| 1. |
- Kristjansdottir, Thordis, et al.
(författare)
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Engineering the carotenoid biosynthetic pathway in Rhodothermus marinus for lycopene production
- 2020
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Ingår i: Metabolic Engineering Communications. - : Elsevier BV. - 2214-0301. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Rhodothermus marinus has the potential to be well suited for biorefineries, as an aerobic thermophile that produces thermostable enzymes and is able to utilize polysaccharides from different 2nd and 3rd generation biomass. The bacterium produces valuable chemicals such as carotenoids. However, the native carotenoids are not established for industrial production and R. marinus needs to be genetically modified to produce higher value carotenoids. Here we genetically modified the carotenoid biosynthetic gene cluster resulting in three different mutants, most importantly the lycopene producing mutant TK-3 (ΔtrpBΔpurAΔcruFcrtB::trpBcrtBT.thermophilus). The genetic modifications and subsequent structural analysis of carotenoids helped clarify the carotenoid biosynthetic pathway in R. marinus. The nucleotide sequences encoding the enzymes phytoene synthase (CrtB) and the previously unidentified 1′,2′-hydratase (CruF) were found fused together and encoded by a single gene in R. marinus. Deleting only the cruF part of the gene did not result in an active CrtB enzyme. However, by deleting the entire gene and inserting the crtB gene from Thermus thermophilus, a mutant strain was obtained, producing lycopene as the sole carotenoid. The lycopene produced by TK-3 was quantified as 0.49 g/kg CDW (cell dry weight).
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| 2. |
- Nordberg Karlsson, Eva, et al.
(författare)
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Metabolic engineering of thermophilic bacteria for production of biotechnologically interesting compounds
- 2020
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Ingår i: Biotechnological applications of extremophilic microorganisms. - : De Gruyter. - 9783110424331 ; , s. 73-96
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Bokkapitel (refereegranskat)abstract
- Many thermophilic bacteria are efficient biomass degraders (producing polysaccharide degrading enzymes and utilizing a great variety of substrates, e.g. lignocellulosic polymers, pentoses, hexoses, as well sugar acids, and sugar alcohols). This makes them interesting organisms as potential cell factories in a circular bioeconomy. Lignocellulosic and marine macroalgal biomasses are regarded as sustainable biorefinery feedstocks for the production of energy carriers and platform and specialty chemicals, thereby meeting impending fossil fuel shortage and counteracting accumulation of greenhouse gasses. However, progress in using thermophilic bacteria that utilize these feedstocks as carbon sources has been hampered by the lack of suitable engineering tools to improve the production profiles of interesting target metabolites as specific synthetic production pathways need to be inserted/modified or existing pathways optimized by metabolic engineering. In this chapter, we review the progress on the use of thermophilic bacteria in metabolic engineering and the available engineering tools and give examples of species for which successful engineering has been accomplished. Today, the majority of thermophilic bacteria targeted for production of compounds of industrial interest by metabolic engineering belong to the phylum Firmicutes (e.g. Thermoanaerobacterium, Caldocellulosiruptor, Geobacillus, and Bacillus), taking advantage of anaerobic catabolic pathways producing organic acids and alcohols. However, there are additional and aerobic species gaining interest concerning biomass degradation and the ability of carbon dioxide fixation as well as production of molecules of interest, and some examples of this are also given.
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| 3. |
- Aevarsson, Arnthór, et al.
(författare)
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Going to extremes - a metagenomic journey into the dark matter of life
- 2021
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968. ; 368:12
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Forskningsöversikt (refereegranskat)abstract
- The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
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| 4. |
- Ahlqvist, Josefin, et al.
(författare)
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Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6
- 2022
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 78:Pt 2, s. 212-227
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Tidskriftsartikel (refereegranskat)abstract
- This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.
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| 5. |
- Ahlqvist, Josefin, et al.
(författare)
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Crystal structure of DNA polymerase I from Thermus phage G20c
- 2022
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Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 78:Pt 11, s. 1384-1398
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Tidskriftsartikel (refereegranskat)abstract
- This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.
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| 6. |
- Allahgholi, Leila, et al.
(författare)
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Composition analysis and minimal treatments to solubilize polysaccharides from the brown seaweed Laminaria digitata for microbial growth of thermophiles
- 2020
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Ingår i: Journal of Applied Phycology. - : Springer Science and Business Media LLC. - 0921-8971 .- 1573-5176. ; 32:3, s. 1933-1947
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Tidskriftsartikel (refereegranskat)abstract
- Brown macroalgae (Phaeophyta) hold high potential as feedstock for biorefineries due to high biomass productivity and carbohydrate content. They are, however, a challenging, unconventional feedstock for microbial refining and several processing problems need to be solved to make them a viable option. Pre-treatment is necessary to enhance accessibility and solubility of the biomass components but should be minimal and mild to assure sustainable and cost-effective processing. Here, two routes to pre-treatLaminaria digitata to release polysaccharides were investigated: hot water pre-treatment by autoclaving (121 °C, 20 min or 60 min) and a two-step extraction with mild acid (0.1 M HCl) followed by alkaline treatment. Hot water pre-treatment resulted in partial extraction of a mixture of polysaccharides consisting of alginate, fucoidan and laminarin. After mild acid pre-treatment, alginate was found in the remaining insoluble residues and was extracted in a second step via alkaline treatment using Na2CO3 (0.15 M) at 80 °C and CaCl2 (10%) for the precipitation. In addition to carbohydrates, a fraction of other components such as proteins, phenolic compounds, minerals and trace elements was detected in the extracts. Cultivation of the thermophilic bacterial strains Rhodothermus marinus DSM 16675 and Bacillus methanolicus MGA3 (ATCC 53907) in media supplemented with the respective extracts resulted in growth of both strains, indicating that they were able to utilize the available carbon source for growth. R. marinus displayed the highest cell density in the medium containing the extract from acid pre-treatment, whereas B. methanolicus growth was highest with the extract from hot water pre-treatment.
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| 7. |
- Allahgholi, Leila, et al.
(författare)
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Exploring a novel β-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides
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Ingår i: Glycobiology. - 1460-2423.
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Tidskriftsartikel (refereegranskat)abstract
- The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses β-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and + 2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming β-1,3 glucan acceptor, making a β-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several β-1,6-linked branches. The modelled structure revealed an active site comprising five subsites: three glycone (-3, -2 and - 1) and two aglycone (+1 and + 2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-β-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-β-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modelled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.
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| 8. |
- Ara, Kazi Zubaida Gulshan, et al.
(författare)
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Characterization and diversity of the complete set of GH family 3 enzymes from Rhodothermus marinus DSM 4253
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. The RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. The β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pNP-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.
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| 9. |
- Gulshan Kazi, Zubaida, et al.
(författare)
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A CGTase with high coupling activity using γ-cyclodextrin isolated from a novel strain clustering under the genus Carboxydocella.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:5, s. 514-523
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Tidskriftsartikel (refereegranskat)abstract
- Cyclodextrin glucanotransferases (CGTases; EC 2.4.1.19) have mainly been characterized for their ability to produce cyclodextrins (CDs) from starch in an intramolecular transglycosylation reaction (cyclization). However, this class of enzymes can also catalyze intermolecular transglycosylation via disproportionation or coupling reactions onto a wide array of acceptors and could therefore be valuable as a tool for glycosylation. In this paper, we report the gene isolation, via the CODEHOP-strategy, expression and characterization of a novel CGTase (CspCGT13) from a Carboxydocella sp. This enzyme is the first glycoside hydrolase isolated from the genus, indicating starch degradation via cyclodextrin production in the Carboxydocella strain. The fundamental reactivities of this novel CGTase are characterized and compared to two commercial CGTases, assayed under identical condition, in order to facilitate interpretation of the results. The comparison showed that the enzyme, CspCGT13, displayed high coupling activity using γ-CD as donor, despite preferentially forming α and β-CD in the cyclization reaction using wheat starch as substrate. Comparison of subsite conservation within previously characterized CGTases showed significant sequence variation in subsite -3 and -7, which may be important for the coupling activity.
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| 10. |
- Kobayashi, Yumi, et al.
(författare)
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Life-cycle assessment of yeast-based single-cell protein production with oat processing side-stream
- 2023
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Ingår i: Science of the Total Environment. - : Elsevier BV. - 1879-1026 .- 0048-9697. ; 873
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Tidskriftsartikel (refereegranskat)abstract
- Production of fish meal and plant-based feed proteins continues to increase to meet the growing demand for seafood, leading to impacts on marine and terrestrial ecosystems. Microbial proteins such as single-cell proteins (SCPs) have been introduced as feed alternatives since they can replace current fish feed ingredients, e.g., soybean, which are associated with negative environmental impacts. Microbial protein production also enables utilization of grain processing side-streams as feedstock sources. This study assesses the environmental impacts of yeast-based SCP using oat side-stream as feedstock (OS-SCP). Life-cycle assessment with a cradle-to-gate approach was used to quantify global warming, freshwater eutrophication, marine eutrophication, terrestrial acidification, land use, and water consumption of OS-SCP production in Finland. Dried and wet side-streams of oat were compared with each other to identify differences in energy consumption and transportation effects. Sensitivity analysis was performed to examine the difference in impacts at various locations and fermentation times. Benchmarking was used to evaluate the environmental impacts of OS-SCP and other feed products, including both conventional and novel protein products. Results highlight the importance of energy sources in quantifying the environmental performance of OS-SCP production. OS-SCP produced with dried side-streams resulted in higher global warming (16.3 %) and water consumption (7.5 %) than OS-SCP produced from wet side-streams, reflecting the energy and water requirements for the drying process. Compared with conventional products, such as soy protein concentrates, OS-SCP resulted in 61 % less land use, while exacerbating the environmental impacts in all the other categories. OS-SCP had more impact on global warming (205-754 %), water consumption (166-1401 %), freshwater eutrophication (118-333 %), and terrestrial acidification (85-340 %) than other novel products, including yeast protein concentrate, methanotrophic bacterial SCP, and insect meal, while lowering global warming (11 %) and freshwater eutrophication (20 %) compared with dry microalgae biomass.
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