| 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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