| 21. |
- McKee, Lauren S., et al.
(författare)
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A GH115 alpha-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan
- 2016
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Ingår i: Biotechnology for Biofuels. - : BioMed Central. - 1754-6834. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. Results: We report the characterisation of a recombinant alpha-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me) GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an alpha-l-arabinofuranosidase (AbfA), and a beta-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Conclusions: Our GH115 alpha-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.
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| 22. |
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| 23. |
- Shin, Jae Ho, 1987, et al.
(författare)
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Molecular docking and linear interaction energy studies give insight to α, β-reduction of enoate groups in enzymes
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Production of adipic acid from renewable sources has been gaining attention in an attempt to move from an oil-based economy to a biobased economy. Metabolic engineering allows microorganisms to produce useful chemicals using renewable resources as carbon sources. We target a theoretical metabolic pathway that relies on conversion of L-lysine to adipic acid. One of the enzymatic steps in this conversion pathway is an α, β-reduction of an unsaturated bond in an enoate moiety and no aerobic enzymes have been identified to specifically make this conversion on 6-amino-trans-2-hexenoic acid. We evaluated Escherichia coli NemA, and Saccharomyces pastorianus Oye1 (Old Yellow Enzyme 1) for their potenstial capability to carry out the desired α, β-reduction. Here, we build homology models for E. coli NemA and perform molecular docking studies of trans-2-hexenoic acid and trans-2-hexenal to the candidate enzyme models. Ligand-enzyme binding stability is assessed by molecular dynamics (MD) simulations. Additionally, linear energy calculations were used to investigate binding stability in solution environment. Here, we propose that NemA and Oye1, both belonging to the Old yellow enzyme family, have large enough catalytic pocket for accommodating enoate moieties but not enough stability to carry out the α, β-reduction. Protein engineering of both NemA and Oye1 would be necessary for these enzymes to perform the targeted reactions efficiently. The results shown in this study provides a useful insight to α, β-reduction reaction potentially crucial in bio-based production of adipic acid.
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| 24. |
- Skoog, Emma, 1983, et al.
(författare)
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Biobased adipic acid – The challenge of developing the production host
- 2018
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 36:8, s. 2248-2263
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Forskningsöversikt (refereegranskat)abstract
- Adipic acid is a platform chemical, and is the most important commercial dicarboxylic acid. It has been targeted for biochemical conversion as an alternative to present chemical production routes. From the perspective of bioeconomy, several kinds of raw material are of interest including the sugar platform (derived from starch, cellulose or hemicellulose), the lignin platform (aromatics) and the fatty acid platform (lipid derived). Two main biochemical-based production schemes may be employed: (i) direct fermentation to adipic acid, or (ii) fermentation to muconic or glucaric acid, followed by chemical hydrogenation (indirect fermentation). This review presents a comprehensive description of the metabolic pathways that could be constructed and analyzes their respective theoretical yields and metabolic constraints. The experimental yields and titers obtained so far are low, with the exception of processes based on palm oil and glycerol, which have been reported to yield up to 50 g and 68 g adipic acid/L, respectively. The challenges that remain to be addressed in order to achieve industrially relevant production levels include solving redox constraints, and identifying and/or engineering enzymes for parts of the metabolic pathways that have yet to be metabolically demonstrated. This review provides new insights into ways in which metabolic pathways can be constructed to achieve efficient adipic acid production. The production host provides the chassis to be engineered via an appropriate metabolic pathway, and should also have properties suitable for the industrial production of adipic acid. An acidic process pH is attractive to reduce the cost of downstream processing. The production host should exhibit high tolerance to complex raw material streams and high adipic acid concentrations at acidic pH.
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| 25. |
- Olofsson, Martin, 1975-, et al.
(författare)
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Combined Effects of Nitrogen Concentration and Seasonal Changes on the Production of Lipids in Nannochloropsis oculata
- 2014
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Ingår i: Marine Drugs. - Basel, Switzerland : MDPI AG. - 1660-3397. ; 12:4, s. 1891-1910
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Tidskriftsartikel (refereegranskat)abstract
- Instead of sole nutrient starvation to boost algal lipid production, we addressed nutrient limitation at two different seasons (autumn and spring) during outdoor cultivation in flat panel photobioreactors. Lipid accumulation, biomass and lipid productivity and changes in fatty acid composition of Nannochloropsis oculata were investigated under nitrogen (N) limitation (nitrate:phosphate N:P 5, N:P 2.5 molar ratio). N. oculata was able to maintain a high biomass productivity under N-limitation compared to N-sufficiency (N:P 20) at both seasons, which in spring resulted in nearly double lipid productivity under N-limited conditions (0.21 g L−1 day−1) compared to N-sufficiency (0.11 g L−1 day−1). Saturated and monounsaturated fatty acids increased from 76% to nearly 90% of total fatty acids in N-limited cultures. Higher biomass and lipid productivity in spring could, partly, be explained by higher irradiance, partly by greater harvesting rate (~30%). Our results indicate the potential for the production of algal high value products (i.e., polyunsaturated fatty acids) during both N-sufficiency and N-limitation. To meet the sustainability challenges of algal biomass production, we propose a dual-system process: Closed photobioreactors producing biomass for high value products and inoculum for larger raceway ponds recycling waste/exhaust streams to produce bulk chemicals for fuel, feed and industrial material.
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| 26. |
- Hong, Kuk-ki, 1976, et al.
(författare)
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Metabolic Engineering of Saccharomyces cerevisiae: A Key Cell Factory Platform for Future Biorefineries
- 2012
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Ingår i: Cellular and Molecular Life Sciences. - : Springer Science and Business Media LLC. - 1420-9071 .- 1420-682X. ; 69:16, s. 2671-2690
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Forskningsöversikt (refereegranskat)abstract
- Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.
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| 27. |
- Sunner, Hampus, 1981, et al.
(författare)
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Fungal Ferulic Acid Esterases – Specificity and Phylogeny
- 2009
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Ingår i: Italic5 Science and Technology of Biomasses Proceedings Book, M Orlandi, C Crestine (Ed.). Italic5/COST conference, Sept 1-4 2009, Varenna, Italy.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Ferulic Acid Esterases (FAE) is a large heterogeneous group of enzymes with activity on esters of hydroxy- and metoxy- substituted cinnamic acid derivatives, such as ferulic acid. These ester bonds occur in the cell walls of plants and are especially common in grasses. As little systematic knowledge has been collected about this group of enzymes and only a few enzymes have been biochemically characterised to date, we have explored the phylogeny of FAEs using bioinformatic tools. We can conclude that the known Ferulic Acid Esterases belong to several evolutionary distant groups, two of which have dozens of highly related sequences, and a few groups with no members other than the known enzyme. The phylogeny also suggests certain similarities of substrate specificity within groups and proposes enzymes, whose biochemical characterisation would be especially informative for our understanding of the FAE families.
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| 28. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies
- 2013
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Ingår i: Cell Factories and Biosustainability (Hilleroed, Denmark, May 5-8 2013).
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The realization of an oil independent economy relies on the development of competitive processes for the production of fuels and chemicals from renewable resources. The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Harsh conditions during the bioconversion of lignocellulose-derived sugars to the desired products drastically hamper cell viability and therefore productivity. Microbial inhibition limits bioprocesses to an extent such that it can be said that understanding and harnessing microbial robustness is a prerequisite for the feasibility of new bioprocess and the production of renewable fuels and chemicals.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.
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| 29. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies
- 2013
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Ingår i: 35th Symposium on Biotechnology for Fuels and Chemicals (Portland, OR. April 29-May 2, 2013).
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The realization of an oil independent economy relies on the development of competitive processes for the production of fuels and chemicals from renewable resources. The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Sugars are released from cellulose and hemicellulose by pretreatment and hydrolysis steps. Harsh conditions result in the formation of a number of compounds, originating from sugars and lignin breakdown and acting as microorganism inhibitors. Weak organic acids, furaldehydes and phenolic compounds are sources of stress for the fermenting microorganism, as they influence cellular metabolism in a number of ways, including direct damage on cellular functions or by perturbations of the cellular energy and redox metabolism. In addition, the product of interest can act as a potent inhibitor. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose-based bioprocesses.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.
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| 30. |
- Westman, Johan
(författare)
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Ethanol production from lignocellulose using high local cell density yeast cultures. Investigations of flocculating and encapsulated Saccharomyces cerevisiae
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Efforts are made to change from 1st to 2nd generation bioethanol production, using lignocellulosics as raw materials rather than using raw materials that alternatively can be used as food sources. An issue with lignocellulosics is that a harsh pretreatment step is required in the process of converting them into fermentable sugars. In this step, inhibitory compounds such as furan aldehydes and carboxylic acids are formed, leading to suboptimal fermentation rates. Another issue is that lignocellulosics may contain a large portion of pentoses, which cannot be fermented simultaneously with glucose by Saccharomyces cerevisiae. In this thesis, high local cell density has been investigated as a means of overcoming these two issues. Encapsulation of yeast in semi-permeable alginate-chitosan capsules increased the tolerance towards furan aldehydes, but not towards carboxylic acids. The selective tolerance can be explained by differences in the concentration of compounds radially through the cell pellet inside the capsule. For inhibitors, gradients will only be formed if the compounds are readily convertible, like the furan aldehydes. Conversion of inhibitors by cells close to the membrane leads to decreased concentrations radially through the cell pellet. Thus, cells closer to the core experience subinhibitory levels of inhibitors and can ferment sugars. Carbohydrate gradients also give rise to nutrient limitations, which in turn trigger a stress response in the yeast, as was observed on mRNA and protein level. The stress response is believed to increase the robustness of the yeast and lead to improved tolerance towards additional stress. Glucose and xylose co-consumption by a recombinant strain, CEN.PK XXX, was also improved by encapsulation. Differences in affinity of the sugar transporters normally result in that glucose is taken up preferentially to xylose. However, when encapsulated, cells in different parts of the capsule experienced high and low glucose concentrations simultaneously. Xylose and glucose could thus be taken up concurrently. This improved the co-utilisation of the sugars by the system and led to 50% higher xylose consumption and 15% higher final ethanol titres. A protective effect by the capsule membrane itself could not be shown. Hence, the interest in flocculation was triggered, as a more convenient way to keep the cells together. To investigate whether flocculation increases the tolerance, like encapsulation, recombinant flocculating yeast strains were constructed and compared with the non-flocculating parental strain. Experiments showed that strong flocculation did not increase the tolerance towards carboxylic acids. However, the tolerance towards a spruce hydrolysate and especially against furfural was indeed increased. The results of this thesis show that high local cell density yeast cultures have the potential to aid against two of the major problems for 2nd generation bioethanol production: inhibitors and simultaneous hexose and pentose utilisation.
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