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M Penttilä

Publications and source records attributed to M Penttilä.

At least 19 recordsLinked to original sources
PubMed2004

The ire1 and ptc2 genes involved in the unfolded protein response pathway in the filamentous fungus Trichoderma reesei.

A signal transduction pathway called the unfolded protein response is activated when increased levels of misfolded proteins or incorrectly assembled subunits accumulate in the endoplasmic reticulum (ER). The expression of several genes for ER-resident foldases and chaperones, as well as genes encoding proteins that are involved in functions associated with the secretory process, are induced by this pathway. This paper describes the cloning and characterisation of genes for two components of the pathway, ire1 and ptc2, from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The data presented demonstrates that the T. reesei genes can complement Saccharomyces cerevisiae mutants that are deficient in the corresponding homologues. The T. reesei IREI protein has intrinsic kinase activity, as revealed by an in vitro autophosphorylation assay. Overexpression of ire1 in a T. reesei strain that expresses a foreign protein (laccase 1 from Phlebia radiata), results in up-regulation of the UPR pathway, as indicated by the increased expression levels of the known UPR target genes bip1 and pdi1. Splicing of the mRNA encoding the transcription factor HAC1 is also observed. Other genes encoding proteins from different parts of the secretory pathway also respond to ire1 overexpression.

Base Sequence↗
PubMed2004

The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and up-regulates its own transcription.

The unfolded protein response (UPR) involves a complex signalling pathway in which the transcription factor HACA plays a central role. Here we report the cloning and characterisation of the hacA gene and its product from Aspergillus niger. ER (endoplasmic reticulum) stress results in the splicing of an unconventional 20-nt intron from the A. niger hacA mRNA, and is associated with truncation of the 5'-end of the hacA mRNA by 230 nt. In this study the UPR was triggered by over expressing tissue plasminogen activator (t-PA), and by treatment of mycelia with dithiothreitol (DTT) or tunicamycin. Overexpression of the processed form of hacA not only led to the up-regulation of bipA, cypB and pdiA--mimicking the UPR--but also led to the up-regulation of the hacA gene itself. In vitro binding assays confirmed that the HACA protein binds to the promoters of genes encoding ER-localised chaperones and foldases, and to the promoter of the hacA gene itself. Finally, a GFP-HACA fusion was shown to localise in the nucleus.

Amino Acid Sequence↗
PubMed2002

Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei.

Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.

Base Sequence↗
PubMed2002

Construction of a flocculent Saccharomyces cerevisiae strain secreting high levels of Aspergillus niger beta-galactosidase.

A flocculent Saccharomyces cerevisiae strain secreting Aspergillus niger beta-galactosidase activity was constructed by transforming S. cerevisiae NCYC869-A3 strain with plasmid pVK1.1 harboring the A. niger beta-galactosidase gene, lacA, under the control of the ADH1 promoter and terminator. Compared to other recombinant S. cerevisiae strains, this recombinant yeast has higher levels of extracellular beta-galactosidase activity. In shake-flask cultures, the beta-galactosidase activity detected in the supernatant was 20 times higher than that obtained with previously constructed strains (Domingues et al. 2000a). In bioreactor culture, with cheese-whey permeate as substrate, a yield of 878.0 nkat/gsubstrate was obtained. The recombinant strain is an attractive alternative to other fungal beta-galactosidase production systems as the enzyme is produced in a rather pure form. Moreover, the use of flocculating yeast cells allows for enzyme production with high productivity in continuous fermentation systems with facilitated downstream processing.

Aspergillus niger↗
PubMed2001

Cloning and expression of a fungal L-arabinitol 4-dehydrogenase gene.

L-Arabinitol 4-dehydrogenase (EC ) was purified from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). It is an enzyme in the L-arabinose catabolic pathway of fungi catalyzing the reaction from L-arabinitol to L-xylulose. The amino acid sequence of peptide fragments was determined and used to identify the corresponding gene. We named the gene lad1. It is not constitutively expressed. In a Northern analysis we found it only after growth on L-arabinose. The gene was cloned and overexpressed in Saccharomyces cerevisiae, and the enzyme activity was confirmed in a cell extract. The enzyme consists of 377 amino acids and has a calculated molecular mass of 39,822 Da. It belongs to the family of zinc-binding dehydrogenases and has some amino acid sequence similarity to sorbitol dehydrogenases. It shows activity toward L-arabinitol, adonitol (ribitol), and xylitol with K(m) values of about 40 mM toward L-arabinitol and adonitol and about 180 mM toward xylitol. No activity was observed with D-sorbitol, D-arabinitol, and D-mannitol. NAD is the required cofactor with a K(m) of 180 microM. No activity was observed with NADP.

Amino Acid Sequence↗
PubMed2001

Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum.

Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls.

Amino Acid Sequence↗
PubMed2001

ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei.

A novel yeast-based method to isolate transcriptional activators was applied to clone regulators binding to the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei (Hypocrea jecorina). This led to the isolation of the cellulase activator ace2 encoding for a protein belonging to the class of zinc binuclear cluster proteins found exclusively in fungi. The DNA-binding domain of ACEII was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and ACEII was shown to bind in vitro to the 5'-GGCTAATAA site present in the cbh1 promoter. This site also contains the proposed binding sequence of the xylanase activator XlnR of Aspergillus niger. Mutation of the GGC triplet abolished ACEII binding. The function of ACEII was studied by analyzing the effects of ace2 deletion in the hypercellulolytic T. reesei strain ALKO2221. Deletion of the ace2 gene led to lowered induction kinetics of mRNAs encoding the major cellulases cellobiohydrolases I and II and endoglucanases I and II and to 30-70% reduced cellulase activity when the fungus was grown on medium containing Solka floc cellulose. The expression level of the gene encoding xylanase was also affected. ace2 deletion led to lowered xyn2 expression in cellulose-induced cultivation. Cellulase induction by sophorose was not affected by ace2 deletion.

Amino Acid Sequence↗
PubMed2001

Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis.

Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots were selected for analysis in order to collect information for future studies, to cover the whole pI range from 3 to 10, and to evaluate information from spots of different intensities. Mass mapping as a rapid, high-throughput method was in most cases sensitive enough for identification. LC/MS/MS was found to be more sensitive and to provide more accurate data, and was very useful when analyzing small amounts of sample. Even one sequence tag acquired by this method could be enough for unambiguous identification, and, in the present case, successfully identified a point mutation.

Amino Acid Sequence↗
PubMed2001

Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability.

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).

Aerobiosis↗
PubMed2001

Trichoderma reesei rho3 a homologue of yeast RH03 suppresses the growth defect of yeast sec15-1 mutation.

The Trichoderma reesei gene, rho3, encoding the functional homologue of the Saccharomyces cerevisiae small GTP-binding protein Rho3p was cloned as a suppressor of the secretion-deficient mutation sec15-1 in yeast. The encoded protein showed 61% amino acid identity to the Rho3 protein. Rescue of the growth defect of a RHO3 disruption strain by an expression vector carrying rho3 cDNA confirmed the functional homology with the S. cerevisiae RHO3 gene. In addition, overproduction of T. reesei RHOIII in this yeast strain appeared to improve the actin organization and chitin localization of the cells. Three putative mutant (rho3Gly20Val alleles of the T. reesei rho3 gene rho3 Thr25Asn, rho3Asp12Ala) were introduced into the wild-type yeast, in yeast with sec15 mutation and in yeast with Rho3p depletion. Cells expressing rho3Gly20Val displayed wild-type growth and those expressing rho3 Thr25Asn and rho3Asp126Ala had a loss-of-function phenotype.

Amino Acid Sequence↗
PubMed2001

The hydrophobins HFBI and HFBII from Trichoderma reesei showing efficient interactions with nonionic surfactants in aqueous two-phase systems.

Fungal hydrophobins are a group of surface active, self-assembling proteins. The filamentous fungus Trichoderma reesei produces two (class II) hydrophobins, HFBI and HFBII. We have studied how these water-soluble hydrophobins behave in two-phase systems using a series of nonionic surfactants with different characteristics. It was found that both hydrophobins, but especially HFBI, had a very high affinity for the surfactants. The highest partitioning coefficient, over 2500, was observed for HFBI with C(11)EO(2). Reducing the disulfides in the protein resulted in a complete loss of affinity for the surfactant, which demonstrates that the interaction is dependent on the disulfide-stabilized conformation. The hydrophobins could be efficiently extracted back from the surfactant phase by addition of alcohols such as isobutanol. Effects of the type of surfactant, temperature, pH, and ionic strength were investigated. The use of this method for purifying the proteins from crude fungal culture supernatants is demonstrated and implications of the protein-polymer interaction are discussed.

Chromatography, High Pressure Liquid↗
PubMed2001

Parameters influencing protein extraction for whole broths in detergent based aqueous two-phase systems.

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed.

Biomass↗
PubMed2001

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei.

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

Base Sequence↗
PubMed2001

Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei.

We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38 degrees C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.

Base Sequence↗
PubMed2000

The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism.

Many yeast species have growth rates on D-xylulose of 25-130% of those on glucose, but for Saccharomyces cerevisiae this ratio is only about 6%. The xylulokinase reaction has been proposed to be the rate-limiting step in the D-xylulose fermentation with S. cerevisiae. Over-expression of xylulokinase encoding XKS1 stimulated growth on D-xylulose in a S. cerevisiae strain to about 20% of the growth rate on glucose and deletion of the gene prevented growth on D-xylulose and D-xylulose metabolism. We have partially purified the xylulokinase and characterised its kinetic properties. It is reversible and will also accept D-ribulose as a substrate.

Fermentation↗
PubMed2000

Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei.

A genetic selection method was developed for the cloning of positive-acting transcriptional regulatory genes in Saccharomyces cerevisiae. The method was applied for the isolation of activators of Trichoderma reesei (Hypocrea jecorina) cellulase genes. Activator genes were isolated from a T. reesei expression cDNA library on the basis of the ability of their translation products to activate transcription from the full-length T. reesei cbh1 promoter coupled to the S. cerevisiae HIS3 gene and to support the growth of the yeast colonies in the absence of histidine. Among the clones obtained was the ace1 gene encoding a novel polypeptide, ACEI, that contains three zinc finger motifs of Cys(2)-His(2) type. Possible ACEI homologues were found among expressed sequence tags of Aspergillus and Neurospora. The ability of ACEI to bind to the cbh1 promoter was further confirmed in the yeast one-hybrid system. In vitro binding and gel mobility shift assays revealed several binding sites for the ACEI protein in the cbh1 promoter. Disruption of the ace1 gene in T. reesei resulted in retarded growth of the fungus on a cellulose-containing medium, on which cellulases are normally highly expressed.

Amino Acid Sequence↗
PubMed2000

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase.

A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.

Amino Acid Sequence↗