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Träfflista för sökning "WFRF:(van Maris Antonius J. A. Professor 1976 ) "

Sökning: WFRF:(van Maris Antonius J. A. Professor 1976 )

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1.
  • Marques, W. L., et al. (författare)
  • Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae
  • 2018
  • Ingår i: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 45, s. 121-133
  • Tidskriftsartikel (refereegranskat)abstract
    • Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h−1 (LmSPase) and 0.06 ± 0.01 h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h−1 and 0.08 ± 0.00 h−1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.
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2.
  • Gabba, M., et al. (författare)
  • Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane
  • 2020
  • Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 118:2, s. 422-434
  • Tidskriftsartikel (refereegranskat)abstract
    • We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.
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3.
  • Guevara-Martínez, Mónica, 1989- (författare)
  • Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
  • 2019
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Microbial bio-based processes have emerged as an alternative to replace fossil-based processes for the production of fuels and chemicals. (R)-3-hydroxybutyrate (3HB) is a medium-value chemical that has gained special attention as a precursor of antibiotics and vitamins, as a monomer for the synthesis of tailor-made polyesters and as a nutritional source for eukaryotic cells. By integrating strain and bioprocess-design strategies the work of this thesis has aimed to improve microbial 3HB production by the well-studied platform organism Escherichia coli (strain AF1000) expressing a thiolase and a reductase from Halomonas boliviensis.Uncoupling growth and product formation by NH4+- or PO43-- limited fed-batch cultivations allowed for 3HB titers of 4.1 and 6.8 g L-1 (Paper I). Increasing the NADPH supply by overexpression of glucose-6-phosphate dehydrogenase (zwf) resulted in 1.7 times higher 3HB yield compared to not overexpressing zwf in NH4+ depleted conditions (Paper II). To increase 3HB production in high-cell density cultures, strain BL21 was selected as a low acetate-forming, 3HB-producing platform. BL21 grown in NH4+ limited fed-batch cultivations resulted in 2.3 times higher 3HB titer (16.3 g L-1) compared to strain AF1000 (Paper III). Overexpression of the native E. coli thioesterase “yciA”, identified as the largest contributor in 3HB-CoA hydrolysis, resulted in 2.6 times higher 3HB yield compared to AF1000 not overexpressing yciA. Overexpressing zwf and yciA in NH4+ depleted fed-batch experiments resulted in 2 times higher total 3HB yield (0.210 g g-1) compared to AF1000 only overexpressing zwf (Paper IV). Additionally, using 3HB as a model product, the bacterial artificial chromosome was presented as a simple platform for performing pathway design and optimization in E. coli (Paper V). While directly relevant for 3HB production, these findings also contribute to the knowledge on how to improve the production of a chemical for the development of robust and scalable processes.
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4.
  • Juergens, H., et al. (författare)
  • Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics
  • 2018
  • Ingår i: Scientific Data. - : Nature Publishing Groups. - 2052-4463. ; 5
  • Tidskriftsartikel (refereegranskat)abstract
    • Open data in science requires precise definition of experimental procedures used in data generation, but traditional practices for sharing protocols and data cannot provide the required data contextualization. Here, we explore implementation, in an academic research setting, of a novel cloud-based software system designed to address this challenge. The software supports systematic definition of experimental procedures as visual processes, acquisition and analysis of primary data, and linking of data and procedures in machine-computable form. The software was tested on a set of quantitative microbial-physiology experiments. Though time-intensive, definition of experimental procedures in the software enabled much more precise, unambiguous definitions of experiments than conventional protocols. Once defined, processes were easily reusable and composable into more complex experimental flows. Automatic coupling of process definitions to experimental data enables immediate identification of correlations between procedural details, intended and unintended experimental perturbations, and experimental outcomes. Software-based experiment descriptions could ultimately replace terse and ambiguous ‘Materials and Methods’ sections in scientific journals, thus promoting reproducibility and reusability of published studies.
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5.
  • Perez-Zabaleta, Mariel, 1987- (författare)
  • Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
  • 2019
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Metabolic engineering and process engineering are two powerful disciplines to design and improve microbial processes for sustainable production of an extensive number of compounds ranging from chemicals to pharmaceuticals. The aim of this thesis was to synergistically combine these two disciplines to improve the production of a model chemical called (R)-3-hydroxybutyrate (3HB), which is a medium-value product with a stereocenter and two functional groups. These features make 3HB an interesting building block, especially for the pharmaceutical industry. Recombinant production of 3HB was achieved by expression of two enzymes from Halomonas boliviensis in the model microorganism Escherichia coli, which is a microbial cell factory with proven track record and abundant knowledge on its genome, metabolism and physiology.Investigations on cultivation strategies demonstrated that nitrogen-depleted conditions had the biggest impact on 3HB yields, while nitrogen-limited cultivations predominantly increased 3HB titers and volumetric productivities. To further increase 3HB production, metabolic engineering strategies were investigated to decrease byproduct formation, enhance NADPH availability and improve the overall 3HB-pathway activity. Overexpression of glucose-6-phosphate dehydrogenase (zwf) increased cofactor availability and together with the overexpression of acyl-CoA thioesterase YciA resulted in a 2.7-fold increase of the final 3HB concentration, 52% of the theoretical product yield and a high specific productivity (0.27 g g-1 h-1). In a parallel strategy, metabolic engineering and process design resulted in an E. coli BL21 strain with the hitherto highest reported volumetric 3HB productivity (1.52 g L-1 h-1) and concentration (16.3 g L-1) using recombinant production. The concepts developed in this thesis can be applied to industrial 3HB production processes, but also advance the knowledge base to benefit design and expansion of the product range of biorefineries.
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6.
  • Valk, Laura C., et al. (författare)
  • Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures : Combined Fermentation and Acetogenesis by the Dominant sp nov "Candidatus Galacturonibacter soehngenii"
  • 2018
  • Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 84:18
  • Tidskriftsartikel (refereegranskat)abstract
    • Agricultural residues such as sugar beet pulp and citrus peel are rich in pectin, which contains galacturonic acid as a main monomer. Pectin-rich residues are underexploited as feedstocks for production of bulk chemicals or biofuels. The anaerobic, fermentative conversion of D-galacturonate in anaerobic chemostat enrichment cultures provides valuable information toward valorization of these pectin-rich feedstocks. Replicate anaerobic chemostat enrichments, with D-galacturonate as the sole limiting carbon source and inoculum from cow rumen content and rotting orange peels, yielded stable microbial communities, which were dominated by a novel Lachnospiraceae species, for which the name "Candidatus Galacturonibacter soehngenii" was proposed. Acetate was the dominant catabolic product, with formate and H-2 as coproducts. The observed molar ratio of acetate and the combined amounts of H-2 and formate deviated significantly from 1, which suggested that some of the hydrogen and CO2 formed during D-galacturonate fermentation was converted into acetate via the Wood-Ljungdahl acetogenesis pathway. Indeed, metagenomic analysis of the enrichment cultures indicated that the genome of "Candidatus G. soehngenii" encoded enzymes of the adapted Entner-Doudoroff pathway for D-galacturonate metabolism as well as enzymes of the Wood-Ljungdahl pathway. The simultaneous operation of these pathways may provide a selective advantage under D-galacturonate-limited conditions by enabling a higher specific ATP production rate and lower residual D-galacturonate concentration than would be possible with a strictly fermentative metabolism of this carbon and energy source. IMPORTANCE This study on D-galacturonate metabolism by open, mixed-culture enrichments under anaerobic, D-galacturonate-limited chemostat conditions shows a stable and efficient fermentation of D-galacturonate into acetate as the dominant organic fermentation product. This fermentation stoichiometry and population analyses provide a valuable baseline for interpretation of the conversion of pectin-rich agricultural feedstocks by mixed microbial cultures. Moreover, the results of this study provide a reference for studies on the microbial metabolism of D-galacturonate under different cultivation regimes.
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7.
  • Björlenius, Berndt, 1963- (författare)
  • Pharmaceuticals – improved removal from municipal wastewater and their occurrence in the Baltic Sea
  • 2018
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Pharmaceutical residues are found in the environment due to extensive use in human and veterinary medicine. The active pharmaceutical ingredients (APIs) have a potential impact in non-target organisms. Municipal wastewater treatment plants (WWTPs) are not designed to remove APIs.In this thesis, two related matters are addressed 1) evaluation of advanced treatment to remove APIs from municipal wastewater and 2) the prevalence and degradation of APIs in the Baltic Sea.A stationary pilot plant with nanofiltration (NF) and a mobile pilot plant with activated carbon and ozonation were designed to study the removal of APIs at four WWTPs. By NF, removal reached 90%, but the retentate needed further treatment. A predictive model of the rejection of APIs by NF was developed based on the variables: polarizability, globularity, ratio hydrophobic to polar water accessible surface and charge. The pilot plants with granular and powdered activated carbon (GAC) and (PAC) removed more than 95% of the APIs. Screening of activated carbon products was essential, because of a broad variation in adsorption capacity. Recirculation of PAC or longer contact time, increased the removal of APIs. Ozonation with 5-7 g/m3 ozone resulted in 87-95% removal of APIs. Elevated activity and transcription of biomarkers indicated presence of xenobiotics in regular effluent. Chemical analysis of APIs, together with analysis of biomarkers, were valuable and showed that GAC-filtration and ozonation can be implemented to remove APIs in WWTPs, with decreased biomarker responses.Sampling of the Baltic Sea showed presence of APIs in 41 out of 43 locations. A developed grey box model predicted concentration and half-life of carbamazepine in the Baltic Sea to be 1.8 ng/L and 1300 d respectively.In conclusion, APIs were removed to 95% by GAC or PAC treatment. The additional treatment resulted in lower biomarker responses than today and some APIs were shown to be widespread in the aquatic environment.
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8.
  • Kuil, Teun, 1993- (författare)
  • Analysis and engineering of central metabolism in Clostridium thermocellum
  • 2023
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • To mitigate climate change, greenhouse gas emissions must be reduced to net-zero in 2050 requiring a drastic transition in today´s energy sector. To achieve this goal, the use of biofuels produced from lignocellulosic feedstocks, including agricultural and forestry residues, is expected to play an important role. The native ability of the anaerobic thermophile Clostridium thermocellum to efficiently degrade lignocellulose makes this microorganism a promising candidate for consolidated bioprocessing of lignocellulosic feedstocks into the biofuel ethanol. However, improvements in ethanol yield, titre, and tolerance are required for industrial implementation. The aim of this thesis was to increase understanding of the central metabolism of C. thermocellum and thereby aid future metabolic engineering and process optimization efforts focused on improving ethanol production from lignocellulosic material. The atypical glycolysis of C. thermocellum uses pyrophosphate (PPi) instead of ATP as phosphoryl donor. This alteration is hypothesized to increase energetic efficiency but simultaneously decrease thermodynamic driving force resulting in lower achievable ethanol titres. As such, improved understanding of the PPi metabolism has both fundamental and applied importance. Knockout studies combined with physiological characterization of four predicted metabolic PPi sources provided valuable insights into the PPi metabolism and demonstrated that the energetic benefits of PPi usage are likely limited. Furthermore, biochemical characterization of the ATP-Pfk from C. thermocellum and other bacteria demonstrated that PPi might be a key allosteric regulator in bacteria with a PPi-dependent glycolysis. The low thermodynamic driving force of the ethanol formation pathway combined with a flexible redox network are key factors that impact ethanol titre, yield, and tolerance in C. thermocellum. Apart from dominant thermodynamic limitations, physiological characterization of wild-type and a non-ethanol producing mutant at various exogenous ethanol concentrations and temperatures demonstrated that biophysical limitations also impact ethanol tolerance. Lowering the cultivation temperature decreased chaotropic effects of ethanol and improved ethanol tolerance. By-product formation and incomplete substrate utilization decrease obtained ethanol yields. To minimize formation of one specific class of by-products, the mechanism behind amino acid secretion in C. thermocellum was investigated. Cellobiose- or ammonium-limited chemostats of wild-type and knockout strains of NADPH-supplying and NADPH-consuming pathways identified catabolic oversupply of NADPH as the main driver behind amino acid secretion. The malate shunt and the ammonium-regulated shift between nitrogen assimilation pathways with differing cofactor specificities were shown to play key roles in NADPH metabolism and amino acid secretion. To improve substrate utilization, laboratory evolution combined with reverse metabolic engineering was used as a tool to provide insights into increased utilization of glucose and fructose. Reproducible and constitutive growth on these hexose sugars was achieved for evolved mutant strains. Additionally, two mutations were identified that are involved in (regulation of) transport or metabolism of these hexose sugars.Together these findings provide valuable insights into the central metabolism of C. thermocellum and aid future optimizations of this organism for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. 
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9.
  • Ljungqvist, Emil E. (författare)
  • Modeling and analysis of the rapid aerobic metabolism of Geobacillus sp. LC300
  • 2024
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • To mitigate climate change, global greenhouse gas emissions must be halved before 2030. To achieve this goal, alternative routes for fuel and chemical production that do not rely on fossil resources must be explored. Industrial biotechnology has been identified as a key technology in this transition, allowing the sustainable valorization of biomass to biofuels and biochemicals. Geobacillus sp. LC300 is a thermophilic microorganism displaying remarkable growth rates and metabolic capabilities, thus showing promise for development into a microbial cell factory for sustainable production of biochemicals. However, the metabolism of the organism is unexplored, and its metabolic requirements and optimal growth conditions unknown. The aim of this thesis was to investigate the fast metabolism of Geobacillus sp. LC300 and thereby evaluate the potential and facilitate the development of the organism as a microbial cell factory. To explore the metabolic landscape of G. sp. LC300, a homology-based genome-scale metabolic model was constructed. By analyzing the model-predicted metabolic pathways, a prototrophy for all amino acids was predicted, along with an auxotrophy for vitamin B12. Analysis of transporters further predicted growth on several carbon sources, and the model showed accurate predictions of intracellular flux distributions and growth yields on both glucose and xylose. This model serves as a crucial tool for understanding the G. sp. LC300’s metabolism and guiding metabolic engineering efforts to optimize it for industrial use. Growth media previously used for the cultivation of G. sp. LC300 contained complex components, such as yeast extract, and was unable to support growth to high cell densities. This complicated quantitative studies of metabolism where controlled conditions and high cell densities are important for quantification of rates and yields. A minimal medium was developed based on the biomass composition predicted by the genome-scale model. In this development, the predicted auxotrophy for vitamin B12 was confirmed, and an additional auxotrophy for biotin revealed. The modified medium supported growth to high cell densities without the addition of complex components. An investigation of the optimal growth conditions of G. sp. LC300 revealed an optimal growth temperature several degrees lower than earlier reported values, providing a more accurate basis for the development of future production process settings. The range of carbon source utilization was further investigated, revealing fast growth on substrates like glycerol and starch that are common byproducts and in waste-streams from industry.To investigate the keys to the rapid substrate consumption rate, growth, and respiration of G. sp. LC300, glucose-limited chemostat cultivations were performed. The cultivations revealed a capacity of fully respiratory growth at a rate higher than the maximum specific growth rate of most other microorganisms, and a lower fraction of substrate consumed by maintenance than E. coli. Proteomics analysis further revealed an unusually low allocation of protein to the central carbon metabolism and translation, made possible by high turnover numbers of these enzymes allowing a larger allocation to respiratory enzymes. Finally, enzyme-constrained modeling indicated limited protein availability as the cause of overflow metabolism at growth rates above critical, with a switch from respiratory to respiro-fermentative pathways. Together, these findings provide insights into the rapid metabolism of G. sp. LC300 and highlights its potential as a microbial cell factory. This work can provide the basis for the development of new production processes that play an important role in the bioeconomy of the future and help circularize greenhouse gas emissions to net-zero.
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10.
  • Verhoeven, Maarten D., et al. (författare)
  • Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive L-arabinose uptake
  • 2018
  • Ingår i: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 18:6
  • Tidskriftsartikel (refereegranskat)abstract
    • Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantarum L-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h(-1) on L-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on L-arabinose in the presence of D-glucose and D-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2(N376T) or Gal(2N376I) substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2(T89I) substitution, which was subsequently also detected in the independently evolved strain IMS0010. In C-14-sugar-transport assays, Gal2(N376S), Gal2(N376T) and Gal(2N376I) substitutions showed a much lower glucose sensitivity of L-arabinose transport and a much higher Km for D-glucose transport than wild-type Gal2. Introduction of the Gal2(N376I) substitution in a non-evolved strain enabled growth on L-arabinose in the presence of D-glucose. Gal2(N376T), T89I and Gal2(T89I) variants showed a lower K-m for L-arabinose and a higher K-m for D-glucose than wild-type Gal2, while reverting Gal2(N376T), T89I to Gal2(N376) in an evolved strain negatively affected anaerobic growth on L-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.
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11.
  • Yayo, Johannes, 1992- (författare)
  • Insights into the metabolism of Clostridium thermocellum for cellulosic ethanol production
  • 2022
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The societal goal of reaching net-zero CO2 emissions requires development of integrated biorefineries to produce biomass-derived fuels and chemicals. For sustainable second-generation bioethanol production, consolidated bioprocessing with the thermophile Clostridium thermocellum is regarded as a promising concept in view of the microorganism’s native ability to efficiently degrade plant cell wall material. However, for industrial implementation, improvements in ethanol titer and yield are needed. The aim of this thesis was to increase knowledge on the metabolism of C. thermocellum and thereby guide future metabolic engineering strategies to maximize the ethanol yield and titer.Yield improvements and fundamental studies into the metabolism of C. thermocellum would benefit from higher utilization of hexose monomers as well as minimized byproduct formation. To investigate underlying mechanisms for growth on glucose and fructose, laboratory evolution in chemostats together with genome sequence-based reverse engineering was applied. This successfully revealed two underlying mutations with (regulatory) roles in metabolism or transport of the monosaccharides. Together, these mutations enable reproducible and constitutive growth and are relevant for follow-up studies into transport and upper glycolysis. Separately, the mechanism behind the surprising byproduct formation of secreted amino acids was investigated by knock-out studies in NADPH-supplying and -consuming pathways. Physiological characterization in cellobiose- or ammonium-limited chemostats of mutant strains, with deletions in the NADPH-forming malate shunt or in the putatively ferredoxin-dependent ammonium assimilation, demonstrated a central role of NADPH in driving amino acid secretion. The findings indicated that electron availability will be crucial for further yield improvements in the NADH-dependent ethanol pathway.Fundamental mechanisms that might contribute to improved ethanol titer were addressed by studying thermodynamic and biophysical limitations. The pyrophosphate (PPi)-dependent glycolysis of C. thermocellum has been hypothesized to increase the overall ATP yield at the expense of the overall driving force. Knock-out studies combined with functional annotation of potential PPi-sources questioned this trade-off and increased knowledge of the PPi metabolism. The chaotropic effect (biophysical toxicity) of ethanol is commonly counteracted by lowering the cultivation temperature. Here, physiological characterization at varying ethanol titers demonstrated improved growth and fermentation at lower temperature. Comparisons to a non-ethanol producing mutant indicated both thermodynamic and biophysical limitations specifically in the ethanol pathway.Overall, these findings suggest that improvements in ethanol yield and titer would benefit from a simplified glycolysis that is engineered for a high driving force. While this work is beneficial for second-generation ethanol production, these findings can also be broadly applicable in the research and development of C. thermocellum as a cell factory for sustainable production of other fuels and chemicals. 
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12.
  • Daga-Quisbert, Jeanett, et al. (författare)
  • Analysis of the microbiome of the Bolivian high-altitude Lake Pastos Grandes
  • 2023
  • Ingår i: FEMS Microbiology Ecology. - : Oxford University Press (OUP). - 0168-6496 .- 1574-6941. ; 99:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Lake Pastos Grandes in Bolivia is mainly composed of salt flats, which are sporadically and only partially submerged during the wet season. In the present study, the chemical composition of water samples of the lake and some influent rivers was determined. We found that it is likely that the lake was influenced by the dilution of metals from ancient evaporites. We performed the first metagenomic studies of this lake. Analyses of shotgun metagenomics revealed that the relative abundances of Burkholderiales and Pseudomonadales were noteworthy in the water samples, whereas the archaea belonging to the Halobacteriales and Cyanobacteria from subsection III had high abundances in the salt flat. The eukaryotes Crustacea and Diatomea exhibited the highest abundances in the water samples. We investigated further the potential effect of human activities on the nitrogen cycle mobilization in the lake and the propagation of antimicrobial resistance genes. This is the first report about the cycle in the lake. Additionally, rifamycin resistance genes and genes related to efflux pumps, which are not considered a hazard when identified in metagenomes, had the uppermost relative abundances in all sampling points. We found that Lake Pastos Grandes hitherto does not show an appreciable influence by anthropogenic actions. The microbiome of Lake Pastos Grandes, including microbial distribution, the nitrogen cycle and antibiotic resistance genes, was analyzed.
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13.
  • Daga-Quisbert, Jeanett, et al. (författare)
  • Assessing water quality of a hypereutrophic alkaline urban lake and its coagulation-treated water using metagenomic analysis
  • 2024
  • Ingår i: Water, Air and Soil Pollution. - : Springer Nature. - 0049-6979 .- 1573-2932. ; 235:6
  • Tidskriftsartikel (refereegranskat)abstract
    • The study on Lake Alalay, an urban alkaline lake facing increasing pollution, focused on the impact of coagulation treatment on its water quality and microbiome. The findings revealed higher nutrient concentrations, specifically phosphate and ammonium, compared to the 2019 benchmark. The lake was found to be dominated by Proteobacteria, followed by Cyanobacteria, with Desulfobacterota thriving in areas with low dissolved oxygen. Arthrospira and Roseobacter, halo-alkali-tolerant photosynthetic bacterial genera, were detected at all sampling points. Local phosphate and oxygen concentration variations led to distinct microbial communities on the lake's surface. Despite these differences, long-term ex-situ studies on water treatment with iron chloride and poly-aluminum chloride reduced the relative abundance of Cyanobacteria, promoting the presence of Proteobacteria and Bacteroidota. However, the coagulants required higher quantities than those typically used in small shallow lakes to precipitate phosphate and improve water quality effectively. Furthermore, the large-scale assay of lake water treatment with coagulants failed to eliminate Vibrio and Acinetobacter multidrug-resistant bacteria. In conclusion, the study underscores the need to prevent the inflow of polluted water into Lake Alalay and implement effective measures to deal with the existing chemical and biological contamination.
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14.
  • Guevara-Martínez, Mónica, 1989-, et al. (författare)
  • The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
  • 2019
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; , s. 1-12
  • Tidskriftsartikel (refereegranskat)abstract
    • Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.
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15.
  • Hörnström, David, 1988- (författare)
  • Engineering and applications of surface displayed tyrosinase on Escherichia coli
  • 2019
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The rise of biotechnology has provided a toolbox to deal with major challenges related to pollution and health. Microbial enzymes constitute powerful macromolecules with applications in environmental technology, and industrial and medical production. The display of enzymes on cellular surfaces promotes external access to reactants, thereby simplifying production and cost-effectiveness of bioprocesses. To this end, a system for the surface display of the oxidative enzyme tyrosinase was developed, optimized and implemented. The first part of the thesis focused on developing tyrosinase surface-display via autotransport-based secretion in Escherichia coli. Initially, the presence of active surface-displayed tyrosinase, catalyzing the oxidation, of L-tyrosine was verified. Next, the components of the surface expression system were systematically engineered to yield an optimized tyrosinase-displaying strain with five times higher biomass-specific tyrosinase activity. The second half of the thesis applied the surface-displayed tyrosinase for wastewater treatment and biosensor development. It was found that the catalyzed oxidation of L- tyrosine resulted in the deposition of melanin at the E. coli cell surface. The resulting melanized cells were used in a membrane bioreactor for adsorption of the pharmaceutical chloroquine from an aqueous solution, with a specific binding capacity of 140 mg/g cells and allowed simple cell regeneration by lowering the pH. In a second application, the tyrosinase- display system was integrated into a genetic circuit with regulated oxidation and production of L-tyrosine in response to specific toxins. By employing the resulting cells in an electrochemical cell, the circuit generated a means to directly and selectively link biological information to an electrical output. Overall, the results in this thesis highlight the functionality of the surface expression methodology and demonstrates its versatile applicability.
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16.
  • Hörnström, David, et al. (författare)
  • Molecular optimization of autotransporter-based tyrosinase surface display
  • 2019
  • Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : ELSEVIER SCIENCE BV. - 0005-2736 .- 1879-2642. ; 1862:2, s. 486-494
  • Tidskriftsartikel (refereegranskat)abstract
    • Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia colt surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.
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17.
  • Jansen, Mickel LA, et al. (författare)
  • Saccharomyces cerevisiae strains for second-generation ethanol production : from academic exploration to industrial implementation
  • 2017
  • Ingår i: FEMS Yeast Research. - : Oxford University Press. - 1567-1364. ; 17:5
  • Tidskriftsartikel (refereegranskat)abstract
    • The recent start-up of several full-scale 'second generation' ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.
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18.
  • Kuil, Teun, et al. (författare)
  • Ethanol tolerance of Clostridium thermocellum : the role of chaotropicity, temperature and pathway thermodynamics on growth and fermentative capacity
  • 2022
  • Ingår i: Microbial Cell Factories. - : Springer Nature. - 1475-2859. ; 21:1
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundClostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures.ResultsTo separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (P-clo1313_2638::ldh* adhE) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE, in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 degrees C for ethanol concentrations >= 10 g L-1 and was able to reach higher maximum OD600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD600 and relative growth rates were higher at 45 and 50 degrees C, compared to 55 degrees C, for ethanol concentrations >= 15 g L-1. For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L-1 at 45 and 50 degrees C compared to 55 degrees C.ConclusionPositive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.
  •  
19.
  • Kuil, Teun, et al. (författare)
  • Ethanol tolerance of Clostridium thermocellum: the role of chaotropicity, temperature and pathway thermodynamics on growth and fermentative capacity
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • BACKGROUND Clostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropicinduced stress resulting in increased membrane uidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures.RESULTS To separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (Pclo1313_2638::ldh* ∆adhE) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE, in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 °C for ethanol concentrations ≥ 10 g L-1 and was able to reach higher maximum OD600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD600and relative growth rates were higher at 45 and 50 °C, compared to 55 °C, for ethanol concentrations ≥ 15 g L-1. For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L-1 at 45 and 50 °C compared to 55 °C.CONCLUSION Positive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions
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20.
  • Kuil, Teun, et al. (författare)
  • Functional Analysis of H+-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum
  • 2022
  • Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 88:4
  • Tidskriftsartikel (refereegranskat)abstract
    • The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 +/- 0.01 h(-1)) and the biomass yield by 38% (0.18 +/- 0.00 g(biomass) g(substrate)-1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk-malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PP, stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi and ATP-dependent glycolysis.
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21.
  • Kuil, Teun, et al. (författare)
  • Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and activity (Vmax > 156 U mgprotein-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and activity (14.5 U mgprotein-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis. 
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22.
  • Kuil, Teun, et al. (författare)
  • Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases
  • 2023
  • Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 743
  • Tidskriftsartikel (refereegranskat)abstract
    • The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and maximum activity (Vmax > 156 U mg-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and maximum activity (14.5 U mg-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.
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23.
  • Lindroos, Magnus, et al. (författare)
  • Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor
  • 2019
  • Ingår i: Journal of Hazardous Materials. - : ELSEVIER SCIENCE BV. - 0304-3894 .- 1873-3336. ; 365, s. 74-80
  • Tidskriftsartikel (refereegranskat)abstract
    • Environmental release and accumulation of pharmaceuticals and personal care products is a global concern in view of increased awareness of ecotoxicological effects. Adsorbent properties make the biopolymer melanin an interesting alternative to remove micropollutants from water. Recently, tyrosinase-surface-displaying Escherichia coli was shown to be an interesting self-replicating production system for melanin-covered cells for batch-wise absorption of the model pharmaceutical chloroquine. This work explores the suitability of these melanin-covered E. coli for the continuous removal of pharmaceuticals from wastewater. A continuous-flow membrane bioreactor containing melanized E. coli cells was used for adsorption of chloroquine from the influent until saturation and subsequent regeneration. At a low loading of cells (10 g/L) and high influent concentration of chloroquine (0.1 mM), chloroquine adsorbed until saturation after 26 +/- 2 treated reactor volumes (39 +/- 3 L). The average effluent concentration during the first 20 h was 0.0018 mM, corresponding to 98.2% removal. Up to 140 +/- 6 mg chloroquine bound per gram of cells following mixed homo- and heterogeneous adsorption kinetics. In situ low pH regeneration released all chloroquine without apparent capacity loss over three consecutive cycles. This shows the potential of melanized cells for treatment of conventional wastewater or highly concentrated upstream sources such as hospitals or manufacturing sites.
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24.
  • Ljungqvist, Emil E., et al. (författare)
  • A chemostat- and enzyme-constrained model-based analysis of the exceptionally high substrate consumption rate and respiratory capacity of Geobacillus sp. LC300
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    •  Geobacillus LC300 is a thermophilic bacterium displaying exceptionally fast growth and substrate utilization rates.  Despite its potential, fundamental understanding of its metabolism and fast growth is lacking. Here, the metabolism of G. sp. LC300 was studied through a combination of chemostat cultivations, proteomics, and enzyme-constrained modeling. Glucose-limited chemostat cultivations revealed an unprecedented respiratory capacity of 48 mmolO2 gDW-1 h-1 and concomitant complete respiratory metabolism until very high growth rates. Respiro-fermentative metabolism, i.e. formation of acetate in addition to respiration, only occurred at growth rates above 1.7 h-1 and above glucose uptake rates of 23 mmolglc gDW-1 h-. Proteome analysis of batch cultures showed an optimization of central carbon metabolism, with high apparent catalytic rates allowing a redistribution of protein resources to respiration and biosynthetic pathways. An enzyme-constrained genome-scale model was constructed, able to accurately simulate chemostat and batch growth. Proteome allocation analysis at varying growth rates was studied in the model, and the overflow metabolism observed at growth rates above 1.7 h-1 was explained by a limited protein supply causing a downregulation of large respiratory enzymes in favor of ATP generation through acetate formation. These insights into G. sp. LC300’s metabolic capabilities enhance our understanding of fast-growing thermophilic microorganisms, which also paves the way for more efficient biomanufacturing applications.
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25.
  • Ljungqvist, Emil E., et al. (författare)
  • Insights into the rapid metabolism of Geobacillus sp. LC300 : unraveling metabolic requirements and optimal growth conditions
  • 2024
  • Ingår i: Extremophiles. - : Springer Nature. - 1431-0651 .- 1433-4909. ; 28:1
  • Tidskriftsartikel (refereegranskat)abstract
    • This study investigated the metabolism of Geobacillus sp. LC300, a promising biorefinery host organism with high substrate utilization rates. A new defined medium was designed and tested that allows for exponential growth to elevated cell densities suitable for quantitative physiological studies. Screening of the metabolic requirements of G. sp. LC300 revealed prototrophy for all essential amino acids and most vitamins and only showed auxotrophy for vitamin B12 and biotin. The effect of temperature and pH on growth rate was investigated, adjusting the optimal growth temperature to several degrees lower than previously reported. Lastly, studies on carbon source utilization revealed a capability for fast growth on several common carbon sources, including monosaccharides, oligosaccharides, and polysaccharides, and the highest ever reported growth rate in defined medium on glucose (2.20 h(-1)) or glycerol (1.95 h(-1)). These findings provide a foundation for further exploration of G. sp. LC300's physiology and metabolic regulation, and its potential use in bioproduction processes.
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26.
  • Perez-Zabaleta, Mariel, 1987-, et al. (författare)
  • Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
  • 2019
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 103:14, s. 5627-5636
  • Tidskriftsartikel (refereegranskat)abstract
    • Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.
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27.
  • Pöschel, Laura, et al. (författare)
  • Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
  • 2023
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Nature. - 0175-7598 .- 1432-0614. ; 107:20, s. 6219-6236
  • Tidskriftsartikel (refereegranskat)abstract
    • Abstract: Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches. Key points:• Substitutions at position F35 of YciAHI changed the productivity of YciA-based release of carboxylic acid products in M. extorquens AM1 and E. coli.• YciAHI F35N and F35L are improved variants for dicarboxylic production of 2-methylsuccinic acid and mesaconic acid with M. extorquens AM1.• In vitro enzyme assays did not reveal superior properties of the optimized protein variants.
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28.
  • Schroeder, Wheaton L., et al. (författare)
  • A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis
  • 2023
  • Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 77, s. 306-322
  • Tidskriftsartikel (refereegranskat)abstract
    • Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.
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29.
  • Sjöberg, Gustav, 1991-, et al. (författare)
  • Characterization of volatile fatty acid utilization in Escherichia coli aiming for robust valorisation of food residues
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Valorisation of food residues would greatly benefit from development of robust processes that create added value compared to current feed- and biogas applications. Recent advances in membrane-bioreactor-based open mixed microbial cultures, enable robust conversion of fluctuating streams of food residues to a mixture of volatile fatty acids (VFAs). In this study, such a mixed stream of VFAs was investigated as a substrate for Escherichia coli, a well studied organism suitable for application in further conversion of the acids into compounds of higher value, and/or that are easier to separate from the aqueous medium. E. coli was cultured in batch on a VFA-rich anaerobic digest of food residues, tolerating up to 40 mM of total VFAs without any reduction in growth rate. In carbon-limited chemostats of E. coli W3110 ΔFadR on a simulated VFA mixture, the straight chain VFAs (C2-C6) in the mixture were readily consumed simultaneously. At the dilution rate 0.1 h-1, mainly acetic-, propionic- and caproic acid were consumed, while consumption of all the provided acids were observed at 0.05 h-1. Interestingly, also the branched isovaleric acid was consumed through a hitherto unknown mechanism. In total, up to 80% of the carbon supplied from VFAs was consumed by the cells, and approximately 2.7% was excreted as nucleotide precursors in the medium. These results suggest that VFAs derived from food residues are a promising substrate for E. coli.
  •  
30.
  • Sjöberg, Gustav, et al. (författare)
  • Characterization of volatile fatty-acid utilization in Escherichia coli aiming for robust valorisation of food residues
  • 2020
  • Ingår i: AMB Express. - : Springer Nature. - 2191-0855. ; 10:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Valorisation of food residues would greatly benefit from development of robust processes that create added value compared to current feed- and biogas applications. Recent advances in membrane-bioreactor-based open mixed microbial cultures, enable robust conversion of fluctuating streams of food residues to a mixture of volatile fatty acids (VFAs). In this study, such a mixed stream of VFAs was investigated as a substrate for Escherichia coli, a well-studied organism suitable for application in further conversion of the acids into compounds of higher value, and/or that are easier to separate from the aqueous medium. E. coli was cultured in batch on a VFA-rich anaerobic digest of food residues, tolerating up to 40 mM of total VFAs without any reduction in growth rate. In carbon-limited chemostats of E. coli W3110 ΔFadR on a simulated VFA mixture, the straight-chain VFAs (C2-C6) in the mixture were readily consumed simultaneously. At a dilution rate of 0.1 h−1, mainly acetic-, propionic- and caproic acid were consumed, while consumption of all the provided acids were observed at 0.05 h−1. Interestingly, also the branched isovaleric acid was consumed through a hitherto unknown mechanism. In total, up to 80% of the carbon from the supplied VFAs was consumed by the cells, and approximately 2.7% was excreted as nucleotide precursors in the medium. These results suggest that VFAs derived from food residues are a promising substrate for E. coli.
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31.
  • Sjöberg, Gustav, 1991- (författare)
  • Engineering short-chain carboxylic-acid metabolism in the model microorganism Escherichia coli
  • 2020
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The ever-increasing concern about carbon dioxide emissions has created an urgent need to develop alternative methods to cheaply and renewably produce materials, chemicals and fuels. The biorefinery is uniquely suited to deliver these products from sustainable biomass. However, cheaply and efficiently converting the dispersed, heterogenous and recalcitrant biomass to useful products requires further technical development. To address some of these challenges, the aim of this thesis was to investigate methods to improve the economic viability of the microbial biorefinery by evaluating short chain carboxylic acids as substrates (volatile fatty acids) and products ((R)-3-hydroxybutyrate, 3HB).Initially, two renewable and cheap sources of carbon were investigated as substrates for E. coli. It was determined that E. coli is a suitable microorganism for valorization of volatile fatty acids derived from food waste. Also, it was shown that lignocellulosic sugars with a composition based on a hydrolysate of wheat straw can be converted to 3HB in E. coli with similar yields and productivities as from pure glucose. To improve the yield of the model product 3HB, and thereby the potential gross profit, substrate depletion was used as a strategy throughout the thesis to control bioprocesses. Specifically, nutrient depletion was shown to decouple growth from 3HB production in nitrogen and phosphorous depleted batches, increasing the yield of 3HB. To further improve 3HB production, metabolic engineering was used to improve the availability of NADPH. Additionally, the bacterial artificial chromosome (BAC) was investigated as a robust single-copy vector for metabolic engineering in E. coli. The expression of a large operon from the BAC was shown to be comparable to chromosomal expression. Then, the specific growth rate, productivity and yield of 3HB producing strains was increased by expression of the 3HB production pathway from the BAC instead of a multi-copy plasmid. Finally, the BAC was shown to be a useful tool for the optimization of enzyme expression levels in metabolic pathways. While directly beneficial for 3HB production, the methods and strategies employed in this thesis are broadly applicable to increase the economic viability of microbial biorefineries.
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32.
  • Sjöberg, Gustav, et al. (författare)
  • Evaluation of enzyme-constrained genome-scale model through metabolic engineering of anaerobic co-production of 2,3-butanediol and glycerol by Saccharomyces cerevisiae
  • 2024
  • Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 82, s. 49-59
  • Tidskriftsartikel (refereegranskat)abstract
    • Enzyme-constrained genome-scale models (ecGEMs) have potential to predict phenotypes in a variety of conditions, such as growth rates or carbon sources. This study investigated if ecGEMs can guide metabolic engineering efforts to swap anaerobic redox-neutral ATP-providing pathways in yeast from alcoholic fermentation to equimolar co-production of 2,3-butanediol and glycerol. With proven pathways and low product toxicity, the ecGEM solution space aligned well with observed phenotypes. Since this catabolic pathway provides only one-third of the ATP of alcoholic fermentation (2/3 versus 2 ATP per glucose), the ecGEM predicted a growth decrease from 0.36 h−1 in the reference to 0.175 h−1 in the engineered strain. However, this <3-fold decrease would require the specific glucose consumption rate to increase. Surprisingly, after the pathway swap the engineered strain immediately grew at 0.15 h−1 with a glucose consumption rate of 29 mmol (g CDW)−1 h−1, which was indeed higher than reference (23 mmol (g CDW)−1 h−1) and one of the highest reported for S. cerevisiae. The accompanying 2,3-butanediol- (15.8 mmol (g CDW)−1 h−1) and glycerol (19.6 mmol (g CDW)−1 h−1) production rates were close to predicted values. Proteomics confirmed that this increased consumption rate was facilitated by enzyme reallocation from especially ribosomes (from 25.5 to 18.5 %) towards glycolysis (from 28.7 to 43.5 %). Subsequently, 200 generations of sequential transfer did not improve growth of the engineered strain, showing the use of ecGEMs in predicting opportunity space for laboratory evolution. The observations in this study illustrate both the current potential, as well as future improvements, of ecGEMs as a tool for both metabolic engineering and laboratory evolution.
  •  
33.
  • Sjöberg, Gustav, et al. (författare)
  • Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3 hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization. 
  •  
34.
  • Sjöberg, Gustav, et al. (författare)
  • Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
  • 2019
  • Ingår i: Journal of Biotechnology. - : ELSEVIER. - 0168-1656 .- 1873-4863. ; 305, s. 43-50
  • Tidskriftsartikel (refereegranskat)abstract
    • In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3-hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization.
  •  
35.
  • Suits, Kristjan, et al. (författare)
  • Overview of the (Smart) Stormwater Management around the Baltic Sea
  • 2023
  • Ingår i: Water. - : MDPI AG. - 2073-4441. ; 15:8, s. 1623-
  • Forskningsöversikt (refereegranskat)abstract
    • In this review paper, we investigate the management of the quality of stormwater in the Baltic Sea region. Current stormwater management practices, standards, and legislation do not accurately depict stormwater quality, resulting in an underestimation of its environmental impact. The digitalization and harmonization of stormwater management through the implementation of e-monitoring (online or continuous monitoring) allow for the collection of data. This data can be used to improve stormwater quality and quantity management, thereby reducing the environmental harm induced by anthropogenic activities. Based on the literature review, supporting tables and matrices are proposed to assist decision-makers and other interested parties in developing and implementing "smart" stormwater management solutions. In this article, we demonstrate that such systems can enhance stormwater management and system performance by leveraging data-driven operation and maintenance. Another advantage of the approach is that it contributes to a healthier urban environment and ecosystem well-being.
  •  
36.
  • VanArsdale, Eric, et al. (författare)
  • A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks
  • 2020
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:5, s. 1117-1128
  • Tidskriftsartikel (refereegranskat)abstract
    • There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples and cells, one can both electronically obtain a redox "portrait" of a biological system and, conversely, program gene expression. Here, we have created a cell-based synthetic biology-electrochemical axis in which engineered cells process molecular cues, producing an output that can be directly recorded via electronics-but without the need for added redox mediators. The process is robust; two key components must act together to provide a valid signal. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPAquinone, which are both redox active. "Catalytic" transducer cells provide for signal-mediated surface expression of tyrosinase. Additionally, "reagent" transducer cells synthesize and export tyrosine, a substrate for tyrosinase. In cocultures, this system enables real-time electrochemical transduction of cell activating molecular cues. To demonstrate, we eavesdrop on quorum sensing signaling molecules that are secreted by Pseudomonas aeruginosa, N-(3-oxododecanoyl)-l-homoserine lactone and pyocyanin.
  •  
37.
  • Yayo, Johannes, et al. (författare)
  • Laboratory Evolution and Reverse Engineering of Clostridium thermocellum for Growth on Glucose and Fructose
  • 2021
  • Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 87:9
  • Tidskriftsartikel (refereegranskat)abstract
    • The native ability of Clostridium thermocellum to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of C. thermocellum, as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of C. thermocellum on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 +/- 0.00 and 0.18 +/- 0.00 g(biomass) g(substrate)(-1), respectively, compared to 0.15 +/- 0.01 g(biomass) g(substrate)(-1) for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, while wild type showed 42 +/- 4 h on glucose and >80 h on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 h with a growth rate of 0.11 +/- 0.01 h(-1) and resulted in immediate growth on glucose at 0.24 +/- 0.01 h(-1). Additional introduction of a G-to-V mutation at position 148 in cbpA resulted in immediate growth on fructose at 0.32 +/- 0.03 h(-1). These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings. IMPORTANCE C. thermocellum is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that C. thermocellum can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing, and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with C. thermocellum using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of C. thermocellum can be engineered to (i) reduce glucose accumulation, (ii) study cellodextrin transport systems in vivo, (iii) allow experiments at >120 g liter(-1) soluble substrate concentration, or (iv) reduce costs for labeling studies.
  •  
38.
  • Yayo, Johannes, et al. (författare)
  • The role of redox-cofactor regeneration and ammonium assimilation in secretion of amino acids as byproducts of Clostridium thermocellum
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Clostridium thermocellum is a cellulolytic thermophile considered for consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated by gene deletions in ammonium-regulated NADPH-supplying and -consuming pathways and physiological characterization in cellobiose- or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased 46-60%, suggesting that secretion of these amino acids balances NADPH surplus from the malate shunt. Unchanged amino acid secretion in Δppdk falsified a previous hypothesis on ammonium-regulated PEP-to-pyruvate flux redistribution. Possible involvement of another NADPH-supplier, namely NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, deletion of glutamate synthase (gogat) in ammonium assimilation resulted in upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, which is supported by product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of NADH needed for ethanol formation. This suggests that metabolic engineering strategies on simplifying redox metabolism and ammonium assimilation can contribute to increased ethanol yields.Importance. Improving the ethanol yield of C. thermocellum is important for industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated, by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data relevant for training genome-scale metabolic models and improving the validity of their predictions, especially considering the reduced degree-of-freedom in redox metabolism of the strains generated here. In addition, this study advances fundamental understanding on mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid development of C. thermocellum for sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment. 
  •  
39.
  • Yayo, Johannes, et al. (författare)
  • The Roles of Nicotinamide Adenine Dinucleotide Phosphate Reoxidation and Ammonium Assimilation in the Secretion of Amino Acids as Byproducts of Clostridium thermocellum
  • 2023
  • Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 89:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or a redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Δppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields.
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