| 1. |
- Nilsson, R. Henrik, 1976, et al.
(författare)
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Mycobiome diversity: high-throughput sequencing and identification of fungi.
- 2019
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Ingår i: Nature reviews. Microbiology. - : Springer Science and Business Media LLC. - 1740-1534 .- 1740-1526. ; 17, s. 95-109
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Forskningsöversikt (refereegranskat)abstract
- Fungi are major ecological players in both terrestrial and aquatic environments by cycling organic matter and channelling nutrients across trophic levels. High-throughput sequencing (HTS) studies of fungal communities are redrawing the map of the fungal kingdom by hinting at its enormous - and largely uncharted - taxonomic and functional diversity. However, HTS approaches come with a range of pitfalls and potential biases, cautioning against unwary application and interpretation of HTS technologies and results. In this Review, we provide an overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis. We also discuss upcoming trends and techniques in the field and summarize recent and noteworthy results from HTS studies targeting fungal communities and guilds. Our Review highlights the need for reproducibility and public data availability in the study of fungal communities. If the associated challenges and conceptual barriers are overcome, HTS offers immense possibilities in mycology and elsewhere.
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| 2. |
- 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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| 3. |
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| 4. |
- Hong, Kuk-ki, 1976
(författare)
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Advancing Metabolic Engineering through Combination of Systems Biology and Adaptive Evolution
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Understanding evolutionary strategies of microorganisms may provide opportunities foradvanced strain development with the aim to produce valuable bio-products from renewablebiomass resources. Through evolutionary processes, microorganisms can attain new traitsassociated with genetic changes that may be useful for the construction of improved strains.Therefore, the characterization of evolutionary strategies may result in identification of themolecular and genetic changes underlying newly obtained traits, and can hereby become anessential step in strain development. However, so far the depth of analysis has limited the rangeof comprehension. This thesis applied genome-wide analyses such as transcriptome, metabolomeand whole-genome sequencing to investigate the evolutionary strategies of the yeastSaccharomyces cerevisiae. Three evolved mutants were independently generated by adaptiveevolution on galactose minimal media to obtain the trait of improved galactose utilization byyeast. Those strains expressed higher galactose utilization rates than a reference strain in terms ofboth maximum specific growth rate and specific galactose uptake rate. Application of thegenome-scale comparative analyses employing engineered strains as controls elucidated uniquechanges obtained by adaptive evolution. Molecular bases referred from the changes oftranscriptome and metabolome were located around galactose metabolism, while genetic basesfrom whole-genome sequencing showed no mutations in those changes. Common mutationsamong the evolved mutants were identified in the Ras/PKA signaling pathway. Those mutationswere placed on the reference strain background and their effects were evaluated by comparisonwith the evolved mutants. One of the site-directed mutants showed even higher specific galactoseuptake rate than the evolved mutants, and just few number of genetic and molecular changes wereenough to recover complete the adaptive phenotype. These results indicate that identification ofkey mutations provide new strategies for further metabolic engineering of strains. In addition, thepleiotropy of obtained phenotype that is improved galactose availability was tested. When thegalactose-evolved mutants were cultured on glucose that is the most favorite carbon source ofyeast, those mutants showed reduction of glucose utilization. Genome-wide analyses and sitedirectedmutagenesis were applied again to understand underlying molecular and genetic bases ofthis trade-off in carbon utilization. The results indicated that loosening of tight glucose regulationwas likely the reason of increased galactose availability. The implications of evolutionarystrategies and the impact of genome-scale analyses on characterization of evolved mutants arediscussed.
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| 5. |
- Radecka, Dorota, et al.
(författare)
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Looking beyond Saccharomyces: the potential of non-conventional yeast species for desirable traits in bioethanol fermentation
- 2015
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:6
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Forskningsöversikt (refereegranskat)abstract
- Saccharomyces cerevisiae has been used for millennia in the production of food and beverages and is by far the most studied yeast species. Currently, it is also the most used microorganism in the production of first-generation bioethanol from sugar or starch crops. Second-generation bioethanol, on the other hand, is produced from lignocellulosic feedstocks that are pretreated and hydrolyzed to obtain monomeric sugars, mainly D-glucose, D-xylose and L-arabinose. Recently, S. cerevisiaerecombinant strains capable of fermenting pentose sugars have been generated. However, the pretreatment of the biomass results in hydrolysates with high osmolarity and high concentrations of inhibitors. These compounds negatively influence the fermentation process. Therefore, robust strains with high stress tolerance are required. Up to now, more than 2000 yeast species have been described and some of these could provide a solution to these limitations because of their high tolerance to the most predominant stress conditions present in a second-generation bioethanol reactor. In this review, we will summarize what is known about the non-conventional yeast species showing unusual tolerance to these stresses, namely Zygosaccharomyces rouxii(osmotolerance), Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha(thermotolerance), Dekkera bruxellensis (ethanol tolerance), Pichia kudriavzevii (furan derivatives tolerance) and Z. bailii (acetic acid tolerance).
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| 6. |
- Razaghi, Ali, et al.
(författare)
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Effects of nitrogen on growth and carbohydrate formation in Porphyridium cruentum
- 2014
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Ingår i: Central European Journal of Biology. - : Walter de Gruyter GmbH. - 1895-104X .- 1644-3632. ; 9:2, s. 156-162
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Tidskriftsartikel (refereegranskat)abstract
- The microalga Porphyridium cruentum (Rhodophyta) has several industrial and pharmaceutical uses, especially for its polysaccharide production. This study aimed to investigate the influence of nitrogen levels as reflected by altered N:P ratios on the production and content of biomass and carbohydrate. N:P molar ratios were altered in batch cultures to range from 1.6 to 50 using the Redfield ratio of 1:16 as reference. Algal growth (estimated as final cell number, biomass concentration and maximum specific growth rate) was negatively affected at low N:P ratios. The optimal N:P ratio for growth was identified at 35-50, with specific growth rates of 0.19 day(-1) and maximum cell concentrations of 59 center dot 10(8) cells L-1 and 1.2 g dry weight of biomass L-1. In addition, variation in cell size was seen. Cells with larger diameters were at higher N:P ratios and smaller cells at lower ratios. The cellular carbohydrate content increased under reduced nitrogen availability. However, because accumulation was moderate at the lowest N:P ratio, 0.4 g per g dry weight biomass compared to 0.24 at the Redfield ratio of 16:1, conditions for increased total carbohydrate formation were identified at the N:P ratios optimal for growth. Additionally, carbohydrates were largely accumulated in late exponential to stationary phase.
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| 7. |
- 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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| 8. |
- Mukherjee, Vaskar, 1986, et al.
(författare)
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Phenotypic landscape of non-conventional yeast species for different stress tolerance traits desirable in bioethanol fermentation
- 2017
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: Non-conventional yeasts present a huge, yet barely exploited, resource of yeast biodiversity for industrial applications. This presents a great opportunity to explore alternative ethanol-fermenting yeasts that are more adapted to some of the stress factors present in the harsh environmental conditions in second-generation (2G) bioethanol fermentation. Extremely tolerant yeast species are interesting candidates to investigate the underlying tolerance mechanisms and to identify genes that when transferred to existing industrial strains could help to design more stress-tolerant cell factories. For this purpose, we performed a high-throughput phenotypic evaluation of a large collection of non-conventional yeast species to identify the tolerance limits of the different yeast species for desirable stress tolerance traits in 2G bioethanol production. Next, 12 multi-tolerant strains were selected and used in fermentations under different stressful conditions. Five strains out of which, showing desirable fermentation characteristics, were then evaluated in small-scale, semi-anaerobic fermentations with lignocellulose hydrolysates. Results: Our results revealed the phenotypic landscape of many non-conventional yeast species which have not been previously characterized for tolerance to stress conditions relevant for bioethanol production. This has identified for each stress condition evaluated several extremely tolerant non-Saccharomyces yeasts. It also revealed multitolerance in several yeast species, which makes those species good candidates to investigate the molecular basis of a robust general stress tolerance. The results showed that some non-conventional yeast species have similar or even better fermentation efficiency compared to S. cerevisiae in the presence of certain stressful conditions. Conclusion: Prior to this study, our knowledge on extreme stress-tolerant phenotypes in non-conventional yeasts was limited to only few species. Our work has now revealed in a systematic way the potential of non-Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.
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| 9. |
- Torello Pianale, Luca, 1995, et al.
(författare)
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Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology
- 2019
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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 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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| 10. |
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