| 1. |
- Munthe, Christian, 1962
(författare)
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Precaution and Ethics: Handling risks, uncertainties and knowledge gaps in the regulation of new biotechnologies
- 2017
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Bok (övrigt vetenskapligt/konstnärligt)abstract
- This volume outlines and analyses ethical issues actualized by applying a precautionary approach to the regulation of new biotechnologies. It presents a novel way of categorizing and comparing biotechnologies from a precautionary standpoint. Based on this, it addresses underlying philosophical problems regarding the ethical assessment of decision-making under uncertainty and ignorance, and discusses how risks and possible benefits of such technologies should be balanced from an ethical standpoint. It argues on conceptual and ethical grounds for a technology neutral regulation as well as for a regulation that not only checks new technologies but also requires old, inferior ones to be phased out. It demonstrates how difficult ethical issues regarding the extent and ambition of precautionary policies need to be handled by such a regulation, and presents an overarching framework for doing so.
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| 2. |
- 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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| 3. |
- Wang, Shule, 1994-, et al.
(författare)
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Effect of H2 as Pyrolytic Agent on the Product Distribution during Catalytic Fast Pyrolysis of Biomass Using Zeolites
- 2018
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Ingår i: Energy & Fuels. - : American Chemical Society (ACS). - 0887-0624 .- 1520-5029. ; 32:8, s. 8530-8536
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Tidskriftsartikel (refereegranskat)abstract
- Bio-oil generated from catalytic fast pyrolysis or hydrotreating processes represents one of the most promising alternatives to liquid fossil fuels. The use of H2 as carrier gas in the pyrolysis of biomass requires further research to study the catalytic fast pyrolysis reactions in the case of using reactive atmosphere. In this work, pyrolysis experiments with lignocellulosic biomass have been performed in a fixed bed reactor in H2 and N2 atmospheres with/without HZSM-5 additions to investigate the influence of the pyrolytic agents during fast pyrolysis of biomass and upgrading of pyrolytic vapors over a zeolitic catalyst. It was found that in a H2 atmosphere, H2 was consumed in both noncatalytic and catalytic pyrolysis processes, respectively. Higher yields of nonaqueous liquids and permanent gases are obtained in a H2 atmosphere compared to a N2 atmosphere. A catalytic pyrolysis process using HZSM-5 in a H2 atmosphere increased the production of polymer aromatic hydrocarbons and suppressed the production of monomer aromatic hydrocarbons compared to similar tests performed in a N2 atmosphere. The results show an overall increased activity of HZSM-5 in the reactive H2 atmosphere compared to a N2 atmosphere.
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| 4. |
- 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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| 5. |
- 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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| 6. |
- Zhou, Yongjin, 1984, et al.
(författare)
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Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 7, s. 11709-11709
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Tidskriftsartikel (refereegranskat)abstract
- Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g/L of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg /L) and fatty alcohols (1.5 g/L), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.
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| 7. |
- Karlsson, Emma, 1983, et al.
(författare)
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In silico and in vitro studies of the reduction of unsaturated α,β bonds of trans-2-hexenedioic acid and 6-amino-trans-2-hexenoic acid – Important steps towards biobased production of adipic acid
- 2018
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 13:2
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Tidskriftsartikel (refereegranskat)abstract
- The biobased production of adipic acid, a precursor in the production of nylon, is of great interest in order to replace the current petrochemical production route. Glucose-rich lignocel-lulosic raw materials have high potential to replace the petrochemical raw material. A number of metabolic pathways have been proposed for the microbial conversion of glucose to adipic acid, but achieved yields and titers remain to be improved before industrial applications are feasible. One proposed pathway starts with lysine, an essential metabolite industrially produced from glucose by microorganisms. However, the drawback of this pathway is that several reactions are involved where there is no known efficient enzyme. By changing the order of the enzymatic reactions, we were able to identify an alternative pathway with one unknown enzyme less compared to the original pathway. One of the reactions lacking known enzymes is the reduction of the unsaturated α,β bond of 6-amino-trans-2-hexenoic acid and trans-2hexenedioic acid. To identify the necessary enzymes, we selected N-ethylmaleimide reductase from Escherichia coli and Old Yellow Enzyme 1 from Saccharomyces pastorianus. Despite successful in silico docking studies, where both target substrates could fit in the enzyme pockets, and hydrogen bonds with catalytic residues of both enzymes were predicted, no in vitro activity was observed. We hypothesize that the lack of activity is due to a difference in electron withdrawing potential between the naturally reduced aldehyde and the carboxylate groups of our target substrates. Suggestions for protein engineering to induce the reactions are discussed, as well as the advantages and disadvantages of the two metabolic pathways from lysine. We have highlighted bottlenecks associated with the lysine pathways, and proposed ways of addressing them.
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| 8. |
- Gontia, Paul, 1984, et al.
(författare)
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Life cycle assessment of bio-based sodium polyacrylate production from pulp mill side streams: Case study of thermo-mechanical and sulfite pulp mills
- 2016
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Ingår i: Journal of Cleaner Production. - : Elsevier BV. - 0959-6526. ; 131, s. 475-484
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Tidskriftsartikel (refereegranskat)abstract
- Sodium polyacrylate (Na-PA) is a super absorbent polymer, which is commonly used in diverse hygiene products. The polymer is currently produced from fossil feedstock and its production consequently leads to adverse environmental impacts. Na-PA production from sugars present in pulp mill side streams can potentially be a successful way to achieve a more sustainable production of this polymer. In order to guide the development of a novel biochemical process for producing Na-PA, a life cycle assessment was done in which Na-PA produced from side streams of thermo-mechanical pulp (TMP) and sulfite pulp mills were compared. Furthermore, a comparison was made with Na-PA produced from fossil resources. The results show that the main determinant of the environmental impact of the bio-based Na-PA production is the free sugar content in the side streams. The lowest environmental impact is achieved by the least diluted side streams. More diluted side streams require larger amounts of energy for concentration, and, if the diluted streams are not concentrated, processes such as hydrolysis and detoxification, and fermentation are the environmental hotspots. Furthermore, the higher the yield of the fermentation process, the lower the environmental impact will be. Lastly, the production of bio-based Na-PA led to a lower global warming potential for some of the considered pulp mill side streams, but all of the other impacts considered were higher, when compared to fossil-based Na-PA production. Therefore, in parallel with efforts to develop a high-yield yeast for the fermentation process, technology developers should focus on low energy concentration processes for the side streams.
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| 9. |
- Brink, Daniel
(författare)
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Understanding and improving microbial cell factories through Large Scale Data-approaches
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Since the advent of high-throughput genome sequencing methods in the mid-2000s, molecular biology has rapidly transitioned towards data-intensive science. Recent technological developments have increased the accessibility of omics experiments by decreasing the cost, while the concurrent design of new algorithms have improved the computational work-flow needed to analyse the large datasets generated. This has enabled the long standing idea of a systems approach to the cell, where molecular phenomena are no longer observed in isolation, but as parts of a tightly regulated cell-wide system. However, large data biology is not without its challenges, many of which are directly related to how to store, handle and analyse ome-wide datasets.The present thesis examines large data microbiology from a middle ground between metabolic engineering and in silico data management. The work was performed in the context of applied microbial lignocellulose valorisation with the end goal of generating improved cell factories for the production of value-added chemicals from renewable plant biomass. Three different challenges related to this feedstock were investigated from a large data-point of view: bacterial catabolism of lignin and its derived aromatic compounds; tolerance of baker’s yeast Saccharomyces cerevisiae to inhibitory compounds in lignocellulose hydrolysate; and the non-fermentable response to xylose in S. cerevisiae engineered for growth on this pentose sugar.The bibliome of microbial lignin catabolism is vast and consists of a long-standing cohort of fundamental microbiology, and a more recent cohort of applied lignin biovalorisation. Here, an online database was created with the long-term ambition of closing the gap between the two and make new connections that can fuel the generation of new knowledge. Whole-genome sequencing was used to investigate the genetic basis for observed phenotypes in bacterial isolates capable of growing on different kinds of lignin-derived aromatics. A whole-genome approach was also used to identify key sequence variants in the genotype of an industrial S. cerevisiae strain evolved for improved tolerance to inhibitors and high temperature. Finally, assessment of the sugar signalome of S. cerevisiae was enabled by the design and validation of a panel of in vivo fluorescent biosensors for single-cell cytometric analysis. It was found that xylose triggered a signal similar to that of low glucose in yeast cells engineered with xylose utilization pathways, and that introduction of deletions previously related to improved xylose utilization altered the signal towards that of high glucose.Taken together, the present thesis illustrates how omics-approaches can aid design of laboratory experiments to increase the knowledge and understanding of microorganisms, and demonstrates the need for a combined knowledge of molecular and computational biology in large-scale data microbiology.
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| 10. |
- Xiros, Charilaos, 1973, et al.
(författare)
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Toward a sustainable biorefinery using high-gravity technology
- 2017
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Ingår i: Biofuels, Bioproducts and Biorefining. - : Wiley. - 1932-1031 .- 1932-104X. ; 11:1, s. 15-27
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Tidskriftsartikel (refereegranskat)abstract
- The realization of process solutions for a sustainable bioeconomy depends on the efficient processing of biomass. High-gravity technology is one important alternative to realizing such solutions. The aims of this work were to expand the knowledge-base on lignocellulosic bioconversion processes at high solids content, to advance the current technologies for production of second-generation liquid biofuels, to evaluate the environmental impact of the proposed process by using life cycle assessment (LCA), and to develop and present a technically, economically, and environmentally sound process at high gravity, i.e., a process operating at the highest possible concentrations of raw material. The results and opinions presented here are the result of a Nordic collaborative study within the framework of the HG Biofuels project. Processes with bioethanol or biobutanol as target products were studied using wheat straw and spruce as interesting Nordic raw materials. During the project, the main scientific, economic, and technical challenges of such a process were identified. Integrated solutions to these challenges were proposed and tested experimentally, using wheat straw and spruce wood at a dry matter content of 30% (w/w) as model substrates. The LCA performed revealed the environmental impact of each of the process steps, highlighting the importance of the enzyme dose used for the hydrolysis of the plant biomass, as well as the importance of the fermentation yield.
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| 11. |
- Aulitto, Martina, 1991, et al.
(författare)
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Seed culture pre-adaptation of Bacillus coagulans MA-13 improves lactic acid production in simultaneous saccharification and fermentation
- 2019
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Lignocellulosic biomass is an abundant and sustainable feedstock, which represents a promising raw material for the production of lactic acid via microbial fermentation. However, toxic compounds that affect microbial growth and metabolism are released from the biomass upon thermochemical pre-treatment. So far, susceptibility of bacterial strains to biomass-derived inhibitors still represents a major barrier to lactic acid production from lignocellulose. Detoxification of the pre-treated lignocellulosic material by water washing is commonly performed to alleviate growth inhibition of the production microorganism and achieve higher production rates. Results In this study, we assessed the feasibility of replacing the washing step with integrated cellular adaptation during pre-culture of Bacillus coagulans MA-13 prior to simultaneous saccharification and lactic acid fermentation of steam exploded wheat straw. Using a seed culture pre-exposed to 30% hydrolysate led to 50% shorter process time, 50% higher average volumetric and 115% higher average specific productivity than when using cells from a hydrolysate-free seed culture. Conclusions Pre-exposure of B. coagulans MA-13 to hydrolysate supports adaptation to the actual production medium. This strategy leads to lower process water requirements and combines cost-effective seed cultivation with physiological pre-adaptation of the production strain, resulting in reduced lactic acid production costs.
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| 12. |
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| 13. |
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| 14. |
- Bonzom, Cyrielle, 1987, et al.
(författare)
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Feruloyl esterases immobilization in mesoporous silica particles
- 2016
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Mesoporous silica materials (MPS) are an interesting choice as support to immobilize enzymes because MPS offer unique properties such as high enzyme loading and tunable pore size. They also provide the enzyme with a sheltered environment therefore reducing loss of function in industrial applications.Feruloyl esterases (FAEs) are naturally hydrolytic enzymes which are known for their action on lignocellulosic material and release ferulic acid (FA) which is bound to plant cell wall materials. Under specific conditions they are also able to perform synthetic reactions. In our work we focused on performing transesterification reactions with FAEs. The synthesis of butyl ferulate (BFA) from methyl ferulate (MFA) was chosen as a model reaction.Reduced water content of the reaction system, needed for the transesterification reaction to happen, can be achieved by replacing buffer with solvents. However, solvents can have a deleterious effect on the biocatalyst. Therefore, the use of ionic liquids instead of solvents was investigated. In addition, the enzymes were immobilized on MPS. In order to achieve a good immobilization yield and a good immobilized activity of the FAEs, several parameters were varied, and enzyme activity and selectivity were assessed. Since the reaction of interest was transesterification, the selectivity of the enzyme was quantified by determining the molar ratio between the product of transesterification reaction and the product of hydrolysis reaction: BFA/FA. Kinetic parameters, stability and reusability of the immobilized biocatalyst were also investigated. We found that the properties of enzyme themselves influence the immobilization process as well as the enzyme performance. Enzymes having a different isoelectric point or bearing different surface modifications such as glycosylations have different behaviors both in terms of enzyme activity and of immobilization performance.
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| 15. |
- Janssen, Mathias, 1973, et al.
(författare)
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Guiding technology development using LCA: The case of bio-based adipic acid production
- 2015
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Ingår i: ISIE Conference 2015.
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Konferensbidrag (refereegranskat)abstract
- Moving from a fossil-based to a bio-based economy requires the development of new technologies for the production of bio-based chemicals and materials. These technologies may become part of novel biorefinery concepts that combine the production of bulk and fine chemicals. This paper presents a life cycle assessment (LCA) of such a novel concept in which forest residues and micro-algae are used as feedstock for the combined production of adipic acid and high value-added chemicals. Adipic acid is mainly used as a precursor in the production of nylon, and its current fossil-based production process emits significant quantities of nitrous oxide (N2O), a highly potent greenhouse gas. There is thus a great potential to reduce the global warming potential of the production of adipic acid, and consequently nylon.The novel biorefinery concept is the main target of a multi-disciplinary R&D project. The concept is in a very early stage of development which mostly consists of experimental lab work. There are some challenges that must be overcome to evaluate the concept using life cycle assessment at this early development stage. Scaling up lab results to an industrially relevant process capacity needs to be addressed in order to e.g. account for changes in yield. This can be done using process modeling and simulation. The total scale of production is another aspect which is important for the assessment of bio-based products, because the environmental impact of biomass production is not linearly dependent on the amount harvested. Furthermore, the results of the assessment need to be communicated in a meaningful way to the stakeholders who have different backgrounds and are often not familiar with LCA. The paper will discuss these challenges for the case of bio-based adipic acid production using the novel biorefinery concept, and will present results from the assessment and compare these to the case of fossil-based adipic acid production.
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| 16. |
- Jiang, Wen, et al.
(författare)
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Liquefaction of lignocellulosic materials and its applications in wood adhesives — A review
- 2018
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Ingår i: Industrial crops and products (Print). - : Elsevier. - 0926-6690 .- 1872-633X. ; 124, s. 325-342
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Tidskriftsartikel (refereegranskat)abstract
- Liquefaction, a useful method of turning whole biomass into liquids, provides advantages for energy andpolymers and finds applications in many sectors. This paper reviews the different liquefaction technologies andrecent advances in the development of sustainable wood adhesives. Current liquefaction technologies includehydrothermal liquefaction (HTL) and moderate acid-catalyzed liquefaction (MACL). HTL produces bio-oils asprimary products, and solid residues and gases as by-products. MACL depends on the solvent types used, whichare grouped to polyhydric alcohols and phenols. Bio-polyols from alcohol liquefaction, phenolated biomass fromphenol liquefaction and phenolic compounds rich-HTL bio-oils have been used in the production of liquefiedbiomass-based adhesives, which have shown competitive properties but face challenges for industrial uses. Yet, abetter understanding of reaction pathways and optimization of the liquefaction processes is needed.
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| 17. |
- Salomon Johansen, Katja, 1969
(författare)
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Oxidoreductases in biomass saccharification processes
- 2015
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Ingår i: Oral presentation, 37th Symposium on biotechnology for fuels and chemicals, San Diego, US 2015.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The inauguration of several commercial scale lignocellulosic ethanol factories within the past few months is a clear sign of the emergence of an industry. The development of an industry is likely to call for further studies of the fundamental principles governing the processes in order to facilitate further optimisation and thus competitive edge. Although impressive improvement in the efficiency of enzyme cocktails for lignocellulose deconstruction has been achieved already, the understanding of this process step is still incomplete. A recent development is the discovery of lytic polysaccharide monooxygenases which has opened a vide avenue for further studies of the mechanism by which plant cell wall are deconstructed in nature as well as in industrial settings. This is truly a new avenue within cellulase research which was in the past exclusively devoted to the study of hydrolases. Other oxidoreductases may also have a role to play. In nature, cellulytic LPMOs work in cooperation with another oxidoreductase, namely cellobiose dehydrogensase, but how the enzymes interact is inadequately described. In addition it has also been reported that laccase and peroxidase may have a positive effect on the saccharification efficiency but the mechanism behind is not understood in detail. The use of oxidoreductases in the production of lignocellulosic ethanol will be discussed in light of current scientific thinking and new insights.
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| 18. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Bottlenecks in lignocellulosic ethanol production: xylose fermentation and cell propagation
- 2017
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Ingår i: European biomass conference 2017, 25th edition, June 12-15; Stockholm, Sweden..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A remaining challenge for the development of economically feasible 2nd generation bio-ethanol is low xylose consumption rate and inhibitor tolerance of the utilized Saccharomyces cerevisiae strains. Yeast starter cultures produced for ethanol production in simultaneous saccharification and co-fermentation (SSCF) processes have to meet high, seemingly conflicting requirements. A high biomass yield during propagation is required to produce the high cell concentrations required for the harsh conditions in the proceeding fermentation. Inhibitor tolerance is essential for producing a highly viable starter culture as well as favorable fermentation kinetics. Short-term adaptation of yeast cultures during propagation has been shown to have a positive effect on pentose conversion as well as inhibitor tolerance. Here we propose a model propagation strategy for evaluating physiology of yeast cultures during propagation. This model propagation strategy will be implemented in a study comparing physiology of yeast cultures with and without exposure to lignocellulosic inhibitors during propagation to assess what molecular mechanisms underlie the short-term adaptation response phenotype. For industry, a better control of yeast properties during propagation will result in an improved and consistent performance of yeast starter cultures for SSCF purposes.
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| 19. |
- Skoog, Emma, 1983
(författare)
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Biobased Adipic Acid - Challenges in Establishing a Cell Factory
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Growing concern regarding climate change calls for sustainable solutions to significantly reduce our dependency on non-renewable fossil-based raw materials. One potential solution is the development of biorefineries where biobased, renewable raw materials are converted into valuable products via enzymatic, chemical or microbial conversion. This work focuses on the microbial production of adipic acid, a precursor in the nylon industry, currently derived from fossil-based raw material. No known naturally occurring microorganism is able to produce adipic acid, and genetic engineering of a suitable host is therefore required. The aim of the work presented in this thesis was to engineer a microorganism for the production of adipic acid from glucose, more specifically, from glucose streams derived from lignocellulosic forest residues. Theoretical evaluation of metabolic pathways for adipic acid production revealed several obstacles, including redox imbalance and the discovery or engineering of enzymes to catalyze novel reactions. Mining of enzyme databases for alternative paths proved fruitful, and the number of biochemical reactions in the lysine pathway employing as yet unidentified enzymes was reduced from three to two, without affecting the thermodynamics of the pathway. A combined approach of in vitro and in silico analysis suggested potential enzyme engineering strategies for one of the reactions, for which there are as yet no identified enzymes, namely, the reduction of unsaturated α,β bonds of 6-aminohex-2-enoic acid and 2-hexenedioic acid. When defining a suitable host for microbial adipic acid production, tolerance to high concentrations of adipic acid (50-100 g L-1) is important to ensure an economically feasible process, preferably at low pH (below 5) to further reduce the overall process cost. Screening of bacteria, yeasts and a filamentous fungus grown in increasing concentrations of adipic acid (0-100 g L-1) and at different pH revealed Candida viswanathii to be a promising host to engineer for adipic acid production. A comparative study of C. viswanathii with Saccharomyces cerevisiae in controlled batch cultivations at increasing adipic acid concentrations (0-95 g L-1) and low pH (pH 4 and pH 5) revealed significant differences in their tolerance to adipic acid; C. viswanathii being able to grow, almost unaffected, under all the conditions investigated, whereas S. cerevisiae was unable to grow at 95 g L-1. Lipid analysis of their cell membranes revealed C. viswanathii to have a thicker and more compact cell membrane, which is probably less permeable to adipic acid.
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| 20. |
- Hao, Nanjing, et al.
(författare)
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Glyconanomaterials for biosensing applications
- 2016
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 76:15, s. 113-130
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Tidskriftsartikel (refereegranskat)abstract
- Nanomaterials constitute a class of structures that have unique physiochemical properties and are excellent scaffolds for presenting carbohydrates, important biomolecules that mediate a wide variety of important biological events. The fabrication of carbohydrate-presenting nanomaterials, glyconanomaterials, is of high interest and utility, combining the features of nanoscale objects with biomolecular recognition. The structures can also produce strong multivalent effects, where the nanomaterial scaffold greatly enhances the relatively weak affinities of single carbohydrate ligands to the corresponding receptors, and effectively amplifies the carbohydrate-mediated interactions. Glyconanomaterials are thus an appealing platform for biosensing applications. In this review, we discuss the chemistry for conjugation of carbohydrates to nanomaterials, summarize strategies, and tabulate examples of applying glyconanomaterials in in vitro and in vivo sensing applications of proteins, microbes, and cells. The limitations and future perspectives of these emerging glyconanomaterials sensing systems are furthermore discussed.
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| 21. |
- Aryapratama, Rio, et al.
(författare)
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Prospective life cycle assessment of bio-based adipic acid production from forest residues
- 2017
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Ingår i: Journal of Cleaner Production. - : Elsevier BV. - 0959-6526. ; 164, s. 434-443
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Tidskriftsartikel (refereegranskat)abstract
- Abstract Environmental concerns related to the production of bulk chemicals are growing. Researchers and technology developers are currently looking into alternative production pathways for such chemicals by utilizing renewable resources, such as lignocellulosic feedstocks. Adipic acid is an example of such a bulk chemical, and its conventional fossil-based production emits significant amounts of N2O, a major greenhouse gas. In this study, a prospective life cycle assessment (LCA) of bio-based adipic acid production from forest residues at an early development stage, situated in Sweden, was conducted. Acid-catalyzed (using SO2) and alkaline (using NaBH4) pretreatment were employed and scenarios and sensitivity analyses were conducted. The potential environmental impacts of this technology under development were compared to those of conventional adipic acid production. The results show that bio-based adipic acid production has a lower impact on global warming, eutrophication and photochemical ozone creation compared to fossil-based production. In contrast, it has a higher impact on acidification. An increased efficiency of mitigating \{N2O\} emissions from the fossil-based production may alter this comparison. Producing bio-based adipic acid using the alkaline pretreatment has a higher environmental impact than producing it using acid-catalyzed pretreatment. Furthermore, if biomass is used to fulfil process energy demands, instead of fossil fuel, the environmental impact of the bio-based production decreases. It is therefore important to reduce the amount of NaBH4 used in the alkaline pretreatment or to lower the environmental impact of its production.
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| 22. |
- Torén, Johan, et al.
(författare)
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Food waste as a resource for bio-based chemicals and materials in Sweden
- 2019
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Waste, also food waste, abound. From an environmental point of view food waste should first and foremost be avoided. However, the waste that inevitably is produced along the food production chain should be utilized to the best of our ability. One option is to produce biobased chemicals and materials from the waste through biological processes. This study looks into what food waste resources are available for such production, industrial fermentation, in Sweden, from waste emanating in primary production all the way through to final consumption. In addition, drivers for waste generation, influencing institution and waste market characteristics are assessed.
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| 23. |
- Teixeira, Paulo, 1990
(författare)
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Engineering Lipid Metabolism for Production of Oleochemicals in Saccharomyces cerevisiae
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oleochemicals are chemicals usually derived from plant oils or animal fat. Large use of plant oil derivatives as replacements for petroleum-derived chemicals brings sustainability issues from extensive cultivation of oil plants in restricted regions. This project studied and developed the baker’s yeast Saccharomyces cerevisiae as a platform for sustainable production of oleochemical precursors. The first part of this work studied the dynamics of free fatty acids (FFAs) production. First, an alternative fatty acid synthesis system based on the reverse β-oxidation pathway was evaluated for its in vivo function but concluding that it was not an efficient route for fatty acid synthesis. The subsequent studies were based on high level production of FFA and secretion to the extracellular medium through removal of acyl-CoA synthase activity by deleting the FAA1-4 genes. This phenotype was coupled to a pathway that converts FFA to fatty alcohols, which allowed the observation that while FFA are more efficiently converted to fatty alcohols during growth on glucose, the production of FFA is highly increased during growth on ethanol. Fine-tuning of FAA1 expression resulted in improved production of fatty alcohols without FFA secretion in this strain. Following up, the pathways leading to FFA formation in a Δfaa1 Δfaa4 background were studied through construction of a strain with a constrained lipid metabolism network. It was observed that upon removing storage lipid formation, phospholipid synthesis had a strong correlation with FFA production and FFA formation was mostly derived from phospholipid hydrolysis. On the second part of this work, S. cerevisiae was engineered for the highest TAG production levels reported so far. This relied on overexpressing genes involved in malonyl-CoA supply and TAG synthesis from acyl-CoA, and removing genes involved in TAG hydrolysis, β-oxidation and glycerol-3-phosphate usage. On a second approach, TAG accumulation properties were further improved in these strains through enhancing lipid droplet assembly processes. This was achieved through expression of perilipins and FIT proteins and through stimulation of ER stress mechanisms. In conclusion, lipid metabolism is an important part of cell homeostasis and engineering this system requires overcoming its tight regulation networks and mastering the processes involved in the physical structural organization of the system. Here this was highlighted using both knowledge-driven studies and engineering approaches, leading to important advancements in the field.
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| 24. |
- Wang, Zhao, 1989-
(författare)
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Recalcitrance of wood to biochemical conversion : feedstock properties, pretreatment, saccharification, and fermentability
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulose is an inexpensive and abundant renewable resource that can be used to produce advanced biofuels, green chemicals, and other bio-based products. Pretreatment and efficient enzymatic saccharification are essential features of bioconversion of lignocellulosic biomass. The aims of the research were to achieve a better understanding of the recalcitrance of woody biomass to bioconversion, to explore different pretreatment techniques that can be used to decrease the recalcitrance of the biomass and improve the digestibility of the cellulose, and to investigate by-products of acid pretreatment that cause enzymes and microorganisms to work less efficiently.The recalcitrance of wood from aspen, birch, and spruce was investigated before and after acid pretreatment. Before pretreatment, birch exhibited the highest recalcitrance, which was attributed to structural factors. After pretreatment, spruce showed the highest recalcitrance, which was attributed to chemical factors, such as high lignin content. Deacetylation of hybrid aspen in planta by a CE5 acetyl xylan esterase decreased the recalcitrance, and the glucose yield of enzymatic saccharification of non-pretreated wood increased with 27%.Pretreatment options based on ionic liquids and steam explosion were further explored. The effects of the anionic constituents of a series of imidazolium-based ionic liquids on pretreatment of aspen and spruce were investigated. [HSO4]− was efficient only for aspen, which was attributed to acid degradation of xylan. [MeCO2]− was efficient for both aspen and spruce, which was attributed to its capability to create a disordered cell wall structure rather than to removal of lignin and hemicellulose. A comparison was made between using sulfuric acid and sulfur dioxide for pretreatment of spruce. Although sulfur dioxide resulted in a pretreatment liquid that was more inhibitory to both enzymes and yeast, it was still superior to pretreatment with sulfuric acid, a phenomenon that was attributed to the particle size of the pretreated material.In a comparison of microbial inhibitors in pretreatment liquids from steam explosion of spruce, formaldehyde was found to be the most important inhibitor of yeast. Enzyme inhibition by catalytically non-productive adsorption to lignins and pseudo-lignin was investigated using quantitative proteomics. The results indicate that protein adsorption to pseudo-lignin can be as extensive as adsorption to real lignin.
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| 25. |
- Li, Xue-Yuan, et al.
(författare)
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Combination of modern plant breeding and enzyme technology to obtain highly enriched erucic acid from Crambe oil
- 2016
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Ingår i: Sustainable Chemical Processes. - : Springer Science and Business Media LLC. - 2043-7129. ; 4
-
Tidskriftsartikel (refereegranskat)abstract
- Background Fatty acids from vegetable oils are useful building blocks for industrial materials. The purpose of this work was to prepare erucic acid with high purity from a vegetable oil. High purity erucic acid is used for the production of erucamide with applications in plastics manufacturing. A newly developed transgenic Crambe line produces seed oil with 68% erucic acid compared to 53% in the wild type oil. Results Further enrichment of erucic acid from Crambe (wild type and transgenic) oil was achieved by selective enzymatic hydrolysis. UsingCandida rugosalipase as catalyst, other fatty acids were preferentially hydrolysed from the triacylglycerols and erucic acid was enriched in the acylglycerol fraction. The highest content of erucic acid achieved in that fraction was 95%. Conclusions The combination of modern plant breeding and enzyme technology is a promising approach for preparation of fatty acids of high purity.
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| 26. |
- Bonzom, Cyrielle, 1987, et al.
(författare)
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Feruloyl esterase immobilization in mesoporous silica: hydrolysis and transesterifications reactions.
- 2015
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Mesoporous silica materials (MPS) are an interesting choice as support to immobilize enzymes because MPS offer unique properties such as high enzyme loading and tunable pore size. They also provide the enzyme with a sheltered environment therefore reducing the risks of denaturation in industrial applications.Immobilization parameters such as pH, buffer and pore size of the material were investigated. Among them, the chemical composition of the buffer as well as its pH proved to be critical resulting in enzyme loadings varying from nearly zero up to 0.025 mgenzyme.mgMPS-1.Selectivity of the enzyme, a feruloyl esterase (FAE), was investigated by quantifying the molar ratio between the transesterification and hydrolysis products, namely butyl ferulate (BFA) and ferulic acid (FA). The reaction of interest was transesterification therefore hydrolysis was an unwanted side-reaction. The immobilization pH and the water content of the reaction were the most influent parameters inducing variation up to 4-fold of the BFA/FA molar ratio.Optimal reaction conditions and kinetic parameters of the free and immobilized enzyme were determined for both hydrolysis and transesterification to determine in which conditions transesterification is prevailing. While optimal pHs were similar for all studied, temperature optimums varied from 25 to 50°C. Interestingly the Km of the FAE was not affected upon immobilization, but the kcat was decreased 10-fold resulting in a lower catalytic efficiency. Km was 100-fold higher for transesterification than for hydrolysis whereas kcat was 100-fold lower; this resulted in a drastic reduction of the catalytic efficiency of the FAE.Stability of the enzyme was evaluated using the hydrolysis reaction. No significant improvement could be observed for the immobilized enzyme. Reusability of the immobilized biocatalyst was determined during 10-cycles of 48h. A decrease in activity was observed during the course of the experiment. In addition a decrease in the BFA/FA molar ratio indicating a shift in enzyme specificity happened.
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| 27. |
- Zhang, Yiming, 1986
(författare)
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Engineering cytosolic acetyl-CoA metabolism in Saccharomyces cerevisiae
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for biochemical production. However, it cannot grow on glucose as the sole carbon source due to the lack of cytosolic acetyl-CoA for lipid biosynthesis. Its growth inability on glucose could be restored through directed evolution, which was explained by an in-frame internal deletion in MTH1 (MTH1-∆T). The MTH1-∆T allele resulted in reduced glucose uptake, which may attenuate the repression of respiratory metabolism. However, it was not clear what mechanism could provide the cells with sufficient precursors for cytosolic acetyl-CoA. Here we investigated this using a Pdc negative strain with MTH1-∆T, IMI076. Our results identified a route relying on Ach1 that could transfer acetyl units from mitochondria to the cytoplasm. Based on the results a new model was proposed, in which acetyl units are shuttled from the mitochondria to the cytoplasm in the form of acetate. In addition, a collection of Pdc negative strains was constructed and one of them was adaptively evolved on glucose via serial transfer. Three independently evolved strains were obtained, which can grow on glucose as the sole carbon source at maximum specific rates of 0.138 h-1, 0.148 h-1, 0.141 h-1, respectively. Several genetic changes were identified in the evolved Pdc negative strains by genome sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1, HXT2, CIT1, and RPD3. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH181D allele restored its growth on glucose at a maximum specific rate of 0.05 h-1 in minimal medium with 2% glucose. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain. In connection with biobased chemical production, it is necessary to engineer the metabolism of cell factories such that the raw material, typically sugars, can be efficiently converted to the product of interest. Although IMI076 could grow on glucose, it was still inefficient at conversion of pyruvate to cytosolic acetyl-CoA. To increase cytosolic acetyl-CoA supply from pyruvate, pyruvate formate lyase and its activating enzyme from Escherichia coli were expressed with two different cofactors, ferredoxin or flavodoxin, and their reductase, respectively, and it was found that the co-expression of either of these cofactors had a positive effect on growth under aerobic conditions, indicating increased activity of PFL. The positive effect on growth was manifested as a higher final biomass concentration and a significant increase in transcription of formate dehydrogenase genes (FDHs). Among the two cofactors reduced flavodoxin was found to be a better electron donor than reduced ferredoxin.
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| 28. |
- Chmielarz, Mikolaj, et al.
(författare)
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FT-NIR: a tool for rapid intracellular lipid quantification in oleaginous yeasts
- 2019
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundLipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming. Therefore, in most cases just endpoint measurements of lipid accumulation are performed and kinetics of intracellular lipid accumulation is difficult to follow. To address this, we created a prediction model using Fourier-transform near-infrared (FT-NIR) spectroscopy. This method allows to measure lipid content in yeast.MethodsThe FT-NIR calibration sets were constructed from spectra of freeze-dried cells of the oleaginous yeasts Rhodotorula toruloides CBS 14, Lipomyces starkeyi CBS 1807 and Yarrowia lipolytica CBS 6114. The yeast cells were obtained from different cultivation conditions. Freeze-dried cell pellets were scanned using FT-NIR in the Multi Purpose Analyser (MPA) from Bruker. The obtained spectra were assigned corresponding to total fat content, obtained from lipid extraction using a modified Folch method. Quantification models using partial least squares (PLS) regression were built, and the calibration sets were validated on independently cultivated samples. The R. toruloides model was additionally tested on Rhodotorula babjevae DBVPG 8058 and Rhodotorula glutinis CBS 2387.ResultsThe R-2 of the FT-NIR model for R. toruloides was 98%, and the root mean square error of cross-validation (RMSECV) was 1.53. The model was validated using a separate set of R. toruloides samples with a root mean square error of prediction (RMSEP) of 3.21. The R-2 of the Lipomyces model was 96%, with RMSECV 2.4 and RMSEP 3.8. The R-2 of the mixed model, including all tested yeast strains, was 90.5%, with RMSECV 2.76 and RMSEP 3.22, respectively. The models were verified by predicting the total fat content in newly cultivated and freeze-dried samples. Additionally, the kinetics of lipid accumulation of a culture were followed and compared with standard lipid extraction methods.ConclusionsUsing FT-NIR spectroscopy, we have developed a faster, less laborious and non-destructive quantification of yeast intracellular lipid content compared to methods using lipid extraction.
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| 29. |
- Nordlander, Eva, et al.
(författare)
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Investigating the possibility of applying an ADM1 based model to a full-scale co-digestion plant
- 2017
-
Ingår i: Biochemical engineering journal. - : Elsevier. - 1369-703X .- 1873-295X. ; 120, s. 73-83
-
Tidskriftsartikel (refereegranskat)abstract
- This study investigated the possibility of using a model based on the anaerobic digestion model no. 1 (ADM1) on a full-scale 4000 m3 digester in order to understand how such theoretical models can be applied to a real industrial process. The industrial scale digester co-digests the organic fraction of municipal solid waste, grease trap sludge, and ley crop silage with varying feed rates and amounts of volatile solids. A year of process data was collected. Biogas flow, methane content/flow, and ammonia nitrogen were the variables that the model was best at predicting (index of agreement at 0.78, 0.61/0.77, and 0.68, respectively). The model was also used to investigate the effect of increasing the volatile solids (VS) concentration entering the digester. According to simulation results, increasing the influent VS concentration will increase biogas and methane outflow (from 1.5 million Nm3 methane to more than 2 million Nm3 methane), but decrease the amounts of biogas/methane per unit of volatile solids (from about 264 Nm3 methane per tonne VS to below 215 Nm3 methane per tonne VS).
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| 30. |
- Barzegar, Hamid Reza, et al.
(författare)
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Spontaneous twisting of a collapsed carbon nanotube
- 2017
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Ingår i: Nano Reseach. - : Tsinghua University Press. - 1998-0124 .- 1998-0000. ; 10:6, s. 1942-1949
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Tidskriftsartikel (refereegranskat)abstract
- We study the collapsing and subsequent spontaneous twisting of a carbon nanotube by in situ transmission electron microscopy (TEM). A custom-sized nanotube is first created in the microscope by selectively extracting shells from a parent multi-walled tube. The few-walled, large-diameter daughter nanotube is driven to collapse via mechanical stimulation, after which the ribbon-like collapsed tube spontaneously twists along its long axis. In situ diffraction experiments fully characterize the uncollapsed and collapsed tubes. The experimental observations and associated theoretical analysis indicate that the origin of the twisting is compressive strain.
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| 31. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
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Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society. However, the substitution of fossil based biochemical with its renewable counterpart requires the production to be significantly more efficient and price competitive. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulose hydrolysate. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. Numerous attempts have been reported to improve the stress tolerance of Saccharomyces cerevisiae by different bioengineering strategies such as deletion/overexpression of genes. However, the inability to achieve a fine balance of the transcriptional expression of the target and the ancillary gene(s) is one of the major factors that impedes the efficiency of many of these strategies. In this project, we apply CRISPR interference (CRISPRi) technology to investigate the potential of fine-tuning the expression of genes that are related to the stress regulation. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using a sgRNA. Strains with altered regulation will be screened for inhibitor tolerance. Furthermore, transcriptomics analysis of tolerant mutants will be conducted to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test-learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate. Here, we will show data on a methodology that we have developed for studying hydrolysate tolerance, adaptation and ethanol production capacity at microscale, directly in lignocellulosic hydrolysates.
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| 32. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2019
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society. However, the substitution of fossil-based chemicals with renewable biochemicals requires the production to be significantly more efficient and price competitive. Remediation of several technical bottlenecks is needed before this can be accomplished. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulosic hydrolysates. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. In this project, we are trying to fine tune the expression of stress related genes to boost the stress tolerance in Saccharomyces cerevisiae using the CRISPR interference (CRISPRi) technology. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using an sgRNA. Using a high-throughput yeast transformation method developed in our laboratory, we are generating a CRISPRi strain library. Each strain in this library has altered regulation for at-least one stress related gene. Next, high-throughput phenotypic evaluation of this library is performed by growing the strains under the exposure of inhibitors relevant to lignocellulosic hydrolysates. Here, we will demonstrate our primary CRISPRi library data. Further, we will explain the high-throughput methodologies for generating the CRISPRi mutants and to study their hydrolysate tolerance, adaptation and ethanol production capacity at microscale. In future, we will perform transcriptomics analysis of the most tolerant mutants to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test-learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate.
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| 33. |
- Grigorenko, L. V., et al.
(författare)
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Exploring the manifestation and nature of a dineutron in two-neutron emission using a dynamical dineutron model
- 2018
-
Ingår i: Physical Review C. - 2469-9985 .- 2469-9993. ; 97:3
-
Tidskriftsartikel (refereegranskat)abstract
- Emission of two neutrons or two protons in reactions and decays is often discussed in terms of "dineutron" or "diproton" emission. The discussion often leans intuitively on something described by Migdal-Watson approximation. In this work we propose a way to formalize situations of dineutron emission. It is demonstrated that properly formally defined dineutron emission may reveal properties which are drastically different from those traditionally expected, and properties which are actually observed in three-body decays.
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| 34. |
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| 35. |
- García-Hidalgo, Javier, et al.
(författare)
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Identification of the two-component guaiacol demethylase system from Rhodococcus rhodochrous and expression in Pseudomonas putida EM42 for guaiacol assimilation
- 2019
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9:1
-
Tidskriftsartikel (refereegranskat)abstract
- A diversity of softwood lignin depolymerization processes yield guaiacol as the main low molecular weight product. This key aromatic compound can be utilized as a carbon source by several microbial species, most of which are Gram positive bacteria. Microbial degradation of guaiacol is known to proceed initially via demethylation to catechol, and this reaction is catalyzed by cytochrome P450 monooxygenases. These enzymes typically require a set of redox partner proteins, whose number and identities were not described until very recently in the case of guaiacol. In this work we identified two proteins involved in guaiacol demethylation by the actinomycete Rhodococcus rhodochrous. Additionally, we constructed four different polycistronic operons carrying combinations of putative redox partners of this guaiacol demethylation system in an inducible expression plasmid that was introduced into the Gram negative host Pseudomonas putida EM42, and the guaiacol consumption dynamics of each resulting strain were analyzed. All the polycistronic operons, expressing a cytochrome P450 together with a putative ferredoxin reductase from R. rhodochrous and putative ferredoxins from R. rhodochrous or Amycolatopsis ATCC 39116 enabled P. putida EM42 to metabolize and grow on guaiacol as the sole carbon source.
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| 36. |
- Ma, Qian, et al.
(författare)
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New fluorescently labeled auxins exhibit promising anti-auxin activity
- 2019
-
Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 48, s. 44-52
-
Tidskriftsartikel (refereegranskat)abstract
- The plant hormone auxin is a key player in the regulation of plant growth and development. Despite numerous studies devoted to understanding its role in a wide spectrum of physiological processes, full appreciation of its function is linked to a comprehensive determination of its spatio-temporal distribution, which plays a crucial role in its mode of action. Conjugation of fluorescent tracers to plant hormones enables sensitive and specific visualization of their subcellular and tissue-specific localization and transport in planta, which represents a powerful tool for plant physiology. However, to date, only a few fluorescently labeled auxins have been developed. We report the synthesis of four novel fluorescently labeled derivatives of indole-3-acetic acid (IAA) in the form of a conjugate with a nitrobenzoxadiazole (NBD) fluorophore together with validation of their biological activity. These compounds, unlike other previously reported auxins fluorescently labeled at N1 position (nitrogen of the indole ring), do not possess auxin activity but rather show dose-dependent inhibition of auxininduced effects, such as primary root growth inhibition, root hair growth and the auxin reporter DR5::GUS expression. Moreover, the study demonstrates the importance of the character of the linker and optimal choice of the labeling site in the preparation of fluorescently labeled auxins as important variables influencing their biological activity and fluorescent properties.
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| 37. |
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| 38. |
- Wilhelmsson, Marcus, 1974, et al.
(författare)
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Preface
- 2016
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Ingår i: Fluorescent Analogs of Biomolecular Building Blocks: Design and Applications. - Hoboken, NJ, USA : John Wiley & Sons, Inc. - 9781119179320 ; , s. xvii -xxii
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Fluorescent Analogs of Biomolecular Building Blocks focuses on the design of fluorescent probes for the four major families of macromolecular building blocks. Compiling the expertise of multiple authors, this book moves from introductory chapters to an exploration of the design, synthesis, and implementation of new fluorescent analogues of biomolecular building blocks, including examples of small-molecule fluorophores and sensors that are part of biomolecular assemblies.
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| 39. |
- Abdelaziz, Omar Y., et al.
(författare)
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Biological valorization of low molecular weight lignin
- 2016
-
Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 34:8, s. 1318-1346
-
Forskningsöversikt (refereegranskat)abstract
- Lignin is a major component of lignocellulosic biomass and as such, it is processed in enormous amounts in the pulp and paper industry worldwide. In such industry it mainly serves the purpose of a fuel to provide process steam and electricity, and to a minor extent to provide low grade heat for external purposes. Also from other biorefinery concepts, including 2nd generation ethanol, increasing amounts of lignin will be generated. Other uses for lignin – apart from fuel production – are of increasing interest not least in these new biorefinery concepts. These new uses can broadly be divided into application of the polymer as such, native or modified, or the use of lignin as a feedstock for the production of chemicals. The present review focuses on the latter and in particular the advances in the biological routes for chemicals production from lignin. Such a biological route will likely involve an initial depolymerization, which is followed by biological conversion of the obtained smaller lignin fragments. The conversion can be either a short catalytic conversion into desired chemicals, or a longer metabolic conversion. In this review, we give a brief summary of sources of lignin, methods of depolymerization, biological pathways for conversion of the lignin monomers and the analytical tools necessary for characterizing and evaluating key lignin attributes.
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| 40. |
- Belgrano, F. D.S., et al.
(författare)
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EPS production by Propionibacterium freudenreichii facilitates its immobilization for propionic acid production
- 2018
-
Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 125:2, s. 480-489
-
Tidskriftsartikel (refereegranskat)abstract
- Aims: Immobilization of microbial cells is a useful strategy for developing high cell density bioreactors with improved stability and productivity for production of different chemicals. Functionalization of the immobilization matrix or biofilm forming property of some strains has been utilized for achieving cell attachment. The aim of the present study was to investigate the production of exopolysaccharide (EPS) by Propionibacterium freudenreichii C.I.P 59.32 and utilize this feature for immobilization of the cells on porous glass beads for production of propionic acid. Methods and Results: Propionibacterium freudenreichii was shown to produce both capsular and excreted EPS during batch cultivations using glucose as carbon source. Different electron microscopy techniques confirmed the secretion of EPS and formation of cellular aggregates. The excreted EPS was mainly composed of mannose and glucose in a 5·3 : 1 g g−1 ratio. Immobilization of the cells on untreated and polyethyleneimine (PEI)-treated Poraver beads in a bioreactor was evaluated. Higher productivity and yield of propionic acid (0·566 g l−1 h−1 and 0·314 g g−1, respectively) was achieved using cells immobilized to untreated beads and EPS production reached 617·5 mg l−1 after 48 h. Conclusion: These results suggest an important role of EPS-producing strains for improving cell immobilization and propionic acid production. Significance and Impact of the Study: This study demonstrates the EPS-producing microbe to be easily immobilized on a solid matrix and to be used in a bioprocess. Such a system could be optimized for achieving high cell density in fermentations without the need for functionalization of the matrix.
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| 41. |
- Brink, Daniel P, et al.
(författare)
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Real-time monitoring of the sugar sensing in Saccharomyces cerevisiae indicates endogenous mechanisms for xylose signaling
- 2016
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 15:183
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Tidskriftsartikel (refereegranskat)abstract
- The sugar sensing and carbon catabolite repression in Baker’s yeast Saccharomyces cerevisiae is governed by three major signaling pathways that connect carbon source recognition with transcriptional regulation. Here we present a screening method based on a non-invasive in vivo reporter system for real-time, single-cell screening of the sugar signaling state in S. cerevisiae in response to changing carbon conditions, with a main focus on the response to glucose and xylose.ResultsThe artificial reporter system was constructed by coupling a green fluorescent protein gene (yEGFP3) downstream of endogenous yeast promoters from the Snf3p/Rgt2p, SNF1/Mig1p and cAMP/PKA signaling pathways: HXT1p/2p/4p; SUC2p, CAT8p; TPS1p/2p and TEF4p respectively. A panel of eight biosensors strains was generated by single copy chromosomal integration of the different constructs in a W303-derived strain. The signaling biosensors were validated for their functionality with flow cytometry by comparing the fluorescence intensity (FI) response in the presence of high or nearly depleted glucose to the known induction/repression conditions of the eight different promoters. The FI signal correlated with the known patterns of the selected promoters while maintaining a non-invasive property on the cellular phenotype, as was demonstrated in terms of growth, metabolites and enzyme activity.ConclusionsOnce verified, the sensors were used to evaluate the signaling response to varying conditions of extracellular glucose, glycerol and xylose by screening in 96-well microtiter plates. We show that these yeast strains, which do not harbor any recombinant pathways for xylose utilization, are lacking a signaling response for extracellular xylose. However, for the HXT2p/4p sensors, a shift in the flow cytometry population dynamics indicated that internalized xylose does affect the signaling. These results suggest that the previously observed effects of this pentose on the S. cerevisiae physiology and gene regulation can be attributed to xylose and not only to a lack of glucose.
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| 42. |
- Chandolias, Konstantinos, 1985-
(författare)
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Lignocellulosic Biorefinery for Biohydrogen and Carboxylic Acids Production in Flexible Membrane Bioreactor and Two-stage System
- 2017
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Ingår i: 7th Nordic Wood Biorefinery Conference. 28-30 March 2017. Stockholm.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic biorefineries can produce numerous biofuels and chemicals via the anaerobic digestion process. Although several works have been recently conducted on this field, the technology is considered new and more research efforts are required towards industrialisation. In this work, wheat straw was digested after hydrolysis with dilute phosphoric acid. The substrate was biologically converted into carboxylic acids and biohydrogen at different OLRs (4.42-17.95 g COD/L.d). The semi-continuous experiments took place at 55 °C, both in reactors with free-cells or mixed free and membrane-encased cells, According to the results, the optimum biohydrogen, acetic and isobutyric acid yields were obtained at OLR of 4.42 g COD/L.d. Moreover, the highest lactic acid production was recorded at OLR of 9.33 g COD/L.d. Furthermore, a reactor containing both free and membrane-encased cells showed 60% higher lactic acid production (at OLR of 13.42 g COD/L.d) in comparison to the conventional free cell reactor. In addition, the production of acetic and isobutyric acid was greatly improved by a two-stage system. The use of both free and encased cells in a flexible membrane system along with the two-stage system for the optimisation of the process is the main novelty of this work.
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| 43. |
- de las Heras, Alejandro Muñoz, et al.
(författare)
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Anaerobic poly-3-d-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase
- 2016
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Poly-3-d-hydroxybutyrate (PHB) that is a promising precursor for bioplastic with similar physical properties as polypropylene, is naturally produced by several bacterial species. The bacterial pathway is comprised of the three enzymes β-ketothiolase, acetoacetyl-CoA reductase (AAR) and PHB synthase, which all together convert acetyl-CoA into PHB. Heterologous expression of the pathway genes from Cupriavidus necator has enabled PHB production in the yeast Saccharomyces cerevisiae from glucose as well as from xylose, after introduction of the fungal xylose utilization pathway from Scheffersomyces stipitis including xylose reductase (XR) and xylitol dehydrogenase (XDH). However PHB titers are still low. Results: In this study the acetoacetyl-CoA reductase gene from C. necator (CnAAR), a NADPH-dependent enzyme, was replaced by the NADH-dependent AAR gene from Allochromatium vinosum (AvAAR) in recombinant xylose-utilizing S. cerevisiae and PHB production was compared. A. vinosum AAR was found to be active in S. cerevisiae and able to use both NADH and NADPH as cofactors. This resulted in improved PHB titers in S. cerevisiae when xylose was used as sole carbon source (5-fold in aerobic conditions and 8.4-fold under oxygen limited conditions) and PHB yields (4-fold in aerobic conditions and up to 5.6-fold under oxygen limited conditions). Moreover, the best strain was able to accumulate up to 14% of PHB per cell dry weight under fully anaerobic conditions. Conclusions: This study reports a novel approach for boosting PHB accumulation in S. cerevisiae by replacement of the commonly used AAR from C. necator with the NADH-dependent alternative from A. vinosum. Additionally, to the best of our knowledge, it is the first demonstration of anaerobic PHB synthesis from xylose.
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| 44. |
- Dishisha, Tarek, et al.
(författare)
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Lactobacillus reuteri NAD(P)H oxidase : Properties and coexpression with propanediol-utilization enzymes for enhancing 3-hydroxypropionic acid production from 3-hydroxypropionaldehyde
- 2019
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656. ; 289, s. 135-143
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Tidskriftsartikel (refereegranskat)abstract
- Lactobacillus reuteri metabolizes glycerol through propanediol-utilization (Pdu) pathway to 1,3-propanediol (1,3-PD) via 3-hydroxypropionaldehyde (3-HPA) as intermediate. In the resting cells, the oxidized co-factor obtained in the reaction is regenerated by simultaneous oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP) using propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). We have earlier shown that the use of resting cells of recombinant Escherichia coli expressing the oxidative pathway gives the highest theoretical yield of 1 mol 3-HP per mol 3-HPA but is limited by cofactor depletion. In the present study, the gene encoding the enzyme NAD(P)H oxidase (LreuNox) that utilizes molecular oxygen as substrate, was isolated from L. reuteri and heterologously overexpressed in E. coli. LreuNox has a pH optimum of 6 and exhibits Vmax of 101.1 ± 2.2 U/mg with NADH, which is 30% higher than that for NADPH. Co-expression of LreuNox with PduP, PduL and PduW in E. coli enhances the biocatalytic lifetime as well as productivity at least two-fold compared to that achieved without co-factor regeneration.
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| 45. |
- Ibrahim, Victor, et al.
(författare)
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Enzymatic monitoring of lignin and lignin derivatives biooxidation.
- 2016
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 1872-8359 .- 0167-7012. ; 120, s. 53-55
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Tidskriftsartikel (refereegranskat)abstract
- Lignin oxidation was enzymatically monitored by measuring methanol released during the reaction. The methanol was oxidized to formaldehyde and hydrogen peroxide, and the latter used to oxidize ABTS to a product measured spectrophotometrically. The efficiency was comparable to the commonly used gas chromatography method. The assay was fast and inexpensive.
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| 46. |
- Lidén, Gunnar
(författare)
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Biomass to transform the chemical industry
- 2016
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Ingår i: Australasian Biotechnology. - 1036-7128. ; 26:3, s. 64-65
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Tidskriftsartikel (refereegranskat)abstract
- The advances that have been made in metabolic engineering of various host organisms today enables a growing number of chemical compounds to be produced from sugars, rather than petroleum. Some of these products, such as propanediol, and lactic and succinic acid, are reaching commercial-scale production.
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| 47. |
- Nasir, Irem, et al.
(författare)
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High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:8
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Tidskriftsartikel (refereegranskat)abstract
- The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions.
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| 48. |
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