| 1. |
- Ask, Magnus, 1983, et al.
(author)
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Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials
- 2013
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 12:87
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Journal article (peer-reviewed)abstract
- Production of bioethanol from lignocellulosic biomass requires the development of robust microorganisms that can tolerate the stressful conditions prevailing in lignocellulosic hydrolysates. Several inhibitors are known to affect the redox metabolism of cells. In this study, Saccharomyces cerevisiae was engineered for increased robustness by modulating the redox state through overexpression of GSH1, CYS3 and GLR1, three genes involved in glutathione (GSH) metabolism. Overexpression constructs were stably integrated into the genome of the host strains yielding five strains overexpressing GSH1, GSH1/CYS3, GLR1, GSH1/GLR1 and GSH1/CYS3/GLR1. Overexpression of GSH1 resulted in a 42% increase in the total intracellular glutathione levels compared to the wild type. Overexpression of GSH1/CYS3, GSH/GLR1 and GSH1/CYS3/GLR1 all resulted in equal or less intracellular glutathione concentrations than overexpression of only GSH1, although higher than the wild type. GLR1 overexpression resulted in similar total glutathione levels as the wild type. Surprisingly, all recombinant strains had a lower [reduced glutathione]:[oxidized glutathione] ratio (ranging from 32--67) than the wild type strain (88), suggesting a more oxidized intracellular environment in the engineered strains. When considering the glutathione half-cell redox potential (Ehc), the difference between the strains was less pronounced. Ehc for the recombinant strains ranged from -225 to -216 mV, whereas for the wild type it was estimated to -225 mV. To test whether the recombinant strains were more robust in industrially relevant conditions, they were evaluated in simultaneous saccharification and fermentation (SSF) of pretreated spruce. All strains carrying the GSH1 overexpression construct performed better than the wild type in terms of maximum ethanol concentration, ethanol yield and furfural and HMF conversion. The strain overexpressing GSH1/GLR1 produced 14.0 g L-1 ethanol in 48 hours corresponding to an ethanol yield on hexoses of 0.17 g g-1, compared to the wild type, which produced 8.2 g L-1 ethanol in 48 hours resulting in an ethanol yield on hexoses of 0.10 g g-1. In this study, we showed that engineering of the redox state by modulating the levels of intracellular glutathione results in increased robustness of S. cerevisiae in SSF of pretreated spruce.
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| 2. |
- Ask, Magnus, 1983, et al.
(author)
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HMF and furfural stress results in drainage of redox and energy charge of Saccharomyces cerevisiae
- 2012
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In: 13th International Congress on Yeasts, Madison, WI, USA.
-
Conference paper (other academic/artistic)abstract
- Bioethanol produced from lignocellulosic raw materials is a promising alternative to fossil fuels and to decrease greenhouse gas emissions, but several challenges still exist. When lignocellulosic biomass is pretreated, a number of undesired degradation products are generated which may act inhibitory on microbial metabolism. Cellular damage response and repair come at an energy cost for the cell, which could be reflected by alterations in (energy) metabolism. The furaldehydes HMF and furfural have received increasing attention recently. They are formed during pretreatment from dehydration of hexoses and pentoses, respectively. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and transcriptome were investigated. Anaerobic chemostat cultivations were performed with the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 with both glucose and xylose as carbon sources. By quantifying the redox cofactors NAD(P)+ and NAD(P)H, the catabolic and anabolic reduction charges could be calculated. It was found that both reduction charges were significantly decreased in the presence of HMF and furfural, showing that HMF and furfural are draining the cells of reductive power. Furthermore, the [ATP]/[ADP] ratio of stressed cells was found to be lower than for non-stressed cells, suggesting that the energy metabolism was affected. Transcriptome analysis revealed that genes involved in xenobiotic transporter activity were significantly enriched among the up-regulated genes. The results from the present study provide valuable insights of how Saccharomyces cerevisiae deals with stress imposed by HMF and furfural, which potentially can result in strategies to improve stress tolerance.
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| 3. |
- Ask, Magnus, 1983, et al.
(author)
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Intracellular redox state as key target for Saccharomyces cerevisiae tolerance to lignocellulosic hydrolysate inhibitors
- 2013
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In: 35th Symposium on Biotechnology for Fuels and Chemicals (April 29-May 2, 2013).
-
Conference paper (other academic/artistic)abstract
- Liberation of sugars monomers from the polysaccharides constituting lignocellulosic biomass requires pretreatment and hydrolysis. Harsh conditions during pretreatment promote the formation of a number of inhibitory compounds, among which the furaldehydes furfural and hydroxymethylfurfural (HMF) have shown to impede growth and limit ethanol productivity of the yeast Saccharomyces cerevisiae. Cellular damage response to such inhibitory molecules and repair come at an energy cost for the cell, which could be reflected by alterations in energy and redox metabolism. In this study, S. cerevisiae cultures where treated with sub-lethal concentrations of furfural and HMF, both in continuous and batch cultivations. In continuous cultures, the inhibitors concentration was as close as possible to lethal, yet allowing steady state. In batch cultivations, the chosen concentration completely inhibited growth, yet allowing growth resumption. Metabolites connected to energy and redox metabolism such as NAD(P)H, NADP+, ATP, ADP and AMP were quantified and transcriptome analysis was performed. The results, along with data from thorough physiological characterisation under the studied conditions, suggested a severe impact of furfural and HMF on energy and redox metabolism. Based on this evidence, new strain with altered redox carriers intracellular concentration were engineered. The new recombinant strains showed higher ethanol productivity in the presence of lignocellulosic hydrolysate inhibitors.
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| 4. |
- Ask, Magnus, 1983, et al.
(author)
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TARGETING THE INTRACELLULAR REDOX STATE IN THE DEVELOPMENT OF MORE ROBUST Saccharomyces cerevisiae STRAINS FOR LIGNOCELLULOSIC BIOETHANOL PRODUCTION
- 2014
-
In: ISSY31: 31ST INTERNATIONAL SPECIALISED SYMPOSIUM ON YEAST.
-
Conference paper (other academic/artistic)abstract
- Bioethanol produced from lignocellulosic raw materials is a promising alternative to fossil fuels and to decrease greenhouse gas emissions, but several challenges still exist. When lignocellulosic biomass is pretreated, a number of undesired degradation products are generated, among which the furaldehydes furfural and hydroxymethylfurfural (HMF) have shown to impede growth and limit ethanol productivity of the yeast Saccharomyces cerevisiae. In the present study, a recombinant, xylose-utilizing S. cerevisiae strain was challenged with sub-lethal concentrations of furfural and HMF in anaerobic batch cultivations. By pulsing furaldehydes in either the glucose or the xylose consumption phase, perturbations in the intracellular NAD(P)H/NAD(P)+ ratios could be demonstrated. A genome-wide study of transcription found that genes related to NADPH-requiring processes, such as nitrogen and sulphur assimilation, were significantly induced. Moreover, the protective metabolite and antioxidant glutathione was identified as the highest scoring reporter metabolite in the transcriptome analysis. S. cerevisiae strains overproducing glutathione were constructed and the resulting strains were evaluated in simultaneous saccharification and fermentation (SSF) of pretreated spruce. The results from the present study provide valuable insights of how S. cerevisiae responds to stress imposed by HMF and furfural and how such information could be used to engineer more robust yeast strains.
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| 5. |
- Ask, Magnus, 1983, et al.
(author)
-
The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae
- 2013
-
In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:22
-
Journal article (peer-reviewed)abstract
- BackgroundPretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium.ResultsBy cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(+/-0.005) to 0.08(+/-0.002) and the anabolic reduction charge decreased from 0.46(+/-0.11) to 0.27(+/-0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(+/-0.002) to 0.85(+/-0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group "Cell rescue, defense and virulence" was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis.ConclusionsThe redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.
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| 6. |
- Ask, Magnus, 1983, et al.
(author)
-
Transcriptional response and alterations in adenonucleotides and redox cofactors in S. cerevisiae upon treatment with HMF and furfural
- 2012
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In: Advanced Biofuels in a Biorefinery Approach, February 28-March 1, Copenhagen, Denmark.
-
Conference paper (other academic/artistic)abstract
- Liberation of sugars monomers from the polysaccharides constituting lignocellulosic biomass requires pretreatment and hydrolysis. Harsh conditions during pretreatment promote the formation of a number of inhibitory compounds, among which the furaldehydes furfural and hydroxymethylfurfural (HMF) have shown to impede growth and limit ethanol productivity of the yeast Saccharomyces cerevisiae.In the present study, a recombinant xylose-utilizing S. cerevisiae strain was challenged with sub-lethal concentrations of furfural and HMF in anaerobic continuous and batch cultivations. The inhibitors concentration was as close as possible to lethal, yet allowing steady state in continuous cultivations. For batch cultivations, the chosen concentration completely inhibited growth, yet allowing growth resumption. Analysis of the transcriptome and the levels of intracellular metabolites connected to energy and redox metabolism was performed in comparison with cells grown in the absence of inhibitors. Exposure to furaldehydes caused a significant alteration of the fermentation products, especially in batch cultivations. Transcriptome analysis revealed that genes involved in xenobiotic transporter activity were significantly enriched among the up-regulated genes upon inhibitors treatment. Furthermore, inhibitors treatment significantly decreased both catabolic and anabolic reduction charges, indicating that HMF and furfural are draining the cells of reductive power during growth. In addition, HMF and furfural caused a reduction in the [ATP]/[ADP] ratio in treated cells, suggesting that the energy metabolism was affected. The results from the present study provide valuable insights into how S. cerevisiae deals with stress imposed by HMF and furfural, which potentially can result in development of strategies to improve stress tolerance during fermentation of wood hydrolysate.
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| 7. |
- Bettiga, Maurizio, 1978, et al.
(author)
-
Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies
- 2013
-
In: Cell Factories and Biosustainability (Hilleroed, Denmark, May 5-8 2013).
-
Conference paper (other academic/artistic)abstract
- The realization of an oil independent economy relies on the development of competitive processes for the production of fuels and chemicals from renewable resources. The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Harsh conditions during the bioconversion of lignocellulose-derived sugars to the desired products drastically hamper cell viability and therefore productivity. Microbial inhibition limits bioprocesses to an extent such that it can be said that understanding and harnessing microbial robustness is a prerequisite for the feasibility of new bioprocess and the production of renewable fuels and chemicals.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.
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| 8. |
- Bettiga, Maurizio, 1978, et al.
(author)
-
Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies
- 2013
-
In: 35th Symposium on Biotechnology for Fuels and Chemicals (Portland, OR. April 29-May 2, 2013).
-
Conference paper (other academic/artistic)abstract
- The realization of an oil independent economy relies on the development of competitive processes for the production of fuels and chemicals from renewable resources. The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Sugars are released from cellulose and hemicellulose by pretreatment and hydrolysis steps. Harsh conditions result in the formation of a number of compounds, originating from sugars and lignin breakdown and acting as microorganism inhibitors. Weak organic acids, furaldehydes and phenolic compounds are sources of stress for the fermenting microorganism, as they influence cellular metabolism in a number of ways, including direct damage on cellular functions or by perturbations of the cellular energy and redox metabolism. In addition, the product of interest can act as a potent inhibitor. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose-based bioprocesses.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.
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| 9. |
- Bettiga, Maurizio, 1978, et al.
(author)
-
Robust yeast strains as prerequisite for feasible biofuels production from renewable biomass resources
- 2013
-
In: FEMS-V congress of European Microbiologists.
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Conference paper (other academic/artistic)abstract
- The extensive research on second-generation ethanol has paved the way to a new concept of industry, where lignocellulosic material is the primary source of sugars for the bio-based production of a number of fuels and chemicals. The technological achievements in biomass pretreatment and hydrolysis allow today to efficiently obtain sugars from cellulose and hemicellulose. However, a number of unwanted compounds, acting as microorganism inhibitors, are released from sugars and lignin breakdown as well. In addition, the product of interest can act as a potent inhibitor. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose-based bioprocesses.Current research carried out by our group aims at investigating the molecular bases of microbial robustness, with a major focus on the yeast Saccharomyces cerevisiae. The molecular targets of different classes of fermentation inhibitors can be identified and used as cues for new strategies to engineer more robust strains. During the presentation, the concept of robustness will be discussed and examples of key features for S. cerevisiae robustness will be presented.
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| 10. |
- Bettiga, Maurizio, 1978, et al.
(author)
-
Yeast physiology studies and metabolic engineering for enhanced robustness
- 2014
-
In: Enzitec 2014- XI Seminário Brasileiro de Tecnologia Enzimática. Barra da Tijuca-Rio de Janeiro, April 14th to 16th, 2014.
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Conference paper (other academic/artistic)abstract
- The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Sugars are released from cellulose and hemicellulose by pretreatment and hydrolysis steps. Harsh conditions during pretreatment promote the formation of a number of inhibitory compounds, among which weak organic acids, furaldehydes and phenolic compounds. In addition, the product of interest can act as a potent inhibitor. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose-based bioprocesses.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.In particular, during this presentation, the following results will be discussed i) the study of redox and energy metabolism as key determinants of tolerance; ii) conversion routes of in S. cerevisiae as a way of detoxification from phenolic compounds; iii) cell membrane engineering as a strategy to achieve enhanced tolerance to weak acids.
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| 11. |
- Adeboye, Peter, 1982, et al.
(author)
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A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae
- 2016
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In: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 212:July 2016, s. 11-19
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Journal article (peer-reviewed)abstract
- AbstractThe conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1 mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae.
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| 12. |
- Adeboye, Peter, 1982, et al.
(author)
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ALD5, PAD1, ATF1 and ATF2 facilitate the catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid in Saccharomyces cerevisiae
- 2017
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 7
-
Journal article (peer-reviewed)abstract
- The ability of Saccharomyces cerevisiae to catabolize phenolic compounds remains to be fully elucidated. Conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid by S. cerevisiae under aerobic conditions was previously reported. A conversion pathway was also proposed. In the present study, possible enzymes involved in the reported conversion were investigated. Aldehyde dehydrogenase Ald5, phenylacrylic acid decarboxylase Pad1, and alcohol acetyltransferases Atf1 and Atf2, were hypothesised to be involved. Corresponding genes for the four enzymes were overexpressed in a S. cerevisiae strain named APT_1. The ability of APT_1 to tolerate and convert the three phenolic compounds was tested. APT_1 was also compared to strains B_CALD heterologously expressing coniferyl aldehyde dehydrogenase from Pseudomonas, and an ald5 Delta strain, all previously reported. APT_1 exhibited the fastest conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid. Using the intermediates and conversion products of each compound, the catabolic route of coniferyl aldehyde, ferulic acid and p-coumaric acid in S. cerevisiae was studied in greater detail.
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| 13. |
- Adeboye, Peter, 1982, et al.
(author)
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Catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid by Saccharomyces cerevisiae yields less toxic products
- 2015
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14:1, s. 149-
-
Journal article (peer-reviewed)abstract
- Background: Lignocellulosic substrates and pulping process streams are of increasing relevance to biorefineries for second generation biofuels and biochemical production. They are known to be rich in sugars and inhibitors such as phenolic compounds, organic acids and furaldehydes. Phenolic compounds are a group of aromatic compounds known to be inhibitory to fermentative organisms. It is known that inhibition of Sacchromyces cerevisiae varies among phenolic compounds and the yeast is capable of in situ catabolic conversion and metabolism of some phenolic compounds. In an approach to engineer a S. cerevisiae strain with higher tolerance to phenolic inhibitors, we selectively investigated the metabolic conversion and physiological effects of coniferyl aldehyde, ferulic acid, and p-coumaric acid in Saccharomyces cerevisiae. Aerobic batch cultivations were separately performed with each of the three phenolic compounds. Conversion of each of the phenolic compounds was observed on time-based qualitative analysis of the culture broth to monitor various intermediate and final metabolites. Result: Coniferyl aldehyde was rapidly converted within the first 24 h, while ferulic acid and p-coumaric acid were more slowly converted over a period of 72 h. The conversion of the three phenolic compounds was observed to involved several transient intermediates that were concurrently formed and converted to other phenolic products. Although there were several conversion products formed from coniferyl aldehyde, ferulic acid and p-coumaric acid, the conversion products profile from the three compounds were similar. On the physiology of Saccharomyces cerevisiae, the maximum specific growth rates of the yeast was not affected in the presence of coniferyl aldehyde or ferulic acid, but it was significantly reduced in the presence of p-coumaric acid. The biomass yields on glucose were reduced to 73 and 54 % of the control in the presence of coniferyl aldehyde and ferulic acid, respectively, biomass yield increased to 127 % of the control in the presence of p-coumaric acid. Coniferyl aldehyde, ferulic acid and p-coumaric acid and their conversion products were screened for inhibition, the conversion products were less inhibitory than coniferyl aldehyde, ferulic acid and p-coumaric acid, indicating that the conversion of the three compounds by Saccharomyces cerevisiae was also a detoxification process. Conclusion: We conclude that the conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid into less inhibitory compounds is a form of stress response and a detoxification process. We hypothesize that all phenolic compounds are converted by Saccharomyces cerevisiae using the same metabolic process. We suggest that the enhancement of the ability of S. cerevisiae to convert toxic phenolic compounds into less inhibitory compounds is a potent route to developing a S. cerevisiae with superior tolerance to phenolic compounds.
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| 14. |
- Adeboye, Peter, 1982, et al.
(author)
-
Conversion of lignin-derived phenolic compounds by Saccharomyces cerevisiae
- 2014
-
In: 36th Symposium on Biotechnology for Fuels and Chemicals, April 2-May 1st, Clearwater Beach, Florids, USA.
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Conference paper (other academic/artistic)abstract
- Lignin breakdown during biomass pretreatment releases a wide array of phenolic compounds in lignocellulose hydrolysates. Phenolic compounds, together with organic acids and furaldehydes are known to be inhibitors of microbial fermentation, thus limiting the efficient bioconversion of lignocellulose biomass. The goal of our study is to improve S. cerevisiae tolerance to phenolic compounds from lignocellulose hydrolysates and investigate its conversion capacities. In particular, we aimed i) to establish a correlation between the phenolic compounds structure and the effect on yeast growth, and ii) to investigate the conversion/detoxification products of selected representative compounds in order to provide strain engineering strategies for enhanced phenolics conversion.First, the effect on S. cerevisiae growth of 13 different phenolic compounds commonly found in lignocellulose hydrolysates was characterized. The compounds could be grouped in three clusters, according to their effect on lag phase duration, specific growth rate and cell density. Next, coniferyl aldehyde, p-coumaric acid and ferulic acid were chosen as representative compounds and their conversion product by S. cerevisiae in aerobic culture in bioreactor were identified and followed throughout the fermentation time. Understanding the effect of different phenolics on yeast and their conversion/ detoxification pathways is the first step not only in strain engineering for enhanced robustness, but also for designing new biorefinery concepts, where the bioconversion of lignin-derived aromatics could potentially be the source of new bio-based chemicals.
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| 15. |
- Adeboye, Peter, 1982, et al.
(author)
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DETOXIFICATION AS A STRATEGY FOR DEVELOPING TOLERANCE IN Saccharomyces cerevisiae TO PHENOLIC COMPOUNDS
- 2014
-
In: ISSY31: 31ST INTERNATIONAL SPECIALISED SYMPOSIUM ON YEAST.
-
Conference paper (other academic/artistic)abstract
- Several phenolic compounds are formed as products of lignin breakdown during pretreatment of lignocellulosic biomass. These phenolic compounds are inhibitory to cell growth and function as biocatalysts in the production of second generation biofuels from degraded lignocellulosic biomass. Our research is focused on developing a Saccharomyces cerevisiae strain with improved resistance to phenolic compounds.As part of our study, we have focused on understanding the ability of S. cerevisiae to tolerate and convert phenolic compounds. We aim to understand the conversion mechanisms of phenolic compounds and adapt the knowledge to the engineering and use of S. cerevisiae on a biotechnological platform for bioethanol production and prospective, novel bio-based chemicals.We have investigated toxicity of various phenolic compounds against S. cerevisiae. Our results showed that phenolic compounds have varied toxicity against S. cerevisiae and the toxicity may be dependent on the structure of the compound involved. Under aerobic batch cultivation conditions, we have also studied the conversion of phenolic compounds by S. cerevisiae using coniferyl aldehyde, ferulic acid and p-coumaric acid as representative phenolic compounds. We compiled a list of conversion products of the three starting compounds under investigation and we proposed a possible conversion pathway, currently being investigated.In this talk, we present the proposed conversion pathway through which S. cerevisiae converts and detoxifies coniferyl aldehyde, ferulic acid and p-coumaric acid under aerobic cultivation condition.
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| 16. |
- Adeboye, Peter, 1982, et al.
(author)
-
Detoxification in Saccharomyces cerevisiae under phenolics stress
- 2013
-
In: Conference on Physiology of Yeast and Filamentous Fungi.
-
Conference paper (other academic/artistic)abstract
- Phenolic compounds, commonly found in woods hydrolysates and biorefinery side streams are products of lignin breakdown during wood pretreatment. They are formed alongside other products such as organic acids and furaldehydes. Phenolic compounds are widely varied and are known to be inhibitory to cell performance, thus making the efficient bioconversion of lignocellulose biomass to products such as bioethanol, a difficult task. As part of our aim at developing robust Saccharomyces cerevisiae for lignocellulosic fermentation, we have studied the interaction of S. cerevisiae cells with a selected subset of phenolic compounds. Three phenolic compounds; 3-methoxy-4-hydroxycinnamaldehyde, 3-methoxy-4-hydroxycinnamic acid and 4-hydroxycinnamic acid, were selected as representative phenolic compounds and model substrates. These substances represent phenolic aldehydes and acids thus providing an opportunity to closely compare different phenolic compound groups on the same –cinnamic- structural background, at the same time they offer a chance to probe the influence of side groups such as the methoxy group on the phenolic compound toxicity. Our studies show that when S. cerevisiae is exposed to the selected phenolic compounds, the cells carry out a process of detoxification that involves several conversion steps in transforming the toxic phenolic compounds to other phenolic compounds with much higher toxicity limits that confirm them to be less toxic. The toxicity limit here has been defined as the concentration at which S. cerevisiae performance in the presence of phenolic compounds is decreased to about 20% in comparison to the control in Yeast minimal Mineral medium without phenolic compounds. Furthermore, products and observed patterns of the conversion indicate that S. cerevisiae likely employs a common conversion route for the different phenolic compounds.
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| 17. |
- Adeboye, Peter, 1982, et al.
(author)
-
Fermentation of Biorefinery Streams
- 2011
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In: Yeast Retreat, Tjärnö, Sweden. August 15-17, 2011..
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Conference paper (other academic/artistic)abstract
- Fermentation of biorefinery streams with S. cerevisiaePeter Adeboye, Eva Albers, Maurizio Bettiga and Lisbeth OlssonOur project aims at developing robust bioprocessing steps for the production of materials and energy from biomass, such as second‐generation ethanol by fermentation with Saccharomyces cerevisiae. We will concentrate on the fermentation of different biorefinery streams, generated by innovative biomass treatments. Fermentability of the substrates generated by the other project partner (Innventia AB) will be investigated, as well as maximum ethanol productivity and yield. Since lignocellulosic material can be a nutrient‐(especially nitrogen‐) poor and challenging substrate for the fermenting microorganism, the impact of different substrates on yeast metabolism will be investigated. Therefore, part of the research efforts of the project will be dedicated to fundamental studies on the effect of exposure to lignocellulose hydrolysate on energy metabolism, redox power homeostasis, cell integrity and viability. In addition, the effects of nutrient limitations will also be considered.
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| 18. |
- Adeboye, Peter, 1982, et al.
(author)
-
FERMENTATION OF BIOREFINERY STREAMS
- 2011
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In: PHD COURSE ON INDUSTRIAL BIOTECHNOLOGY FOR LIGNOCELLULOSE BASED PROCESSED.
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Conference paper (other academic/artistic)abstract
- Side streams generated from pulping processes have been of interest in the generation of alternative fuels due to the various wood compositional residues such as fermentable sugars leached out with it during the pulping process. These streams, due to their composition of fermentable sugars and other wood carbohydrate residues and the potential to ferment such carbohydrate residues in them for bioethanol production are in that case Biorefinery streams. Although these streams contain several growth inhibitory compounds such as furfural, numerous phenolic derivatives of lignin, several organic acids and are also known to be nutrient- (especially nitrogen-) poor thus constituting a challenging type of substrates for the fermenting microorganism, these traits however make for interesting grey areas for research on cell response to stress . Using biorefinery streams generated by innovative biomass treatments, our project aims at developing robust bioprocessing steps for the production of materials and energy, such as second-generation ethanol by fermentation with Saccharomyces cerevisiae. Fermentability of the substrates generated by the other project partner (Innventia AB) will be investigated, as well as maximum ethanol productivity and yield. The impact of different substrates on yeast metabolism will be investigated. Therefore, part of the research efforts of the project will be dedicated to fundamental studies on the effect of exposure to lignocellulose hydrolysate on energy metabolism, redox power homeostasis, cell integrity and viability. In addition, the effects of nutrient limitations will also be considered.
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| 19. |
- Adeboye, Peter, 1982, et al.
(author)
-
In situ conversion of phenolic compounds as a tool to phenolic tolerance development by S. cerevisiae
- 2015
-
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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| 20. |
- Adeboye, Peter, 1982, et al.
(author)
-
The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulosic hydrolysates
- 2014
-
In: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4:46, s. 1-10
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Journal article (peer-reviewed)abstract
- We investigated the severity of the inhibitory effects of 13 phenolic compounds usually found in spruce hydrolysates (4-hydroxy-3-methoxycinnamaldehyde, homovanilyl alcohol, vanillin, syringic acid, vanillic acid, gallic acid, dihydroferulic acid, p-coumaric acid, hydroquinone, ferulic acid, homovanillic acid, 4-hydroxybenzoic acid and vanillylidenacetone). The effects of the selected compounds on cell growth, biomass yield and ethanol yield were studied and the toxic concentration threshold was defined for each compound. Using Ethanol Red, the popular industrial strain of Saccharomyces cerevisiae, we found the most toxic compound to be 4-hydroxy-3-methoxycinnamaldehyde which inhibited growth at a concentration of 1.8 mM. We also observed that toxicity did not generally follow a trend based on the aldehyde, acid, ketone or alcohol classification of phenolic compounds, but rather that other structural properties such as additional functional groups attached to the compound may determine its toxicity. Three distinctive growth patterns that effectively clustered all the compounds involved in the screening into three categories. We suggest that the compounds have different cellular targets, and that. We suggest that the compounds have different cellular targets and inhibitory mechanisms in the cells, also compounds who share similar pattern on cell growth may have similar inhibitory effect and mechanisms of inhibition.
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| 21. |
- 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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| 22. |
- Ask, Magnus, 1983, et al.
(author)
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Pulsed addition of HMF and furfural to batch-grown xylose-utilizing Saccharomyces cerevisiae results in different physiological responses in glucose and xylose consumption phase
- 2013
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:181
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Journal article (peer-reviewed)abstract
- Pretreatment of lignocellulosic biomass generates a number of undesired degradation products that can inhibit microbial metabolism. Two of these compounds, the furan aldehydes 5-hydroxymethylfurfural (HMF) and 2-furaldehyde (furfural), have been shown to be an impediment for viable ethanol production. In the present study, HMF and furfural were pulse-added during either the glucose or the xylose consumption phase in order to dissect the effects of these inhibitors on energy state, redox metabolism, and gene expression of xylose-consuming Saccharomyces cerevisiae.Pulsed addition of 3.9?g?L-1 HMF and 1.2?g?L-1 furfural during either the glucose or the xylose consumption phase resulted in distinct physiological responses. Addition of furan aldehydes in the glucose consumption phase was followed by a decrease in the specific growth rate and the glycerol yield, whereas the acetate yield increased 7.3-fold, suggesting that NAD(P)H for furan aldehyde conversion was generated by acetate synthesis. No change in the intracellular levels of NAD(P)H was observed 1?hour after pulsing, whereas the intracellular concentration of ATP increased by 58%. An investigation of the response at transcriptional level revealed changes known to be correlated with perturbations in the specific growth rate, such as protein and nucleotide biosynthesis. Addition of furan aldehydes during the xylose consumption phase brought about an increase in the glycerol and acetate yields, whereas the xylitol yield was severely reduced. The intracellular concentrations of NADH and NADPH decreased by 58 and 85%, respectively, hence suggesting that HMF and furfural drained the cells of reducing power. The intracellular concentration of ATP was reduced by 42% 1?hour after pulsing of inhibitors, suggesting that energy-requiring repair or maintenance processes were activated. Transcriptome profiling showed that NADPH-requiring processes such as amino acid biosynthesis and sulfate and nitrogen assimilation were induced 1?hour after pulsing.The redox and energy metabolism were found to be more severely affected after pulsing of furan aldehydes during the xylose consumption phase than during glucose consumption. Conceivably, this discrepancy resulted from the low xylose utilization rate, hence suggesting that xylose metabolism is a feasible target for metabolic engineering of more robust xylose-utilizing yeast strains.
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| 23. |
- Bertacchi, Stefano, et al.
(author)
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Camelina sativa meal hydrolysate as sustainable biomass for the production of carotenoids by Rhodosporidium toruloides
- 2020
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 13:1
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Journal article (peer-reviewed)abstract
- Background: As the circular economy advocates a near total waste reduction, the industry has shown an increased interest toward the exploitation of various residual biomasses. The origin and availability of biomass used as feedstock strongly affect the sustainability of biorefineries, where it is converted in energy and chemicals. Here, we explored the valorization of Camelina meal, the leftover residue from Camelina sativa oil extraction. In fact, in addition to Camelina meal use as animal feed, there is an increasing interest in further valorizing its macromolecular content or its nutri- tional value. Results: Camelina meal hydrolysates were used as nutrient and energy sources for the fermentation of the carot- enoid-producing yeast Rhodosporidium toruloides in shake flasks. Total acid hydrolysis revealed that carbohydrates accounted for a maximum of 31 ± 1.0% of Camelina meal. However, because acid hydrolysis is not optimal for sub- sequent microbial fermentation, an enzymatic hydrolysis protocol was assessed, yielding a maximum sugar recovery of 53.3%. Separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and SSF preceded by presaccharification of Camelina meal hydrolysate produced 5 ± 0.7, 16 ± 1.9, and 13 ± 2.6 mg/L of carotenoids, respectively. Importantly, the presence of water-insoluble solids, which normally inhibit microbial growth, correlated with a higher titer of carotenoids, suggesting that the latter could act as scavengers. Conclusions: This study paves the way for the exploitation of Camelina meal as feedstock in biorefinery processes. The process under development provides an example of how different final products can be obtained from this side stream, such as pure carotenoids and carotenoid-enriched Camelina meal, can potentially increase the initial value of the source material. The obtained data will help assess the feasibility of using Camelina meal to generate high value- added products.
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| 24. |
- Bettiga, Maurizio, 1978, et al.
(author)
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Biogas production as a contributor to the biorefinery concept
- 2011
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In: “First International Conference on Biogas Microbiology”, Leipzig, Germany, September 14-16, 2011.
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Conference paper (other academic/artistic)abstract
- The Industrial Biotechnology group at Chalmers University of Technology, in Gothenburg, Sweden, wants to be at the frontier of the discovery of new biotechnologies, which may become fundamental contributors to the energy infrastructure and green chemistry industry of the world of tomorrow. Our research is part of the Chalmers Energy Initiative and our initial focus is towards sustainable bioenergy production. Within the concept of biorefinery, lignocellulosic raw materials and waste streams are upgraded to a portfolio of fuel and chemical products, which may vary according to the initial nature of the substrate and the local needs. Anaerobic digestion could play a crucial role towards the feasibility of biorefining processes. Our research is devoted at investigating the challenges and opportunities of integrating biogas production with other fuels and chemicals from lignocellulose materials.
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| 25. |
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