| 1. |
- Lindahl, Lina, 1984, et al.
(author)
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Membrane engineering for reduced acetic acid stress: insights from Zygosaccharomyces bailii
- 2015
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In: Oral presentation at 12th Yeast Lipid Conference, May 20-22 2015, Ghent, Belgium.
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Conference paper (other academic/artistic)abstract
- The high concentration of acetic acid released during pretreatment of lignocellulose raw material is a major obstacle to the microbial production of bio-based products. Acetic acid enters the cell mainly by passive diffusion across the plasma membrane and inhibits yeast by mechanisms such as reduction of intracellular pH, accumulation of the acetate anion, and by signaling effects triggering cell death. Through extensive characterization of the acetic acid tolerant yeast Zygosaccharomyces bailii, we have identified the cell membrane as a target for strain engineering with potential to increase acetic acid tolerance in Saccharomyces cerevisiae. We propose membrane permeability as a key component for Z. bailii’s acetic acid tolerance. We have previously shown that Z. bailii has a unique ability to remodel its plasma membrane upon acetic acid stress, to strongly increase its fraction of complex sphingolipids, at the expense of a drastic reduction of glycerophospholipids1. Here we further demonstrate the involvement of complex sphingolipids in acetic acid tolerance by decreasing sphingolipid synthesis using the drug myriocin, and characterize the acetic acid tolerance in terms of growth and intracellular pH. Furthermore we show the impact of complex sphingolipids on membrane physical properties using in silico membrane simulations. Ongoing membrane engineering of S. cerevisiae can potentially give additional strength to our findings. References 1 Lindberg et al. (2013), Lipidomic Profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii Reveals Critical Changes in Lipid Composition in Response to Acetic Acid Stress, PLoS One 8: e73936.
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| 2. |
- Lindahl, Lina, 1984, et al.
(author)
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THE INFLUENCE OF MEMBRANE COMPOSTION ON ACETIC ACID PERMEABILITY AND POTENTIALLY ACETIC ACID TOLERANCE
- 2014
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In: ISSY31: 31st International Specialised Symposium on Yeast.
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Other publication (other academic/artistic)abstract
- Compounds entering the cell do so either by passive diffusion over the plasma membrane or through transporters in the membrane. The specific lipid composition of the plasma membrane influences both the passive diffusion rate but also the activity of membrane proteins. Acetic acid, a major hurdle in fermentation processes using lignocellulosic material, is believed to pass through the membrane in its protonated from mainly by passive diffusion [1]. Sterols and sphingolipids are lipid classes thought to contribute to membrane rigidity. Sterols are often found to be involved in stress resistance [2, 3] and in our previous work sphingolipids were pointed at as an important constituent of the plasma membrane of the yeast Zygosaccharomyces bailii, known to be very tolerant to acetic acid, suggesting a possible link between acetic acid tolerance and sphingolipid relative abundance in the membrane [4]. Here we will provide supporting evidence of the importance of sphingolipids and sterols in acetic acid membrane permeability. We have combined biochemistry techniques with in silico membrane modeling to answer the question how membrane engineering can be used to decrease acetic acid membrane permeability. [1] Verduyn et al. Yeast (1992) 501-517. [2] Alexandre et al. FEMS Microbiology Letters (1994) 124:17-22. [3] Liu et al. Journal of Applied Microbiology (2013) 114:482-491. [4] Lindberg et al. PlosONE (2003) 8(9): e73936.
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| 3. |
- Adeboye, Peter, 1982, et al.
(author)
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In situ conversion of phenolic compounds as a tool to phenolic tolerance development by S. cerevisiae
- 2015
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Conference paper (other academic/artistic)abstract
- Phenolic compounds in hydrolysates are degradation products from the lignin component of wood. They are diverse in nature and they account for some of the inhibitory activities observed during lignocellulosic fermentation. S. cerevisiae possesses the ability to convert some phenolic compounds. We are currently studying the interaction between S. cerevisiae and selected phenolic compounds namely; coniferyl aldehyde, ferulic acid and p-coumaric acid to understand the ability of S. cerevisiae to convert the selected compounds. Preliminary results show that the three phenolic compounds are being converted into several other less inhibitory phenolic compounds common to the three compounds. We hypothesised a conversion route and engineered S. cerevisiae strains to test the hypothesis, the preliminary result shows faster conversion in an engineered strain.
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| 4. |
- Ask, Magnus, 1983, et al.
(author)
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Physiological studies of Saccharomyces cerevisiae for increased tolerance against furfural and HMF – two common inhibitors in lignocellulosic hydrolysate
- 2011
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In: 5th EU-Summer School Proteomic Basics, Brixen, Italy.
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Conference paper (other academic/artistic)abstract
- The use of fossil fuels in the transport sector has a significant impact on the environment through the emission of greenhouse gases such as carbon dioxide. Bioethanol is one alternative that has shown potential to at least partly replace fossil fuels. Today’s bioethanol is mainly produced from sugar cane and corn with the yeast Saccharomyces cerevisiae as production organism. Since these raw materials compete with food production, new feedstocks have to be found. A promising alternative is to make use of forest and agricultural residues, so called lignocellulosic materials. Nevertheless, there are many challenges with using lignocellulosic materials for bioethanol production. Since they are recalcitrant to decomposition, harsh conditions have to be used to break down the materials. These conditions tend to produce byproducts that can be inhibitory for the production organism, resulting in lower process productivity. Low productivity is one of the main factors affecting the feasibility of lignocellulosic ethanol production processes. Hydroxymethylfurfural (HMF) and furfural are two compounds that have received a lot of attention during the last years. By studying the effect of these inhibitory compounds on the energy metabolism of S. cerevisiae, the aim of the project is to increase the understanding of the mechanisms by which these inhibitors affect the microorganism. More specifically, the inhibitors effect is studied by quantifying intracellular key compounds such as NADH, NAD+, sugar phosphates and the adenine nucleotide pool. In addition, the biochemical data on intracellular concentrations will be integrated with transcriptomic data. In the future, this knowledge will be used to produce strains that are more tolerant to the process conditions.
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| 5. |
- Cámara, Elena, 1985, et al.
(author)
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CRISPR interference technology for development of more tolerant industrial yeast strains (Milan)
- 2019
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Conference paper (other academic/artistic)abstract
- Second generation bioethanol using lignocellulosic biomass as raw material is a promising alternative to bioethanol produced from sugar-based feedstocks. In addition to sugars, lignocellulosic hydrolysates also contain inhibitors that impair microbial growth. One way to tackle the low productivities is to develop new strains with increased tolerance towards inhibitors. Over the past few years, different CRISPR technologies have been developed to accelerate the construction of new strains. The CRISPR interference (CRISPRi) technology utilizes a catalytically inactive Cas9 (dCas9) to modulate the expression of genes targeted by a sgRNA, allowing the alteration of essential genes and the manipulation of multiple traits without altering the target sequence. In the present work, our goal was to use CRISPRi to improve the inhibitor tolerance of a polyploid industrial yeast strain. We explored different strategies to overcome the challenges of implementing CRISPRi in an industrial strain. As a proof of concept, the expression of a gene encoding a fluorescent protein was modulated using dCas9 with different activation or repression domains. Changes in fluorescence were measured by flow cytometry and changes in expression were verified by qPCR, validating the use of CRISPRi for alteration of gene expression in an industrial yeast strain. Subsequently, a number of genes previously identified to be involved in inhibitor tolerance were selected as targets for CRISPRi. The performance of the novel strains during growth in the presence of different inhibitors was analysed in a high-throughput platform, leading to identification of strains where the altered gene expression led to improved tolerance. This work shows that the CRISPRi technology can be used to accelerate the development of more robust, industrial production hosts.
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| 6. |
- Hüttner, Silvia, 1984, et al.
(author)
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Fungal Glucuronoyl and Feruloyl Esterases for Wood Processing and Phenolic Acid Ester/Sugar Ester Synthesis
- 2015
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In: Biotrans 2015, Vienna, Austria, 26-30 July 2015.
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Conference paper (other academic/artistic)abstract
- Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) and glucuronoyl esterases(GEs, E.C. 3.1.1.-, CAZy family CE15) are involved in the degradation of plantbiomass by hydrolysing ester linkages in plant cell walls, and thus have potential use inbiofuel production from lignocellulosic materials and in biorefinery applications withthe aim of developing new wood-based compounds [1, 2]. GEs and FAEs are present inthe genomes of a wide range of fungi and bacteria.Under conditions of low water content, these enzymes can also carry out(trans)esterification reactions, making them promising biocatalysts for the modificationof compounds with applications in the food, cosmetic and pharmaceutical industry.Compared to the chemical process, enzymatic synthesis can be carried out under lowerprocess temperatures (50-60°C) and results in fewer side products, thus reducing theenvironmental impact.We characterised new FAE and GE enzymes from mesophilic, thermophilic and coldtolerantfilamentous fungi produced in Pichia pastoris. The enzymes were characterisedfor both their hydrolytic abilities on various model substrates (methyl ferulate, pNPferulate)- for potential applications in deconstruction of lignocellulosic materials andextraction of valuable compounds - as well as for their biosynthetic capacities. Wetested and optimised the FAEs’ transesterification capabilities on ferulate esters in a 1-butanol-buffer system, with the aim of using the most promising candidates for theproduction of antioxidant compounds with improved hydrophobic or hydrophilicproperties, such as prenyl ferulate, prenyl caffeate, glyceryl ferulate and 5-O-(transferuloyl)-arabinofuranose.
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| 7. |
- Karlsson, Emma, 1983, et al.
(author)
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METABOLIC ENGINEERING OF Saccharomyces cerevisiae FOR PRODUCTION OF ADIPIC ACID FROM RENEWABLE SOURCES
- 2014
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Conference paper (other academic/artistic)abstract
- Adipic acid is a six carbon long dicarboxylic acid, considered to be the most important synthetic dicarboxylic acid annually produced, according to the International Energy Agency (IEA). The global production of adipic acid had in 2010 a volume of 2.8 million tonnes, for a total market price of 4.9 billion USD. The current production of adipic acid relies on non-renewable fossil raw materials, leading to emission of the greenhouse gases carbon dioxide and N2O. In addition, the production starts from benzene, whose use has several health related negative implications. This project aims to create a greener process for production of adipic acid developing a fermentation-based process using Swedish domestic renewable raw materials, such as forest residues and/or algae. These materials will be used to establish a biorefinery, wherein the fermentation process for the biosynthesis of adipic acid will represent the core process. Our current strategy is based on the generation of genetically modified strains of the yeast Saccharomyces cerevisiae, harbouring heterologous enzymatic activities allowing the conversion of lysine into adipic acid. This system is our first choice and will also work as proof-of-concept for bio-based production of adipic acid. Here we present the metabolic engineering strategy we are pursuing, based on two possible metabolic pathways for conversion of lysine into adipic acid. Preliminary results on the effect of adipic acid on S. cerevisiae physiology, lysine uptake, the expression of the heterologous genes of choice, and the conversion of lysine into adipic acid precursors are presented.
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| 8. |
- Koppram, Rakesh, 1986, et al.
(author)
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A novel process configuration of Simultaneous Saccharification and Fermentation for bioethanol production at high solid loadings
- 2012
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In: Advanced Biofuels in a Biorefinery Approach, February 28 - March 1, 2012, Copenhagen, Denmark.
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Conference paper (other academic/artistic)abstract
- Simultaneous saccharification and fermentation (SSF) is a process option for lignocellulosic bioethanol production that has proven to have several advantages compared to separate hydrolysis and fermentation. The economical viability and commercialization of cellulose-to-ethanol demands the process to work under high-solid loadings to result in high sugar yield and final ethanol titer in S. cerevisiae based SSF process. In a conventional batch SSF process practical limitations to high-solid loadings include, poor mixing and accessibility of enzymes to substrates and high inhibitors concentration that reduces the yeast viability and metabolism. In order to overcome these limitations, we propose a novel SSF process configuration involving feeding of substrate, enzyme and yeast. It is possible to overcome mixing issues associated with a batch SSF at high-solid loadings by a feed of substrate, enzyme and yeast. The feed of freshly cultivated yeast throughout the fermentation process ensures active metabolic state of yeast. In addition, the substrate feed ensures low inhibitors concentration at any given time point increasing the survival ability of yeast compared to a batch SSF. The enzyme feed ensures slow release of glucose providing an opportunity for xylose consuming yeast strain to co-consume xylose together with glucose. The aim of the current work is to understand how different combinations of feeding strategies influence the outcome of the SSF process. In the longer perspective, we aim at deducing an optimized SSF process that can handle very high-solid loadings with efficient hydrolysis and fermentation process at low enzyme and yeast loadings, respectively.
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| 9. |
- Lindberg, Lina, 1984, et al.
(author)
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Investigation of weak organic acid tolerance mechanisms by lipidomic profiling of Saccharomyces cerevisiae and Zygosaccaromyces bailii
- 2012
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In: Life Science Engineering Area of Advance Conference. From Human health to Biosustainability – Future challenges for Life Science at Chalmers.Gothenburg, Sweden.November 19, 2012.
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Conference paper (other academic/artistic)abstract
- During pretreatment of lignocellulose raw material, compounds such as furaldehydes, phenolics and weak organic acids, severely inhibiting Saccharomyces cerevisiae, are released. Decrease of intracellular pH after diffusion through the plasma membrane is thought to be one of the effects mediating the cellular toxicity of weak organic acids.The aim of the present study is to investigate the relationship between plasma membrane composition and acid tolerance, in order to develop a strategy for engineering a S. cerevisiae strain more tolerant to acetic acid. Zygosaccharomyces bailii, a well-known food spoilage yeast, is highly tolerant to acetic acid and will be used as a model for weak organic acid tolerance.A complete lipidomic profiling of S. cerevisiae and Z. bailii in the presence and absence of acetic acid will be carried out using LC-MS/MS. Similarities and differences in the two profiles will be correlated with acid tolerance.
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| 10. |
- Mapelli, Valeria, 1978, et al.
(author)
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Metabolism of selenium in Saccharomyces cerevisiae and improved biosynthesis of bioactive organic Se-compounds
- 2010
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In: 4th Conference on Physiology of Yeast and Filamentous Fungi (PYFF4).
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Conference paper (other academic/artistic)abstract
- Selenium (Se) is an essential element for many organisms as it is present under the form of Se-cysteine in Se-proteins. The main sources of Se for animals are edible plants able to accumulate Se from the soil in inorganic and organic forms. Some of the Se organic forms bioavailable for animals have been proven to have cancer-preventing effects if regularly introduced into the diet. Since Se content in plants is highly susceptible to environmental factors, the intake of Se is often insufficient to result in beneficial effects. Therefore, the use of Se-enriched yeast as food supplement is made available to avoid Se shortage. The yeast Saccharomyces cerevisiae does not require Se as essential element, but is able to metabolise and accumulate Se. Due to the very similar properties of Se and sulphur (S), S- and Se-compounds share the same assimilation and metabolic routes, but the competition is in favour of S-species, as the high reactivity of Se leads to the formation of toxic compounds. Due to the delicate balance between beneficial and toxic effects of Se, the study of Se metabolism in yeast is a crucial point towards the establishment of a yeast cell factory for the production of bioactive Se-compounds. This work presents a successful strategy for the improved biosynthesis of bioactive Se-compounds in S. cerevisiae. Mapping of Se metabolome and study of yeast physiology in the presence of Se represent the essential basis of the present approach that couples metabolic engineering and bioprocess optimization towards the production of organic Se-molecule with high anti-cancer potential in yeast.
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