| 1. |
- Lindahl, Lina, 1984, et al.
(författare)
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Alcohols enhance the rate of acetic acid diffusion in S. cerevisiae: biophysical mechanisms and implications for acetic acid tolerance
- 2018
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Ingår i: Microbial Cell. - : Shared Science Publishers OG. - 2311-2638. ; 5:1, s. 42-55
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Tidskriftsartikel (refereegranskat)abstract
- Microbial cell factories with the ability to maintain high productivity in the presence of weak organic acids, such as acetic acid, are required in many industrial processes. For example, fermentation media derived from lignocellulosic biomass are rich in acetic acid and other weak acids. The rate of diffusional entry of acetic acid is one parameter determining the ability of microorganisms to tolerance the acid. The present study demonstrates that the rate of acetic acid diffusion in S. cerevisiae is strongly affected by the alcohols ethanol and n-butanol. Ethanol of 40 g/L and n-butanol of 8 g/L both caused a 65% increase in the rate of acetic acid diffusion, and higher alcohol concentrations caused even greater increases. Molecular dynamics simulations of membrane dynamics in the presence of alcohols demonstrated that the partitioning of alcohols to the head group region of the lipid bilayer causes a considerable increase in the membrane area, together with reduced membrane thickness and lipid order. These changes in physiochemical membrane properties lead to an increased number of water molecules in the membrane interior, providing biophysical mechanisms for the alcohol-induced increase in acetic acid diffusion rate. nbutanol affected S. cerevisiae and the cell membrane properties at lower concentrations than ethanol, due to greater and deeper partitioning in the membrane. This study demonstrates that the rate of acetic acid diffusion can be strongly affected by compounds that partition into the cell membrane, and highlights the need for considering interaction effects between compounds in the design of microbial processes.
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| 2. |
- Mazurkewich, Scott, 1982, et al.
(författare)
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Understanding enzyme-substrate interactions in Carbohydrate Esterase Family 15
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Carbohydrate Esterase Family 15 (CE15) is a rather small family, comprising approximately 200 members, which was established in CAZy (www.cazy.org) in 2006. The family was created following the characterization of a glucuronoyl esterase (GE) from the fungus Schizophyllum commune [1], which was shown to cleave methyl moieties ester-linked to the O6 position of glucuronic acid. CE15 enzymes are proposed to cleave ester linkages between lignin and glucuronoxylan, so-called lignin-carbohydrate complexes (LCCs), which are important features in biomass recalcitrance. We recently characterized ten new GEs from three bacterial species and solved the structures of two of these, essentially doubling both the biochemical and structural data available for the family [2]. An in-depth understanding of how CE15 enzymes interact with their complex substrates is still lacking, as only one structure with a monosaccharide ligand has been solved to date [3]. To address this, we have pursued solving new GE structures and obtaining protein-ligand complex structures. The studies have resulted in a novel structure exhibiting features with prominent inserts surrounding the active site, suggesting different specificities between bacterial and fungal GEs. In addition, we have solved the first structures of a CE15 enzyme with larger ligands, which gives direct evidence of how these enzymes interact with the different parts of its proposed physiological LCC substrates. Combined with kinetic characterizations, these new investigations greatly add to the knowledge of enzyme-substrate interactions in CE15 and enhances how these enzymes may act in natural conditions, which could aid in industrial biomass conversion. ______________ [1] Španiková S.; Biely P. FEBS Lett. 2006, 580, 4597-4601. [2] Arnling Bååth J.; Mazurkewich S.; Knudsen R.M.; Poulsen J.N.; Olsson L.; Lo Leggio L.; Larsbrink J. Biotechnol Biofuels. 2018, 11, 213-226. [3] Charavgi M.D.; Dimarogona M.; Topakas E.; Christakopoulos P.; Chrysina E.D. Acta Crystallogr. D Biol. Crystallogr. 2013, 69, 63-73.
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| 3. |
- Trivellin, Cecilia, 1993, et al.
(författare)
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Robustness quantification of a mutant library screen revealed key genetic markers in yeast
- 2024
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Microbial robustness is crucial for developing cell factories that maintain consistent performance in a challenging environment such as large-scale bioreactors. Although tools exist to assess and understand robustness at a phenotypic level, the underlying metabolic and genetic mechanisms are not well defined, which limits our ability to engineer more strains with robust functions. Results: This study encompassed four steps. (I) Fitness and robustness were analyzed from a published dataset of yeast mutants grown in multiple environments. (II) Genes and metabolic processes affecting robustness or fitness were identified, and 14 of these genes were deleted in Saccharomyces cerevisiae CEN.PK113-7D. (III) The mutants bearing gene deletions were cultivated in three perturbation spaces mimicking typical industrial processes. (IV) Fitness and robustness were determined for each mutant in each perturbation space. We report that robustness varied according to the perturbation space. We identified genes associated with increased robustness such as MET28, linked to sulfur metabolism; as well as genes associated with decreased robustness, including TIR3 and WWM1, both involved in stress response and apoptosis. Conclusion: The present study demonstrates how phenomics datasets can be analyzed to reveal the relationship between phenotypic response and associated genes. Specifically, robustness analysis makes it possible to study the influence of single genes and metabolic processes on stable microbial performance in different perturbation spaces. Ultimately, this information can be used to enhance robustness in targeted strains.
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| 4. |
- Ask, Magnus, 1983, et al.
(författare)
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The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae
- 2013
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:22
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundPretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium.ResultsBy cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(+/-0.005) to 0.08(+/-0.002) and the anabolic reduction charge decreased from 0.46(+/-0.11) to 0.27(+/-0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(+/-0.002) to 0.85(+/-0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group "Cell rescue, defense and virulence" was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis.ConclusionsThe redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.
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| 5. |
- Anasontzis, George E, 1980, et al.
(författare)
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Screening Natural Resources for Enzymes With Wood Degrading and Wood Modifying Properties
- 2011
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Ingår i: Italic6/COST conference, 5–8 September 2011, Viterbo, Italy.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The production of high added value compounds from forest and agricultural biomass has become one of the main targets of contemporary carbohydrates research. The renewability of the biomass, the potential use of waste residues and the complete or partial biodegradability of the products have made the whole approach an attractive perspective towards the sustainable and green ideal. However, most of the already developed biomass separation and modification processes are based on chemical reactions at extreme conditions that are costly and often harmful for the environment. Enzymatic and microbial catalyzed processes present an interesting alternative. The development and discovery of novel biological approaches in the modification, degradation and separation of wood biomass is one of the main activities of the Industrial Biotechnology Group at Chalmers University of Technology, also as part of the Wallenberg Wood Science Center (WWSC).Presently, we pursue this aim through a triple approach: •Μultiple enzymatic screening of phytopathogenic and wood degrading filamentous fungi, such as Trametes hirsuta and Penicillium pinophilum, as well as screening newly isolated microorganisms. We seek enzymes with industrially interesting activities and unique properties, such as reactivity under extreme conditions.•Microorganisms efficient in degrading lignocellulose produce enzyme in response to the environmental conditions. In collaboration with Associate Professor Gianni Panagiotou, Center for Biological Sequence Analysis, DTU, we are looking for sequenced, but still unclassified proteins, which are related to the degradation of plant biomass using information from transcriptomics analysis of Aspergillus oryzae grown on different carbon sources.•Novel enzymes can only be identified by new methods. We investigate the properties of synthetic model compounds that can simulate the natural substrates and the implementation of different analytical methods for the identification of the sometimes complex and singular enzymatic activities. In collaboration with Associate Professor Paul Christakopoulos, BIOtechMASS Unit, School of Chemical Engineering, National Technical University of Athens, we also attempt to isolate model compounds from plant cell wall material.
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| 6. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Plasma membrane as a crucial player in acetic acid effect on yeast
- 2017
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Ingår i: IMYA12- 12th International Meeting on Yeast Apoptosis, Bari, Italy • 14-18 May 2017.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Weak organic acids such as formic, acetic or lactic acid are known inhibitors of microbial growth and fermentation. Acetic acid toxicity to yeast cells has been explained by different theories, involving specific signaling effects triggering an active cell death program, reduction of intracellular pH and acetate anion accumulation. Regardless of the fact whether the actual effect of acetate involves one of these mechanisms or a combination thereof, acetic acid inhibits yeast metabolism and affects yeast viability. This has a high impact on the feasibility of the new generation of fermentation processes, based on the naturally acetic acid-rich lignocellulosic substrates. It is therefore highly desirable to obtain a strain with increased capacity of coping with high acetic acid concentrations in the fermentation medium. Acetic acid is thought to be internalized by yeast cells in its undissociated form, by crossing the hydrophobic barrier of plasma membrane. Thus, in our work we focused on the investigation of membrane properties and how these influence the tolerance of yeast to acetic acid. First, we demonstrated with lipidomics analysis of membrane lipids that the yeast Zygosaccharomyces bailii, showing extraordinary tolerance to acetic acid, has a plasma membrane which is rich in sphingolipids. Next, we combined membrane molecular dynamics and in vivo measurements to confirm the specific role of sphingolipids in altering the permeability of plasma membrane to acetic acid. Finally, we investigated the effect of alcohols on the acetic acid permeation rate through the membrane. Our ultimate goal is to engineer the membrane composition of an industrial yeast strain towards reduced permeability, in order to obtain higher acetic acid tolerance.
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| 7. |
- Mapelli, Valeria, 1978, et al.
(författare)
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Biotechnology for production of bioactive seleno compounds and study of their influence on mouse metabolome
- 2011
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Ingår i: Natural Products Chemistry, Biology and Medicine IV Aug 28 - Sept 2, Acquafredda di Maratea, Italy.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Organic seleno compounds are recognized as effective anti-oxidant agents and their bioactive role in prevention of certain forms of cancer has been suggested via in vitro studies and clinical trials. Among these compounds, Seleno-methyselenocysteine (SeMCys) and γ-glutamyl-SeMCys (γ-glu-SeMCys) are the most bioactive and the latter is the preferred storage form of selenium in Se-accumulator plants thanks to their Se-methyltransferase. Therefore, Se-accumulator edible plants such as Brassicaceae and Allioideae are the main source of SeMCys and γ-glu-SeMCys in the human diet. However, seasonal and environmental factors highly affect the content and the bioavailability of these bioactive compounds. A strategy to by-pass this problem and prevent selenium shortage in human diet is the production of Se-enriched yeast (Se-yeast) to be used as food supplement. In this work we show a biotechnological approach for production Se-yeast featured by higher content of SeMCys and γ-glu-SeMCys. Coupling of metabolic engineering and bioprocess optimization resulted in a Se-yeast with 24-fold increase of SeMCys levels, compared to commercial Se-yeast. The actual effect of the produced yeast has been evaluated in an animal study. In particular, as specific Se-compounds are known to activate phase II enzymes via the electrophile-responsive element (EpRE), this response was studied in transgenic mice expressing the luciferase gene under EpRE control. We observed no effect on regulation of EpRE, either overall or hepatic, by the different Se-supplements. Paradoxically, a decrease was observed in intestinal EpRE transactivation upon supplementation of the Se-yeast produced. The overall effect of the diet supplemented with Se-yeast on mouse metabolism is currently being evaluated by metabolome analysis of liver samples from the transgenic mice.
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| 8. |
- Granchi, Zoraide, et al.
(författare)
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Genome Sequence of the Thermophilic Biomass-Degrading Fungus Malbranchea cinnamomea FCH 10.5
- 2017
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Ingår i: Genome Announcements. - 2169-8287. ; 5:33
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Tidskriftsartikel (refereegranskat)abstract
- We report here the annotated draft genome sequence of the thermophilic biomass-degrading fungus Malbranchea cinnamomea strain FCH 10.5, isolated from compost at a waste treatment plant in Vietnam. The genome sequence contains 24.96 Mb with an overall GC content of 49.79% and comprises 9,437 protein-coding genes.
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| 9. |
- Maertens, Jeroen, 1990, et al.
(författare)
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Molecular-dynamics-simulation-guided membrane engineering allows the increase of membrane fatty acid chain length in Saccharomyces cerevisiae
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The use of lignocellulosic-based fermentation media will be a necessary part of the transition to a circular bio-economy. These media contain many inhibitors to microbial growth, including acetic acid. Under industrially relevant conditions, acetic acid enters the cell predominantly through passive diffusion across the plasma membrane. The lipid composition of the membrane determines the rate of uptake of acetic acid, and thicker, more rigid membranes impede passive diffusion. We hypothesized that the elongation of glycerophospholipid fatty acids would lead to thicker and more rigid membranes, reducing the influx of acetic acid. Molecular dynamics simulations were used to predict the changes in membrane properties. Heterologous expression of Arabidopsis thaliana genes fatty acid elongase 1 (FAE1) and glycerol-3-phosphate acyltransferase 5 (GPAT5) increased the average fatty acid chain length. However, this did not lead to a reduction in the net uptake rate of acetic acid. Despite successful strain engineering, the net uptake rate of acetic acid did not decrease. We suggest that changes in the relative abundance of certain membrane lipid headgroups could mitigate the effect of longer fatty acid chains, resulting in a higher net uptake rate of acetic acid.
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| 10. |
- Arnling Bååth, Jenny, 1987, et al.
(författare)
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A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds
- 2016
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Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 590:16, s. 2611-2618
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Tidskriftsartikel (refereegranskat)abstract
- The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and 31P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.
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