cloned gene contained DNA sequences which were complementary to the amino-acid sequences of tryptic peptides found in invertase purified from A. niger. The suc1 invertase gene can be used as a dominant selectable marker for the transformation of Trichoderma strains.     

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

Summary The 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.     

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans

Summary Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.Deceased April 30, 1988     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): Indications of a pectin lyase gene family in A. niger

Summary Using the previously cloned Aspergillus niger N756 pectin lyase D gene as a probe, the corresponding pelD gene has been isolated from a genomic library of the loboratory strain A. niger N400. This gene encodes PLD, previously described as PLI, which is one of the two major pectin lyases isolated from the commeriial pectinase preparation Ultrazym^®. Heterologous hybridization of the A. niger N400 genomic library with the pelD gene led to the isolation of another five genes: pelA, B, C, E, and F. These genes differ in their hybridization patterns with probes containing either the entire pelD gene, or 5 or 3 parts thereof. By partial sequencing, and expression in an A. niger transformant containing multiple copies of the pelA gene, we show that this gene, which hybridizes strongest with the pelD gene, encodes the other major pectin lyase from Ultrazym^®, PLII.     

Transformation of Trichoderma viride using the Neurospora crassa pyr4 gene and its use in the expression of a Taka-amylase A gene from Aspergillus oryzae

Summary A pyrG mutant of Trichoderma viride, a very efficient cellulase producer, was isolated from among 5-fluoroorotic acid-resistant mutants. The mutation was complemented with the pyr4 gene of Neurospora crassa and used as a selection marker for the transformation of T. viride. A plasmid vector, pDJB1-Taa, carrying both the pyr4 gene and a gene encoding Taka-amylase A from Aspergillus oryzae, was constructed and introduced into protoplasts of T. viride pyrG^-. The transformation frequency was 1–10 transformants (3 on average) per g DNA. One transformant showed highly elevated -amylase production (about 17 times higher than the recipient level) and the integration of more than one copy of the Taka-amylase gene.     

Multiple copies of the amdS gene of Aspergillus nidulans cause titration of trans-acting regulatory proteins

Summary It has been established that a plasmid containing the amdS gene of Aspergillus nidulans may be used to transform amdS⁺ strains by selecting for increased utilization of acetamide as sole nitrogen source. Analysis of transformants has shown that multiple tandem copies of the plasmid can be integrated into the chromosome, commonly at sites other than the amdS locus. While the transformed phenotype was relatively stable through mitotic and meiotic divisions evidence was found for variation in plasmid copy number presumably due to unequal recombination events. Expression of the integrated amdS genes was related to copy number, and the amdS RNA produced was similar in size to wild-type RNA. Evidence for titration of the product of the regulatory gene amdR by multiple copies of amdS was found. No titration of the product of the areA gene was observed, and amdS expression was still dependent on areA function. Multiple copies of the amdI9 mutation resulted in poor growth on acetate. This was not observed in the case of the amdS⁺ gene. The cis-acting amdI9 mutation causes increased facB dependent acetate induction of amdS expression. Titration of the facB gene produce by amdI9 DNA, but not by amdS⁺ DNA, therefore suggested that the mutation results in increased affinity for the facB gene product.     

Regulatory region of the Aspergillus nidulans argB gene

Summary We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC). These deletions comprise the 5 upstream sequence of the argB gene. The pro^– arg^– strain of A. nidulans was transformed with the above plasmids. Several arg⁺ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared. It was concluded that nucleotides located between –150 and –50 by upstream of the argB gene are significant for its cross-pathway regulation. This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast.Abbreviations bp base pairs - kb 10³ base pairs - OTC ornithine carbamoyltransferase - ORF open reading frame     

Location and nucleotide sequence of the pre-apocytochrome f gene on the Oenothera hookeri plastid chromosome (Euoenothera plastome I)

Summary The gene for pre-apocytochrome f has been mapped by blot hybridization on a 2.4 kbp HindIII fragment of the circular plastid chromosome of Oenothera hookeii employing probes from the corresponding spinach gene. The gene is located distal to the gene for the ATP synthase subunit alpha, at the border of the 45 kbp inversion that distinguishes spinach and Oenothera plastid chromosomes. Both genes are transcribed in the same direction. Nucleotide sequence analysis reveals a single open reading frame encoding 318 amino acids of which 285 comprise the mature polypeptide and another 33 residues represent probably a N-terminal signal sequence. The putative pre-sequence is 2 residues shorter than those known from the spinach, wheat and pea protein. The deduced amino acid sequences of f cytochromes from the four plant species show over 80% conservation, maintaining the structural characteristics of the protein.     

Organization and nucleotide sequence of the broad bean chloroplast genes trnL-UAG,ndhF and two unidentified open reading frames

Summary We have determined the nucleotide sequence of a 6.9 kbp BamHI-Xbal fragment of broad bean chloroplasts. Part of this fragment (subfragment BglII-CIal) is known to contain three tRNA genes (trnL-CAA, trnlrUAA and trnF). We have now further identified a gene coding for the third tRNA^Leu isoacceptor (trnL-UAG) which is located close to trnF. The BamHI-Xbal fragment also contains the gene for subunit 5 of NADH dehydrogenase (ndhF) and two unidentified open reading frames (ORFx and ORF48). ORFx shares a high sequence homology with the long reading frames of tobacco (ORF1708), spinach (ORF2131), and liverwort (ORF2136), while ORF48 shares sequence homology with ORF69 of liverwort and ORF55 of tobacco.     

Transformation of Aspergillus flavus: construction of urate oxidase-deficient mutants by gene disruption

Summary A transformation procedure based on the complementation of a genetic defect was developed using a nitrate reductase-deficient mutant of Aspergillus flavus. The initial transformation efficiency was improved 40-fold by combining factors in a planned experimental program. Although low, this transformation rate was sufficient to obtain transformants in which the urate oxidase-encoding gene (uaZ) was disrupted in a gene replacement experiment. These new uaZ^- strains were unable to utilize uric acid as the unique nitrogen source and could be reversed directly to the wild-type phenotype in second order transformation experiments using a urate oxidase-expressing vector.     

Cloning of a gene associated with aflatoxin B1 biosynthesis in Aspergillus parasiticus

Summary A cosmid library was constructed by inserting genomic DNA isolated from a wild-type aflatoxin-producing strain of Aspergillus parasiticus (SU-1) into a cosmid vector containing an homologous nitrate reductase (niaD) gene as a selectable marker. One cosmid was isolated which complemented an aflatoxin-deficient, nitrate-nonutilizing mutant strain, A. parasiticus B62 (nor-1, niaD), to aflatoxin production. Deletion and complementation analyses showed that, a 1.7 kb BglII-SphI DNA fragment isolated form this cosmid was responsible for renewed aflatoxin production. Northern hybridization analyses revealed that the major RNA transcribed from this DNA fragment, was 1.4 kilonucleotides in size. Genetic complementation, proved to be a useful strategy for cloning a gene associated with aflatoxin biosynthesis in A. parasiticus.     

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae

Summary A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5-phosphate decarboxylase (pyrG) and the vector pA04-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular PA04-2 resulted in the appearance of Pyr⁺ transformants at a frequency of up to 20 per g of DNA, whereas with linear pA04-2 up to 200 transformants per g DNA were obtained. In 75 % of the Pyr⁺ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pA04-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding -galactosidase, and uidA, encoding -glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.