| 1. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Plasma membrane as a crucial player in acetic acid effect on yeast
- 2017
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Ingår i: IMYA12- 12th International Meeting on Yeast Apoptosis, Bari, Italy • 14-18 May 2017.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Weak organic acids such as formic, acetic or lactic acid are known inhibitors of microbial growth and fermentation. Acetic acid toxicity to yeast cells has been explained by different theories, involving specific signaling effects triggering an active cell death program, reduction of intracellular pH and acetate anion accumulation. Regardless of the fact whether the actual effect of acetate involves one of these mechanisms or a combination thereof, acetic acid inhibits yeast metabolism and affects yeast viability. This has a high impact on the feasibility of the new generation of fermentation processes, based on the naturally acetic acid-rich lignocellulosic substrates. It is therefore highly desirable to obtain a strain with increased capacity of coping with high acetic acid concentrations in the fermentation medium. Acetic acid is thought to be internalized by yeast cells in its undissociated form, by crossing the hydrophobic barrier of plasma membrane. Thus, in our work we focused on the investigation of membrane properties and how these influence the tolerance of yeast to acetic acid. First, we demonstrated with lipidomics analysis of membrane lipids that the yeast Zygosaccharomyces bailii, showing extraordinary tolerance to acetic acid, has a plasma membrane which is rich in sphingolipids. Next, we combined membrane molecular dynamics and in vivo measurements to confirm the specific role of sphingolipids in altering the permeability of plasma membrane to acetic acid. Finally, we investigated the effect of alcohols on the acetic acid permeation rate through the membrane. Our ultimate goal is to engineer the membrane composition of an industrial yeast strain towards reduced permeability, in order to obtain higher acetic acid tolerance.
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| 2. |
- Lindahl, Lina, 1984, et al.
(författare)
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Alcohols enhance the rate of acetic acid diffusion in S. cerevisiae: biophysical mechanisms and implications for acetic acid tolerance
- 2018
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Ingår i: Microbial Cell. - : Shared Science Publishers OG. - 2311-2638. ; 5:1, s. 42-55
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Tidskriftsartikel (refereegranskat)abstract
- Microbial cell factories with the ability to maintain high productivity in the presence of weak organic acids, such as acetic acid, are required in many industrial processes. For example, fermentation media derived from lignocellulosic biomass are rich in acetic acid and other weak acids. The rate of diffusional entry of acetic acid is one parameter determining the ability of microorganisms to tolerance the acid. The present study demonstrates that the rate of acetic acid diffusion in S. cerevisiae is strongly affected by the alcohols ethanol and n-butanol. Ethanol of 40 g/L and n-butanol of 8 g/L both caused a 65% increase in the rate of acetic acid diffusion, and higher alcohol concentrations caused even greater increases. Molecular dynamics simulations of membrane dynamics in the presence of alcohols demonstrated that the partitioning of alcohols to the head group region of the lipid bilayer causes a considerable increase in the membrane area, together with reduced membrane thickness and lipid order. These changes in physiochemical membrane properties lead to an increased number of water molecules in the membrane interior, providing biophysical mechanisms for the alcohol-induced increase in acetic acid diffusion rate. nbutanol affected S. cerevisiae and the cell membrane properties at lower concentrations than ethanol, due to greater and deeper partitioning in the membrane. This study demonstrates that the rate of acetic acid diffusion can be strongly affected by compounds that partition into the cell membrane, and highlights the need for considering interaction effects between compounds in the design of microbial processes.
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| 3. |
- Lindahl, Lina, 1984, et al.
(författare)
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Membrane engineering for reduced acetic acid stress: insights from Zygosaccharomyces bailii
- 2015
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Ingår i: Oral presentation at 12th Yeast Lipid Conference, May 20-22 2015, Ghent, Belgium.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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| 4. |
- Lindahl, Lina, 1984, et al.
(författare)
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Membrane engineering of S. cerevisiae targeting sphingolipid metabolism
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 7, s. 41868-
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Tidskriftsartikel (refereegranskat)abstract
- The sustainable production of fuels and chemicals using microbial cell factories is now well established. However, many microbial production processes are still limited in scale due to inhibition from compounds that are present in the feedstock or are produced during fermentation. Some of these inhibitors interfere with cellular membranes and change the physicochemical properties of the membranes. Another group of molecules is dependent on their permeation rate through the membrane for their inhibition. We have investigated the use of membrane engineering to counteract the negative effects of inhibitors on the microorganism with focus on modulating the abundance of complex sphingolipids in the cell membrane of Saccharomyces cerevisiae. Overexpression of ELO3, involved in fatty acid elongation, and AUR1, which catalyses the formation of complex sphingolipids, had no effect on the membrane lipid profile or on cellular physiology. Deletion of the genes ORM1 and ORM2, encoding negative regulators of sphingolipid biosynthesis, decreased cell viability and considerably reduced phosphatidylinositol and complex sphingolipids. Additionally, combining ELO3 and AUR1 overexpression with orm1/2? improved cell viability and increased fatty acyl chain length compared with only orm1/2?. These findings can be used to further study the sphingolipid metabolism, as well as giving guidance in membrane engineering.
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| 5. |
- Lindahl, Lina, 1984, et al.
(författare)
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Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii
- 2016
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:4, s. 744-753
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Tidskriftsartikel (refereegranskat)abstract
- Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition, and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingolipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid, and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid, was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress
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| 6. |
- Lindahl, Lina, 1984, et al.
(författare)
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THE INFLUENCE OF MEMBRANE COMPOSTION ON ACETIC ACID PERMEABILITY AND POTENTIALLY ACETIC ACID TOLERANCE
- 2014
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Ingår i: ISSY31: 31st International Specialised Symposium on Yeast.
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Annan publikation (övrigt vetenskapligt/konstnärligt)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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| 7. |
- Lindahl, Lina, 1984
(författare)
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Towards membrane engineering as a tool in cell factory design: A case study on acetic acid tolerance in Saccharomyces cerevisiae
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The sustainable production of fuels, chemicals, and materials using renewable resources is a necessity if we are to reduce our ecological footprint and the rate of climate change. Lignocellulosic biomass, the major constituent of plant cell walls, is a renewable raw material with great potential due to its high abundance. The conversion of lignocellulosic material into desired products using microorganisms is a promising option, although many microbial production processes fail to reach the titers required for process economy due to cellular inhibition. The inhibitory action of some compounds is related to the physiochemical properties of the cell membrane. Inhibitors may enter the cell by passive diffusion through the lipid bilayer of the cell membrane, or may inhibit cells by partition in the lipid bilayer, altering the membrane properties. The aim of the research described in this thesis was to evaluate the possibility of engineering the lipid composition of the cell membrane to create microbial cell factories with maintained production capacity when exposed to compounds whose mechanism of inhibition relates to the physiochemical properties of the membrane. Attempts were made to increase the tolerance of Saccharomyces cerevisiae to the lignocellulose-derived inhibitor acetic acid, by engineering the cell membrane in order to reduce the rate of acetic acid diffusion. Studies of the acetic-acid-tolerant yeast Zygosaccharomyces bailii revealed that its high tolerance relies on its ability to remodel the cell membrane lipid composition so as to greatly increase the fraction of sphingolipids. Further evidence that sphingolipids reduce the rate of acetic acid diffusion was obtained by molecular dynamics simulations of model membranes with increasing fraction of sphingolipids. The lipid metabolism of S. cerevisiae was then engineered in an attempt to increase the fraction of sphingolipids in the cell membrane. However, sphingolipid synthesis was unchanged or decreased in these strains. The effect of sphingolipids on acetic acid tolerance in S. cerevisiae could therefore not be elucidated, but insight was gained into sphingolipid regulation. To understand the variation in membrane permeation, in particular the extent to which compounds partitioning in the cell membrane change the rate of acetic acid diffusion, the effects of ethanol and n-butanol were investigated. It was found that target titers in ethanol and n‑butanol production significantly increased the rate of acetic acid diffusion; n-butanol having a stronger effect than ethanol. Molecular dynamics simulations were then used to suggest mechanisms for the experimental observations.
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| 8. |
- Slunge, Daniel, 1968, et al.
(författare)
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The implementation of the substitution principle in European chemical legislation: a comparative analysis
- 2023
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Ingår i: Environmental Sciences Europe. - : Springer. - 2190-4707 .- 2190-4715. ; 35
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Tidskriftsartikel (refereegranskat)abstract
- Background: The substitution of hazardous chemicals with safer alternatives is an important objective in European chemical policy, but implementation has been slower than expected. We conduct a comprehensive analysis and comparison of the implementation of the substitution principle in European regulations for pesticides, biocides, and industrial chemicals. Specifically, we examine and compare the criteria and processes associated with the identification of candidates for substitution and the assessment of alternatives. Results: We find only minor differences in the criteria applied to identify candidates for substitution amongst pesticides, biocides, and industrial chemicals, but larger differences concerning the processes used. While all substances that are to be approved as a pesticide and biocide are systematically evaluated against the established criteria for substitution, the substitution process for industrial chemicals only focuses on those substances identified as substances of very high concern. The main reason candidates for substitution remain on the market is the lack of identified safer chemical alternatives and the insufficient consideration of non-chemical alternatives, caused, at least to a large extent, by the comparatively weak incentives provided by current regulations. Conclusions: The systematic approach for the identification of industrial substances of very high concern (SVHC) under ECHAs “Integrated Regulatory Strategy” is much welcome. However, no final conclusion on SVHC properties or the need for regulatory action has been drawn for approximately 90% of the REACH-registered substances, as often even basic hazard and exposure data are missing. Hence, at least a screening-level evaluation of SVHC properties should become a mandatory part of the substance registration under REACH. To reduce the risk of strategic behaviour in the search for alternatives to industrial chemicals identified as SVHC, a setup in which regulatory authorities play a larger role as information and knowledge brokers should be considered. Investments in innovation as well as improved sharing of information and a better distribution of the workloads amongst European authorities might also improve the identification of safer alternatives. However, without stronger incentives, making it more costly for companies to continue using hazardous substances relative to safer alternatives, initiatives to promote substitution are likely to have limited success.
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