| 1. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Characterization of a novel non-GMO yeast for future lignocellulosic bioethanol production
- 2014
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Ingår i: ISSY31: 31ST International Specialised Symposium on Yeast.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- CHARACTERIZATION OF A NOVEL NON-GMO YEAST FOR FUTURE LIGNOCELLULOSIC BIOETHANOL PRODUCTIONCecilia Geijer1, David Moreno1, Elia Tomas Pejo1, 2, Lisbeth Olsson11 Industrial Biotechnology , Department of Chemical and Biological Engineering Chalmers University of Technology, Gothenburg, Sweden2Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), SpainContact details: cecilia.geijer@chalmers.seConcerns about climate change and the uncertainty about future fuel supply make renewable biofuels, such as bioethanol, attractive alternatives to fossil fuels in the short/medium term. Lignocellulosic biomass (for example spruce, wheat straw and corn stover) is an abundant raw material that can be utilized to produce ethanol with the help of a fermenting microorganism. Traditionally the yeast Saccharomyces cerevisiae is used for industrial ethanol production. S. cerevisiae can be metabolically engineered to consume xylose (the second to glucose most prevalent monosaccharide in lignocellulose). However, despite many years of intensive research, it can still not ferment xylose in a satisfying way which affects the overall ethanol yield negatively. We have isolated a non-genetically modified (non-GMO) yeast species (here called C5-yeast) that has the natural ability to efficiently produce ethanol from glucose and xylose. The aim of the project is to further characterize the growth and fermentation capacities of this novel microorganism to elucidate its’ potential for lignocellulosic bioethanol production. We can show that besides glucose and xylose, the C5-yeast can also consume the pentose arabinose and the disaccharide cellobiose; both present in lignocellulosic hydrolysates. The C5-yeast rapidly converts the inhibitory sugar degradation products HMF and furfural formed during the conversion of lignocellulosic material into fermentable sugars.
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| 2. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Evolutionary engineered strains of Saccharomyces cerevisiae for efficient lignocellulosic bioethanol production
- 2014
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Ingår i: 36th Symposium on Biotechnology for Fuels and Chemicals.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic biomass is an abundant raw material that can be utilized to produce ethanol with the help of Saccharomyces cerevisiae; a promising alternative to today’s energy sources. Conversion of lignocellulosic material (cellulose, hemicellulose and lignin) into fermentable sugars including both hexoses and pentoses results in formation of inhibitory compounds such as acetic acid, furan aldehydes and phenolics that are known to inhibit the yeasts’ metabolic processes. The aims of this study were to i) generate S. cerevisiae strains that can readily convert glucose and xylose into ethanol in the presence of inhibitory compounds, and ii) elucidate the underlying genetic changes of importance for the improved properties of the generated strains. For these purposes, a strain of S. cerevisiae containing genes for xylose reductase, xylitol dehydrogenase and xylulokinase was used. The strain was subjected to mutagenesis followed by evolutionary engineering (repetitive batch and chemostat cultivation), which resulted in populations with improved ethanol yield, improved xylose conversion rate and increased inhibitor tolerance. The complex combination of different genetic alterations in the evolved populations will now be revealed using a DNA/RNA sequencing approach. The acquired knowledge of proteins and pathways important for efficient lignocellulosic bioethanol production will then hopefully allow directed engineering for further improvement of yeast performance.
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| 3. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Unraveling the potential of non-conventional yeasts in biotechnology
- 2022
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Cost-effective microbial conversion processes of renewable feedstock into biofuels and biochemicals are of utmost importance for the establishment of a robust bioeconomy. Conventional baker's yeast Saccharomyces cerevisiae, widely employed in biotechnology for decades, lacks many of the desired traits for such bioprocesses like utilization of complex carbon sources or low tolerance towards challenging conditions. Many non-conventional yeasts (NCY) present these capabilities, and they are therefore forecasted to play key roles in future biotechnological production processes. For successful implementation of NCY in biotechnology, several challenges including generation of alternative carbon sources, development of tailored NCY and optimization of the fermentation conditions are crucial for maximizing bioproduct yields and titers. Addressing these challenges requires a multidisciplinary approach that is facilitated through the 'YEAST4BIO' COST action. YEAST4BIO fosters integrative investigations aimed at filling knowledge gaps and excelling research and innovation, which can improve biotechnological conversion processes from renewable resources to mitigate climate change and boost transition towards a circular bioeconomy. In this perspective, the main challenges and research efforts within YEAST4BIO are discussed, highlighting the importance of collaboration and knowledge exchange for progression in this research field.
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| 4. |
- Moreno, A. D., et al.
(författare)
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Candida intermedia CBS 141442: A novel glucose/xylose co-fermenting isolate for lignocellulosic bioethanol production
- 2020
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Ingår i: Energies. - : MDPI AG. - 1996-1073 .- 1996-1073. ; 13:20
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Tidskriftsartikel (refereegranskat)abstract
- The present study describes the isolation of the novel strain Candida intermedia CBS 141442 and investigates the potential of this microorganism for the conversion of lignocellulosic streams. Different C. intermedia clones were isolated during an adaptive laboratory evolution experiment under the selection pressure of lignocellulosic hydrolysate and in strong competition with industrial, xylose-fermenting Saccharomyces cerevisiae cells. Isolates showed different but stable colony and cell morphologies when growing in a solid agar medium (smooth, intermediate and complex morphology) and liquid medium (unicellular, aggregates and pseudohyphal morphology). Clones of the same morphology showed similar fermentation patterns, and the C. intermedia clone I5 (CBS 141442) was selected for further testing due to its superior capacity for xylose consumption (90% of the initial xylose concentration within 72 h) and the highest ethanol yields (0.25 ± 0.02 g ethanol/g sugars consumed). Compared to the well-known yeast Scheffersomyces stipitis, the selected strain showed slightly higher tolerance to the lignocellulosic-derived inhibitors when fermenting a wheat straw hydrolysate. Furthermore, its higher glucose consumption rates (compared to S. stipitis) and its capacity for glucose and xylose co-fermentation makes C. intermedia CBS 141442 an attractive microorganism for the conversion of lignocellulosic substrates, as demonstrated in simultaneous saccharification and fermentation processes.
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| 5. |
- Moreno, David, 1986, et al.
(författare)
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An engineered Saccharomyces cerevisiae for cost-effective lignocellulosic bioethanol production: process performance and physiological insights
- 2015
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Ingår i: 37th Symposium on Biotechnology for Fuels and Chemicals.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The success in the commercialization of lignocellulosic bioethanol relies on the development of microorganisms with efficient hexose and pentose fermentation and tolerance towards inhibitory by-products (acetic acid, furan aldehydes and phenolics) generated during biomass processing. Traditionally, the yeast Saccharomyces cerevisiae is the preferred microorganism for industrial ethanol production. Many years of research and development have been conducted to develop S. cerevisiae strains suitable for fermenting lignocellulosic-based streams. S. cerevisiae is robust and ferment glucose efficiently, but it has been proved to be difficult to genetically modify for efficient xylose fermentation. In this work, a xylose-fermenting S. cerevisiae strain was subjected to evolutionary engineering, boosting its robustness and xylose fermentation capacity. The evolved strain was able to ferment a non-diluted enzymatic hydrolysate (representing 23% (w/w) dry matter of steam-exploded wheat straw), reaching ethanol titers higher than 5% (w/w) after 48 h. Within the first 24 h, glucose and xylose were co-consumed with rates of 3.1 and 0.7 g/L h, respectively, and converted to ethanol with yields corresponding to 93% of the theoretical. In addition, once glucose was depleted, xylose was consumed with a similar rate until reducing 70% of its initial concentration (36 h after inoculation). Besides investigating the fermentation parameters, the differences in gene expression levels and enzymatic activities of xylose-assimilating pathway were analyzed. These analyses will be the foundation for understanding the improved phenotype and the physiological mechanisms for efficient xylose fermentation, after which potential targets for subsequent metabolic engineering may be identified.
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| 6. |
- Moreno, David, 1986, et al.
(författare)
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Isolation and evolution of a novel non-saccharomyces xylose-fermenting strain for lignocellulosic bioethanol production
- 2014
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Ingår i: ISSY31: 31ST International Specialised Symposium on Yeast.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- ISOLATION AND EVOLUTION OF A NOVEL NON-SACCHAROMYCES XYLOSE-FERMENTING STRAIN FOR LIGNOCELLULOSIC BIOETHANOL PRODUCTIONAntonio D. Moreno1, Cecilia Geijer1, Elia Tomás-Pejó1,2, Lisbeth Olsson1.1Chalmers University of Technology, Department of Chemical and Biological Engineering, Industrial Biotechnology Group, Göteborg, Sweden. 2Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), Spain.Contact e-mail: davidmo@chalmers.seThe economical success of lignocellulosic bioethanol requires the fermentation of all available sugars obtained during the process. Being the major pentose sugar in lignocellulose, the fermentation of xylose is, therefore, considered essential. The fermentative yeast Saccharomyces cerevisiae is the most promising candidate for lignocellulosic bioethanol production due to its excellent glucose fermentation capability, high ethanol tolerance and resistance to inhibitors presented in lignocellulosic streams. Nevertheless, the wild type S. cerevisae is not able to ferment xylose and all of the purpose-engineered Saccharomyces strains (genetically modified microorganisms (GMO)) are still far away from an economically viable lignocellulosic ethanol production. By chance, we have discovered a non-Saccharomyces xylose-fermenting yeast (here called C5-yeast), which shows a great potential to be used for bioethanol production from lignocellulosic streams. Unlike xylose-fermenting Saccharomyces strains, the C5-yeast is not genetically modified and its use by industries can aid in finding less legislative problems when reaching the market. In the present work, the C5-yeast was isolated from a xylose-fermenting population and evolutionary engineered to enhance its fermentation abilities and robustness. During the isolation process, three different morphologies (smooth, flat and wrinkled) of the C5-yeast were found when growing the xylose-fermenting population in plates with minimal media and xylose as a sole carbon source. Among all morphologies, flat-C5-yeast showed the highest xylose consumption rates (>90% after 72 h) and the highest ethanol conversion yields (≈50% of the theoretical considering glucose and xylose) during the fermentation of wheat straw hydrolysates. The isolated flat-C5-yeast was selected for evolutionary engineering in order to enhance its sugar conversion yields and the tolerance towards the inhibitory compounds that are present in the hydrolysate. Although further characterization is needed, an evolved C5-yeast could be considered as a suitable fermentative strain for lignocellulosic bioethanol production.
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| 7. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
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Yeast reveals unexpected roles and regulatory features of aquaporins and aquaglyceroporins
- 2014
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Ingår i: Biochimica et Biophysica Acta. General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006 .- 0006-3002. ; 1840:5, s. 1482-1491
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Forskningsöversikt (refereegranskat)abstract
- Background: The yeast Saccharomyces cerevisiae provides unique opportunities to study roles and regulation of aqua/glyceroporins using frontline tools of genetics and genomics as well as molecular cell and systems biology. Scope of review: S. cerevisiae has two similar orthodox aquaporins. Based on phenotypes mediated by gene deletion or overexpression as well as on their expression pattern, the yeast aquaporins play important roles in key aspects of yeast biology: establishment of freeze tolerance, during spore formation as well as determination of cell surface properties for substrate adhesion and colony formation. Exactly how the aquaporins perform those roles and the mechanisms that regulate their function under such conditions remain to be elucidated. S. cerevisiae also has two different aquaglyceroporins. While the role of one of them, Yfl054c, remains to be determined, Fps1 plays critical roles in osmoregulation by controlling the accumulation of the osmolyte glycerol. Fpsl communicates with two osmo-sensing MAPK signalling pathways to perform its functions but the details of Fps1 regulation remain to be determined. Major conclusions: Several phenotypes associated with aqua/glyceroporin function in yeasts have been established. However, how water and glycerol transport contribute to the observed effects is not understood in detail. Also many of the basic principles of regulation of yeast aqua/glyceroporins remain to be elucidated. General significance: Studying the yeast aquaporins and aquaglyceroporins offers rich insight into the life style, evolution and adaptive responses of yeast and rewards us with discoveries of unexpected roles and regulatory mechanisms of members of this ancient protein family. This article is part of a Special Issue entitled Aquaporins. (c) 2013 Elsevier B.V. All rights reserved.
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| 8. |
- Cámara, Elena, 1985, et al.
(författare)
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Data mining of Saccharomyces cerevisiae mutants engineered for increased tolerance towards inhibitors in lignocellulosic hydrolysates
- 2022
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 57
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Forskningsöversikt (refereegranskat)abstract
- The use of renewable plant biomass, lignocellulose, to produce biofuels and biochemicals using microbial cell factories plays a fundamental role in the future bioeconomy. The development of cell factories capable of efficiently fermenting complex biomass streams will improve the cost-effectiveness of microbial conversion processes. At present, inhibitory compounds found in hydrolysates of lignocellulosic biomass substantially influence the performance of a cell factory and the economic feasibility of lignocellulosic biofuels and chemicals. Here, we present and statistically analyze data on Saccharomyces cerevisiae mutants engineered for altered tolerance towards the most common inhibitors found in lignocellulosic hydrolysates: acetic acid, formic acid, furans, and phenolic compounds. We collected data from 7971 experiments including single overexpression or deletion of 3955 unique genes. The mutants included in the analysis had been shown to display increased or decreased tolerance to individual inhibitors or combinations of inhibitors found in lignocellulosic hydrolysates. Moreover, the data included mutants grown on synthetic hydrolysates, in which inhibitors were added at concentrations that mimicked those of lignocellulosic hydrolysates. Genetic engineering aimed at improving inhibitor or hydrolysate tolerance was shown to alter the specific growth rate or length of the lag phase, cell viability, and vitality, block fermentation, and decrease product yield. Different aspects of strain engineering aimed at improving hydrolysate tolerance, such as choice of strain and experimental set-up are discussed and put in relation to their biological relevance. While successful genetic engineering is often strain and condition dependent, we highlight the conserved role of regulators, transporters, and detoxifying enzymes in inhibitor tolerance. The compiled meta-analysis can guide future engineering attempts and aid the development of more efficient cell factories for the conversion of lignocellulosic biomass.
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| 9. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(författare)
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Genomic and transcriptomic analysis of Candida intermedia reveals genes for utilization of biotechnologically important carbon sources
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A future biobased society relies on efficient industrial microorganisms that can convert all sugars from agricultural, forestry and industrial waste streams into fuels, chemicals and materials. To be able to tailor-make such potent cell factories, we need a far better understanding of the proteins responsible for the assimilation of biotechnologically important carbon sources including pentoses, disaccharides and oligomers. The yeast Candida intermedia, known for its superior growth on xylose owing to its efficient uptake and conversion systems, can also utilize a range of other important carbon sources such as cellobiose, galactose and lactose. The aim of this project was to identify the genomic determinants for the utilization of these mono- and disaccharides in our in-house isolated C. intermedia strain CBS 141442. Genome sequencing and transcriptional (RNA seq) data analysis during growth in defined medium supplemented with glucose, xylose, galactose, lactose or cellobiose, revealed numerous distinct clusters of coregulated genes. By scanning the CBS 141442 genome for genes encoding Major Facilitator Superfamily (MFS) sugar transporters, and the RNA-seq dataset for the corresponding transcriptional profiles, we identified several novel genes encoding putative xylose transporters and multiple Lac12-like transporters likely involved in the uptake of disaccharides in C. intermedia. We also found that the yeast possesses no less than three genes encoding aldose reductases with different transcriptional profiles, and heterologous expression of the genes in Saccharomyces cerevisiae showed that the aldose reductases have different substrate and co-factor specificities, suggesting diverse physiological roles. Taken together, the results of this study provide insights into the mechanisms underlying carbohydrate metabolism in C. intermedia, and reveals several genes with potential future applications in cell factory development.
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| 10. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(författare)
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Genomic and transcriptomic analysis of Candida intermedia reveals important genes for xylose utilization
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The urgency to reduce carbon emissions and to lower our dependence on oil makes it necessary to strive towards a more sustainable bio-based economy, where energy, chemicals, materials and food are produced from renewable resources. Lignocellulosic biomass constitutes a great source of raw material for such a future bio-based economy since it is widely available, relatively inexpensive and do not compete with food and feed production. The pentose D-xylose, the second most prevalent sugar in lignocellulose after glucose, is an underutilized resource, in large due to the inefficient fermentation of this sugar by the most industrially relevant microorganisms (e.g. Saccharomyces cerevisiae). Thus, development of microorganisms that can ferment all lignocellulosic sugars is of foremost importance for economically viable production processes. Native xylose-utilizing yeasts represent a major source of knowledge and genes for xylose uptake and assimilation that can be transferred to S. cerevisiae. The yeast Candida intermedia is an interesting candidate to characterize further, as it displays a high xylose transport capacity and multiple xylose reductases, of which one appears to prefer NADH over NAPDH. Furthermore, the C. intermedia strain CBS 141442, isolated in the liquid fraction of wheat straw hydrolysate in our laboratory as a contaminant of a xylose fermenting population of S. cerevisiae, is capable of glucose and xylose co-fermentation under certain conditions. The aim of this study was to elucidate the genetic features that are the basis of the xylose utilization capacity of C. intermedia CBS141442. PacBio sequencing and de novo assembly of the genome revealed a haploid yeast with a genome size of 13.2 Mb and a total of 5936 protein-coding genes spread over seven chromosomes. In order to gain insight on the genes involved in the utilization of xylose, we analysed the changes in the transcriptome of C. intermedia CBS141442 during growth in xylose and glucose (as reference condition). Cells were collected in mid-exponential phase at the maximum growth rate when no metabolites were accumulating. The total RNA was extracted and cDNA libraries were prepared after polyA selection. Each sample was sequenced in an Illumina HiSeq2500 system with an average cover of 5-20 million reads. The analysis of the differential expression data lead to the identification of two new genes potentially encoding xylose transporters and no less than three xylose reductases genes with different expression patterns. The xylose reductase genes were heterologously expressed in S. cerevisiae to determine their co-factor preferences and substrate specificities. Whereas two of them are strictly NADPH-dependent, the third can use both co-factors and shows preference for NADH. The heterologous expression of this gene can improve the capacity of S. cerevisiae to ferment xylose, and thus contribute to a more efficient use of lignocellulosic biomass.
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| 11. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(författare)
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Split-marker recombination for efficient targeted gene deletions in Candida intermedia
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Candida intermedia is a non-conventional yeast species with a natural ability to produce ethanol from xylose, making it an attractive non-GMO alternative for lignocellulosic biomass conversion in biorefineries and/or gene donor to Saccharomyces cerevisiae to improve its xylose fermentation capacity. We have de novo genome sequenced the C. intermedia strain CBS 141442, previously isolated in our lab, which allows us to study the yeast at a genomic and molecular level. The aim of this project was to develop a molecular toolbox for C. intermedia to enable also targeted genome editing and subsequent mutant phenotyping. C. intermedia is a haploid yeast belonging to the CTG clade of fungal species, and thus requires drug-resistant markers adapted for the alternative codon usage of these organisms. Transformation of linearized plasmid containing the CaNAT1 marker flanked by the TEF1 promoter and terminator from Ashbya gossypii [1] resulted in hundreds of Nourseothricin-resistant transformants. We then constructed an ADE2-deletion cassette, where the CaNAT1 marker was fused to the upstream and downstream sequences (1000bp) of CiADE2. Transformations resulted in less than 1% of ade2 mutants with the characteristic red pigmentation, which indicates that the non-homologous end joining pathway (NHEJ) is dominant over the homologous recombination (HR) pathway in this yeast. Using the cell cycle inhibitor hydroxyurea to arrest cells in the S-phase has been shown to improve the HR/NHEJ ratio in other yeasts [2], and increased the ADE2 deletion efficiency to 4% in C. intermedia. To further improve the targeted deletion rate, we applied the "split-marker” strategy previously developed for Saccharomyces cerevisiae [3]. Here, the selectable marker gene is truncated in two different fragments, and the gene is not functional until homologous recombination takes place between the two overlapping parts of the fragments. The truncated marker gene fragments were flanked by homologous sequences (1000 bp) upstream and downstream of the target gene using fusion PCR, thereby avoiding a tedious cloning step. This approach increased the targeted gene disruption of ADE2 to 56%. As proof of concept, the method was also used to delete KU70, the xylose reductase gene XYL1_2 as well as a large gene cluster in C. intermedia, with allele-specific HR efficiencies between 87 and 100%. The split-marker approach for targeted gene-disruptions will pave the way for high throughput genetic analysis in C. intermedia as well as in other yeasts where NHEJ is the predominant form of recombination.
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| 12. |
- Fischer, Gerhard, 1978, et al.
(författare)
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Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism.
- 2009
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1545-7885 .- 1544-9173. ; 7:6
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 A resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.
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| 13. |
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| 14. |
- Geijer, Cecilia, 1980, et al.
(författare)
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De novo genome sequencing of the yeast Candida intermedia
- 2015
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Ingår i: 32nd International Specialized Symposium on Yeasts.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The urgency to reduce carbon emissions and to lower our dependence on oil makes it necessary to strive towards a more sustainable, bio-based economy where energy, chemicals, materials and food are produced from renewable resources. Lignocellulosic biomass constitutes a great source of raw material for such a future bio-based economy since it is widely available, relatively inexpensive and do not compete with food and feed production. Saccharomyces cerevisiae is commonly used for bioethanol production and displays excellent glucose fermenting skills, but metabolic engineering is needed to allow consumption and fermentation of xylose (the second to glucose most prevalent sugar in lignocellulose). As an alternative to S. cerevisiae, microorganisms that naturally ferment xylose can be used. An unexpected discovery in our lab allowed us to isolate a clone of the non-conventional, xylose fermenting yeast species Candida intermedia. The aim of this project is to sequence the genome of C. intermedia as well as to develop a molecular toolbox to allow genetic manipulations of this yeast. PacBio sequencing and de novo assembly of the genome revealed a haploid yeast with a genome size of 13.2 Mb and a total of 5216 genes spread over seven chromosomes. Future activities include identification of genes involved in uptake and fermentation of sugars derived from lignocellulosic biomass, and subsequent deletion/overexpression of interesting candidate genes to improve the fermentation capacity of the yeast.
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| 15. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Functional analysis of the Ashbya gossypii Fps1 homolog
- 2010
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Ingår i: FEBS Journal. - 1742-464X .- 1742-4658. ; 277:Supplement 1
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Saccharomyces cerevisiae aquaglyceroporin Fps1 plays a central role in yeast osmoregulation, controlling the intracellular level of the compatible solute glycerol. When a cell encounter hyperosmotic conditions, Fps1 closes rapidly to ensure retention and accumulation of glycerol. In adaptation to lower external osmolarity, Fps1 opens again to release glycerol and hence turgor pressure. Mutants lacking Fps1 can not withstand a hypo-osmotic shock as well as wild type cells. Fps1 has unusually long N- and C-terminal extensions, and regulatory domains that are crucial for the gating mechanism have been identified on both termini. Fps1 also facilitates passive uptake of other small molecules such as arsenite and acidic acid. The filamentous fungi Ashbya gossypii Fps1 homolog (AgFps1) has shorter termini than Fps1. The aim of this study is to determine the function of AgFps1 by heterologous expression in S. cerevisiae fps1Δ mutants, and to study the physiological role of AgFps1 by deletion analysis in Ashbya gossypii. We can show that heterologous expression of AgFps1 in S. cerevisiae can substitute for Fps1 by releasing excessive glycerol upon a hypo-osmotic shock. AgFps1 expressed in S. cerevisiae appears to be hyperactive under hyperosmotic conditions, and exchanging the N- and C-terminal extensions for the corresponding termini of Fps1 is not sufficient to generate a regulated channel. Further, successful deletion of AgFPS1 renders an Ashbya gossypii mutant more resistant to arsenite than wild type fungi, indicating that AgFps1 transports arsenite.
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| 16. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Genomic and transcriptomic analysis of Candida intermedia reveals the genetic determinants for its xylose-converting capacity
- 2020
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. Results To understand the genetic determinants that underlie the metabolic properties of C. intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. Conclusions In the present study, we performed the first genomic and transcriptomic analysis of C. intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.
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| 17. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Initiation of the transcriptional response to hyperosmotic shock correlates with the potential for volume recovery.
- 2013
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Ingår i: The FEBS journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:16, s. 3854-67
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Tidskriftsartikel (refereegranskat)abstract
- The control of activity and localization of transcription factors is critical for appropriate transcriptional responses. In eukaryotes, signal transduction components such as mitogen-activated protein kinase (MAPK) shuttle into the nucleus to activate transcription. It is not known in detail how different amounts of nuclear MAPK over time affect the transcriptional response. In the present study, we aimed to address this issue by studying the high osmolarity glycerol (HOG) system in Saccharomyces cerevisiae. We employed a conditional osmotic system, which changes the period of the MAPK Hog1 signal independent of the initial stress level. We determined the dynamics of the Hog1 nuclear localization and cell volume by single-cell analysis in well-controlled microfluidics systems and compared the responses with the global transcriptional output of cell populations. We discovered that the onset of the initial transcriptional response correlates with the potential of cells for rapid adaptation; cells that are capable of recovering quickly initiate the transcriptional responses immediately, whereas cells that require longer time to adapt also respond later. This is reflected by Hog1 nuclear localization, Hog1 promoter association and the transcriptional response, but not Hog1 phosphorylation, suggesting that a presently uncharacterized rapid adaptive mechanism precedes the Hog1 nuclear response. Furthermore, we found that the period of Hog1 nuclear residence affects the amplitude of the transcriptional response rather than the spectrum of responsive genes.
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| 18. |
- Geijer, Cecilia, 1980
(författare)
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Osmoregulation at different stages of the yeast life cycle
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ability to adapt to changing and potentially harmful conditions in the surrounding environment is crucial for fitness and survival of all living cells; in particular unicellular organisms, since they are frequently exposed to stress factors such as heat, drought, nutritional starvation and toxic substances. The aim of this thesis is to determine how cells respond to osmotic and nutritional changes in the environment and how downstream targets of signalling cascades are regulated. Water is fundamental to life, and all cells must be able to adapt to fluctuations in water availability to maintain cellular water homeostasis. In bakers’ yeast Saccharomyces cerevisiae, the High Osmolarity Glycerol (HOG) pathway is activated upon conditions of high osmolarity, and the pathway coordinates the responses needed to counteract loss of volume and turgor pressure. These actions include glycerol accumulation, ion efflux and transcriptional and translational changes. In this thesis, the osmotic stress response is characterized using a conditional osmotic system. We show that the period of Hog1 activation affects the transcriptional output in a quantitative rather than qualitative way. The analysis also sheds light on an initial adaptation process involving regain of volume through accumulation of compatible osmolytes, which precedes Hog1 nuclear accumulation and the transcriptional response. The S. cerevisiae aquaglyceroporin Fps1 plays an important role during osmotic stress as a regulator of the intracellular glycerol concentration. A decrease in external osmolarity leads to water inflow and cell swelling, and Fps1 activity is vital under this condition for rapid release of excessive glycerol to lower the cells’ turgor pressure. During a hyperosmotic shock, glycerol flux through Fps1 must be decreased; if not, the cells have great difficulties to accumulate glycerol and hence show osmosensitivity. The exact mechanisms behind Fps1 regulation are still unknown, but regulatory domains on both cytoplasmic termini have been identified. Here, the importance of the Fps1 transmembrane core in restricting glycerol flux is described, and we show that the termini alone are not sufficient to regulate channel activity. We have also studied an orthodox aquaporin that is important for freeze and thaw resistance in the yeast Pichia pastoris. The activity of this aquaporin was shown to be regulated by a combination of phosphorylation and mechanosensitivity. Finally, osmotic regulation throughout the yeast developmental pathways of sporulation and germination is briefly discussed. We have determined the transcriptional changes occurring during yeast spore germination and the analysis revealed a sequential upregulation of different subprograms that we can link to specific transcription factors. Although qualitatively similar responses, the transcriptional output of spores in response to glucose is not as pronounced as to rich growth medium, suggesting that spores can sense nutrient starvation early on in the quickening process.
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| 19. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Saccharomyces cerevisiae Spore Germination
- 2010
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Ingår i: Dormancy and Resistance in Harsh Environments, Topics in Current Genetics, Volume 21/2010; editors: Esther Lubzens, Joan Cerda and Melody Clark. - Berlin Heidelberg : Springer. - 9783642124211 ; , s. 29-41
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Saccharomyces cerevisiae spore germination is the process in which dormant spores resume growth. Upon exposure to glucose and other essential nutrients, the spore gradually loses its spore characteristics and starts acquiring properties of a vegetative cell. Translation and transcription are initiated early in the germination process. Global gene expression analysis has revealed that germination can be divided into two stages prior to the first cell cycle. During the first stage, the transcriptional programme resembles the general response of yeast cells to glucose. During the second stage, the spores sense and respond also to other nutrients than glucose. In addition, genes involved in conjugation are upregulated in germinating spores and mating is initiated before the first mitotic cell cycle. Here, we review the current understanding of the cellular rearrangements and the genes and proteins involved in germination.
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| 20. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Time course gene expression profiling of yeast spore germination reveals a network of transcription factors orchestrating the global response
- 2012
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Background Spore germination of the yeast Saccharomyces cerevisiae is a multi-step developmental path on which dormant spores re-enter the mitotic cell cycle and resume vegetative growth. Upon addition of a fermentable carbon source and nutrients, the outer layers of the protective spore wall are locally degraded, the tightly packed spore gains volume and an elongated shape, and eventually the germinating spore re-enters the cell cycle. The regulatory pathways driving this process are still largely unknown. Here we characterize the global gene expression profiles of germinating spores and identify potential transcriptional regulators of this process with the aim to increase our understanding of the mechanisms that control the transition from cellular dormancy to proliferation. Results Employing detailed gene expression time course data we have analysed the reprogramming of dormant spores during the transition to proliferation stimulated by a rich growth medium or pure glucose. Exit from dormancy results in rapid and global changes consisting of different sequential gene expression subprograms. The regulated genes reflect the transition towards glucose metabolism, the resumption of growth and the release of stress, similar to cells exiting a stationary growth phase. High resolution time course analysis during the onset of germination allowed us to identify a transient up-regulation of genes involved in protein folding and transport. We also identified a network of transcription factors that may be regulating the global response. While the expression outputs following stimulation by rich glucose medium or by glucose alone are qualitatively similar, the response to rich medium is stronger. Moreover, spores sense and react to amino acid starvation within the first 30 min after germination initiation, and this response can be linked to specific transcription factors. Conclusions Resumption of growth in germinating spores is characterized by a highly synchronized temporal organisation of up- and down-regulated genes which reflects the metabolic reshaping of the quickening spores.
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| 21. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Yeast Aquaglyceroporins Use the Transmembrane Core to Restrict Glycerol Transport
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:28, s. 23562-23570
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Tidskriftsartikel (refereegranskat)abstract
- Aquaglyceroporins are transmembrane proteins belonging to the family of aquaporins, which facilitate the passage of specific uncharged solutes across membranes of cells. The yeast aquaglyceroporin Fps1 is important for osmoadaptation by regulating intracellular glycerol levels during changes in external osmolarity. Upon high osmolarity conditions, yeast accumulates glycerol by increased production of the osmolyte and by restricting glycerol efflux through Fps1. The extended cytosolic termini of Fps1 contain short domains that are important for regulating glycerol flux through the channel. Here we show that the transmembrane core of the protein plays an equally important role. The evidence is based on results from an intragenic suppressor mutation screen and domain swapping between the regulated variant of Fps1 from Saccharomyces cerevisiae and the hyperactive Fps1 ortholog from Ashbya gossypii. This suggests a novel mechanism for regulation of glycerol flux in yeast, where the termini alone are not sufficient to restrict Fps1 transport. We propose that glycerol flux through the channel is regulated by interplay between the transmembrane helices and the termini. This mechanism enables yeast cells to fine-tune intracellular glycerol levels at a wide range of extracellular osmolarities.
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| 22. |
- Laukkonen Ravn, Jonas, 1987, et al.
(författare)
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Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
- 2024
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 23:85
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Tidskriftsartikel (refereegranskat)abstract
- Background The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.
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| 23. |
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| 24. |
- Moreno, David, 1986, et al.
(författare)
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Effect of oxygen on the fermentation performance of Candida intermedia: a study case for lignocellulosic bioethanol production
- 2015
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Ingår i: 32nd International Specialized Symposium on Yeasts.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Microbial robustness is considered one of the remaining challenges for a cost-effective lignocellulosic bioethanol production. This concept stands for the efficient conversion of all sugars present in lignocellulose while dealing with the inhibitory compounds generated during biomass processing (furan derivatives, short chain organic acids and phenolic compounds) and fermentation (ethanol). In this context, efficient xylose conversion is crucial as it represents the second most abundant sugar in lignocellulosic biomass. We have isolated a clone of the non-conventional xylose-fermenting yeast species Candida intermedia that shows great potential for being a non-genetically modified alternative for lignocellulosic bioethanol production. To understand its potential for industrial use, we performed a thorough physiological investigation of the strain. In the present work, the fermentation performance of the isolated clone was evaluated in glucose/xylose medium under different oxygen-limiting conditions to promote ethanol production. When oxygen was supplied with a flow rate of 1 vvm and a concentration ranging from 1% to 21% in the gas flow, a xylose consumption rate of 0.10-0.78 g/L h was observed. After glucose depletion, ethanol concentration remained constant and only xylitol (0.34-0.61 g/g) and cell biomass were further produced. When no oxygen was supplied, a maximum xylose consumption rate of 0.53 ± 0.04 g/L h was observed. Furthermore, almost 90% of the initial xylose concentration was consumed within the first 48 h and ethanol was produced continuously, even after glucose depletion. Regarding xylitol accumulation, a yield of 0.14 ± 0.04 g/g was found at 48 h of fermentation. In brief, we can conclude that C. intermedia requires low oxygen concentrations for triggering ethanol production, and that the presence of even low amounts of oxygen further on in the process has a detrimental effect in the conversion of xylose to ethanol.
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| 25. |
- Moreno, David, 1986, et al.
(författare)
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Evolutionary engineered Candida intermedia exhibits improved xylose utilization and robustness to lignocellulose-derived inhibitors and ethanol
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 103:3, s. 1405-1416
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Tidskriftsartikel (refereegranskat)abstract
- The development of robust microorganisms that can efficiently ferment both glucose and xylose represents one of the major challenges in achieving a cost-effective lignocellulosic bioethanol production. Candida intermedia is a non-conventional, xylose-utilizing yeast species with a high-capacity xylose transport system. The natural ability of C. intermedia to produce ethanol from xylose makes it attractive as a non-GMO alternative for lignocellulosic biomass conversion in biorefineries. We have evaluated the fermentation capacity and the tolerance to lignocellulose-derived inhibitors and the end product, ethanol, of the C. intermedia strain CBS 141442 isolated from steam-exploded wheat straw hydrolysate. In a mixed sugar fermentation medium, C. intermedia CBS 141442 co-fermented glucose and xylose, although with a preference for glucose over xylose. The strain was clearly more sensitive to inhibitors and ethanol when consuming xylose than glucose. C. intermedia CBS 141442 was also subjected to evolutionary engineering with the aim of increasing its tolerance to inhibitors and ethanol, and thus improving its fermentation capacity under harsh conditions. The resulting evolved population was able to ferment a 50% (v/v) steam-exploded wheat straw hydrolysate (which was completely inhibitory to the parental strain), improving the sugar consumption and the final ethanol concentration. The evolved population also exhibited a better tolerance to ethanol when growing in a xylose medium supplemented with 35.5 g/L ethanol. These results highlight the potential of C. intermedia CBS 141442 to become a robust yeast for the conversion of lignocellulose to ethanol.
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| 26. |
- Moreno, David, 1986, et al.
(författare)
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Increasing the tolerance of the non-conventional yeast Candida intermedia to ethanol and lignocellulose-derived inhibitors
- 2016
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Ingår i: 4th Symposium on Biotechnology Applied to Lignocelluloses.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The necessity of providing ‘robust microorganisms’ – defined as the ability to efficiently ferment all the available sugars (both hexoses and pentoses) and to cope with the main stressors present during the fermentation process (including biomass-derived products and ethanol) – represents one of the major challenges for a cost-effective lignocellulosic bioethanol production. The yeast Saccharomyces cerevisiae is the preferred fermentative microorganism in the current bioethanol industry due to its superior fermentation capacity of hexose sugars and its tolerance to several inhibitory compounds. The main disadvantage of S. cerevisiae is, however, its inability to ferment xylose, the second most abundant sugar in lignocellulose (>30% of the total sugar). Metabolic and evolutionary engineering methods have been applied to allow xylose fermentation in S. cerevisiae. Still, xylose-fermenting S. cerevisiae strains lack an efficient xylose-to-ethanol conversion system and issues such as low xylose uptake rates and conversion yields, redox imbalance and the lack of simultaneous use of glucose and xylose are important parameters that still need to be optimized. As an alternative to genetically modified S. cerevisiae strains, non-conventional, native xylose-utilizing yeasts such as the Scheffersomyces species S. stipitis and S. shehatae, Spathaspora passalidarum and various Candida species (C. tropicalis, C. guilliermondii or C. intermedia) have been considered for the fermentation of pentose sugars. These yeasts have, however, a modest tolerance to lignocellulose-derived inhibitors and ethanol, which limits their applicability. Candida intermedia is a xylose-fermenting yeast species that encompasses a high capacity xylose transport system. This trait makes C. intermedia attractive for being a non-GMO alternative in the lignocellulosic bioethanol industry. In the present work, the ethanol tolerance and the fermentation capacity in the presence of lignocellulose-derived inhibitors of an in-house isolated C. intermedia strain was evaluated. The isolated strain showed a medium-tolerance towards lignocellulose-derived inhibitors, being more sensitive when using xylose as a carbon source. The ethanol concentration above which there is no growth was estimated to be 42 g/L when growing in glucose and 55 g/L when growing in xylose. The isolated strain was subjected to evolutionary engineering with the aim of increasing its tolerance towards both lignocellulose-derived inhibitors and ethanol. The obtained evolved population was able to ferment a lignocellulosic hydrolysate (steam-exploded wheat straw), not fermentable by the isolated strain. Furthermore, the evolved population produced higher biomass concentration (7.5-fold higher OD600nm values) when growing in the presence of 36 g/L ethanol, compared to the parental strain. These results highlight the potential of C. intermedia to become a robust yeast microorganism for the lignocellulose-to-ethanol conversion.
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| 27. |
- Palmgren, Madelene, et al.
(författare)
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Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins
- 2017
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Ingår i: Journal of Membrane Biology. - : Springer Science and Business Media LLC. - 0022-2631 .- 1432-1424. ; 250:6, s. 629-639
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Tidskriftsartikel (refereegranskat)abstract
- Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.
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| 28. |
- Peri, Kameshwara Venkata Ramana, 1990, et al.
(författare)
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Regulation of lactose and galactose growth: Insights from a unique metabolic gene cluster in Candida intermedia
- 2023
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia. Through genomic and transcriptomic analyses and deletion mutant phenotyping, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster (LAC12, LAC4) for lactose uptake and hydrolysis, the conserved GAL cluster (GAL1, GAL7, GAL10) for galactose catabolism, and a unique “GALLAC” cluster. This novel GALLAC cluster, which has evolved through gene duplication and divergence, proved indispensable for C. intermedia’s growth on lactose and galactose. The cluster contains the transcriptional activator gene LAC9, second copies of GAL1 and GAL10 and the XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Notably, the regulatory network in C. intermedia, governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the (ga)lactose regulons in Saccharomyces cerevisiae, Kluyveromyces lactis and Candida albicans. Moreover, although lactose and galactose metabolism are closely linked in C. intermedia, our results also point to important regulatory differences. This study paves the way to a better understanding of lactose and galactose metabolism in C. intermedia and provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products.
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| 29. |
- Peri, Kameshwara Venkata Ramana, 1990, et al.
(författare)
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Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia
- 2023
-
Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 23
-
Tidskriftsartikel (refereegranskat)abstract
- Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.
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| 30. |
- Peri, Kameshwara Venkata Ramana, 1990, et al.
(författare)
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Understanding gene cluster interactions enables cell factory application of non-conventional yeast Candida intermedia
- 2023
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lactose-rich cheese whey is an abundant industrial side stream that can be converted into value-added products using lactose-assimilating yeasts. Whereas the dairy yeasts Kluyveromyces lactis and marxianus have been well studied in terms of their lactose-metabolising traits, most other lactose-assimilating yeast species have not yet been characterized at a genomic and molecular level. The aim of this project was to elucidate the genetic determinants behind the lactose metabolism in one such yeast, Candida intermedia, and thereafter demonstrate the yeast’s potential as a cell factory for production of metabolites from cheese whey. Through comparative growth assays, we found that C. intermedia is one of the top ten among 36 tested lactose-growing ascomycetous yeast, ranked on growth rates in lactose containing media. Transcriptomic analysis revealed that in addition to the well conserved LAC and GAL metabolic gene clusters for (ga)lactose metabolism, C. intermedia also contains a third gene cluster that we refer to as the GALLAC cluster, which is unique to this yeast and essential for its (ga)lactose metabolism. Through targeted genome editing we have confirmed and assigned physiological functions to individual genes in the three clusters and revealed close cluster interdependence. Using the acquired knowledge, we have managed to engineer a C. intermedia that overproduces the sugar alcohol galactitol from lactose. Subsequent strain improvement led to an increased productivity and a >95% galactitol yield from the galactose moiety of lactose. Our work sheds light on gene clusters dynamics and lactose metabolism in C. intermedia. We envision that C. intermedia can be used as a new model organism for deciphering evolutionary aspects of lactose metabolism in ascomycetous yeast as well as a cell factory for production of added-value chemicals using lactose-rich industrial side streams as raw material.
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| 31. |
- Ravn, Jonas, 1987, et al.
(författare)
-
CAZyme prediction in ascomycetous yeast genomes guides discovery of novel xylanolytic species with diverse capacities for hemicellulose hydrolysis
- 2021
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 14:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Ascomycetous yeasts from the kingdom fungi inhabit every biome in nature. While filamentous fungi have been studied extensively regarding their enzymatic degradation of the complex polymers comprising lignocellulose, yeasts have been largely overlooked. As yeasts are key organisms used in industry, understanding their enzymatic strategies for biomass conversion is an important factor in developing new and more efficient cell factories. The aim of this study was to identify polysaccharide-degrading yeasts by mining CAZymes in 332 yeast genomes from the phylum Ascomycota. Selected CAZyme-rich yeasts were then characterized in more detail through growth and enzymatic activity assays. Results: The CAZyme analysis revealed a large spread in the number of CAZyme-encoding genes in the ascomycetous yeast genomes. We identified a total of 217 predicted CAZyme families, including several CAZymes likely involved in degradation of plant polysaccharides. Growth characterization of 40 CAZyme-rich yeasts revealed no cellulolytic yeasts, but several species from the Trichomonascaceae and CUG-Ser1 clades were able to grow on xylan, mixed-linkage β-glucan and xyloglucan. Blastobotrys mokoenaii, Sugiyamaella lignohabitans, Spencermartinsiella europaea and several Scheffersomyces species displayed superior growth on xylan and well as high enzymatic activities. These species possess genes for several putative xylanolytic enzymes, including ones from the well-studied xylanase-containing glycoside hydrolase families GH10 and GH30, which appear to be attached to the cell surface. B. mokoenaii was the only species containing a GH11 xylanase, which was shown to be secreted. Surprisingly, no known xylanases were predicted in the xylanolytic species Wickerhamomyces canadensis, suggesting that this yeast possesses novel xylanases. In addition, by examining non-sequenced yeasts closely related to the xylanolytic yeasts, we were able to identify novel species with high xylanolytic capacities. Conclusions: Our approach of combining high-throughput bioinformatic CAZyme-prediction with growth and enzyme characterization proved to be a powerful pipeline for discovery of novel xylan-degrading yeasts and enzymes. The identified yeasts display diverse profiles in terms of growth, enzymatic activities and xylan substrate preferences, pointing towards different strategies for degradation and utilization of xylan. Together, the results provide novel insights into how yeast degrade xylan, which can be used to improve cell factory design and industrial bioconversion processes.
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| 32. |
- Ravn, Jonas, 1987, et al.
(författare)
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Yeasts Have Evolved Divergent Enzyme Strategies To Deconstruct and Metabolize Xylan
- 2023
-
Ingår i: Microbiology spectrum. - 2165-0497. ; 11:3
-
Tidskriftsartikel (refereegranskat)abstract
- Together with bacteria and filamentous fungi, yeasts actively take part in the global carbon cycle. Over 100 yeast species have been shown to grow on the major plant polysaccharide xylan, which requires an arsenal of carbohydrate active enzymes. However, which enzymatic strategies yeasts use to deconstruct xylan and what specific biological roles they play in its conversion remain unclear. In fact, genome analyses reveal that many xylan-metabolizing yeasts lack expected xylanolytic enzymes. Guided by bioinformatics, we have here selected three xylan-metabolizing ascomycetous yeasts for in-depth characterization of growth behavior and xylanolytic enzymes. The savanna soil yeast Blastobotrys mokoenaii displays superior growth on xylan thanks to an efficient secreted glycoside hydrolase family 11 (GH11) xylanase; solving its crystal structure revealed a high similarity to xylanases from filamentous fungi. The termite gut-associated Scheffersomyces lignosus, in contrast grows more slowly, and its xylanase activity was found to be mainly cell surface-associated. The wood-isolated Wickerhamomyces canadensis, surprisingly, could not utilize xylan as the sole carbon source without the addition of xylooligosaccharides or exogenous xylanases or even co-culturing with B. mokoenaii, suggesting that W. canadensis relies on initial xylan hydrolysis by neighboring cells. Furthermore, our characterization of a novel W. canadensis GH5 subfamily 49 (GH5_49) xylanase represents the first demonstrated activity in this subfamily. Our collective results provide new information on the variable xylanolytic systems evolved by yeasts and their potential roles in natural carbohydrate conversion. IMPORTANCE Microbes that take part in the degradation of the polysaccharide xylan, the major hemicellulose component in plant biomass, are equipped with specialized enzyme machineries to hydrolyze the polymer into monosaccharides for further metabolism. However, despite being found in virtually every habitat, little is known of how yeasts break down and metabolize xylan and what biological role they may play in its turnover in nature. Here, we have explored the enzymatic xylan deconstruction strategies of three underexplored yeasts from diverse environments, Blastobotrys mokoenaii from soil, Scheffersomyces lignosus from insect guts, and Wickerhamomyces canadensis from trees, and we show that each species has a distinct behavior regarding xylan conversion. These findings may be of high relevance for future design and development of microbial cell factories and biorefineries utilizing renewable plant biomass.
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| 33. |
- Schaubeder, Jana B., et al.
(författare)
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Xylan-cellulose thin film platform for assessing xylanase activity
- 2022
-
Ingår i: Carbohydrate Polymers. - : Elsevier BV. - 0144-8617. ; 294
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatic degradation of plant polysaccharide networks is a complex process that involves disrupting an intimate assembly of cellulose and hemicelluloses in fibrous matrices. To mimic this assembly and to elucidate the efficiency of enzymatic degradation in a rapid way, models with physicochemical equivalence to natural systems are needed. Here, we employ xylan-coated cellulose thin films to monitor the hydrolyzing activity of an endo-1,4-β-xylanase. In situ surface plasmon resonance spectroscopy (SPRS) revealed a decrease in xylan areal mass ranging from 0.01 ± 0.02 to 0.52 ± 0.04 mg·m−2. The extent of digestion correlates to increasing xylanase concentration. In addition, ex situ determination of released monosaccharides revealed that incubation time was also a significant factor in degradation (P > 0.01). For both experiments, atomic force microscopy confirmed the removal of xylans from the cellulose thin films. We provide a new model platform that offers nanoscale sensitivity for investigating biopolymer interactions and their susceptibility to enzymatic hydrolysis.
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| 34. |
- Šuchová, Katarína, et al.
(författare)
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Cellulose- and xylan-degrading yeasts: Enzymes, applications and biotechnological potential
- 2022
-
Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 59
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Forskningsöversikt (refereegranskat)abstract
- Microbes and their carbohydrate-active enzymes are central for depolymerization of complex lignocellulosic polysaccharides in the global carbon cycle. Their unique abilities to degrade and ferment carbohydrates are also utilized in many industrial processes such as baking, brewing and production of biofuels and drugs. Effective degradation and utilization of cellulose and hemicelluloses is important for the shift towards green bioeconomy, and requires microbes equipped with proper sets of carbohydrate-active enzymes (CAZymes). Knowledge of cellulolytic and xylanolytic CAZymes has mainly been generated from bacteria and filamentous fungi, while yeasts have been largely overlooked and may represent an untapped resource in natural CAZymes with industrial relevance. Cellulose and xylan-degrading yeasts with the ability to ferment saccharides are also promising candidates for consolidated bioprocesses (CBPs), as they can degrade lignocellulose and utilize its constituents to produce desired products at the same time. Cellulolytic yeasts able to utilize insoluble crystalline cellulose are rare while xylanolytic yeasts are rather widespread in nature. The lack of particular enzymes in yeasts can be remediated by introducing the missing enzymes into strains having outstanding product-forming attributes. In this review, we provide a comprehensive overview of the cellulose- and xylan-degrading ascomycetous and basidiomycetous yeasts known to date. We describe how these yeasts can be identified through bioprospecting and bioinformatic approaches and summarize available growth and enzymatic assays for strain characterization. Known and predicted CAZymes are extensively analyzed, both in individual species and in a phylogenetic perspective. We also describe the strategies used for construction of recombinant cellulolytic and xylanolytic strains as well as current applications for polysaccharide-degrading yeasts. Finally, we discuss the great potential of these yeasts as industrial cell factories, identify open research questions and provide suggestions for future investigations.
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| 35. |
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