Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei

A cDNA encoding for the glucoamylase P enzyme (GAMP) of the fungus Hormoconis resinae was introduced into the cellulolytic filamentous fungus Trichoderma reesei under the control of the promoter of the major cellulase gene (cbh1) of Trichoderma. The transforming vector plasmid used was found to be integrated into the genome of T. reesei at various locations and in multiple copies. The size of the GAMP secreted by Trichoderma varied because of different glycosylation patterns. The best transformant strains secreted about 700 mg/l of active GAMP, which is 20-fold more than obtained with H. resinae.     

Presence,transcription and translation of cellobiohydrolase genes in several Trichoderma species

Eight different species of Trichoderma (T. virgatum, T. longibrachiatum, T. harzianum, T. pseudokoningii, T. polysporum, T. koningii, T. todica, T. saturnisporum), and three strains of T. reesei [QM 6a (wildtype), QM 9123 and QM 9414 (derived mutants)] were found to contain single copies of the cellobiohydrolase genes cbh1 and cbh2 in their genome. This was demonstrated by hybridization of the respective chromosomal DNAs with the corresponding gene fragments of T. reesei QM 9414. According to the relative position of cbh1 and cbh2 in Southern blots, T. harzianum, T. virgatum and T. saturnisporum were clearly distinguishable as unique species. Despite the presence of both cbh genes, these species did not form detectable cellobiohydrolase (CBH) I or II, or exhibit any cellulase activity. All other taxa were identical with respect to the genomic position of cbh1, formed two groups with respect to the position of cbh2, and produced varying amounts of CBH I and II. In all cases CBH I and II production correlated with the relative amount of cbh1- and cbh2-mRNA found. This was particularly true for the three strains of T. reesei, which secreted different amounts of CBH I and II, their efficiency to transcribe cbh1 and cbh2 having been increased as a result of mutation for higher cellulase production.Communicated by J.R. Schweyen     

Genomic organization of a cellulase gene family in Phanerochaete chrysosporium

Summary Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family.     

Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae

A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies. Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues. Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37–48%. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals

Trichoderma reesei was transformed to hygromycin B resistance using a novel vector, which contains the E. coli hygromycin B phosphotransferase gene (hph) fused between promoter and terminator elements of the homologous Trichoderma pkil (coding for pyruvate kinase) and cbh2 (coding for cellobiohydrolase II) genes, respectively. Transformation frequencies of over 1800–2500 transformants/g DNA were obtained, which is a 15–20-fold increase over that with pAN7-1, which contains hph between A. nidulans expression signals. Mitotically-stable transformants contained the hph gene and the regulatory sequences of the pkil promoter and the cbh2 terminator integrated into the genome. Evidence for preferentially ectopic integration is given.     

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 heterologous overexpression of <Emphasis Type="Italic">hsp23</Emphasis>, a small heat-shock protein gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis>, confers thermotolerance to <Emphasis Type="Italic">T. harzianum</Emphasis>

An EST showing high values of identity with genes coding for small heat shock proteins (sHSPs) was selected from an EST library collection of Trichoderma virens T59. The cDNA gene (hsp23) with a sequence size of 645 bp long was amplified by PCR. The expression of this gene was evaluated in cultures grown at temperatures ranging from 4 to 41°C. An increased level of expression was detected when the fungus was grown at extreme temperatures (4, 10 or 41°C). A high-expression level was also observed when the fungus was grown in 10% ethanol for 4 h. The hsp23 gene was present as a unique copy in the T. virens genome, and a homologous gene was also present in other five investigated Trichoderma species. Strain T. harzianum T34 was transformed with the hsp23 gene from T. virens T59 under the control of the pki (pyruvate kinase) promoter from T. reesei and the ble (phleomycin resistance) gene as selection marker. Statistically significant differences were detected between the strains T34 and two selected transformants in the biomass quantities obtained after heat shock treatment and in the colony diameters after incubation at 4°C for 2 months.     

Construction and characterization of a haploid strain of Saccharomyces cerevisiae that completely lacks all genomic CYH2 sequences

Summary A diploid strain of the yeast Saccharomyces cerevisiae has been constructed that has one copy of the ribosomal protein gene CYH2 completely deleted and replaced with the TRP1 gene using the method of Rothstein (1983). There are only small differences in growth rate and no detectable difference in steady state level of CYH2 mRNA between the diploid that is heterozygous for the CYH2 deletion and the parent diploid with two normal copies of this gene. This suggests that the diploid must partially compensate for the loss of one CYH2 gene. Tetrad dissection shows that haploid spores lacking the CYH2 gene cannot germinate. The lethality of this deletion can be rescued by a CYH2 cDNA on a low copy vector. Haploids which lack the genomic copy of the CYH2 gene, but contain a plasmid copy of the CYH2 cDNA are able to grow normally. These CYH2 deleted yeast haploids should be useful to analyze mutationally altered CYH2 genes and genes homologous to CYH2 from other organisms without interference from a genomic copy.     

Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene,XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant

Summary A P. stipitis cDNA library in gt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.     

Expression of tyrosinase in vegetative cultures of Neurospora crassa transformed with a metallothionein promoter/protyrosinase fusion gene

Summary Wild-type Neurospora crassa, strain Singapore, was transformed with a N. crassa metallothionein promoter/protyrosinase fusion gene. Transformants produced tyrosinase during vegetative growth, as determined by Western analyses and activity assays. This is in sharp contrast to wild-type strains, where this enzyme is only expressed in situations of starvation or sexual differentiation. Complete integration of a 400 bp metallothionein promoter-fragment leads to constitutive expression of protyrosinase, whereas a 3.6 kb promoter-fragment conferred copper inducibility on the reporter gene in four transformants. A transformant with high constitutive tyrosinase levels was able to produce melanin on complete medium agar plates supplemented with 1 mg/ml L-tyrosine.     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.     

Integrative and replicative genetic transformation of Aureobasidium pullulans

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1- (TEF1) adjacent to the 5 end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmB^R) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41 bp of homologous DNA has also been shown to transform A. pullulans to HmB^R. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmB^R transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7–8 kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8 kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmB^R A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4 kb) was unable to transform A. pullulans to HmB^R.     

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.     

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.     

Isolation and characterization of a Neurospora glucose-repressible gene

Summary Using differential hybridization, the cDNA copy of a Neurospora gene coding for an abundant glucose-repressible mRNA (grg-l) has been isolated. The cDNA was used to clone the genomic copy, and both were sequenced. The cDNA is nearly full length and contains putative translational start and termination codons. Conceptual translation indicates that grg-1 mRNA could direct the synthesis of a 7,000 molecular weight polypeptide. The genomic clone, contained in an 1,888 by PvuII fragment, encompasses the entire cDNA as well as 838 by of 5 and 369 bp of 3 flanking sequence. Comparison of the cDNA and genomic clones revealed the presence of two short introns in potential protein-coding sequences. grg-1 message levels were found to increase within minutes following the onset of glucose deprivation and rise 50 fold during the first 90 min of derepression.     

The cloning and sequencing of thealcB gene,coding for alcohol dehydrogenase II,inAspergillus nidulans

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in gt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5 region of the gene is discussed in relation to the regulation of the gene.     

Isolation and characterization of the 3-phosphoglycerate kinase gene (pgk) from the filamentous fungus Trichoderma reesei

Summary The 3-phosphoglycerate kinase gene (pgk) from Trichoderma reesei was isolated by hybridization with the corresponding Saccharomyces cerevisiae PGK gene. The 1,545 nt long nucleotide sequence of the cloned gene codes for a 416 amino acid protein. The coding sequence contains two introns of 219 and 75 nt, respectively, at positions identical to those corresponding genes from the other filamentous fungi Aspergillus nidulans and Penicillum chrysogenum. This gene codes for two mRNAs of about 1.65 kb and 1.85 kb. The PGK protein of Trichoderma shows extensive homology to the PGKs of other fungi A. nidulans (77%), P. chrysogenum (73%) and Saccharomyces cerevisiae (69%). However, the PGKs of the two other filamentous fungi, A. nidulans and P. chrysogenum, seem to be more closely related to each other than to the T. reesei enzyme.Abbreviations bp Base pair - nt nucleotide - kb kilobase