An analytical method for the determination of betaine (trimethylglycine) from hair.

Hair fibres have no active renewable protective mechanisms yet hair (wool and fur) provides an essential protective function. Therefore, it is possible that hair contains a number of internal components to maintain fibre and protein structure and function through a range of normal environmental conditions. Trimethylglycine (betaine) provides cellular and protein protection during times of high osmotic stress and low water concentrations. These conditions are very pertinent to everyday cosmetic procedures such as shampooing, bleach or perming. Therefore, we have conducted preliminary investigations to establish the presence of natural betaine in hair as a possible ingredient to provide protection and maintain protein function. Using an high performance liquid chromatography (HPLC) technique designed for the analysis of betaine in blood and urine, we have demonstrated, via simple hot water extraction, a measurable level of betaine in blood and human hair. Further, we have demonstrated, in principle, the possibility of adding betaine back to the fibre via cosmetic products such as shampoos.

Proteins associated with the cell envelope of Trichoderma reesei: a proteomic approach.

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.

Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei.

Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.

Overexpression of the Aspergillus niger pH 2.5 acid phosphatase gene in a heterologous host Trichoderma reesei.

An Aspergillus gene coding for a pH 2.5 acid phosphatase enzyme was successfully overexpressed in Trichoderma reesei under the strong main cellobiohydrolase I (cbh 1) promoter. The best transformants produced up to 240 times more of the acid phosphatase than the Aspergillus strain from which the phosphatase gene was originally isolated. The recombinant enzyme was effectively secreted into the culture medium both by its own and the cbh 1 secretion signal. The heterologous pH 2.5 acid phosphatase enzyme produced by the Trichoderma transformants was seen as four protein bands of about 55-66 kD resulting from variable glycosylation in Trichoderma. The activity of the recombinant enzyme was not affected. Enzyme preparations rich in both cellulose and phytate hydrolysing enzymes are of interest in the animal feed industry.

Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency.

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A + T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A + T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed.

Involvement of CIF1 (GGS1/TPS1) in osmotic stress response in Saccharomyces cerevisiae.

The transcriptional responses of the osmotically induced genes ALD2, CTT1, ENA1, GPD1, HSP12 and HSP104, were studied in Saccharomyces cerevisiae strains differing in CIF1 gene function following application of osmotic stress. The CIF1 gene (allelic to GGS1 and TPS1) encodes a subunit of the trehalose synthase complex that affects trehalose synthesis. Recent work has implicated this gene in various signalling events in the cell, including transcriptional response to heat-shock treatment. Because many genetic factors can influence S. cerevisiae osmoresponse, we have compared the expression of osmotically induced genes and glycerol production in isogenic strains differing only in functionality of CIF1, growing logarithmically on galactose medium. When cultures were exposed to 0.8 M NaCl or 1.5 M sorbitol the cif1 strain showed greatly reduced transcription of osmotically induced genes compared to the wild type. These treatments did not affect viability of the yeast strains. Treatment with 0.3 M NaCl produced no significant differences in transcription of these genes in CIF1 or cif1 strains. Treatment with 0.6 M sorbitol induced small but reproducible differences, with gene expression higher in the CIF1 strain compared to the cif1 mutant. When cultures were treated with 0.3 M NaCl or 0.6 M sorbitol for 1 h, glycerol production was similar for both strains, but after 3 h of the same treatment, total glycerol production was higher in the CIF1 strain. When cultures were treated with 0.8 M NaCl for 3 h, the wild type strain produced more glycerol than the mutant strain. Both strains produced similar amounts of glycerol following exposure to 1.5 M sorbitol for 3 h, although the wild type strain showed enhanced ability to retain glycerol inside the cell. The results are discussed in the context of the possible role that the CIF1 gene product has in response to osmotic stress.

Expression of Barley Endopeptidase B in Trichoderma reesei.

The gene for barley endopeptidase B (EPB) has been expressed in the filamentous fungus Trichoderma reesei from the cbh1 promoter. The EPB signal sequence allowed secretion of over 90% of the recombinant protein. Yields reached about 500 mg of immunoreactive protein per liter and exceeded values for any other protein derived from a higher eukaryotic organism produced in T. reesei.

Mycoparasitic species of Trichoderma produce lectins.

Culture filtrates and mycelial extracts of two mycoparasitic Trichoderma species were tested for the presence of lectins, by haemagglutination with human and marsupial erythrocytes. In Trichoderma viride, haemagglutinating activity was present in both mycelial extracts and culture filtrate. While secreted lectins were only detected after 6 days of growth, the presence of mycelium-associated lectins was first noted in 3-day-old cultures. Agglutinating activity was also demonstrated in the mycelium of 6-, 9- and 13-day-old cultures of Trichoderma harzianum. In this species, however, lectins were not secreted. In all instances, haemagglutination was inhibited by N-acetylgalactosamine and related sugars. This is the first report on the occurrence of lectins in Trichoderma spp.

Evidence that the Saccharomyces cerevisiae CIF1 (GGS1/TPS1) gene modulates heat shock response positively.

The CIF1 gene (also called GGS1/TPS1) encodes a protein of the trehalose synthase complex that affects trehalose accumulation and general glucose sensing by Saccharomyces cerevisiae cells. There is considerable debate as to whether CIF1-dependent trehalose accumulation is a determinant in heat shock-acquired thermotolerance. Thermosensitivity of cif1 mutants could alternatively, or also, be related to gene expression-signalling defects in such strains. Because many signal-dependent factors are involved in stress protection and repair in yeast, we have compared the expression of various stress response and heat shock genes in 'isogenic' CIF1 and cif1 strains growing exponentially in galactose medium. Transcription of CTT1, CIF1, HSP26, HSP82, HSP104, SSA4 and UB14 was notably lower in the cif1 mutant following heat shock. Moreover, a single copy of chromosomally integrated HSP104-lacZ fusion gave up to 5.5-fold more heat shock induction in the CIF1 strain compared to the cif1 mutant. We conclude that reduced heat shock-acquired thermotolerance in cif1-deletion mutants growing exponentially on galactose is more likely to result from a general reduction in expression of stress response and heat shock genes, than simply or solely through deficiency of trehalose accumulation. The possible role of CIF1 in modulating stress response is discussed.

On the safety of Trichoderma reesei.

Trichoderma reesei has a long history of safe use in industrial-scale enzyme production. Applications of cellulases and xylanases produced by this fungus are found in food, animal feed, pharmaceutical, textile and pulp and paper industries. T. reesei is non-pathogenic for man and it has been shown not to produce fungal toxins or antibiotics under conditions used for enzyme production. During recent years genetic engineering techniques have also been used to improve the industrial production strains of T. reesei and, in addition, considerable experience of safe use of recombinant T. reesei strains in industrial scale has accumulated. Thus, T. reesei can be generally considered not only a safe production organism of its natural enzymes but also a safe host for other harmless gene products.

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes.

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.

The cloning and sequencing of the genes encoding phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger var. awamori.

The genes encoding phytase (EC 3.1.3.8) and pH 2.5-optimum acid phosphatase (EC 3.1.3.2) have been cloned and sequenced from Aspergillus niger var. awamori. The translated nucleotide sequences yielded polypeptides of 467 and 479 amino acids (aa) for phytase and acid phosphatase, respectively. The genes were isolated using oligodeoxyribonucleotide probes based on the aa sequences of the purified proteins. Recombinant A. niger var. awamori strains carrying additional copies of the gene sequences demonstrated elevated enzyme activities.

Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei.

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.

Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene.

The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner. We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases. The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I. The best conserved region is found at the C terminus and shows about 70% homology. The data suggest that the two enzymes have arisen from a common ancestor by gene duplication. Despite this, the intron positions have not been conserved in these genes which both contain two short introns. The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I. Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.