SwePub - sökning: WFRF:(Mapelli Valeria 1978)

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1.	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.
2.	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.
3.	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.
4.	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.
5.	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.
6.	Ask, Magnus, 1983, et al. (författare) Transcriptional response and alterations in adenonucleotides and redox cofactors in S. cerevisiae upon treatment with HMF and furfural 2012 Ingår i: Advanced Biofuels in a Biorefinery Approach, February 28-March 1, Copenhagen, Denmark. 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.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.
7.	Bettiga, Maurizio, 1978, et al. (författare) Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies 2013 Ingår i: Cell Factories and Biosustainability (Hilleroed, Denmark, May 5-8 2013). Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
8.	Bettiga, Maurizio, 1978, et al. (författare) Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies 2013 Ingår i: 35th Symposium on Biotechnology for Fuels and Chemicals (Portland, OR. April 29-May 2, 2013). Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
9.	Bettiga, Maurizio, 1978, et al. (författare) Robust yeast strains as prerequisite for feasible biofuels production from renewable biomass resources 2013 Ingår i: FEMS-V congress of European Microbiologists. Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
10.	Bettiga, Maurizio, 1978, et al. (författare) Yeast physiology studies and metabolic engineering for enhanced robustness 2014 Ingår i: Enzitec 2014- XI Seminário Brasileiro de Tecnologia Enzimática. Barra da Tijuca-Rio de Janeiro, April 14th to 16th, 2014. Konferensbidrag (övrigt vetenskapligt/konstnärligt)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.
11.	Mapelli, Valeria, 1978, et al. (författare) Viability Study of the Use of Cast Iron Open Cell Foam as Microbial Fuel Cell Electrodes 2013 Ingår i: Advanced Engineering Materials. - : Wiley. - 1527-2648 .- 1438-1656. ; 15:3, s. 112-117 Tidskriftsartikel (refereegranskat)abstract Nowadays, the development of new green technologies has been promoted worldwide both by public andprivate institutions. In this context the research on microbial fuel cells (MFC) represents a promisingalternative to carbon based energy sources. Unfortunately, this technology has been always affected by too lowcurrent density output for allowing an intensive application in the industrial and civil field. The study dealswith this limitation and focuses on the implementation of metallic sponges, specifically cast iron based, aselectrodes, aiming at increasing the exposed surface and thus the current density at the MFC anode. Cast ironwas selected because of its low toxicity for the microorganisms, however its high melting point carries severalproblems for the manufacture process. Parallel to this, the realization of electrodes using foamed metals impliesfurther issues related to the generation of correct pore size distribution and adequate bacterial activity. Forinstance, the metal foams are expected to be open-cell type, so that there can be an efficientmass transport alsoto the inner regions of the electrode. In order to control these parameters the metal sponges are produced byinfiltration of cast iron on ceramic beds. Combining previous data with themeasurements of power generationefficiency the authors conclude the study attempting to design MFCs with metal foamed electrodes.
12.	Mombelli, Davide, et al. (författare) Performance of Metal Foams as MFC Electrodes and Characterization of Waste Streams from Pulp and Paper plants in Bioelectrochemical Systems 2012 Ingår i: Communication in Agricultural and Applied Sciences, Ghent University. ; 77:2, s. 129- Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Optimization of electrode structure and materials is a subject taken into high consideration to improve the performance of microbial fuel cell (MFC) systems. In this work we show the use of novel electrodes made of metal alloys with a peculiar foam structure. The foam structure of the electrodes, realized according to a proprietary process, is intended to increase the electrode surface available to the microorganisms for the formation of the biofilm. Cast iron (GS800) and stainless steel (304) foams have been used as electrodes in MFC systems run with pure culture of Shewanella oneidensis. In general, foam electrodes showed better performance compared to the massive ones, in terms of current density at the anode. Metal foams with different porosities (i.e. 10, 20 and 30 pores per inch) were compared, showing that the pore density is a critical feature, clearly influencing the overall MFC output and the kinetics of the voltage generation throughout the run. These differences were potentially related to differences in the mass transfer and in the dynamics of the anode colonization by S. oneidensis, which were both affected by the varying porosities.Furthermore, MFC with stainless steel electrodes performed better than the ones using cast iron electrodes, most probably due to changes occurring on the cast iron surface affected by oxidation and corrosion phenomena.Concomitantly to MFC experiments aiming to test metal foam electrodes, we performed a study on the possible use of waste streams from a Swedish pulp and paper plant, exploiting the endogenous microbial populations present in the stream. Analysis of the chemical composition of the waste stream and of the microbial populations thriving in this environment was performed and followed by the analysis of dynamic changes occurring within a bioelectrochemical system. This exploratory study on the use of waste streams from pulp and paper plants aimed to set the basis for a future integration of bioelectrochemical systems into a wider biorefinery concept.
13.	Mombelli, Davide, et al. (författare) Viability study of the use of cast iron open cell foam as microbial fuel cell electrodes. 2011 Ingår i: MetFoam2011, Sept. 18-21 2011, BEXCO, Busan, Korea. Konferensbidrag (refereegranskat)abstract Nowadays, the development of new green technologies has been promoted worldwide both by public and private institutions. In this context the research on microbial fuel cells (MFC) represents a promising alternative to carbon based energy sources. Unfortunately, this technology has been always affected by too low current density for allowing an intensive application in the industrial and civil field. The study deals with this limitation and focuses on the implementation of metallic foams, specifically cast iron based, as electrodes, increasing the exposed surface and thus the activity of the bacterial population. The pig iron was selected because of its low toxicity for the microorganisms and their metabolism, however its high melting point carries several problems for the manufacture process. Parallel to this, the realization of electrodes using foamed metals implies further issues related to the generation of correct pores size distribution and adequate bacterial activity. For instance, the metal foams are expected to be open-cell type, so that the mass transport might reach also the inner regions. In order to control these parameters the metal foams are produced by infiltration of cast iron on ceramic beds. Combining the previous data with the measurements of power generation efficiency the authors conclude the study attempting to design MFCs with metal foamed electrodes.
14.	Busti, Stefano, et al. (författare) Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast 2016 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6, s. Art. no. 27942- Tidskriftsartikel (refereegranskat)abstract Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage.
15.	Karlsson, Emma, 1983, et al. (författare) Adipic acid tolerance screening for potential adipic acid production hosts 2017 Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16:20 Tidskriftsartikel (refereegranskat)abstract Biobased processes for the production of adipic acid are of great interest to replace the current environmentally detrimental petrochemical production route. No efficient natural producer of adipic acid has yet been identified, but several approaches for pathway engineering have been established. Research has demonstrated that the microbial production of adipic acid is possible, but the yields and titres achieved so far are inadequate for commercialisation. A plausible explanation may be intolerance to adipic acid. Therefore, in this study, selected microorganisms, including yeasts, filamentous fungi and bacteria, typically used in microbial cell factories were considered to evaluate their tolerance to adipic acid. Results: Screening of yeasts and bacteria for tolerance to adipic acid was performed in microtitre plates, and in agar plates for A. niger in the presence of adipic acid over a broad range of concentration (0-684 mM). As the different dissociation state(s) of adipic acid may influence cells differently, cultivations were performed with at least two pH values. Yeasts and A. niger were found to tolerate substantially higher concentrations of adipic acid than bacteria, and were less affected by the undissociated form of adipic acid than bacteria. The yeast exhibiting the highest tolerance to adipic acid was Candida viswanathii, showing a reduction in maximum specific growth rate of no more than 10-15% at the highest concentration of adipic acid tested and the tolerance was not dependent on the dissociation state of the adipic acid. Conclusions: Tolerance to adipic acid was found to be substantially higher among yeasts and A. niger than bacteria. The explanation of the differences in adipic acid tolerance between the microorganisms investigated are likely related to fundamental differences in their physiology and metabolism. Among the yeasts investigated, C. viswanathii showed the highest tolerance and could be a potential host for a future microbial cell factory for adipic acid.
16.	Karlsson, Emma, 1983, et al. (författare) In silico and in vitro studies of the reduction of unsaturated α,β bonds of trans-2-hexenedioic acid and 6-amino-trans-2-hexenoic acid – Important steps towards biobased production of adipic acid 2018 Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 13:2 Tidskriftsartikel (refereegranskat)abstract The biobased production of adipic acid, a precursor in the production of nylon, is of great interest in order to replace the current petrochemical production route. Glucose-rich lignocel-lulosic raw materials have high potential to replace the petrochemical raw material. A number of metabolic pathways have been proposed for the microbial conversion of glucose to adipic acid, but achieved yields and titers remain to be improved before industrial applications are feasible. One proposed pathway starts with lysine, an essential metabolite industrially produced from glucose by microorganisms. However, the drawback of this pathway is that several reactions are involved where there is no known efficient enzyme. By changing the order of the enzymatic reactions, we were able to identify an alternative pathway with one unknown enzyme less compared to the original pathway. One of the reactions lacking known enzymes is the reduction of the unsaturated α,β bond of 6-amino-trans-2-hexenoic acid and trans-2hexenedioic acid. To identify the necessary enzymes, we selected N-ethylmaleimide reductase from Escherichia coli and Old Yellow Enzyme 1 from Saccharomyces pastorianus. Despite successful in silico docking studies, where both target substrates could fit in the enzyme pockets, and hydrogen bonds with catalytic residues of both enzymes were predicted, no in vitro activity was observed. We hypothesize that the lack of activity is due to a difference in electron withdrawing potential between the naturally reduced aldehyde and the carboxylate groups of our target substrates. Suggestions for protein engineering to induce the reactions are discussed, as well as the advantages and disadvantages of the two metabolic pathways from lysine. We have highlighted bottlenecks associated with the lysine pathways, and proposed ways of addressing them.
17.	Karlsson, Emma, 1983, et al. (författare) METABOLIC ENGINEERING OF Saccharomyces cerevisiae FOR PRODUCTION OF ADIPIC ACID FROM RENEWABLE SOURCES 2014 Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Adipic acid is a six carbon long dicarboxylic acid, considered to be the most important synthetic dicarboxylic acid annually produced, according to the International Energy Agency (IEA). The global production of adipic acid had in 2010 a volume of 2.8 million tonnes, for a total market price of 4.9 billion USD. The current production of adipic acid relies on non-renewable fossil raw materials, leading to emission of the greenhouse gases carbon dioxide and N2O. In addition, the production starts from benzene, whose use has several health related negative implications. This project aims to create a greener process for production of adipic acid developing a fermentation-based process using Swedish domestic renewable raw materials, such as forest residues and/or algae. These materials will be used to establish a biorefinery, wherein the fermentation process for the biosynthesis of adipic acid will represent the core process. Our current strategy is based on the generation of genetically modified strains of the yeast Saccharomyces cerevisiae, harbouring heterologous enzymatic activities allowing the conversion of lysine into adipic acid. This system is our first choice and will also work as proof-of-concept for bio-based production of adipic acid. Here we present the metabolic engineering strategy we are pursuing, based on two possible metabolic pathways for conversion of lysine into adipic acid. Preliminary results on the effect of adipic acid on S. cerevisiae physiology, lysine uptake, the expression of the heterologous genes of choice, and the conversion of lysine into adipic acid precursors are presented.
18.	Koppram, Rakesh, 1986, et al. (författare) The Presence of Pretreated Lignocellulosic Solids from Birch during Saccharomyces cerevisiae Fermentations Leads to Increased Tolerance to Inhibitors - A Proteomic Study of the Effects 2016 Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 11:2 Tidskriftsartikel (refereegranskat)abstract The fermentation performance of Saccharomyces cerevisiae in the cellulose to ethanol conversion process is largely influenced by the components of pretreated biomass. The insoluble solids in pretreated biomass predominantly constitute cellulose, lignin, and -to a lesser extent-hemicellulose. It is important to understand the effects of water-insoluble solids (WIS) on yeast cell physiology and metabolism for the overall process optimization. In the presence of synthetic lignocellulosic inhibitors, we observed a reduced lag phase and enhanced volumetric ethanol productivity by S. cerevisiae CEN. PK 113-7D when the minimal medium was supplemented with WIS of pretreated birch or spruce and glucose as the carbon source. To investigate the underlying molecular reasons for the effects of WIS, we studied the response of WIS at the proteome level in yeast cells in the presence of acetic acid as an inhibitor. Comparisons were made with cells grown in the presence of acetic acid but without WIS in the medium. Altogether, 729 proteins were detected and quantified, of which 246 proteins were significantly up-regulated and 274 proteins were significantly down-regulated with a fold change >= 1.2 in the presence of WIS compared to absence of WIS. The cells in the presence of WIS up-regulated several proteins related to cell wall, glycolysis, electron transport chain, oxidative stress response, oxygen and radical detoxification and unfolded protein response; and down-regulated most proteins related to biosynthetic pathways including amino acid, purine, isoprenoid biosynthesis, aminoacyl-tRNA synthetases and pentose phosphate pathway. Overall, the identified differentially regulated proteins may indicate that the likelihood of increased ATP generation in the presence of WIS was used to defend against acetic acid stress at the expense of reduced biomass formation. Although, comparative proteomics of cells with and without WIS in the acetic acid containing medium revealed numerous changes, a direct effect of WIS on cellular physiology remains to be investigated.
19.	Lambrughi, Matteo, et al. (författare) Conformational gating in ammonia lyases 2020 Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 1872-8006 .- 0304-4165. ; 1864:7 Tidskriftsartikel (refereegranskat)abstract Background: Ammonia lyases are enzymes of industrial and biomedical interest. Knowledge of structure-dynamics-function relationship in ammonia lyases is instrumental for exploiting the potential of these enzymes in industrial or biomedical applications. Methods: We investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. At this scope, we used microsecond all-atom molecular dynamics simulations, analyzed with dimensionality reduction techniques, as well as in terms of contact networks and correlated motions. Results: We identify two regulatory elements in the MAL structure, i.e., the β5-α2 loop and the helix-hairpin-loop subdomain. These regulatory elements undergo conformational changes switching from ‘occluded’ to ‘open’ states. The rearrangements are coupled to changes in the accessibility of the active site. The β5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of tunnels from the protein surface to the catalytic site, making the active site more accessible to the substrate when they are in an open state. Conclusions: Our work pinpoints a sequential mechanism, in which the helix-hairpin-loop subdomain of MAL needs to break a subset of intramolecular interactions first to favor the displacement of the β5-α2 loop. The coupled conformational changes of these two elements contribute to modulate the accessibility of the catalytic site. General significance: Similar molecular mechanisms can have broad relevance in other ammonia lyases with similar regulatory loops. Our results also imply that it is important to account for protein dynamics in the design of variants of ammonia lyases for industrial and biomedical applications.
20.	Lambrughi, Matteo, et al. (författare) DEAMINATION REACTIONS AS PART OF THE METABOLIC PATHWAY FOR THE PRODUCTION OF ADIPIC ACID 2018 Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Deamination of lysine, β-lysine or 2-aminoadipic acid are enzymatic reactions that have great biotechnological interest as part of the metabolic pathways for the production of adipic acid. The enzymatic activity necessary to catalyze the mentioned deamination reactions is defined as ammonia lyase (EC 4.3.1.-) and cleaves the bond between the amine group and adjacent carbon. Whereas ammonia lyases able to act on the target substrates lysine, β-lysine or 2-aminoadipic acid have not been identified so far, ammonia lyases able to act on other amino acids have long been known. We selected 3-methylaspartate-ammonia lyase (MAL, EC 4.3.1.2) as enzyme that potentially could be made active on the target substrates. Three MAL were recombinantly expressed and purified and the activity tested towards the substrates, but no activity was observed. Different MAL single mutant variants potentially able to catalyze the desired reactions were designed using a computational approach, but unfortunately, no activity towards the target substrates was detected. In order to better understand the substrate scope and catalytic mechanism of MAL, different compunds with similar structure to the natural substrate were tried as substrates. MAL showed activity towards aspartic acid, but no towards the other substrates indicating the narrow specificity of the enzyme. Inhibition studies showed that β-lysine was a competitive inhibitor suggesting that the amino group of the substrate need to be in the β-carbon for the binding. 2-aminoadipic acid was shown to be a non-competitive inhibitor. Finally, docking experiments were carried out to understand if the target substrates fit in the catalytic pocket. The study provides a deeper knowledge of the substrate scope and inhibitors of MAL and analyze if and how the target substrates could be deaminated by MAL. Moreover, the study establishes reliable methods for the detection of deamination activity of lysine, β-lysine and 2-aminoadipic acid.
21.	Lorantfy, Bettina, 1986, et al. (författare) Characterization of the respiratory physiology of Lactococcus lactis for starter culture production with improved acidification capacity 2015 Ingår i: Microbial Stress: From Molecules to Systems. Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Commercial freeze-dried starter cultures for cheese making are produced mainly via anaerobic batch processes. A recent discovery has shown that some lactic acid bacteria (LAB) are able to sustain respiration under aerobic conditions when hemin is added to the growth medium, since it completes the electron transport chain for respiration, which is otherwise defective [1]. Respiration is energetically beneficial: compared to fermentation, under respiratory conditions the biomass yield is higher and a different by-product pattern is observed. However, it is also important to consider whether the different metabolism can affect the performance of the starter culture. Thus, this project investigates LAB respiratory physiology, aiming to clarify the molecular reasons behind the milk acidification capacity of the respiratory culture. Our bioreactor results demonstrate that with hemin addition, cells switch from the fermentation to respiration only in the late exponential phase of growth. Although presence of oxygen is an additional stress for LAB, in the presence of hemin under aerobic conditions cells have surprisingly better fermentation behaviour, i.e. higher lactate yield before the respiratory switch. Therefore, we hypothesize that with improved fermentation a certain energy threshold is achieved for the respiratory switch. This energy requirement might be related to the intake of the hemin, however this aspect needs further investigation, as hemin transport into the cells has not been characterized yet.Reference: [1] Lechardeur D et al Curr Opin Biotechnol 2011,22(2):143
22.	Lorantfy, Bettina, 1986, et al. (författare) Presence of galactose in precultures induces lacS and leads to short lag phase in lactose-grown Lactococcus lactis cultures 2019 Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 46:1, s. 33-43 Tidskriftsartikel (refereegranskat)abstract Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacSgene, known to encode a lactose permease with affinity for galactose, a putative galactose–lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures.
23.	Lorantfy, Bettina, 1986, et al. (författare) What induces respiration in lactic acid bacteria? Characterization of respiratory metabolism of Lactococcus lactis in bioreactors for production of starter cultures with improved acidification capacity 2015 Ingår i: RAFT 11 Recent Advances in Fermentation Technology. Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Commercial freeze-dried lactic acid bacteria starter cultures for cheese making are produced mainly via anaerobic batch fermentations. Recently, it has been shown that some Lactococcus lactis species are able to sustain respiration under aerobic conditions when hemin is added to the growth medium, since it completes the electron transport chain for respiration, which is otherwise defective. Respiration is energetically beneficial and under respiratory conditions, higher biomass yield is obtained together with a changed by-product pattern, compared to fermentation [1]. So far it has not been studied how the different culture conditions and thereby different metabolism affect the starter culture performance. In this project, we investigate respiratory culture conditions, and the effect on the milk acidification capacity of the culture. Since Lactococcus lactis is a fastidious microorganism, a rich chemically defined medium was developed to support the nutrient requirements, and was applied for bioreactor cultivations with quantitative approaches. The product profile and on-line gas analysis revealed that with hemin addition at the start of the process, cells switch to respiratory metabolism only in the second phase of growth, after an initial mixed-acid fermentative phase. To characterize the observed respiratory switch, a multivariate study was performed: a set of bioreactor batch experiments were carried out with different initial sugar concentrations under anaerobic, aerobic, and respiratory conditions. The results indicate that hemin addition together with some yet not defined threshold must be met in order to induce the respiratory metabolic state of the culture.[1] Lechardeur D et al Curr Opin Biotechnol 2011,22(2):143‐149
24.	Mapelli, Valeria, 1978, et al. (författare) Biosyntheis of Bioactive Seleno-Compunds in Saccharomyces cerevisiae via Metabolic and Bioprocess Engineering 2010 Ingår i: 14th International Biotechnology Symposium and Exhibition (IBS 2010). Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Selenium (Se) is an essential element for many organisms as it is present under the form of Se-cysteine in Se-proteins. The main sources of Se for animals are edible plants able to accumulate Se from the soil and store it under organic forms. Some of Se organic forms bioavailable for animals, such as Se-methyl-selenocysteine (SeMCys), have been proven to have cancer-preventing effects if regularly introduced into the diet. Since Se content in plants is highly susceptible to environmental factors, the intake of Se is often insufficient to result in beneficial effects. Therefore, the use of Se-enriched yeast as food supplement is made available to avoid Se shortage. The yeast Saccharomyces cerevisiae does not require Se as essential element, but is able to metabolise and accumulate Se. This work shows a study of Se-metabolism in yeast that lead to the definition of a metabolic engineering strategy and to an optimized bioprocess to increase the levels of beneficial Se-compounds in yeast. After a preliminary study on Se uptake dynamics and Se-metabolite profile in yeast, a recombinant yeast strain was generated, by introducing heterologous genes from plants belonging to Brassicaceae and Fabaceae families. Due to the delicate balance between toxic and beneficial effects of Se, the cultivation process was carefully optimized in terms of medium composition and Se feeding. The established bioprocess allowed minimizing the toxicity of Se and redirecting Se fluxes towards the biosynthesis of SeMCys at the same time.The results obtained demonstrate that the coupling of genetic engineering strategies with optimization of cultivation system is a promising approach for the establishment of a yeast cell factory for the production of yeast enriched in health promoting Se-compounds.
25.	Mapelli, Valeria, 1978 (författare) Biosynthesis of bioactive seleno-compounds in Saccharomyces cerevisiae 2012 Ingår i: Proceedings of the 25th VH-Yeast Conference. Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract Selenium (Se) is an essential element for many organisms as it is present under the form of Se-cysteine in Se-proteins. The main sources of Se for animals are edible plants able to accumulate Se from the soil and store it under organic forms. Some of Se organic forms bioavailable for animals, such as Se-methyl-selenocysteine (SeMCys), have been proven to have cancer-preventing effects if regularly introduced into the diet. Since Se content in plants is highly susceptible to environmental factors, the intake of Se is often insufficient to result in beneficial effects. Therefore, the use of Se-enriched yeast as food supplement is made available to avoid Se shortage. The yeast Saccharomyces cerevisiae does not require Se as essential element, but is able to metabolise and accumulate Se. This work shows a study of Se-metabolism in yeast that lead to the definition of a metabolic engineering strategy and to an optimized bioprocess to increase the levels of beneficial Se-compounds in yeast. After a preliminary study on Se uptake dynamics and Se-metabolite profile in yeast, a recombinant yeast strain was generated, by introducing heterologous genes from plants belonging to Brassicaceae and Fabaceae families. Due to the delicate balance between toxic and beneficial effects of Se, the cultivation process was carefully optimized in terms of medium composition and Se feeding via optimization of a fed-batch process characterized by dual limitation. The established bioprocess allowed minimizing the toxicity of Se and redirecting Se fluxes towards the biosynthesis of SeMCys at the same time.The results obtained demonstrate that the coupling of genetic engineering strategies with optimization of cultivation system is a promising approach for the establishment of a yeast cell factory for the production of yeast enriched in health promoting Se-compounds.

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