45 results on '"Penttila M"'
Search Results
2. A consensus yeast metabolic network obtained from a community approach to systems biology
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Herrgard, M.J., Swainston, N., Dobson, P., Dunn, W.B., Arga, K.Y., Arvas, M., Bluthgen, N., Borger, S., Costenoble, E.R., Heinemann, M., Hucka, M., Li, P., Liebermeister, W., Mo, M.L., Oliveira, A.P., Petranovic, D., Pettifer, S., Simeonides, E., Smallbone, K., Spasi, I., Weichart, D., Brent, R., Broomhead, D.S., Westerhoff, H.V., Kirdar, B., Penttila, M., Klipp, E., Paton, N., Palsson, B.O., Sauer, U., Oliver, S.G., Mendes, P., Nielsen, J., Kell, D.B., and Molecular Cell Physiology
- Subjects
ComputingMethodologies_PATTERNRECOGNITION - Abstract
Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms. © 2008 Nature Publishing Group.
- Published
- 2008
3. How to create economic incentives in occupational safety and health: A practical guide
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Elsler, D., Heyer, A., Kuhl, K., Eeckelaert, L., Chatzigiannoglou, C., Maier, A., Cuervo, M., Frusteri, L., Charalambous, A., Molinaro, R., Steiger, O., Brummer, E., Penttila, M., Petrisic, N., Vanadzins, I., Benedetti, F., Karadeniz, O., Treutlein, D., Tompa, E., Kohstall, T., Nicot, A.M., Tynkkynen, M., Kruger, H., Wittig, K., Stadnik, M., Jones, C., Epegui, H., Lunde-Jensen, P., Ottati, M., Pecillo-Pacek, M., Greef, M.de, Mierlo, M. van, Maya Rubio, M.I., Kahr, J., and Sapir, M.
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Organisation ,WH - Work & Health ,Work and Employment ,Workplace ,Healthy Living ,BSS - Behavioural and Societal Sciences ,ComputingMilieux_MISCELLANEOUS - Abstract
This Guide on Economic Incentives Schemes is intended to serve as a practical and user-friendly guide to help incentive providers to create or optimise their own economic incentive schemes. Incentives schemes should not only reward past results of good OSH management (such as low accident numbers), but should also reward specific prevention efforts that aim to reduce future accidents and ill-health. Therefore the expert group suggested the development of compilations of innovative and evidence-based preventive solutions, starting with the three sectors construction, health care and HORECA.
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- 2011
4. Efficacy of desloratadine in persistent allergic rhinitis - a GA²LEN study
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Bousquet, J, Bachert, C, Canonica, Gw, Mullol, J, Van Cauwenberge, P, Jensen, Cb, Fokkens, Wj, Ring, J, Keith, P, Gopalan, G, Lorber, R, Zuberbier, T, 2 Study Group: Antepara I, Accept, Bálint, B, Barbosa, M, Bindslev Jensen, C, Blanco, C, Bousquet, Pj, Campos, Á, Camps, Pj, Castel Branco, G, Cheema, A, Chieira, C, Chivato, T, Cirillo, A, Daikhes, N, del Carpio, J, di Lorenzo, G, Erisen, L, Farouz, Jc, Fokkens, W, Gambarelli, J, Gering, R, Goryachkina, L, Guilherme, A, Hébert, J, Ilyina, N, Jääskeläinen, T, Joki Erkkila VP, Kalogermitros, D, Karci, B, Kasche, D, Klimek, L, Knight, A, Koistinen, T, Külahi, I, Lebeaupin, B, Lindskog, T, Lopatin, A, Magyar, P, Malek, T, Manconi, P, Meischner, K, Merk, H, Morete, A, Moscato, G, Mösges, R, Önerci, M, Ortolan, D, Pasch, N, Pastorello, Ea, Penttila, M, Pucci, S, Quercia, O, Rantaiso, E, Rinne, J, Roger, A, Rolla, Giovanni, Romano, A, Romberg, K, Rouanet Bousquet, L, Ryazantsev, S, Salo, S, Sancinena, O, Sanquer, F, Sauvan Pistof, C, Sidiropoulos, I, Sidorenko, I, Sussman, G, Szalai, Z, Szilasi, M, Tilling, B, Tosoni, C, Troise, C, Tutkun, A, Vacca, A, Vinge, I, Vourdas, D, Wessel, F, Yang, W, and Zuberbier, T.
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Adult ,Male ,Histamine H1 Antagonists, Non-Sedating ,allergic rhinitis ,Rhinitis, Allergic, Perennial ,desloratadine ,Rhinitis, Allergic, Seasonal ,Loratadine ,Middle Aged ,Severity of Illness Index ,anti histamine drugs ,Disease Progression ,Quality of Life ,Humans ,Female ,Follow-Up Studies ,Pain Measurement - Abstract
The ARIA (Allergic Rhinitis and its Impact on Asthma) guidelines proposed a classification for allergic rhinitis based on the duration of symptoms (intermittent or persistent) rather than on the time of allergen exposure (seasonal or perennial). There had been no placebo-controlled, randomized, clinical trial of desloratadine (DL) in patients with persistent allergic rhinitis to date.To assess the efficacy and safety of DL in patients with persistent allergic rhinitis based on the ARIA classification.Patients 12 years of age and older with persistent allergic rhinitis were assessed over 85 days of treatment with DL 5 mg once daily (n = 360) or placebo (n = 356). The primary endpoint was the AM/PM reflective total 5-symptom score (T5SS) averaged over days 1-29. Secondary endpoints included AM/PM instantaneous T5SS and individual symptoms, therapeutic response, symptom severity assessed by a visual analogue scale and quality of life.The mean reduction in AM/PM reflective T5SS was significantly greater with DL than placebo over days 1-29 (-3.76 vs. -2.87, p0.001) and on each individual day (p0.05). The mean AM instantaneous T5SS was significantly reduced with DL compared with placebo as early as day 2 (-1.90 vs. -1.46; p0.001). The therapeutic response and improvement in quality of life were significantly greater with DL than placebo (p0.001 for each). The frequency of treatment-related adverse events was low and similar between DL (10.0%) and placebo (8.4%).This study showed DL to be effective and safe in the treatment of persistent allergic rhinitis.
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- 2009
5. Influence of Growth Temperature on the Production of Antibody Fab Fragments in Different Microbes: A Host Comparative Analysis
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Dragosits, M, Frascotti, G, Bernard Granger, L, Vazquez, F, Giuliani, M, Baumann, K, Rodriguez Carmona, E, Tokkanen, J, Parrilli, E, Wiebe, M, Kunert, R, Maurer, M, Gasser, B, Sauer, M, Branduardi, P, Pakula, T, Saloheimo, M, Penttila, M, Ferrer, P, Tutino, M, Villaverde, A, Porro, D, Mattanovich, D, Mattanovich, D., FRASCOTTI, GIANNI, BRANDUARDI, PAOLA, PORRO, DANILO, Dragosits, M, Frascotti, G, Bernard Granger, L, Vazquez, F, Giuliani, M, Baumann, K, Rodriguez Carmona, E, Tokkanen, J, Parrilli, E, Wiebe, M, Kunert, R, Maurer, M, Gasser, B, Sauer, M, Branduardi, P, Pakula, T, Saloheimo, M, Penttila, M, Ferrer, P, Tutino, M, Villaverde, A, Porro, D, Mattanovich, D, Mattanovich, D., FRASCOTTI, GIANNI, BRANDUARDI, PAOLA, and PORRO, DANILO
- Abstract
Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes. © 2010 American Institute of Chemical Engineers.
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- 2011
6. Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains
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Canelas, A.B., Harrison, N., Fazio, A., Zhang, J.Z., Pitkanen, J-P., van den Brink, J., Bakker, B.M., Bogner, L., Bouwman, J., Castrillo, J.I., Cankorur, A., Chumnanpuen, P., Daran-Lapujade, P., Dikicioglu, D., van Eunen, K., Ewald, J.C., Heijnen, J.J., Kirdar, B., Mattila, I., Mensonides, F.I.C., Niebel, A., Penttila, M., Pronk, J.T., Reuss, M., Salusjarvi, L., Sauer, U., Sherman, D., Siemann-Herzberg, M., Westerhoff, H., de Winde, J., Petranovic, D., Oliver, S.G., Workman, C., Zamboni, N., Nielsen, J., Canelas, A.B., Harrison, N., Fazio, A., Zhang, J.Z., Pitkanen, J-P., van den Brink, J., Bakker, B.M., Bogner, L., Bouwman, J., Castrillo, J.I., Cankorur, A., Chumnanpuen, P., Daran-Lapujade, P., Dikicioglu, D., van Eunen, K., Ewald, J.C., Heijnen, J.J., Kirdar, B., Mattila, I., Mensonides, F.I.C., Niebel, A., Penttila, M., Pronk, J.T., Reuss, M., Salusjarvi, L., Sauer, U., Sherman, D., Siemann-Herzberg, M., Westerhoff, H., de Winde, J., Petranovic, D., Oliver, S.G., Workman, C., Zamboni, N., and Nielsen, J.
- Abstract
he field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism., he field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism.
- Published
- 2010
7. Characterization of paracetamol agglomerates by image analysis and strength measurement
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Alander, E. M., Uusi-Penttila, M. S., Rasmuson, Åke C., Alander, E. M., Uusi-Penttila, M. S., and Rasmuson, Åke C.
- Abstract
Paracetamol is crystallized in different solvents and techniques are developed and used to characterize the product. The product particles from three different solvent compositions: ethylene glycol, acetone and an acetone-water mixture (30-70 wt.%) have been examined. Product properties visually observed are quantified by image analysis and evaluation of measured image descriptors with Principal Component Analysis (PCA). The agglomerate strength has been determined by crushing single agglomerates. Depending on the solvent, the content of single crystals and agglomerates differ. Agglomerates differ by the number and size of crystals grown together, as well as by the strength., QC 20100525
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- 2003
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8. Experimental study for agglomeration behaviour of paracetamol in acetone-toluene-water systems
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Uus-Penttila, M. S., Rasmuson, Åke C., Uus-Penttila, M. S., and Rasmuson, Åke C.
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Agglomeration behaviour of paracetamol was studied in acetone-toluene-water systems. The aim was to experimentally determine how the solvent composition influences the agglomeration tendency of paracetamol. It was found that for the chosen solvent system there is one main region where paracetamol agglomerates: the region with large amounts of acetone (>65 wt%) and very small amounts of water (<4 wt%). The same behaviour can be observed both within the one-phase region and within the two-phase region. The experimental agglomeration results were compared with molecular simulations from literature. Both methods indicate that it is more likely for paracetamol crystals to agglomerate in organic systems than in aqueous systems., QC 20100525
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- 2003
9. Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)phosphate aqueous two-phase system
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Collen, A., Penttila, M., Stalbrand, H., Tjerneld, F., Veide, Andres, Collen, A., Penttila, M., Stalbrand, H., Tjerneld, F., and Veide, Andres
- Abstract
Endoglucanases (EGI) (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)(2), (WP)(4) or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)(4) extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)(4) fused to the catalytic module and a short sequence of the linker [EGI(core-P5)(WP)(4)] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)(4) tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran syst, QC 20100525
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- 2002
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10. Optimization of cDNA-AFLP experiments using genomic sequence data
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Kivioja, T., primary, Arvas, M., additional, Saloheimo, M., additional, Penttila, M., additional, and Ukkonen, E., additional
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- 2005
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11. Production of ethanol from -arabinose by containing a fungal -arabinose pathway
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RICHARD, P, primary, VERHO, R, additional, PUTKONEN, M, additional, LONDESBOROUGH, J, additional, and PENTTILA, M, additional
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- 2003
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12. The 3-D structure of a Trichoderma reesei b-mannanase from glycoside hydrolase family 5
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Sabini, E., primary, Schubert, H., additional, Murshudov, G., additional, Wilson, K.S., additional, Siika-Aho, M., additional, and Penttila, M., additional
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- 2000
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13. Transcrition of mannose metabolism genes relates to protein secretion in Trichoderma reesei
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Kruszewska, J. S., primary, Saloheimo, M., additional, Penttila, M., additional, and Palamarczyk, G., additional
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- 2000
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14. Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin
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Williams, M. G., primary, Wilsher, J., additional, Nugent, P., additional, Mills, A., additional, Dhanaraj, V., additional, Fabry, M., additional, Sedlacek, J., additional, Uusitalo, J. M., additional, Penttila, M. E., additional, Pitts, J. E., additional, and Blundell, T. L., additional
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- 1997
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15. Enhanced Expression of Endochitinase in Trichoderma harzianum with the cbh1 Promoter of Trichoderma reesei
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Margolles-Clark, E, primary, Harman, G E, additional, and Penttila, M, additional
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- 1996
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16. Production of functional IgM Fab fragments by
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EDQVIST, J, primary, KERANEN, S, additional, PENTTILA, M, additional, STRROABY, K, additional, and KNOWLES, J, additional
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- 1991
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17. The comparative efficacy and safety of clarithromycin and amoxycillin in the treatment of outpatients with acute maxillary sinusitis
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Karma, P., primary, Pukander, J., additional, Penttila, M., additional, Ylikoski, J., additional, Savolainen, S., additional, Olen, L., additional, Melen, I., additional, and Loth, S., additional
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- 1991
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18. Agglomeration of Paracetamol during Crystallization in Pure and Mixed Solvents
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Alander, E. M., Uusi-Penttila, M. S., and Rasmuson, A. C.
- Abstract
The agglomeration of paracetamol during crystallization has been investigated. It is shown that the agglomeration behavior depends on the solvent composition. The following solvent systems were used in isothermal desupersaturation experiments: five different acetone−toluene−water mixtures and the pure solvents acetone, 2-propanol, acetic acid, and ethylene glycol. Sieving, image analysis processed by principal component analysis, and agglomerate strength measurements were used to characterize the product particles. Mixtures with a high concentration of acetone were found to produce a highly agglomerated product with strong agglomerates. In contrast, products from crystallization in ethylene glycol, 2-propanol, acetic acid, and acetone−toluene−water mixtures having a high concentration of water contained not only agglomerates but also a significant fraction of single crystals. Furthermore, the agglomerates formed in these solvents were much weaker than those produced in mixtures with a high content of acetone. The results were correlated with the polarity and the viscosity of the solvents.
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- 2004
19. The Hydrophobins HFBI and HFBII from Trichoderma reesei Showing Efficient Interactions with Nonionic Surfactants in Aqueous Two-Phase Systems
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Linder, M., Selber, K., Nakari-Setala, T., Qiao, M., Kula, M.-R., and Penttila, M.
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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 EO2 . 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.- Published
- 2001
20. Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-@a-d-mannosidase
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Maras, M., Callewaert, N., Piens, K., Claeyssens, M., Martinet, W., Dewaele, S., Contreras, H., Dewerte, I., Penttila, M., and Contreras, R.
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- 2000
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21. In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei
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Maras, M., Bruyn, A. De, Vervecken, W., Uusitalo, J., Penttila, M., Busson, R., Herdewijn, P., and Contreras, R.
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- 1999
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22. Liquid−Liquid Equilibria of Selected Dibasic Ester + Water + Solvent Ternary Systems
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Uusi-Penttila, M., Richards, R. J., Blowers, P., Torgerson, B. A., and Berglund, K. A.
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Experimental liquid−liquid equilibria for various dibasic ester + solvent + water systems were obtained at 297 K. These systems are suggested as possible substitutes in applications where chlorocarbons and aromatic hydrocarbons are used. The dibasic esters can also be used as novel solvents in separation techniques.
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- 1996
23. Applications of the molecular biology of Trichoderma reesei
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Knowles, J., Penttila, M., Harkki, A., Helena Nevalainen, Teeri, T. T., Saloheimo, M., and Uusitalo, J.
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- 1989
24. Spectroscopically Determined Dielectric Constants for Various Esters
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Uusi-Penttila, M. S., Richards, R. J., Torgerson, B. A., and Berglund, K. A.
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Polarity of a solvent can be defined by the Onsager function or the Debye function, which both are functions of the static dielectric constant. Polarity has also been shown to be related to the solvatochromic shifts of the absorption and fluorescence spectra. In this study, the emission maxima of the probe molecule Nile Red were taken in different solvents of known dielectric constants, and relationships between the emission maxima and the Onsager and Debye functions were established. These relationships were used to estimate dielectric constants for various environmentally benign esters.
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- 1997
25. Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates
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Häkkinen Mari, Arvas Mikko, Oja Merja, Aro Nina, Penttilä Merja, Saloheimo Markku, and Pakula Tiina M
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Carbohydrate active enzymes ,Cellulase ,Hemicellulase ,Lignocellulose ,Transcriptome ,Transcriptional profiling ,Gene regulation ,Wheat ,Spruce ,Bagasse ,Biorefinery ,Microbiology ,QR1-502 - Abstract
Abstract Background Trichoderma reesei is a soft rot Ascomycota fungus utilised for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. About 30 carbohydrate active enzymes (CAZymes) of T. reesei have been biochemically characterised. Genome sequencing has revealed a large number of novel candidates for CAZymes, thus increasing the potential for identification of enzymes with novel activities and properties. Plenty of data exists on the carbon source dependent regulation of the characterised hydrolytic genes. However, information on the expression of the novel CAZyme genes, especially on complex biomass material, is very limited. Results In this study, the CAZyme gene content of the T. reesei genome was updated and the annotations of the genes refined using both computational and manual approaches. Phylogenetic analysis was done to assist the annotation and to identify functionally diversified CAZymes. The analyses identified 201 glycoside hydrolase genes, 22 carbohydrate esterase genes and five polysaccharide lyase genes. Updated or novel functional predictions were assigned to 44 genes, and the phylogenetic analysis indicated further functional diversification within enzyme families or groups of enzymes. GH3 β-glucosidases, GH27 α-galactosidases and GH18 chitinases were especially functionally diverse. The expression of the lignocellulose degrading enzyme system of T. reesei was studied by cultivating the fungus in the presence of different inducing substrates and by subjecting the cultures to transcriptional profiling. The substrates included both defined and complex lignocellulose related materials, such as pretreated bagasse, wheat straw, spruce, xylan, Avicel cellulose and sophorose. The analysis revealed co-regulated groups of CAZyme genes, such as genes induced in all the conditions studied and also genes induced preferentially by a certain set of substrates. Conclusions In this study, the CAZyme content of the T. reesei genome was updated, the discrepancies between the different genome versions and published literature were removed and the annotation of many of the genes was refined. Expression analysis of the genes gave information on the enzyme activities potentially induced by the presence of the different substrates. Comparison of the expression profiles of the CAZyme genes under the different conditions identified co-regulated groups of genes, suggesting common regulatory mechanisms for the gene groups.
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- 2012
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26. Lipid production in batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon carbohydrates
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Wiebe Marilyn G, Koivuranta Kari, Penttilä Merja, and Ruohonen Laura
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Lipid ,Rhodosporidium toruloides ,Bio- and renewable diesel ,Fed-batch ,High cell density ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Microbial lipids are a potential source of bio- or renewable diesel and the red yeast Rhodosporidium toruloides is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates. Methods R. toruloides was grown in batch and fed-batch cultures in 0.5 L bioreactors at pH 4 in chemically defined, nitrogen restricted (C/N 40 to 100) media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. Lipid was extracted from the biomass using chloroform-methanol, measured gravimetrically and analysed by GC. Results Lipid production was most efficient with glucose (up to 25 g lipid L−1, 48 to 75% lipid in the biomass, at up to 0.21 g lipid L−1 h−1) as the sole carbon source, but high lipid concentrations were also produced from xylose (36 to 45% lipid in biomass). Lipid production was low (15–19% lipid in biomass) with arabinose as sole carbon source and was lower than expected (30% lipid in biomass) when glucose, xylose and arabinose were provided simultaneously. The presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells. High cell densities were obtained in both batch (37 g L−1, with 49% lipid in the biomass) and fed-batch (35 to 47 g L−1, with 50 to 75% lipid in the biomass) cultures. The highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed. However, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply. Conclusions Lipid production in R. toruloides was lower from arabinose and mixed carbohydrates than from glucose or xylose. Although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. Constant feeding was better than intermittent feeding. The feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of C5 sugars did.
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- 2012
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27. Correlation of gene expression and protein production rate - a system wide study
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Arvas Mikko, Pakula Tiina, Smit Bart, Rautio Jari, Koivistoinen Heini, Jouhten Paula, Lindfors Erno, Wiebe Marilyn, Penttilä Merja, and Saloheimo Markku
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).
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- 2011
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28. High level secretion of cellobiohydrolases by Saccharomyces cerevisiae
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Ahlgren Simon, Thorngren Naomi, la Grange Daniël C, Siika-aho Matti, Voutilainen Sanni P, Koivula Anu, Froehlich Allan, Wiswall Erin, McBride John, Brevnova Elena, den Haan Riaan, Ilmén Marja, Mellon Mark, Deleault Kristen, Rajgarhia Vineet, van Zyl Willem H, and Penttilä Merja
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biofuels ,cellulolytic yeast ,UPR ,Fuel ,TP315-360 ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. Results We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. Conclusions Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.
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- 2011
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29. The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30
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Pakula Tiina, Limón M Carmen, Saloheimo Markku, and Penttilä Merja
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Microbiology ,QR1-502 - Abstract
Abstract Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1.
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- 2011
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30. Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) using filamentous fungi
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Wiebe Marilyn G, Mojzita Dominik, Hilditch Satu, Ruohonen Laura, and Penttilä Merja
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Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-threo-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion. Results Keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) accumulated in the culture supernatant when Trichoderma reesei Δlga1 and Aspergillus niger ΔgaaC were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate l-1 was produced from 20 g D-galacturonate l-1 and A. niger ΔgaaC produced 15.0 g keto-deoxy-L-galactonate l-1 from 20 g polygalacturonate l-1, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate [g D-galacturonate consumed]-1. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g l-1 intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in A. niger ΔgaaC, but decreased in T. reesei. Conclusions Bioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both A. niger ΔgaaC and T. reesei Δlga1, although production (titre, volumetric and specific rates) was better with A. niger than T. reesei. A. niger was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto-deoxy-L-galactonate accumulated intracellularly, concentrations above ~12 g l-1 were exported to the culture supernatant. Lysis may have contributed to the release of keto-deoxy-L-galactonate from T. reesei mycelia.
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- 2010
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31. Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties
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Penttilä Merja, Oja Merja, Pakula Tiina, Arvas Mikko, Vitikainen Marika, and Saloheimo Markku
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Trichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general. Results We carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30) using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor) on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production. Conclusions aCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.
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- 2010
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32. Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae
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Wiebe Marilyn G, Pitkänen Juha-Pekka, Toivari Mervi, Rintala Eija, Ruohonen Laura, and Penttilä Merja
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background The industrially important yeast Saccharomyces cerevisiae is able to grow both in the presence and absence of oxygen. However, the regulation of its metabolism in conditions of intermediate oxygen availability is not well characterised. We assessed the effect of oxygen provision on the transcriptome and proteome of S. cerevisiae in glucose-limited chemostat cultivations in anaerobic and aerobic conditions, and with three intermediate (0.5, 1.0 and 2.8% oxygen) levels of oxygen in the feed gas. Results The main differences in the transcriptome were observed in the comparison of fully aerobic, intermediate oxygen and anaerobic conditions, while the transcriptome was generally unchanged in conditions receiving different intermediate levels (0.5, 1.0 or 2.8% O2) of oxygen in the feed gas. Comparison of the transcriptome and proteome data suggested post-transcriptional regulation was important, especially in 0.5% oxygen. In the conditions of intermediate oxygen, the genes encoding enzymes of the respiratory pathway were more highly expressed than in either aerobic or anaerobic conditions. A similar trend was also seen in the proteome and in enzyme activities of the TCA cycle. Further, genes encoding proteins of the mitochondrial translation machinery were present at higher levels in all oxygen-limited and anaerobic conditions, compared to fully aerobic conditions. Conclusion Global upregulation of genes encoding components of the respiratory pathway under conditions of intermediate oxygen suggested a regulatory mechanism to control these genes as a response to the need of more efficient energy production. Further, cells grown in three different intermediate oxygen levels were highly similar at the level of transcription, while they differed at the proteome level, suggesting post-transcriptional mechanisms leading to distinct physiological modes of respiro-fermentative metabolism.
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- 2009
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33. Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae
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Penttilä Merja, Pitkänen Juha-Pekka, Soliymani Rabah, Kankainen Matti, Salusjärvi Laura, and Ruohonen Laura
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Microbiology ,QR1-502 - Abstract
Abstract Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.
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- 2008
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34. Transcription of hexose transporters of Saccharomyces cerevisiae is affected by change in oxygen provision
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Ruohonen Laura, Tamminen Anu, Wiebe Marilyn G, Rintala Eija, and Penttilä Merja
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Microbiology ,QR1-502 - Abstract
Abstract Background The gene family of hexose transporters in Saccharomyces cerevisiae consists of 20 members; 18 genes encoding transporters (HXT1-HXT17, GAL2) and two genes encoding sensors (SNF3, RGT2). The effect of oxygen provision on the expression of these genes was studied in glucose-limited chemostat cultivations (D = 0.10 h-1, pH 5, 30°C). Transcript levels were measured from cells grown in five steady state oxygen levels (0, 0.5, 1, 2.8 and 20.9% O2), and from cells under conditions in which oxygen was introduced to anaerobic cultures or removed from cultures receiving oxygen. Results The expression pattern of the HXT gene family was distinct in cells grown under aerobic, hypoxic and anaerobic conditions. The transcription of HXT2, HXT4 and HXT5 was low when the oxygen concentration in the cultures was low, both under steady state and non-steady state conditions, whereas the expression of HXT6, HXT13 and HXT15/16 was higher in hypoxic than in fully aerobic or anaerobic conditions. None of the HXT genes showed higher transcript levels in strictly anaerobic conditions. Expression of HXT9, HXT14 and GAL2 was not detected under the culture conditions studied. Conclusion When oxygen becomes limiting in a glucose-limited chemostat cultivation, the glucose uptake rate per cell increases. However, the expression of none of the hexose transporter encoding genes was increased in anaerobic conditions. It thus seems that the decrease in the moderately low affinity uptake and consequently the relative increase of high affinity uptake may itself allow the higher specific glucose consumption rate to occur in anaerobic compared to aerobic conditions.
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- 2008
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35. Comparison of protein coding gene contents of the fungal phyla Pezizomycotina and Saccharomycotina
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Ussery David, Saloheimo Markku, Mitchell Alex, Kivioja Teemu, Arvas Mikko, Penttila Merja, and Oliver Stephen
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Several dozen fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. In addition comparative genomics has been used to analyse specific sub groups of fungi. Notably, analysis of the phylum Saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. However, little has been done to gain a comprehensive comparative view to the fungal kingdom. We have carried out a computational genome wide comparison of protein coding gene content of Saccharomycotina and Pezizomycotina, which include industrially important yeasts and filamentous fungi, respectively. Results Our analysis shows that based on genome redundancy, the traditional model organisms Saccharomyces cerevisiae and Neurospora crassa are exceptional among fungi. This can be explained by the recent genome duplication in S. cerevisiae and the repeat induced point mutation mechanism in N. crassa. Interestingly in Pezizomycotina a subset of protein families related to plant biomass degradation and secondary metabolism are the only ones showing signs of recent expansion. In addition, Pezizomycotina have a wealth of phylum specific poorly characterised genes with a wide variety of predicted functions. These genes are well conserved in Pezizomycotina, but show no signs of recent expansion. The genes found in all fungi except Saccharomycotina are slightly better characterised and predicted to encode mainly enzymes. The genes specific to Saccharomycotina are enriched in transcription and mitochondrion related functions. Especially mitochondrial ribosomal proteins seem to have diverged from those of Pezizomycotina. In addition, we highlight several individual gene families with interesting phylogenetic distributions. Conclusion Our analysis predicts that all Pezizomycotina unlike Saccharomycotina can potentially produce a wide variety of secondary metabolites and secreted enzymes and that the responsible gene families are likely to evolve fast. Both types of fungal products can be of commercial value, or on the other hand cause harm to humans. In addition, a great number of novel predicted and known enzymes are found from all fungi except Saccharomycotina. Therefore further studies and exploitation of fungal metabolism appears very promising.
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- 2007
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36. Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions
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Bhattacharyya Anamitra, Rautio Jari, Sauer Michael, Maurer Michael, Gasser Brigitte, Saloheimo Markku, Penttilä Merja, and Mattanovich Diethard
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. Results Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. Conclusion Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.
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- 2007
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37. Physiological evaluation of the filamentous fungus Trichoderma reesei in production processes by marker gene expression analysis
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Penttilä Merja, Söderlund Hans, Kivioja Teemu, Bailey Michael, Rautio Jari J, and Saloheimo Markku
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Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Biologically relevant molecular markers can be used in evaluation of the physiological state of an organism in biotechnical processes. We monitored at high frequency the expression of 34 marker genes in batch, fed-batch and continuous cultures of the filamentous fungus Trichoderma reesei by the transcriptional analysis method TRAC (TRanscript analysis with the aid of Affinity Capture). Expression of specific genes was normalised either with respect to biomass or to overall polyA RNA concentration. Expressional variation of the genes involved in various process relevant cellular functions, such as protein production, growth and stress responses, was related to process parameters such as specific growth and production rates and substrate and dissolved oxygen concentrations. Results Gene expression of secreted cellulases and recombinant Melanocarpus albomyces laccase predicted the trends in the corresponding extracellular enzyme production rates and was highest in a narrow "physiological window" in the specific growth rate (μ) range of 0.03 – 0.05 h-1. Expression of ribosomal protein mRNAs was consistent with the changes in μ. Nine starvation-related genes were found as potential markers for detection of insufficient substrate feed for maintaining optimal protein production. For two genes induced in anaerobic conditions, increasing transcript levels were measured as dissolved oxygen decreased. Conclusion The data obtained by TRAC supported the usefulness of focused and intensive transcriptional analysis in monitoring of biotechnical processes providing thus tools for process optimisation purposes.
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- 2007
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38. Transcriptional monitoring of steady state and effects of anaerobic phases in chemostat cultures of the filamentous fungus Trichoderma reesei
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Penttilä Merja, Wiebe Marilyn, Smit Bart A, Rautio Jari J, and Saloheimo Markku
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Chemostat cultures are commonly used in production of cellular material for systems-wide biological studies. We have used the novel TRAC (transcript analysis with aid of affinity capture) method to study expression stability of approximately 30 process relevant marker genes in chemostat cultures of the filamentous fungus Trichoderma reesei and its transformant expressing laccase from Melanocarpus albomyces. Transcriptional responses caused by transient oxygen deprivations and production of foreign protein were also studied in T. reesei by TRAC. Results In cultures with good steady states, the expression of the marker genes varied less than 20% on average between sequential samples for at least 5 or 6 residence times. However, in a number of T. reesei cultures continuous flow did not result in a good steady state. Perturbations to the steady state were always evident at the transcriptional level, even when they were not measurable as changes in biomass or product concentrations. Both unintentional and intentional perturbations of the steady state demonstrated that a number of genes involved in growth, protein production and secretion are sensitive markers for culture disturbances. Exposure to anaerobic conditions caused strong responses at the level of gene expression, but surprisingly the cultures could regain their previous steady state quickly, even after 3 h O2 depletion. The main effect of producing M. albomyces laccase was down-regulation of the native cellulases compared with the host strain. Conclusion This study demonstrates the usefulness of transcriptional analysis by TRAC in ensuring the quality of chemostat cultures prior to costly and laborious genome-wide analysis. In addition TRAC was shown to be an efficient tool in studying gene expression dynamics in transient conditions.
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- 2006
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39. Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae
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Suortti Tapani, Valkonen Mari, Saloheimo Markku, Lanthaler Karin, Pakula Tiina, Arvas Mikko, Robson Geoff, and Penttilä Merja
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Secretion stress is caused by compromised folding, modification or transport of proteins in the secretory pathway. In fungi, induction of genes in response to secretion stress is mediated mainly by the unfolded protein response (UPR) pathway. This study aims at uncovering transcriptional responses occurring in the filamentous fungi Trichoderma reesei exposed to secretion stress and comparing these to those found in the yeast Saccharomyces cerevisiae. Results Chemostat cultures of T. reesei expressing human tissue plasminogen activator (tPA) and batch bioreactor cultures treated with dithiothreitol (DTT) to prevent correct protein folding were analysed with cDNA subtraction and cDNA-amplified fragment length polymorphism (AFLP) experiments. ESTs corresponding to 457 unique genes putatively induced under secretion stress were isolated and the expression pattern of 60 genes was confirmed by Northern analysis. Expression of these genes was also studied in a strain over-expressing inositol-requiring enzyme 1 (IREI) protein, a sensor for the UPR pathway. To compare the data with that of S. cerevisiae, published transcriptome profiling data on various stress responses in S. cerevisiae was reanalysed. The genes up-regulated in response to secretion stress included a large number of secretion related genes in both organisms. In addition, analysis of T. reesei revealed up regulation of the cpc1 transcription factor gene and nucleosomal genes. The induction of the cpcA and histone gene H4 were shown to be induced also in cultures of Aspergillus nidulans treated with DTT. Conclusion Analysis of the genes induced under secretion stress has revealed novel features in the stress response in T. reesei and in filamentous fungi. We have demonstrated that in addition to the previously rather well characterised induction of genes for many ER proteins or secretion related proteins also other types of responses exist.
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- 2006
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40. Accumulation of mast cells in the shoulder regions of human aortic and coronary atheromas
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Kovanen, P.T., Kaartinen, M., and Penttila¨, M.
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- 1994
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41. Influence of growth temperature on the production of antibody Fab fragments in different microbes:A host comparative analysis
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Lise Bernard-Granger, Jaana Tokkanen, Danilo Porro, Martin Dragosits, Gianni Frascotti, Tiina Pakula, Diethard Mattanovich, Kristin Baumann, Escarlata Rodríguez-Carmona, Michael Maurer, Maria Luisa Tutino, Renate Kunert, Paola Branduardi, Maria Giuliani, Merja Penttilä, Felícitas Vázquez, Michael Sauer, Pau Ferrer, Antonio Villaverde, Brigitte Gasser, Marilyn G. Wiebe, Ermenegilda Parrilli, Markku Saloheimo, Dragosits, M, Frascotti, G, Bernard Granger, L, Vázquez, F, Giuliani, M, Baumann, K, Rodríguez Carmona, E, Tokkanen, J, Parrilli, Ermenegilda, Wiebe, Mg, Kunert, R, Maurer, M, Gasser, B, Sauer, M, Branduardi, P, Pakula, T, Saloheimo, M, Penttilä, M, Ferrer, P, Tutino, MARIA LUISA, Villaverde, A, Porro, D, Mattanovich, D., Vazquez, F, Rodriguez Carmona, E, Parrilli, E, Wiebe, M, Penttila, M, Tutino, M, and Mattanovich, D
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Trichoderma reesei ,Saccharomyces cerevisiae ,Microbial metabolism ,Enzyme-Linked Immunosorbent Assay ,Chemostat ,medicine.disease_cause ,Microbiology ,Pseudoalteromonas haloplanktis ,Pichia pastoris ,Immunoglobulin Fab Fragments ,Species Specificity ,Yeasts ,medicine ,Escherichia coli ,Bacteria ,biology ,recombinant protein production, chemostat, Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli, Pseudoalteromonas haloplanktis ,Temperature ,biology.organism_classification ,CHIM/11 - CHIMICA E BIOTECNOLOGIA DELLE FERMENTAZIONI ,Recombinant protein production ,Biochemistry ,Protein folding ,Biotechnology - Abstract
Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes. © 2010 American Institute of Chemical Engineers.
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- 2011
42. Overexpression of an endochitinase gene (ThEn-42) in Trichoderma atroviride for increased production of antifungal enzymes and enhanced antagonist action against pathogenic fungi
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Shiping Deng, Gary E. Harman, Matteo Lorito, Merja Penttilä, Deng, S, Lorito, Matteo, Penttila, M, and Harman, G. E.
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Antifungal Agents ,Bioengineering ,Cellulase ,Applied Microbiology and Biotechnology ,Biochemistry ,Polymerase Chain Reaction ,Synergistic interaction ,Microbiology ,Trichoderma spp ,Gene Expression Regulation, Fungal ,Spore germination ,Transgenes ,Pest Control, Biological ,Molecular Biology ,Plant Diseases ,chemistry.chemical_classification ,Trichoderma ,Penicillium digitatum ,biology ,Models, Genetic ,Chitinases ,Chitinase ,Biocontrol ,Proteins ,General Medicine ,Fungi imperfecti ,biology.organism_classification ,Spore ,Culture Media ,Enzyme ,chemistry ,Genetic Techniques ,biology.protein ,Gene expression ,Biotechnology - Abstract
Trichoderma is one of the most promising biocontrol agents against plant fungal diseases. In this study, a transgenic strain of Trichoderma atroviride was characterized. The transgenic strain contains an endochitinase gene (ThEn-42) driven by the cellulase promoter cbh1 of T. reesei for overexpression of ThEn-42. The culture filtrates of the transformant and the parental strain grown in eight different media were evaluated for chitinase and antifungal enzyme production based on activity gels, protein profiles, and antifungal activities. Results demonstrated that chitinases are important components and synergistic interactions play a key role in the antagonistic action of T. atroviride. Moreover, altering medium nutrient concentration and composition led to enhanced production of antifungal enzymes, a potential strategy for mass production. Two of the culture filtrates contained almost pure endochitinase, and could be excellent commercial sources for this enzyme. Several culture filtrates were highly antifungal. Two filtrates were so effective in biocontrol of a fungal pathogen, Penicillium digitatum, that they not only inhibited spore germination but destroyed the spores completely when 20 μl of culture filtrate (corresponding to approximately 104 μg of total protein) was applied in a total volume of 150 μl (approximately 0.7 mg protein ml-1).
- Published
- 2007
43. Simulated and measured piezoelectric energy harvesting of dynamic load in tires.
- Author
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Staaf H, Matsson S, Sepheri S, Köhler E, Daoud K, Ahrentorp F, Jonasson C, Folkow P, Ryynänen L, Penttila M, and Rusu C
- Abstract
From 2007 in US and from 2022 in EU it is mandatory to use TPMS monitoring in new cars. Sensors mounted in tires require a continuous power supply, which currently only is from batteries. Piezoelectric energy harvesting is a promising technology to harvest energy from tire movement and deformation to prolong usage of batteries and even avoid them inside tires. This study presents a simpler method to simultaneous model the tire deformation and piezoelectric harvester performance by using a new simulation approach - dynamic bending zone. For this, angular and initial velocities were used for rolling motion, while angled polarization was introduced in the model for the piezoelectric material to generate correct voltage from tire deformation. We combined this numerical simulation in COMSOL Multiphysics with real-life measurements of electrical output of a piezoelectric energy harvester that was mounted onto a tire. This modelling approach allowed for 10 times decrease in simulation time as well as simpler investigation of systems parameters influencing the output power. By using experimental data, the simulation could be fine-tuned for material properties and for easier extrapolation of tire deformation with output harvested energy from simulations done at low velocity to the high velocity experimental data., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Cristina Rusu reports financial support was provided by 10.13039/501100000780European Commission. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)
- Published
- 2024
- Full Text
- View/download PDF
44. Controllable coacervation of recombinantly produced spider silk protein using kosmotropic salts.
- Author
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Mohammadi P, Jonkergouw C, Beaune G, Engelhardt P, Kamada A, Timonen JVI, Knowles TPJ, Penttila M, and Linder MB
- Subjects
- Animals, Fibroins genetics, Fibroins metabolism, Microfluidics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Salts chemistry, Fibroins chemistry, Recombinant Proteins chemistry, Salts metabolism, Spiders chemistry
- Abstract
Recent developments suggest that the phase transition of natural and synthetic biomacromolecules represents an important and ubiquitous mechanism underlying structural assemblies toward the fabrication of high-performance materials. Such a transition results in the formation of condensed liquid droplets, described as condensates or coacervates. Being able to effectively control the assembly of such entities is essential for tuning the quality and their functionality. Here we describe how self-coacervation of genetically engineered spidroin-inspired proteins can be preceded by a wide range of kosmotropic salts. We studied the kinetics and mechanisms of coacervation in different conditions, from direct observation of initial phase separation to the early stage of nucleation/growth and fusion into large fluid assemblies. We found that coacervation induced by kosmotropic salts follows the classical nucleation theory and critically relies on precursor clusters of few weak-interacting protein monomers. Depending on solution conditions and the strength of the supramolecular interaction as a function of time, coacervates with a continuum of physiochemical properties were observed. We observed similar characteristics in other protein-based coacervates, which include having a spherical-ellipsoid shape in solution, an interconnected bicontinuous network, surface adhesion, and wetting properties. Finally, we demonstrated the use of salt-induced self-coacervates of spidroin-inspired protein as a cellulosic binder in dried condition., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
45. Comparison of protein coding gene contents of the fungal phyla Pezizomycotina and Saccharomycotina.
- Author
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Arvas M, Kivioja T, Mitchell A, Saloheimo M, Ussery D, Penttila M, and Oliver S
- Subjects
- Algorithms, Cluster Analysis, Computational Biology, Databases, Genetic, Fungal Proteins chemistry, Fungal Proteins metabolism, Genetic Variation, Genome, Fungal, Genomics, Open Reading Frames, Phylogeny, Principal Component Analysis, Protein Structure, Tertiary, Sequence Analysis, Protein, Software, Ascomycota classification, Ascomycota genetics, Fungal Proteins genetics, Genes, Fungal
- Abstract
Background: Several dozen fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. In addition comparative genomics has been used to analyse specific sub groups of fungi. Notably, analysis of the phylum Saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. However, little has been done to gain a comprehensive comparative view to the fungal kingdom. We have carried out a computational genome wide comparison of protein coding gene content of Saccharomycotina and Pezizomycotina, which include industrially important yeasts and filamentous fungi, respectively., Results: Our analysis shows that based on genome redundancy, the traditional model organisms Saccharomyces cerevisiae and Neurospora crassa are exceptional among fungi. This can be explained by the recent genome duplication in S. cerevisiae and the repeat induced point mutation mechanism in N. crassa. Interestingly in Pezizomycotina a subset of protein families related to plant biomass degradation and secondary metabolism are the only ones showing signs of recent expansion. In addition, Pezizomycotina have a wealth of phylum specific poorly characterised genes with a wide variety of predicted functions. These genes are well conserved in Pezizomycotina, but show no signs of recent expansion. The genes found in all fungi except Saccharomycotina are slightly better characterised and predicted to encode mainly enzymes. The genes specific to Saccharomycotina are enriched in transcription and mitochondrion related functions. Especially mitochondrial ribosomal proteins seem to have diverged from those of Pezizomycotina. In addition, we highlight several individual gene families with interesting phylogenetic distributions., Conclusion: Our analysis predicts that all Pezizomycotina unlike Saccharomycotina can potentially produce a wide variety of secondary metabolites and secreted enzymes and that the responsible gene families are likely to evolve fast. Both types of fungal products can be of commercial value, or on the other hand cause harm to humans. In addition, a great number of novel predicted and known enzymes are found from all fungi except Saccharomycotina. Therefore further studies and exploitation of fungal metabolism appears very promising.
- Published
- 2007
- Full Text
- View/download PDF
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