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Sökning: WFRF:(Olsson Lisbeth 1963)

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1.
  • Adeboye, Peter, 1982, et al. (författare)
  • Catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid by Saccharomyces cerevisiae yields less toxic products
  • 2015
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14:1, s. 149-
  • Tidskriftsartikel (refereegranskat)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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2.
  • Lindahl, Lina, 1984, et al. (författare)
  • Membrane engineering of S. cerevisiae targeting sphingolipid metabolism
  • 2017
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 7, s. 41868-
  • 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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3.
  • Lindahl, Lina, 1984, et al. (författare)
  • THE INFLUENCE OF MEMBRANE COMPOSTION ON ACETIC ACID PERMEABILITY AND POTENTIALLY ACETIC ACID TOLERANCE
  • 2014
  • Ingår i: ISSY31: 31st International Specialised Symposium on Yeast.
  • 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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4.
  • Matsakas, Leonidas, et al. (författare)
  • A novel hybrid organosolv: Steam explosion method for the efficient fractionation and pretreatment of birch biomass
  • 2018
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance saccharification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractionation into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction. Results: In this article, a hybrid method combining the cooking and fractionation of conventional organosolv pretreatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/wbiomassH2SO4was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum. Conclusions: The novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive discharge at the end of pretreatment had a positive effect on enzymatic saccharification, resulting in high hydrolyzability of the pretreated solids and elevated ethanol titers in the following high-gravity SSF. To the best of our knowledge, the ethanol concentration obtained with this method is the highest so far for birch biomass.
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5.
  • Matsakas, Leonidas, et al. (författare)
  • Lignin-first biomass fractionation using a hybrid organosolv – Steam explosion pretreatment technology improves the saccharification and fermentability of spruce biomass
  • 2019
  • Ingår i: Bioresource Technology. - : Elsevier. - 0960-8524 .- 1873-2976. ; 273, s. 521-528
  • Tidskriftsartikel (refereegranskat)abstract
    • For a transition to a sustainable society, fuels, chemicals, and materials should be produced from renewable resources. Lignocellulosic biomass constitutes an abundant and renewable feedstock; however, its successful application in a biorefinery requires efficient fractionation into its components; cellulose, hemicellulose and lignin. Here, we demonstrate that a newly established hybrid organosolv – steam explosion pretreatment can effectively fractionate spruce biomass to yield pretreated solids with high cellulose (72% w/w) and low lignin (delignification up to 79.4% w/w) content. The cellulose-rich pretreated solids present high saccharification yields (up to 61% w/w) making them ideal for subsequent bioconversion processes. Moreover, under high-gravity conditions (22% w/w) we obtained an ethanol titer of 61.7 g/L, the highest so far reported for spruce biomass. Finally, the obtained high-purity lignin is suitable for various advanced applications. In conclusion, hybrid organosolv pretreatment could offer a closed-loop biorefinery while simultaneously adding value to all biomass components.
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6.
  • Nyberg, Lena, 1979, et al. (författare)
  • A single-step competitive binding assay for mapping of single DNA molecules
  • 2012
  • Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 417:1, s. 404-408
  • Tidskriftsartikel (refereegranskat)abstract
    • Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.
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7.
  • Springett, Jane, 1952-, et al. (författare)
  • Närsjukvård : bakgrund, erfarenheter och pilotstudie
  • 2005
  • Rapport (övrigt vetenskapligt/konstnärligt)abstract
    • Närsjukvård är ett centralt begrepp i ett förändringsarbete som för genomförs i nordöstra Skåne för att utveckla hälso- och sjukvårdsväsendet. Det ingår därmed som en av de centrala delarna av Region Skånes vision om hälso- och sjukvård: Skånsk livskraft – vård och hälsa. Syftet med denna rapport är att ge en bakgrund till begreppet (Del A) och att presentera en del preliminära rön beträffande hur olika aktörer i nordöstra Skåne uppfattar begreppet (Del B). Del A ger en översikt kring ursprunget till begreppet Närsjukvård inom ramen för de förändringar i hälso- och sjukvården som sker i Sverige i stort. Den beskriver sedan vilka slags förändringar som har planerats på politisk nivå och som nu håller på att genomföras under detta paraplybegrepp, nationellt, regionalt och lokalt. För detta syfte används statliga dokument och publicerade utvärderingsstudier i stor utsträckning som källmaterial. Denna del ska därför inte ses som en heltäckande översikt. Del B inriktas på att belysa hur långt förverkligandet av idén om Närsjukvård har kommit inom regionen. Avsnittet är en kartläggning av olika aktörers förståelse av Närsjukvård i den nordöstra delen av Region Skåne. Forskningsfrågorna inriktades på hur folk pratade om Närsjukvård, det vill säga på hur de förstod och använde begreppet.
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8.
  • Adeboye, Peter, 1982, et al. (författare)
  • A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae
  • 2016
  • Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 212:July 2016, s. 11-19
  • Tidskriftsartikel (refereegranskat)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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9.
  • Adeboye, Peter, 1982, et al. (författare)
  • ALD5, PAD1, ATF1 and ATF2 facilitate the catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid in Saccharomyces cerevisiae
  • 2017
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 7
  • Tidskriftsartikel (refereegranskat)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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10.
  • Adeboye, Peter, 1982, et al. (författare)
  • Conversion of lignin-derived phenolic compounds by Saccharomyces cerevisiae
  • 2014
  • Ingår i: 36th Symposium on Biotechnology for Fuels and Chemicals, April 2-May 1st, Clearwater Beach, Florids, USA.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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11.
  • Adeboye, Peter, 1982, et al. (författare)
  • DETOXIFICATION AS A STRATEGY FOR DEVELOPING TOLERANCE IN Saccharomyces cerevisiae TO PHENOLIC COMPOUNDS
  • 2014
  • Ingår i: ISSY31: 31ST INTERNATIONAL SPECIALISED SYMPOSIUM ON YEAST.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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12.
  • Adeboye, Peter, 1982, et al. (författare)
  • Detoxification in Saccharomyces cerevisiae under phenolics stress
  • 2013
  • Ingår i: Conference on Physiology of Yeast and Filamentous Fungi.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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13.
  • Adeboye, Peter, 1982, et al. (författare)
  • Fermentation of Biorefinery Streams
  • 2011
  • Ingår i: Yeast Retreat, Tjärnö, Sweden. August 15-17, 2011..
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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14.
  • Adeboye, Peter, 1982, et al. (författare)
  • FERMENTATION OF BIOREFINERY STREAMS
  • 2011
  • Ingår i: PHD COURSE ON INDUSTRIAL BIOTECHNOLOGY FOR LIGNOCELLULOSE BASED PROCESSED.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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15.
  • Adeboye, Peter, 1982, et al. (författare)
  • In situ conversion of phenolic compounds as a tool to phenolic tolerance development by S. cerevisiae
  • 2015
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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16.
  • Adeboye, Peter, 1982, et al. (författare)
  • The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulosic hydrolysates
  • 2014
  • Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4:46, s. 1-10
  • Tidskriftsartikel (refereegranskat)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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17.
  • Albers, Eva, 1966, et al. (författare)
  • Comparison of industrial xylose fermentation with yeast performed at different process scale
  • 2012
  • Ingår i: 13th International Congress on Yeasts, ICY 2012, August 26-30, Madison, USA.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Second generation of bioethanol production with yeast from lignocellulosic material may contribute to a sustainable production of energy. However, the commercialization of cellulose-to-ethanol remains challenging due to various limitations in process technology and microbial physiology. Despite that the technical progress lately has come far, lignocellulose bioethanol production is still not well established in full production scale. Production scale demands large financial investments and to minimize the risk knowledge about cellular performance of the yeast as response to conditions of large scale is needed. Large scale may impose specific conditions that normally are not present in smaller scale. Such conditions are then needed to be identified and mimicked in smaller scale to obtain crucial scaling-up data. In this project, we wanted to establish scalable cultivation processes and compare the performance at different scales. Experiments were performed at three process scales: lab (1.5 l), process development unit (15 l) and demonstration (10 m3) scales, with an industrial recombinant xylose fermenting Saccharomyces cerevisiae strain and corn cob, bagasse, and spruce lignocellulosic material. It was found that separate fermentation and SSF experiments could be reproducible at all scales. An ethanol level could be obtained above 4 % which is the threshold for feasible down-stream processing. Demonstration scale experiments on xylose-rich liquid of pre-treated corn cobs resulted in a 90% conversion of xylose to ethanol and on the slurry in SSF cultivation an ethanol yield of 0.44 g/g xylose was obtained.
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18.
  • Albers, Eva, 1966, et al. (författare)
  • Development and large scale performance of efficient xylose fermenting yeast strains
  • 2011
  • Ingår i: Seventh International Conference on Renewable Resources and Biorefineries, Belgium.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Fermentation at large industrial scale poses several challenges for the fermenting microorganism to handle. Thus, for an efficient production it is desirable to have robust and efficient strains which can cope with the specific conditions in the process. For bioethanol production by yeast from lignocellulosic material the substrate for growth constitutes one of the largest challenges due to its mixture of sugars and content of inhibitory compounds. Wild-type strains of Saccharomyces cerevisiae can only convert hexose sugars but not the pentoses, xylose and arabinose, which may be present in lignocellulosic material. However, strains have been genetically modified to allow for xylose conversion, but their performance is needed to be improved in terms of rate and efficiency. During the pre-treatment of lignocellulosic material inhibitory compounds are formed; furans, phenolics and organic acids. In an industrial setting, a robust strain back ground (industrial yeast strains) is a prerequisite, in which earlier pentose fermenting traits should be incorporated and further adaptation to the inhibitory compounds need to follow. In the present project, we have used directed evolution to simultaneously improve the inhibitor tolerance and xylose conversion capability of recombinant yeast strains with an industrial background. Improved yeast strains resulting from several strategies were evaluated and one of the best strains with high ethanol production, good xylose utilization capacity, and low xylitol formation was selected for evaluation in larger scale. Fermentations on pre-treated corn cobs were performed with good results regarding ethanol production and xylose utilization both in process development unit scale (15 l) and demonstration scale (10 m3).
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19.
  • Albers, Eva, 1966, et al. (författare)
  • Development of industrial yeast strains for efficient xylose fermentation in lignocellulosic material
  • 2012
  • Ingår i: 13th International Congress on Yeasts, ICY 2012, August 26-30, Madison, USA.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Fermentation at large industrial scale poses several challenges for the fermenting microorganism to handle. Thus, for an efficient production it is desirable to have robust and efficient strains, which can cope with the specific conditions in the process. For bioethanol production by yeast from lignocellulosic material, one of the largest challenges is the mixture of sugars and the content of inhibitory compounds in the material. Wild-type strains of Saccharomyces cerevisiae can only convert hexose sugars but not the pentoses, xylose and arabinose, which may be present in these materials. However, strains have been genetically modified to allow for xylose conversion, but their performance need to be improved in terms of rate and efficiency. During the pre-treatment of lignocellulosic material the inhibitory compounds are formed; furans, phenolics and organic acids. In an industrial setting, a robust strain back ground (industrial yeast strains) is a prerequisite, in which first pentose fermenting traits should be incorporated and further improvement of the tolerance to inhibitory compounds need to follow. In the present project, we have used directed evolution to simultaneously improve the inhibitor tolerance and xylose conversion capability of recombinant yeast strains with an industrial background. The strains showed increased xylose utilization and ethanol production which was for some strains coupled to decreased xylitol formation. The resulting properties of the strains are highly dependent on the mode of directed evolution applied, which may also give rise to quite a number of clones with different properties.
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20.
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21.
  • Aldaeus, Fredrik, et al. (författare)
  • Characterization of pulp with high enzymatic hydrolyzability
  • 2014
  • Ingår i: 13th European Workshop on Lignocellulosics and Pulp (EWLP 2014) book of abstracts.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Conversion of biomass to biofuels is currently an area that attracts large interest, and lignocellulosic biomass offers the abundance and environmental attributes that can potentially support large-scale biofuel production as an alternative to petroleum-based transportation fuel.In a recent project, Innventia has developed wood based pulps optimized for conversion to biofuels. These novel pulps were produced to target a high level of enzymatic hydrolyzability. To assess the hydrolyzability of these pulps, a laboratory protocol has been established usingan enzyme mixture containing Celluclast 1.5L and Novozyme 188 with an activity of 10 FPU/g pulp (Andersen 2007). Results obtained using this protocol are assumed to be relevant for industrial conditions. In addition to assessment of the produced pulps, the results havebeen compared to commercial cellulose substrates and pulps of a variety of grades.Furthermore, supramolecular properties – specific surface area and average pore size – were determined by an in-house method utilizing solid state nuclear magnetic resonance (Larsson et al. 2013). Kappa numbers, limiting viscosities, ISO-brightness and carbohydrate compositions were determined using standard methods. Molecular mass distributions of cellulose tricarbanilates were determined by size exclusion chromatography with tetrahydrofuran mobile phase (Drechsler et al. 2000).The presentation will discuss the influence of chemical, macromolecular and supramolecular properties of commercial and novel pulp grades on the enzymatic hydrolyzability. Theprotocol used to assess of enzymatic hydrolyzability will be proposed as a benchmark test.
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22.
  • Aldaeus, Fredrik, et al. (författare)
  • The supramolecular structure of cellulose-rich wood pulps can be a determinative factor for enzymatic hydrolysability
  • 2015
  • Ingår i: Cellulose. - : Springer Science and Business Media LLC. - 0969-0239 .- 1572-882X. ; 22:6, s. 3991-4002
  • Tidskriftsartikel (refereegranskat)abstract
    • The enzymatic hydrolysability of three industrial pulps, five lab made pulps, and one microcrystalline cellulose powder was assessed using commercial cellulolytic enzymes. To gain insight into the factors that influence the hydrolysability, a thorough characterization of the samples was done, including their chemical properties (cellulose content, hemicellulose content, lignin content, and kappa number), their macromolecular properties (peak molar mass, number-average molar mass, weight-average molar mass, polydispersity, and limiting viscosity) and their supramolecular properties (fibre saturation point, specific surface area, average pore size, and crystallinity). The hydrolysability was assessed by determination of initial conversion rate and final conversion yield, with conversion yield defined as the amount of glucose in solution per unit of glucose in the substrate. Multivariate data analysis revealed that for the investigated samples the conversion of cellulose to glucose was mainly dependent on the supramolecular properties, such as specific surface area and average pore size. The molar mass distribution, the crystallinity, and the lignin content of the pulps had no significant effect on the hydrolysability of the investigated samples.
  •  
23.
  • Anasontzis, George E, 1980, et al. (författare)
  • Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris
  • 2014
  • Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 30:3, s. 728-735
  • Tidskriftsartikel (refereegranskat)abstract
    • Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. (c) 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728-735, 2014
  •  
24.
  • Anasontzis, George E, 1980, et al. (författare)
  • Enzyme Discovery Platform
  • 2012
  • Ingår i: WWSC annual conference, 21-23 November 2012, Uddevalla, Sweden.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Biomass treatment for the separation of its components and the modification of their properties requires a wide range of enzymes. Efficiency matters too, so multiple enzymes of the same activity need to be tested before they can be applied in a larger scale. We have launched the implementation of a strategic plan for the discovery of novel enzymes, starting from the isolation of new fungal strains from Vietnam and leading to the cloning and characterization of the enzymes of interest.
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25.
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26.
  •  
27.
  •  
28.
  • Anasontzis, George E, 1980, et al. (författare)
  • Investigating the Yet-Unknown Biomass Degrading and Modifying Enzymes of Aspergillus oryzae
  • 2012
  • Ingår i: Science and Technology Day, 27 March 2012, Göteborg, Sweden.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • The association of plant cell wall components with A. oryzae’s extracellular enzyme machinery was investigated by analyzing the transcriptome profile in relation to the chemical structure of the carbon sources. Strain RIB40 of A. oryzae was cultured on various carbon sources, namely cellohexaose, mannohexaose, xylopentaose, arabinoheptaose, glucohexaose, glucosyl maltotriosyl maltotriose, galactosyl mannotriose, turanose and sophorose, and the transcribed genes were determined with DNA microarrays. The statistically significant genes were selected and potential novel hydrolases were identified.Presently, we aim at the heterologous expression and characterization of seven different hypothetical and non classified proteins of A. oryzae, which could prove to be useful tools in the wood biomass separation and modification process. This work represents a novel way of integrating computational chemical biology and classical enzyme research for improving lignocellulose bioconversion.
  •  
29.
  • Anasontzis, George E, 1980, et al. (författare)
  • Investigating the yet-unknown biomass degrading and modifying enzymes of Aspergillus oryzae
  • 2011
  • Ingår i: WWSC Workshop, 29 November - 1 December, Södertuna, Gnesta, Sweden.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • The associations of plant cell wall components with A. oryzae’s extracellular enzyme machinery were investigated from a chemical perspective using an integrated analysis of the transcriptome profile. We were able to identify novel hydrolases, which we heterologous expressed for subsequent characterization. This work represents a novel way of integrating computational chemical biology and classical enzyme research for improving lignocellulose bioconversion. In this work, we aim at the heterologous expression and characterization of seven different hypothetical and non classified proteins of A. oryzae, which could prove to be useful tools in the wood biomass separation and modification process.
  •  
30.
  •  
31.
  • Anasontzis, George E, 1980, et al. (författare)
  • Rice straw hydrolysis using secretomes from novel fungal isolates from Vietnam
  • 2017
  • Ingår i: Biomass and Bioenergy. - : Elsevier BV. - 1873-2909 .- 0961-9534. ; 99, s. 11-20
  • Tidskriftsartikel (refereegranskat)abstract
    • With a production of 39 million metric tons each year, rice is one of the main agricultural products of Vietnam. Thus, rice straw is a significant by-product, whose use in a biorefinery process would contribute to the bio-based transformation of the Vietnamese and South East Asian economy. In order to find novel efficient enzyme mixtures for the hydrolysis of rice straw and other agricultural residues, we took advantage of the rapidly evolving biodiversity of Vietnam and screened 1100 new fungal isolates from soil and decaying plant tissues for their CMCase activity. We selected 36 strains and evaluated them for their cellulases, xylanases, and accessory enzymes activities. Most of these isolates belonged to the genera Aspergillus and Trichoderma. We identified a few promising isolates, such as A. brunneoviolaceus FEC 156, A. niger FEC 130 and FEC 705, and A. tubingensis FEC 98, FEC 110 and FEC 644, whose produced enzyme mixtures released a mass fraction of the sugar content of alkali-treated rice straw higher than 20%, compared to 10% for Trichoderma reesei RUT C-30. We verified that the black Aspergilli are particularly efficient in their saccharification ability. We also identified strains that although they produced low amounts of cellulases and xylanases, their enzyme mixtures had high saccharification efficiencies, indicating the importance of the synergy effect, rather than the amount of enzymes available. Our results highlight the intra-species variation, especially in the Trichoderma genus, regarding the biomass degradation characteristics and the associated range of enzymatic activities.
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32.
  • Anasontzis, George E, 1980, et al. (författare)
  • Screening Natural Resources for Enzymes With Wood Degrading and Wood Modifying Properties
  • 2011
  • Ingår i: Italic6/COST conference, 5–8 September 2011, Viterbo, Italy.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • The production of high added value compounds from forest and agricultural biomass has become one of the main targets of contemporary carbohydrates research. The renewability of the biomass, the potential use of waste residues and the complete or partial biodegradability of the products have made the whole approach an attractive perspective towards the sustainable and green ideal. However, most of the already developed biomass separation and modification processes are based on chemical reactions at extreme conditions that are costly and often harmful for the environment. Enzymatic and microbial catalyzed processes present an interesting alternative. The development and discovery of novel biological approaches in the modification, degradation and separation of wood biomass is one of the main activities of the Industrial Biotechnology Group at Chalmers University of Technology, also as part of the Wallenberg Wood Science Center (WWSC).Presently, we pursue this aim through a triple approach: •Μultiple enzymatic screening of phytopathogenic and wood degrading filamentous fungi, such as Trametes hirsuta and Penicillium pinophilum, as well as screening newly isolated microorganisms. We seek enzymes with industrially interesting activities and unique properties, such as reactivity under extreme conditions.•Microorganisms efficient in degrading lignocellulose produce enzyme in response to the environmental conditions. In collaboration with Associate Professor Gianni Panagiotou, Center for Biological Sequence Analysis, DTU, we are looking for sequenced, but still unclassified proteins, which are related to the degradation of plant biomass using information from transcriptomics analysis of Aspergillus oryzae grown on different carbon sources.•Novel enzymes can only be identified by new methods. We investigate the properties of synthetic model compounds that can simulate the natural substrates and the implementation of different analytical methods for the identification of the sometimes complex and singular enzymatic activities. In collaboration with Associate Professor Paul Christakopoulos, BIOtechMASS Unit, School of Chemical Engineering, National Technical University of Athens, we also attempt to isolate model compounds from plant cell wall material.
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33.
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34.
  • Anasontzis, George E, 1980, et al. (författare)
  • Screening the tropical fungal biodiversity of Vietnam for biomass modifying enzymes, with secretome and transcriptome analyses
  • 2013
  • Ingår i: 27th Fungal Genetics Conference.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • In the bio-based economy concept, the current hydrocarbon fuels and non-biodegradable plastics will be replaced by new products which will derive from natural and renewable resources. The synthesis of such biofuels and biochemicals is still challenged by the difficulties to cost efficiently degrade lignocellulosic materials to fermentable sugars or to isolate the intact polymers. Biomass degrading and modifying enzymes play an integral role both in the separation of the polymers from the wood network, as well as in subsequent modifications, prior to further product development. The type of application usually defines the conditions where the reactions should take place. Thus, novel enzymes with variable combined properties, such as different thermotolerance, pH range of activity, substrate specificity and solvent tolerance, still need to be discovered and developed to achieve the highest possible efficiency in each occasion. We took advantage of the rapidly evolving and high biodiversity of the tropics and have been screening various isolates for their cellulases and hemicellulases activities. Promising strains were then cultivated in bioreactors with different carbon sources, such as wheat bran, spruce and avicel and their biomass degrading capacity was analysed through cross species protein identification of their secretome with TMT. Information on the genes involved in the different stages of the fermentation and the carbon source will be acquired with next generation sequencing of the total transcriptome. Interesting transcripts will then be used to heterologously clone and express the respective genes and identify their role in the degradation process.
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35.
  • Andersen, Natalija, et al. (författare)
  • Hydrolysis of cellulose using mono-component enzymes shows synergy during hydrolysis of phosphoric acid swollen cellulose (PASC), but competition on Avicel
  • 2008
  • Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; :42, s. 362-370
  • Tidskriftsartikel (refereegranskat)abstract
    • To study the synergy between the three groups of cellulolytic enzymes, 20 mixtures of different mole percentage of Humicola insolens Cel45A (EG V) and Cel6A (CBH II), and Penicillium brasilianum Cel3A (β-glucosidase) were used to hydrolyze Avicel and phosphoric acid swollen cellulose/Avicel (PASC). In contrast to previous studies, where β-glucosidase was either not added or added in excess, we here focus on engineering binary, as well as, ternary cellulase mixtures (including a range of different mol% of Cel3A) for maximal total sugar production. Precise hydrolysis pattern based on the concentration of soluble hydrolysis products (glucose to cellohexaose measured by HPLC) was determined. The importance of proper assay selection for hydrolysis products detection was illustrated. It was found that degree of synergy (DS) for degradation of PASC were generally larger than 1 (indicating cooperativity between the enzymes), increasing as the hydrolysis proceeded. DS of binary exo-/endo-glucanase mixtures, decreased as the mol% of Cel45A increased. In contrast to hydrolysis of PASC, DS values during degradation of Avicel were less then 1, indicating inhibition of the involved enzymes. Thus, our data point to competition for the same binding sites between endo- and exo-glucanases, and preferential absorbance of exo-glucanases on crystalline substrates.
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36.
  • Arnling Bååth, Jenny, 1987, et al. (författare)
  • A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds
  • 2016
  • Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 590:16, s. 2611-2618
  • Tidskriftsartikel (refereegranskat)abstract
    • The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and 31P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.
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37.
  • Arnling Bååth, Jenny, 1987, et al. (författare)
  • Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion
  • 2018
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background Lignocellulose is highly recalcitrant to enzymatic deconstruction, where the recalcitrance primarily results from chemical linkages between lignin and carbohydrates. Glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15) have been suggested to play key roles in reducing lignocellulose recalcitrance by cleaving covalent ester bonds found between lignin and glucuronoxylan. However, only a limited number of GEs have been biochemically characterized and structurally determined to date, limiting our understanding of these enzymes and their potential exploration. Results Ten CE15 enzymes from three bacterial species, sharing as little as 20% sequence identity, were characterized on a range of model substrates; two protein structures were solved, and insights into their regulation and biological roles were gained through gene expression analysis and enzymatic assays on complex biomass. Several enzymes with higher catalytic efficiencies on a wider range of model substrates than previously characterized fungal GEs were identified. Similarities and differences regarding substrate specificity between the investigated GEs were observed and putatively linked to their positioning in the CE15 phylogenetic tree. The bacterial GEs were able to utilize substrates lacking 4-OH methyl substitutions, known to be important for fungal enzymes. In addition, certain bacterial GEs were able to efficiently cleave esters of galacturonate, a functionality not previously described within the family. The two solved structures revealed similar overall folds to known structures, but also indicated active site regions allowing for more promiscuous substrate specificities. The gene expression analysis demonstrated that bacterial GE-encoding genes were differentially expressed as response to different carbon sources. Further, improved enzymatic saccharification of milled corn cob by a commercial lignocellulolytic enzyme cocktail when supplemented with GEs showcased their synergistic potential with other enzyme types on native biomass. Conclusions Bacterial GEs exhibit much larger diversity than fungal counterparts. In this study, we significantly expanded the existing knowledge on CE15 with the in-depth characterization of ten bacterial GEs broadly spanning the phylogenetic tree, and also presented two novel enzyme structures. Variations in transcriptional responses of CE15-encoding genes under different growth conditions suggest nonredundant functions for enzymes found in species with multiple CE15 genes and further illuminate the importance of GEs in native lignin–carbohydrate disassembly.
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38.
  • Arnling Bååth, Jenny, 1987, et al. (författare)
  • Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation
  • 2018
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Galactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. GGM can be hydrolyzed into fermentable sugars, or used as a polymer in films, gels, and food additives. Endo-β-mannanases, which can be found in the glycoside hydrolase families 5 and 26, specifically cleave the mannan backbone of GGM into shorter oligosaccharides. Information on the activity and specificity of different mannanases on complex and acetylated substrates is still lacking. The aim of this work was to evaluate and compare the modes of action of two mannanases from Cellvibrio japonicus (CjMan5A and CjMan26A) on a variety of mannan substrates, naturally and chemically acetylated to varying degrees, including naturally acetylated spruce GGM. Both enzymes were evaluated in terms of cleavage patterns and their ability to accommodate acetyl substitutions. Results: CjMan5A and CjMan26A demonstrated different substrate preferences on mannan substrates with distinct backbone and decoration structures. CjMan5A action resulted in higher amounts of mannotriose and mannotetraose than that of CjMan26A, which mainly generated mannose and mannobiose as end products. Mass spectrometric analysis of products from the enzymatic hydrolysis of spruce GGM revealed that an acetylated hexotriose was the shortest acetylated oligosaccharide produced by CjMan5A, whereas CjMan26A generated acetylated hexobiose as well as diacetylated oligosaccharides. A low degree of native acetylation did not significantly inhibit the enzymatic action. However, a high degree of chemical acetylation resulted in decreased hydrolyzability of mannan substrates, where reduced substrate solubility seemed to reduce enzyme activity. Conclusions: Our findings demonstrate that the two mannanases from C. japonicus have different cleavage patterns on linear and decorated mannan polysaccharides, including the abundant and industrially important resource spruce GGM. CjMan26A released higher amounts of fermentable sugars suitable for biofuel production, while CjMan5A, producing higher amounts of oligosaccharides, could be a good candidate for the production of oligomeric platform chemicals and food additives. Furthermore, chemical acetylation of mannan polymers was found to be a potential strategy for limiting the biodegradation of mannan-containing materials.
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39.
  • Arnling Bååth, Jenny, 1987, et al. (författare)
  • Structure-function analyses reveal that a glucuronoyl esterase from Teredinibacter turnerae interacts with carbohydrates and aromatic compounds
  • 2019
  • Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:16, s. 6635-6644
  • Tidskriftsartikel (refereegranskat)abstract
    • Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages found between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these recalcitrant resources. However, details on how GEs interact with their natural substrates are sparse, calling for thorough structurefunction studies. Presented here is the structure and biochemical characterization of a GE, TtCE15A, from the bacterium Teredinibacter turnerae, a symbiont of wood-boring shipworms. To gain deeper insight into enzyme-substrate interactions, inhibition studies were performed with both the WT TtCE15A and variants in which we, by using site-directed mutagenesis, substituted residues suggested to have key roles in binding to or interacting with the aromatic and carbohydrate structures of its uronic acid ester substrates. Our results support the hypothesis that two aromatic residues (Phe-174 and Trp- 376), conserved in bacterial GEs, interact with aromatic and carbohydrate structures of these substrates in the enzyme active site, respectively. The solved crystal structure of TtCE15A revealed features previously not observed in either fungal or bacterial GEs, with a large inserted N-terminal region neighboring the active site and a differently positioned residue of the catalytic triad. The findings highlight key interactions between GEs and complex lignin-carbohydrate ester substrates and advance our understanding of the substrate specificities of these enzymes in biomass conversion.
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40.
  • Arnling Bååth, Jenny, 1987, et al. (författare)
  • Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica
  • 2022
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 119:2, s. 470-481
  • Tidskriftsartikel (refereegranskat)abstract
    • Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure–function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.
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41.
  • Ask, Magnus, 1983, et al. (författare)
  • A comparison between simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) of spruce and giant reed using two Saccharomyces cerevisiae strains
  • 2010
  • Ingår i: Society for Industrial Microbiology, 60th annual meeting.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • For significant fermentative conversion of lignocellulose to ethanol, the yeast Saccharomyces cerevisiae has proved to be a robust organism, albeit inter-strain variations may have a big influence on process performance. In this study, two S. cerevisiae strains were evaluated for their ability to ferment two different lignocellulosic raw materials, giant reed and spruce at 10 % water insoluble solids (WIS). One industrial strain, Ethanol Red, and one laboratory strain carrying the XR/XDH pathway, VTT C-10880, were used. The process concept may also affect the choice of the most suitable strain. Therefore, two principal process concepts, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were evaluated.The ethanol yield on giant reed based on total soluble sugars in the SHF was higher for VTT C-10880 than for Ethanol Red. On spruce, the yield of ethanol was higher for Ethanol Red. In SSF of giant reed, VTT C-10880 performed better in terms of the ethanol yield based on total sugars in fibres and liquid. However, the ethanol yield on spruce was higher for Ethanol Red than for VTT C-10880, which only produced a minor amount of ethanol. Spruce was more inhibitory than giant reed. Ethanol Red is more robust and converted the inhibitory substances in the pretreated materials faster, and is therefore a suitable industrial strain background for fermentation of both spruce and giant reed. Interestingly, VTT C-10880 performed better in SHF than SSF, primarily due to better xylose conversion in SHF.
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42.
  • Ask, Magnus, 1983, et al. (författare)
  • Challenges in enzymatic hydrolysis and fermentation of pretreated Arundo donax revealed by a comparison between SHF and SSF
  • 2012
  • Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; 47:10, s. 1452-1459
  • Tidskriftsartikel (refereegranskat)abstract
    • The perennial herbaceous crop Arundo donax is a potential feedstock for second-generation bioethanol production. In the present work, two different process options were investigated for the conversion of two differently steam-pretreated batches of A. donax. The pretreated raw material was converted to ethanol with a xylose-consuming Saccharomyces cerevisiae strain, VTT C-10880, by applying either separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). The highest overall ethanol yield and final ethanol concentration were achieved using SHF (0.27 g g(-1) and 20.6 g L-1 compared to 0.24 g g(-1) and 19.0 g L-1 when SSF was used). The performance of both SHF and SSF was improved by complementing the cellulolytic enzymes with hemicellulases. The higher amount of acetic acid in one of the batches was shown to strongly affect xylose consumption in the fermentation. Only half of the xylose was consumed when batch 1 (high acetic acid) was fermented, compared to that 94% of the xylose was consumed in fermentation of batch 2 (lower acetic acid). Furthermore, the high amount of xylooligomers present in the pretreated materials considerably inhibited the enzymatic hydrolysis. Both the formation of xylooligomers and acetic acid thus need to be considered in the pretreatment process in order to achieve efficient conversion of A. donax to ethanol.
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43.
  • Ask, Magnus, 1983, et al. (författare)
  • Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials
  • 2013
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 12:87
  • Tidskriftsartikel (refereegranskat)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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44.
  • Ask, Magnus, 1983, et al. (författare)
  • HMF and furfural stress results in drainage of redox and energy charge of Saccharomyces cerevisiae
  • 2012
  • Ingår i: 13th International Congress on Yeasts, Madison, WI, USA.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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45.
  • Ask, Magnus, 1983, et al. (författare)
  • Intracellular redox state as key target for Saccharomyces cerevisiae tolerance to lignocellulosic hydrolysate inhibitors
  • 2013
  • Ingår i: 35th Symposium on Biotechnology for Fuels and Chemicals (April 29-May 2, 2013).
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
  •  
46.
  • Ask, Magnus, 1983, et al. (författare)
  • Physiological studies of Saccharomyces cerevisiae for increased tolerance against furfural and HMF – two common inhibitors in lignocellulosic hydrolysate
  • 2011
  • Ingår i: 5th EU-Summer School Proteomic Basics, Brixen, Italy.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
  •  
47.
  • Ask, Magnus, 1983, et al. (författare)
  • 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
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:181
  • Tidskriftsartikel (refereegranskat)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.
  •  
48.
  • Ask, Magnus, 1983, et al. (författare)
  • TARGETING THE INTRACELLULAR REDOX STATE IN THE DEVELOPMENT OF MORE ROBUST Saccharomyces cerevisiae STRAINS FOR LIGNOCELLULOSIC BIOETHANOL PRODUCTION
  • 2014
  • Ingår i: ISSY31: 31ST INTERNATIONAL SPECIALISED SYMPOSIUM ON YEAST.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
  •  
49.
  • Ask, Magnus, 1983, et al. (författare)
  • The effect of pretreatment harshness on separate hydrolysis and fermentation of giant reed by a xylose-consuming Saccharomyces cerevisiae strain
  • 2011
  • Ingår i: World Congress on Industrial Biotechnology and Bioprocessing, May 8 - 11, 2011, Toronto, Canada.
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Bioethanol produced from lignocellulosic feedstocks has received increased attention during the last years. To make lignocellulosic biomass susceptible to enzymatic hydrolysis, the materials first have to be pretreated. The pretreatment is often performed under harsh conditions, which release a number of compounds that can be inhibitory for enzymatic hydrolysis and the subsequent fermentation. Xylooligomers and acetic acid are two compounds that are potential inhibitors of enzymatic hydrolysis and fermentation, respectively. The final concentration of these compounds is highly dependent on the pretreatment conditions. In this study, two different pretreatments with different harshness were performed on giant reed. The influence of the resulting material composition on enzymatic hydrolysis was then investigated. The enzymatic hydrolysis was performed at 10 % (w/w) water insoluble solid concentration (WIS) with Celluclast 1.5L and Novozyme 188 with and without the addition of HTec which is acting on hemicellulose. During the harsher pretreatment, more xylooligomers were produced which were found to have a negative effect on the enzymatic hydrolysis. One of the hydrolysates contained a substantially higher concentration of acetic acid. To investigate the effect of this, the hydrolysed giant reed was fermented with a laboratory Saccharomyces cerevisiae strain, VTT C-10880, carrying the XR/XDH pathway. It was found that the acetic acid had a significant negative effect on the xylose consumption. By supplementing the less harsh pretreated material with the same amount of acetic acid, a similar decrease in xylose consumption was observed, indicating that acetic acid is limiting xylose fermentation in this case.
  •  
50.
  • Ask, Magnus, 1983, et al. (författare)
  • The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae
  • 2013
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:22
  • Tidskriftsartikel (refereegranskat)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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