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  • 1.
    Azeem, Muhammad
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry. Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan.
    Barba Aliaga, Marina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Borg-Karlson, Anna-Karin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Organic chemistry. Division of Organic Chemistry, Institute of Technology, Tartu University, Tartu 50411, Estonia.
    Terenius, O.
    Broberg, A.
    Rajarao, Gunaratna Kuttuva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Heterobasidion-growth inhibiting Bacillus subtilis A18 exhibits medium- and age-dependent production of lipopeptides2019In: Microbiology Research, ISSN 0944-5013, E-ISSN 1618-0623, Vol. 223-225, p. 129-136Article in journal (Refereed)
    Abstract [en]

    Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps.

  • 2.
    Björlenius, Berndt
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Pharmaceuticals – improved removal from municipal wastewater and their occurrence in the Baltic Sea2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 3.
    Björlenius, Berndt
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Ripszám, M.
    Haglund, P.
    Lindberg, R. H.
    Tysklind, M.
    Fick, J.
    Pharmaceutical residues are widespread in Baltic Sea coastal and offshore waters – Screening for pharmaceuticals and modelling of environmental concentrations of carbamazepine2018In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 633, p. 1496-1509Article in journal (Refereed)
    Abstract [en]

    The consumption of pharmaceuticals worldwide coupled with modest removal efficiencies of sewage treatment plants have resulted in the presence of pharmaceuticals in aquatic systems globally. In this study, we investigated the environmental concentrations of a selection of 93 pharmaceuticals in 43 locations in the Baltic Sea and Skagerrak. The Baltic Sea is vulnerable to anthropogenic activities due to a long turnover time and a sensitive ecosystem in the brackish water. Thirty-nine of 93 pharmaceuticals were detected in at least one sample, with concentrations ranging between 0.01 and 80 ng/L. One of the pharmaceuticals investigated, the anti-epileptic drug carbamazepine, was widespread in coastal and offshore seawaters (present in 37 of 43 samples). In order to predict concentrations of pharmaceuticals in the sub-basins of the Baltic Sea, a mass balance-based grey box model was set up and the persistent, widely used carbamazepine was selected as the model substance. The model was based on hydrological and meteorological sub-basin characteristics, removal data from smaller watersheds and wastewater treatment plants, and statistics relating to population, consumption and excretion rate of carbamazepine in humans. The grey box model predicted average environmental concentrations of carbamazepine in sub-basins with no significant difference from the measured concentrations, amounting to 0.57-3.2 ng/L depending on sub-basin location. In the Baltic Sea, the removal rate of carbamazepine in seawater was estimated to be 6.2 10(-9) s(-1) based on a calculated half-life time of 3.5 years at 10 degrees C, which demonstrates the long response time of the environment to measures phasing out persistent or slowly degradable substances such as carbamazepine. Sampling, analysis and grey box modelling were all valuable in describing the presence and removal of carbamazepine in the Baltic Sea.

  • 4. Bracher, J. M.
    et al.
    Verhoeven, M. D.
    Wisselink, H. W.
    Crimi, B.
    Nijland, J. G.
    Driessen, A. J. M.
    Klaassen, P.
    Van Maris, Antonius J.A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft University of Technology, Netherlands.
    Daran, J. -MG.
    Pronk, J. T.
    The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae2018In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, no 1, article id 63Article in journal (Refereed)
    Abstract [en]

    Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (K m = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10-3 and 1.8 g L-1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L-1 l-arabinose and 20 g L-1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

  • 5. Bracher, Jasmine M.
    et al.
    Martinez-Rodriguez, Oscar A.
    Dekker, Wijb JC
    Verhoeven, Maarten D.
    van Maris, Antonius
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Pronk, Jack T.
    Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains2019In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 19, no 1, article id foy104Article in journal (Refereed)
    Abstract [en]

    Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7-10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L-1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N-2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose.

  • 6.
    Brechmann, Nils Arnold
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Eriksson, Per-Olov
    Eriksson, Kristofer
    Oscarsson, Sven
    Buijs, Jos
    Shokri, Atefeh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Hjälm, Göran
    Chotteau, Véronique
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.
    Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture2019In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033Article in journal (Refereed)
    Abstract [en]

    High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development

    of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified

    CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding

    capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an

    IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step

    from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were

    obtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scale

    purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,

    where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein

    A capture step, the mAb purity was similar to the one obtained by column chromatography, while

    the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead

    mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient

    single step, which directly connected the culture to the downstream process without cell clarification.

    Purification of mAb directly from non-clarified cell broth without cell separation can provide

    significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.

  • 7.
    Brännström, Sara
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Finnveden, Maja
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Johansson, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Itaconate based polyesters: Selectivity and performance of esterification catalysts2018In: European Polymer Journal, ISSN 0014-3057, E-ISSN 1873-1945, Vol. 103, p. 370-377Article in journal (Refereed)
    Abstract [en]

    The performance of different esterification catalysts was studied for the use in synthesis of renewable polyesters from dimethyl itaconate (DMI), dimethyl succinate (DMS) and 1,4-butanediol (BD). Itaconic acid and derivatives such as DMI are interesting monomers because of their multiple functionalities and previous work has shown great potential. However, the multiple functionalities also pose challenges to avoid side reactions such as thermally initiated, premature, radical crosslinking and/or isomerization of the 1,1-disubstituted unsaturation. Additionally, the two carboxylic acids have inherently different reactivity. One key factor to control reactions with IA is to understand the performance of different catalysts. In this study, six esterification catalysts were investigated; immobilized Candida antarctica lipase B (CalB), titanium(IV)butoxide (Ti(OBu)4), p-toluenesulfonic acid (pTSA), sulfuric acid (H2SO4), 1,8-diazabicycloundec-7-ene (DBU), and 1,5,7-triazabicyclodec-5-ene (TBD). CalB and Ti(OBu)4 were selected for further characterization with appreciable differences in catalytic activity and selectivity towards DMI. CalB was the most effective catalysts and was applied at 60 °C while Ti(OBu)4 required 160 °C for a reasonable reaction rate. CalB was selective towards DMS and the non-conjugated side of DMI, resulting in polyesters with itaconate-residues mainly located at the chain ends, while Ti(OBu)4 showed low selectivity, resulting in polyesters with more randomly incorporated itaconate units. Thermal analysis of the polyesters showed that the CalB-catalyzed polyesters were semi-crystalline, whereas the Ti(OBu)4-catalyzed polyesters were amorphous, affirming the difference in monomer sequence. The polyester resins were crosslinked by UV-initiated free radical polymerization and the material properties were evaluated and showed that the crosslinked materials had similar material properties. The films from the polyester resins catalyzed by CalB were furthermore completely free from discoloration whereas the film made from the polyester resins catalyzed with Ti(OBu)4 had a yellow color, caused by the catalyst. Thus, it has been shown that CalB can be used to attain sustainable unsaturated polyesters resins for coating applications, exhibiting equally good properties as resins obtained from traditional metal-catalysis.

  • 8.
    Brännström, Sara
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Finnveden, Maja
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Razza, Nicolo
    Politecn Torino, Dept Appl Sci & Technol, Corso Duca Abruzzi 24, I-10129 Turin, Italy..
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Sangermano, Marco
    Politecn Torino, Dept Appl Sci & Technol, Corso Duca Abruzzi 24, I-10129 Turin, Italy..
    Johansson, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Tailoring Thermo-Mechanical Properties of Cationically UV-Cured Systems by a Rational Design of Vinyl Ether Ester Oligomers using Enzyme Catalysis2018In: Macromolecular Chemistry and Physics, ISSN 1022-1352, E-ISSN 1521-3935, Vol. 219, no 21, article id 1800335Article in journal (Refereed)
    Abstract [en]

    There is a demand for new sustainable polymeric materials. Vinyl ethers are, in this context, attractive oligomers since they polymerize fast, are non-toxic, and can be polymerized under ambient conditions. The availability of vinyl ether oligomers is, however, currently limited due to difficulties in synthesizing them without using tedious synthesis routes. This work presents the synthesis of a series of vinyl ether ester oligomers using enzyme catalysis under solvent-free conditions and the subsequent photoinduced cationic polymerization to form polymer thermosets with T(g)s ranging from -10 to 100 degrees C. The whole process is very efficient as the synthesis takes less than 1 h with no need for purification and the crosslinking is complete within 2 min.

  • 9.
    Chen, Shan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Stability and inactivation mechanisms of two transaminases2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the past decades, more and more enzymes are employed as biocatalysts in industrial processes because of their advantages, such as high efficiency, substrate selectivity and stereoselectivity. Among them, amine transaminases (ATAs) are pyridoxal 5’-phosphate (PLP) dependent enzymes. ATAs have gained attention for their excellent performance in chiral amine synthesis, and their broad substrate acceptance. However, the low operational stability of amine transaminases still limits their application in industry.

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) has been selected for further investigation for its relatively low operational stability. Co-solvents and various additives have been added to the enzyme storage solution to improve its storage stability at various temperatures. Co-lyophilization of Cv-ATA with surfactants has been applied to improve its enzymatic activity in neat organic solvents.

    As a PLP-dependent dimeric enzyme, the Cv-ATA is not primarily inactivated due to tertiary structural changes. Instead, both dimer dissociation and PLP release may affect the enzyme stability. Therefore, the inactivation pathway of the Cv-ATA during operational conditions was explored. The unfolding of the enzyme was detected by several methods, and the detection of fluorescence intensity spectrum of tryptophan is extensively applied for its high sensitivity. The phosphate group of PLP can be coordinated into the phosphate group binding cup, which may influence the enzyme structural stability. Therefore, the effect of both PLP and inorganic phosphate ions (present in phosphate buffer) on the enzyme stability was explored.

    The amine transaminase from Vibrio fluvialis (Vf-ATA) is another amine transaminase, which catalyses the same biocatalytic reaction and has a similar substrate scope as Cv-ATA. However, there is still a lack of data on the stability of Vf-ATA. Consequently, the operational stability of Vf-ATA in various environments was studied.

  • 10.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum2018In: Molecular Catalysis, ISSN 2468-8231, Vol. 446, p. 115-123Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5’-phosphate (PLP)dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer.The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.

  • 11.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Characterization of the operational stability of a transaminase from Vibrio fluvialisManuscript (preprint) (Other academic)
  • 12.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Inactivation pathway underlying the operational instability of an amine transaminase from Chromobacterium violaceumManuscript (preprint) (Other academic)
  • 13.
    Chen, Shan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Characterization of the stability of Vibrio fluvialis JS17 amine transaminase2018In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 282, p. 10-17Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Vibrio fluvialis (Vf-ATA) is an attractive enzyme with applications within Biocatalysis for the preparation of chiral amines. Various catalytic properties of Vf-ATA have been investigated, but a biophysical characterization of its stability has been lacking. Today, the industrial application of Vf-ATA is limited by its low operational stability. In order to enhance the knowledge regarding the structural stability of ATAs, general characterizations of different ATAs are required. In this work, the stability of Vf-ATA was explored. First, the affinity between enzyme and pyridoxal-5’-phosphate (PLP) (KD value of 7.9 ΌM) was determined. Addition of PLP to enzyme preparations significantly improved the enzyme thermal stability by preventing enzyme unfolding. With the aim to understand if this was due to the PLP phosphate group coordination into the phosphate group binding cup, the effect of phosphate buffer on the enzyme stability was compared to HEPES buffer. Low concentrations of phosphate buffer showed a positive effect on the enzyme initial activity, while higher phosphate buffer concentrations prevented cofactor dissociation. Additionally, the effects of various amine or ketone substrates on the enzyme stability were explored. All tested amines caused a concentration dependent enzyme inactivation, while the corresponding ketones showed no or stabilizing effects. The enzyme inactivation due to the presence of amine can be connected to the formation of PMP, which forms in the presence of amines in the absence of ketone. Since PMP is not covalently bound to the enzyme, it could readily leave the enzyme upon formation. Exploring the different stability effects of cofactor, substrates, additives and buffer system on ATAs seems to be important in order to understand and improve the general performance of ATAs.

  • 14.
    Dorau, Robin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gorbe, Tamas
    Humble, Maria Svedendahl
    Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 4, p. 338-346Article in journal (Refereed)
    Abstract [en]

    Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36%), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

  • 15.
    Eklöf, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO).
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 82009In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, no 4, p. 1029-1036Article in journal (Refereed)
  • 16.
    Feng, Zhaoxuan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Odelius, Karin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Rajarao, Gunaratna Kuttuva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Hakkarainen, Minna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Polymer Technology.
    Microwave carbonized cellulose for trace pharmaceutical adsorption2018In: Chemical Engineering Journal, ISSN 1385-8947, E-ISSN 1873-3212, Vol. 346, p. 557-566Article in journal (Refereed)
    Abstract [en]

    A promising sustainable strategy to valorize cellulose to high-value adsorbents for trace pharmaceuticals, like diclofenac sodium (DCF), in the water is demonstrated. Carbon nanospheres (CN) as the DCF adsorbent were derived from cellulose through a one-pot microwave-assisted hydrothermal carbonization method. CN exhibited efficient DCF removal (100% removal of 0.001 mg/mL DCF in 30 s and 59% removal of 0.01 mg/mL DCF in 1 h). The adsorption kinetics and isotherm data were well-fitted with the pseudo-second-order kinetic model and Langmuir model, respectively. The adsorption process was endothermic and spontaneous as confirmed by the thermodynamic parameters. Multiple characterization techniques including SEM/EDS, FTIR, FTIR-imaging and zeta potential were applied to qualitatively investigate the adsorption process. π-π stacking and hydrogen bonding were proposed as the dominant adsorption interactions. CN also demonstrated effective adsorption capacity towards three other commonly-detected contaminants in the wastewater including ketoprofen (KP), benzophenone (BZP), and diphenylamine (DPA), each bearing partial structural similarity with DCF. The affinity of the contaminants towards CN followed the order DPA > BZP > DCF > KP, which could be explained by the different configurations and chemical units. It was speculated that for DCF and KP, the steric hindrance and electrostatic repulsion produced by dissociated carboxyl groups can impede the adsorption process as compared to DPA and BZP. This methodology could offer further insights into the drug adsorption on the cellulose-derived carbon adsorbents and the use of bioderived carbons for treatment of wastewaters contaminated with pharmaceuticals.

  • 17. Fernández-Niño, Miguel
    et al.
    Pulido, Sergio
    Stefanoska, Despina
    Pérez, Camilo
    González-Ramos, Daniel
    van Maris, Antonius JA
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Marchal, Kathleen
    Nevoigt, Elke
    Swinnen, Steve
    Identification of novel genes involved in acetic acid tolerance of Saccharomyces cerevisiae using pooled-segregant RNA sequencing2018In: FEMS yeast research, Vol. 18, no 8Article in journal (Refereed)
    Abstract [en]

    Acetic acid tolerance of the yeast Saccharomyces cerevisiae is manifested in several quantifiable parameters, of which the duration of the latency phase is one of the most studied. It has been shown recently that the latter parameter is mostly determined by a fraction of cells within the population that resumes proliferation upon exposure to acetic acid. The aim of the current study was to identify genetic determinants of the difference in this parameter between the highly tolerant strain MUCL 11987-9 and the laboratory strain CEN. PK113-7D. To this end, a combination of genetic mapping and pooled-segregant RNA sequencing was applied as a new approach. The genetic mapping data revealed four loci with a strong linkage to strain MUCL 11987-9, each containing still a large number of genes making the identification of the causal ones by traditional methods a laborious task. The genes were therefore prioritized by pooled-segregant RNA sequencing, which resulted in the identification of six genes within the identified loci showing differential expression. The relevance of the prioritized genes for the phenotype was verified by reciprocal hemizygosity analysis. Our data revealed the genes ESP1 and MET22 as two, so far unknown, genetic determinants of the size of the fraction of cells resuming proliferation upon exposure to acetic acid.

  • 18.
    Finnveden, Maja
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Brännström, Sara
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Johansson, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Novel sustainable synthesis of vinyl ether ester building blocks, directly from carboxylic acids and the corresponding hydroxyl vinyl ether, and their photopolymerization2018In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 8, no 44, p. 24716-24723Article in journal (Refereed)
    Abstract [en]

    Increased environmental awareness has led to a demand for sustainable, bio-based materials. Consequently, the development of new benign synthesis pathways utilizing a minimum of reaction steps and available bio-based building blocks is needed. In the present study, vinyl ether alcohols and functional carboxylic acids were used to synthesize bifunctional vinyl ether esters using the immobilized enzyme Candida antarctica lipase B as a catalyst. Vinyl ethers are attractive alternatives to (meth)acrylates due to low allergenic hazards, low toxicity, and fast polymerization; however, difficult synthesis limits the monomer availability. The synthesis was performed in one-pot and the described method was successful within a broad temperature range (22-90 degrees C) and in various organic solvents as well as in the bulk. The synthesis of different vinyl ether esters reached high conversions (above 90%) after less than 1 h and products were purified by removing the enzyme by filtration using only small amounts of acetone. This approach is a straightforward route to reach monomers with multiple types of functionalities that can be used as different photo-curable thermoset resins. In this work, this was demonstrated by polymerizing the monomers with cationic and radical UV-polymerization. By changing the functional carboxylic acids, the architecture of the final polymer can be tailored, herein demonstrated by two examples. In the developed versatile method, carboxylic acids can be used directly as acyl donors, constituting a more sustainable alternative to the carboxylic acid derivatives used today.

  • 19.
    Guevara-Martínez, Mónica
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Perez-Zabaleta, Mariel
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Quillaguamán, Jorge
    Faculty of Science and Technology, Center of Biotechnology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
    Larsson, Gen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Superseded Departments (pre-2005), Biotechnology.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli2019In: Applied Microbiology and Biotechnology, p. 1-12Article in journal (Refereed)
    Abstract [en]

    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.

  • 20.
    Gullfot, Fredrika
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    von Schantz, Laura
    Karlsson, Eva Nordberg
    Ohlin, Mats
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module2010In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 78, no 3, p. 785-789Article in journal (Refereed)
  • 21. Hakkaart, Xavier DV
    et al.
    Pronk, Jack T
    van Maris, Antonius JA
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    A Simulator-Assisted Workshop for Teaching Chemostat Cultivation in Academic Classes on Microbial Physiology2017In: Journal of Microbiology & Biology Education, Vol. 18, no 3Article in journal (Refereed)
    Abstract [en]

    Understanding microbial growth and metabolism is a key learning objective of microbiology and biotechnology courses, essential for understanding microbial ecology, microbial biotechnology and medical microbiology. Chemostat cultivation, a key research tool in microbial physiology that enables quantitative analysis of growth and metabolism under tightly defined conditions, provides a powerful platform to teach key features of microbial growth and metabolism. Substrate-limited chemostat cultivation can be mathematically described by four equations. These encompass mass balances for biomass and substrate, an empirical relation that describes distribution of consumed substrate over growth and maintenance energy requirements (Pirt equation), and a Monod-type equation that describes the relation between substrate concentration and substrate-consumption rate. The authors felt that the abstract nature of these mathematical equations and a lack of visualization contributed to a suboptimal operative understanding of quantitative microbial physiology among students who followed their Microbial Physiology B.Sc. courses. The studio-classroom workshop presented here was developed to improve student understanding of quantitative physiology by a set of question-guided simulations. Simulations are run on Chemostatus, a specially developed MATLAB-based program, which visualizes key parameters of simulated chemostat cultures as they proceed from dynamic growth conditions to steady state. In practice, the workshop stimulated active discussion between students and with their teachers. Moreover, its introduction coincided with increased average exam scores for questions on quantitative microbial physiology. The workshop can be easily implemented in formal microbial physiology courses or used by individuals seeking to test and improve their understanding of quantitative microbial physiology and/or chemostat cultivation.

  • 22.
    Hallberg, Martin
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Leitner, C.
    Haltrich, D.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 341, no 3, p. 781-796Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8 Angstrom resolution to R and R-free values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three U helices. The homotetramer buries a large internal cavity of roughly 15,000 Angstrom(3), from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.

  • 23. Holmquist, M.
    et al.
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Berglund, P.
    Mid Sweden University.
    Clausen, I. G.
    Patkar, S.
    Svendsen, A.
    Hult, K.
    Lipases from Rhizomucor miehei and Humicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity1993In: Journal of Protein Chemistry, ISSN 02778033 (ISSN), Vol. 12, no 6, p. 749-757Article in journal (Refereed)
    Abstract [en]

    The homologous lipases from Rhizomucor miehei and Humicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed the S- enantiomer of 1-heptyl 2-methyldecanoate (R. miehei: E(S) = 8.5; H. lanuginosa: E(S) = 10.5), but the R-enantiomer of phenyl 2-methyldecanoate (E(R) = 2.9). Chemical arginine specific modification of the R. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E(R) = 2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E(S) = 2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E(S) = 1.9) and increased the enantioselectivity with the aromatic ester (E(R) = 4.4) as substrates. The mutation, Glu 87 Ala, in the lid of the H. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E(S) = 17.4) and a decreased enantioselectivity with the phenyl ester (E(R) = 2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.

  • 24.
    Hörnström, David
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Molecular optimization of autotransporter-based tyrosinase surface display2019In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1862, no 2, p. 486-494Article in journal (Refereed)
    Abstract [en]

    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.

  • 25.
    Jahic, Mehmedalija
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Gustavsson, Malin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Jansen, Ann-Katrin
    Martinelle, Mats
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology. KTH, Superseded Departments (pre-2005), Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes2003In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 102, no 1, p. 45-53Article in journal (Refereed)
    Abstract [en]

    A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degreesC during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.

  • 26. Juergens, H.
    et al.
    Niemeijer, M.
    Jennings-Antipov, L. D.
    Mans, R.
    Morel, J.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Pronk, J. T.
    Gardner, T. S.
    Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics2018In: Scientific Data, E-ISSN 2052-4463, Vol. 5, article id 180195Article in journal (Refereed)
    Abstract [en]

    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.

  • 27.
    Land, Henrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Campillo-Brocal, Jonatan C.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svedendahl Humble, Maria
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase – An Evaluation of the Proline Rule as a Method for Enzyme Stabilization2019In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 10, p. 1297-1304Article in journal (Refereed)
    Abstract [en]

    Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. We hereby present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60°C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3°C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the Proline rule was deemed successful at increasing the stability of this enzyme.

  • 28.
    Lu, Ke
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Yang, Liyun
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Ergonomics.
    Abtahi, F.
    Lindecrantz, Kaj
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Rödby, K.
    Seoane, F.
    Wearable cardiorespiratory monitoring system for unobtrusive free-living energy expenditure tracking2019In: IFMBE Proceedings, Springer, 2019, no 1, p. 433-437Conference paper (Refereed)
    Abstract [en]

    In this work, we want to introduce combined heart rate and respiration monitoring for more accurate energy expenditure tracking on free-living subjects. We have developed a wearable cardiorespiratory monitoring system with unobtrusive heart rate measurement and ventilation estimation function for this purpose. The system is based on a garment with integrated textile electrodes for one-lead electrocardiogram and impedance pneumography measurements. A pilot experiment has been performed to prove the concept and to evaluate the characteristics of heart rate and ventilation estimated by our system in relation to energy expenditure. In the experiment, ventilation shows a better linearity in relation to the energy expenditure at the low intensity region than heart rate. Based on these characteristics, a model combining heart rate and ventilation for energy expenditure estimation is proposed which shows a significantly lower estimation error than the heart rate only model.

  • 29. Marques, W. L.
    et al.
    Mans, R.
    Henderson, R. K.
    Marella, E. R.
    Horst, J. T.
    Hulster, E. D.
    Poolman, B.
    Daran, J. -M
    Pronk, J. T.
    Gombert, A. K.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, p. 121-133Article in journal (Refereed)
    Abstract [en]

    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.

  • 30. Marques, Wesley Leoricy
    et al.
    van der Woude, Lara Ninon
    Luttik, Marijke AH
    van den Broek, Marcel
    Nijenhuis, Janine Margriet
    Pronk, Jack T
    van Maris, Antonius JA
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Department of Biotechnology, Delft University of Technology, Delft, Netherlands.
    Mans, Robert
    Gombert, Andreas K
    Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter2018In: Yeast, Vol. 35, no 12, p. 639-652Article in journal (Refereed)
    Abstract [en]

    Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (mu = 0.19 h(-1) and mu = 0.08 h(-1), respectively). All five mutants displayed sucrose-induced proton uptake (13-50 mu mol H+ (g biomass)(-1) min(-1)). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1(I209F C265F G326C)) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h(-1)) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.

  • 31.
    Master, Emma
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Rudsander, Ulla
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Zhou, Welin
    Henriksson, Hongbin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Divne, Christina
    KTH, Superseded Departments (pre-2005), Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Denman, Stuart
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Wilson, David
    Teeri, Tuula
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 31, p. 10080-10089Article in journal (Refereed)
    Abstract [en]

    PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30degreesC and pH 6.0, the k(cat) for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (k(cat)/K-m) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.

  • 32.
    Neubauer, P.
    et al.
    Tech Univ Berlin, Dept Biotechnol, Berlin, Germany..
    Golson, R.
    BioSilta Oy, Oulu, Finland..
    Neubauer, A.
    BioSilta Oy, Oulu, Finland..
    Ukkonen, K.
    BioSilta Oy, Oulu, Finland..
    Krause, M.
    BioSilta Oy, Oulu, Finland..
    Tegel, Hanna
    KTH, School of Biotechnology (BIO).
    Ottosson, J.
    KTH, School of Biotechnology (BIO).
    Larsen, M. Wittrup
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Vasala, A.
    BioSilta Oy, Oulu, Finland..
    Using EnBase (TM) to enhance recombinant protein production2009In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 25, p. S190-S190Article in journal (Other academic)
  • 33.
    Ostman, Marcus
    et al.
    Umea Univ, Dept Chem, SE-90187 Umea, Sweden..
    Björlenius, Berndt
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Fick, Jerker
    Umea Univ, Dept Chem, SE-90187 Umea, Sweden..
    Tysklind, Mats
    Umea Univ, Dept Chem, SE-90187 Umea, Sweden..
    Effect of full-scale ozonation and pilot-scale granular activated carbon on the removal of biocides, antimycotics and antibiotics in a sewage treatment plant2019In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 649, p. 1117-1123Article in journal (Refereed)
    Abstract [en]

    Several micropollutants show low removal efficiencies in conventional sewage treatment plants, and therefore enter the aquatic environment. To reduce the levels of micropollutants in sewage effluent, and thereby the effects on biota, a number of extra treatment steps are currently being evaluated. Two such techniques are ozonation and adsorption onto activated carbon. In this study, we investigated the efficiency of Sweden's first full-scale ozonation treatment plant at removing a number of antibiotics, antimycotics and biocides. The effect of adding granular activated carbon (GAC) on a pilot scale and pilot-scale ozonation were also evaluated. The conventional treatment (13,000 PE) with the add-on of full-scale ozonation (0.55 g O-3/g Total organic carbon (TOC)) was able to remove most of the studied compounds (>90%), except for benzotriazoles and fluconazole (<50%). Adsorption on GAC on a pilot scale showed a higher removal efficiency than ozonation (>80% for all studied compounds). Three types of GAC were evaluated and shown to have different removal efficiencies. In particular, the GAC with the smallest particle sizes exhibited the highest removal efficiency. The results demonstrate that it is important to select an appropriate type of carbon to achieve the removal goal for specific target compounds.

  • 34. Papapetridis, Ioannis
    et al.
    Goudriaan, Maaike
    Vitali, María Vázquez
    Keijzer, Nikita A
    Broek, Marcel
    van Maris, Antonius
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft University of Technology, The Netherlands.
    Pronk, Jack T.
    Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield2018In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, no 1, article id 17Article in journal (Refereed)
    Abstract [en]

    Background: Reduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wildtype S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD(+) balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield. Results: Multi-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD(+)-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield. Conclusions: The metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first-and second-generation bioethanol production.

  • 35.
    Papapetridis, Ioannis
    et al.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Verhoeven, Maarten D.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Wiersma, Sanne J.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Goudriaan, Maaike
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Pronk, Jack T.
    Delft Univ Technol, Dept Biotechnol, Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae2018In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 18, no 6, article id foy056Article in journal (Refereed)
    Abstract [en]

    Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose-xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83(G673T) allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L-1 glucose and 10 g L-1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.

  • 36. Pohl, J.
    et al.
    Björlenius, Berndt
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Brodin, T.
    Carlsson, G.
    Fick, J.
    Larsson, D. G. J.
    Norrgren, L.
    Örn, S.
    Effects of ozonated sewage effluent on reproduction and behavioral endpoints in zebrafish (Danio rerio)2018In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 200, p. 93-101Article in journal (Refereed)
    Abstract [en]

    Pharmaceutical residues and other micro-contaminants may enter aquatic environments through effluent from sewage treatment plants (STPs) and could cause adverse effects in wild fish. One strategy to alleviate this situation is to improve wastewater treatment by ozonation. To test the effectiveness of full-scale wastewater effluent ozonation at a Swedish municipal STP, the added removal efficiency was measured for 105 pharmaceuticals. In addition, gene expression, reproductive and behavioral endpoints were analyzed in zebrafish (Danio rerio) exposed on-site over 21 days to ozonated or non-ozonated effluents as well as to tap water. Ozone treatment (7 g O3/m3) removed pharmaceuticals by an average efficiency of 77% in addition to the conventional treatment, leaving 11 screened pharmaceuticals above detection limits. Differences in biological responses of the exposure treatments were recorded in gene expression, reproduction and behavior. Hepatic vitellogenin gene expression was higher in male zebrafish exposed to the ozonated effluent compared to the non-ozonated effluent and tap water treatments. The reproductive success was higher in fish exposed to ozonated effluent compared to non-ozonated effluent and to tap water. The behavioral measurements showed that fish exposed to the ozonated STP effluent were less active in swimming the first minute after placed in a novel vessel. Ozonation is a capable method for removing pharmaceuticals in effluents. However, its implementation should be thoroughly evaluated for any potential biological impact. Future research is needed for uncovering the factors which produced the in vivo responses in fish.

  • 37.
    Quehenberger, Julian
    et al.
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Reichenbach, Tom
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Baumann, Niklas
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Rettenbacher, Lukas
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Spadiut, Oliver
    Research Division Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Faculty of Technical Chemistry, TU Wien, Vienna, 1060, Austria.
    Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum.2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 1, article id E185Article in journal (Refereed)
    Abstract [en]

    While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

  • 38.
    Reichenbach, Tom
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Kalyani, Dayanand
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gandini, Rosaria
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Svartström, Olov
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Aspeborg, Henrik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Structural and biochemical characterization of the Cutibacterium acnes exo-β-1,4-mannosidase that targets the N-glycan core of host glycoproteins.2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 9, article id e0204703Article in journal (Refereed)
    Abstract [en]

    Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host's immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-β-1,4-mannosidase with activity on the β-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway.

  • 39.
    Ruggieri, Federica
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. SARom Biostruct AB, SE-22381 Lund, Sweden.
    van Langen, Luuk M.
    Logan, Derek T.
    Walse, Björn
    Berglund, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Transaminase-catalyzed racemization with potential for dynamic kinetic resolutions2018In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 10, no 21, p. 5026-5032Article in journal (Refereed)
    Abstract [en]

    Dynamic kinetic resolution (DKR) reactions in which a stereoselective enzyme and a racemization step are coupled in one‐pot would represent powerful tools for the production of enantiopure amines through enantioconvergence of racemates. The exploitation of DKR strategies is currently hampered by the lack of effective, enzyme‐compatible and scalable racemization strategies for amines. In the present work, the proof of concept of a fully biocatalytic method for amine racemization is presented. Both enantiomers of the model compound 1‐methyl‐3‐phenylpropylamine could be racemized in water and at room temperature using a couple of wild‐type, non‐proprietary, enantiocomplementary amine transaminases and a minimum amount of pyruvate/alanine as a co‐substrate couple. The biocatalytic simultaneous parallel cascade reaction presented here poses itself as a customizable amine racemization system with potential for the chemical industry in competition with traditional transition‐metal catalysis.

  • 40.
    Schwarz, Hubert
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Zhan, Caijuan
    KTH, School of Engineering Sciences (SCI). KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Malm, Magdalena
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Field, Raymond
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Turner, Richard
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Sellick, Christopher
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Varley, Paul
    Biopharmaceutical Development, MedImmune, Cambridge, UK.
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Chotteau, Véronique
    KTH, School of Engineering Sciences (SCI).
    Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant ErythropoietinManuscript (preprint) (Other academic)
    Abstract [en]

    Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines.

    In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 x 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes. 

  • 41.
    Sjöberg, Gustav
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Guevara-Martínez, Mónica
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Metabolic engineering applications of the Escherichia coli bacterial artificial chromosomeManuscript (preprint) (Other academic)
    Abstract [en]

    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. 

  • 42.
    Torron, Susana
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Johansson, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Malmström, Eva
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Fogelström, Linda
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Hult, Karl
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Martinelle, Mats
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Telechelic polyesters and polycarbonates prepared by enzymatic catalysis2017In: Handbook of Telechelic Polyesters, Polycarbonates, and Polyethers, Pan Stanford Publishing Pte. Ltd. , 2017, p. 29-64Chapter in book (Other academic)
    Abstract [en]

    The majority of polyesters and polycarbonates are traditionally synthesized through conventional metal-based catalysis. Although effective, due to environmental concerns, their substitution for other more environmentally friendly alternatives has received increasing interest during the last decades. The search for catalytic systems that also allow milder reaction conditions has been intensified, owing to 30the unwanted side reactions, for example, backbone scissoring, that the metal-based catalysts may cause [1]. In this context, enzymes are anticipated as suitable alternatives [2,3,4,5,6,7,-8]. 

  • 43.
    Valk, Laura C.
    et al.
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    Frank, Jeroen
    Radboud Univ Nijmegen, Soehngen Inst Anaerob Microbiol, Nijmegen, Netherlands..
    de la Torre-Cortes, Pilar
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    van 't Hof, Max
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    Pronk, Jack T.
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    van Loosdrecht, Mark C. M.
    Delft Univ Technol, Dept Biotechnol, Delft, Netherlands..
    Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures: Combined Fermentation and Acetogenesis by the Dominant sp nov "Candidatus Galacturonibacter soehngenii"2018In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 84, no 18, article id UNSP e01370-18Article in journal (Refereed)
    Abstract [en]

    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.

  • 44.
    van Maris, Antonius Jeroen Adriaan
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Pronk, Jacobus Thomas
    Medina, Victor Gabriel Guadalupe
    Wisselink, Hendrik Wouter
    Recombinant yeast expressing rubisco and phosphoribulokinase2018Patent (Other (popular science, discussion, etc.))
  • 45.
    Verhoeven, Maarten D.
    et al.
    Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Bracher, Jasmine M.
    Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Nijland, Jeroen G.
    Univ Groningen, Groningen Biomol Sci & Biotechnol Inst GBB, Dept Mol Microbiol, Nijenborgh 7, NL-9747 AG Groningen, Netherlands..
    Bouwknegt, Jonna
    Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Daran, Jean-Marc G.
    Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Driessen, Arnold J. M.
    Univ Groningen, Groningen Biomol Sci & Biotechnol Inst GBB, Dept Mol Microbiol, Nijenborgh 7, NL-9747 AG Groningen, Netherlands..
    van Maris, Antonius J. A.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherland.
    Pronk, Jack T.
    Delft Univ Technol, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft, Netherlands..
    Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive L-arabinose uptake2018In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 18, no 6, article id foy062Article in journal (Refereed)
    Abstract [en]

    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.

  • 46. Verhoeven, Maarten D
    et al.
    de Valk, Sophie C
    Daran, Jean-Marc G
    van Maris, Antonius
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology. Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, Delft, 2629, Netherlands.
    Pronk, Jack T
    Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains2018In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 18, no 8, article id foy075Article in journal (Refereed)
    Abstract [en]

    D-glucose, D-xylose and L-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three 'specialist' Saccharomyces cerevisiae strains. A D-glucose- and L-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented D-xylose in the presence of 20 g L-1 of D-glucose and L-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose non-fermenting laboratory strain, rapidly and simultaneously converted D-glucose and L-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved 'generalist' strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1 D-glucose, 10 g L-1 D-xylose and 5 g L-1 L-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.

  • 47. Wang, H.
    et al.
    Sikora, P.
    Rutgersson, C.
    Lindh, M.
    Brodin, T.
    Björlenius, Berndt
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Larsson, D. G. J.
    Norder, H.
    Differential removal of human pathogenic viruses from sewage by conventional and ozone treatments2018In: International journal of hygiene and environmental health (Print), ISSN 1438-4639, E-ISSN 1618-131X, Vol. 221, no 3, p. 479-488Article in journal (Refereed)
    Abstract [en]

    Sewage contains a mixed ecosystem of diverse sets of microorganisms, including human pathogenic viruses. Little is known about how conventional as well as advanced treatments of sewage, such as ozonation, reduce the environmental spread of viruses. Analyses for viruses were therefore conducted for three weeks in influent, after conventional treatment, after additional ozonation, and after passing an open dam system at a full-scale treatment plant in Knivsta, Sweden. Viruses were concentrated by adsorption to a positively charged filter, from which they were eluted and pelleted by ultracentrifugation, with a recovery of about 10%. Ion Torrent sequencing was used to analyze influent, leading to the identification of at least 327 viral species, most of which belonged to 25 families with some having unclear classification. Real-time PCR was used to test for 21 human-related viruses in inlet, conventionally treated, and ozone-treated sewage and outlet waters. The viruses identified in influent and further analyzed were adenovirus, norovirus, sapovirus, parechovirus, hepatitis E virus, astrovirus, pecovirus, picobirnavirus, parvovirus, and gokushovirus. Conventional treatment reduced viral concentrations by one to four log10, with the exception of adenovirus and parvovirus, for which the removal was less efficient. Ozone treatment led to a further reduction by one to two log10, but less for adenovirus. This study showed that the amount of all viruses was reduced by conventional sewage treatment. Further ozonation reduced the amounts of several viruses to undetectable levels, indicating that this is a promising technique for reducing the transmission of many pathogenic human viruses. 

  • 48.
    Xu, Xin
    et al.
    Macquarie Univ, Dept Mol Sci, Sydney, NSW 2109, Australia.
    Williams, Thomas C
    Macquarie Univ, Dept Mol Sci, Sydney, NSW 2109, Australia ; CSIRO Synthet Biol Future Sci Platform, Canberra, ACT 2601, Australia.
    Divne, Christina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Pretorius, Isak S
    Macquarie Univ, Dept Mol Sci, Sydney, NSW 2109, Australia.
    Paulsen, Ian T
    Macquarie Univ, Dept Mol Sci, Sydney, NSW 2109, Australia.
    Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance.2019In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 12, article id 97Article in journal (Refereed)
    Abstract [en]

    Background: Propionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. However, PA inhibits yeast growth at concentrations well below the titers typically required for a commercial bioprocess.

    Results: Adaptive laboratory evolution (ALE) with PA concentrations ranging from 15 to 45 mM enabled the isolation of yeast strains with more than threefold improved tolerance to PA. Through whole genome sequencing and CRISPR-Cas9-mediated reverse engineering, unique mutations in TRK1, which encodes a high-affinity potassium transporter, were revealed as the cause of increased propionic acid tolerance. Potassium supplementation growth assays showed that mutated TRK1 alleles and extracellular potassium supplementation not only conferred tolerance to PA stress but also to multiple organic acids.

    Conclusion: Our study has demonstrated the use of ALE as a powerful tool to improve yeast tolerance to PA. Potassium transport and maintenance is not only critical in yeast tolerance to PA but also boosts tolerance to multiple organic acids. These results demonstrate high-affinity potassium transport as a new principle for improving organic acid tolerance in strain engineering.

  • 49.
    Yang, Liyun
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Ergonomics. IMM, Karolinska Institutet.
    Lu, Ke
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Forsman, Mikael
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Ergonomics. IMM, Karolinska Institutet.
    Lindecrantz, Kaj
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Seoane, Fernando
    Ekblom, Örjan
    GIH, The Swedish School of Sport and Health.
    Eklund, Jörgen
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Ergonomics.
    Evaluation of physiological workload assessment methods using heart rate and accelerometry for a smart wearable system2019In: Ergonomics, ISSN 0014-0139, E-ISSN 1366-5847Article in journal (Other academic)
    Abstract [en]

    Work metabolism (WM) can be accurately estimated by oxygen consumption (VO2), which is commonly assessed by heart rate (HR) in field studies. However, the VO2–HR relationship is influenced by individual capacity and activity characteristics. The purpose of this study was to evaluate three models for estimating WM compared with indirect calorimetry, during simulated work activities. The techniques were: the HR-Flex model; HR branched model, combining HR with hip-worn accelerometers (ACC); and HR + arm-leg ACC model, combining HR with wrist- and thigh-worn ACC. Twelve participants performed five simulated work activities and three submaximal tests. The HR + arm-leg ACC model had the overall best performance with limits of agreement (LoA) of −3.94 and 2.00 mL/min/kg, while the HR-Flex model had −5.01 and 5.36 mL/min/kg and the branched model, −6.71 and 1.52 mL/min/kg. In conclusion, the HR + arm-leg ACC model should, when feasible, be preferred in wearable systems for WM estimation.

  • 50.
    Zhan, Caijuan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Hydrodynamics considerations in cells systems from ocean flow to perfusion cultivation process2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Microorganisms and animal cells are grown surrounded by fluid, which is providing them with nutrients and removing their waste products. In nature and industry processes, cells/microbes can be subject to aggressive environments, such as turbulent flow or shear flow. Hydrodynamics force generated in these flows can affect the distribution of cells/microbes and even lead to cell damage. Understanding the mechanism and exploring the effect of hydrodynamic force in these environments could make the prediction of cells’ hydrodynamic response more systematic. In pharmaceutical industry, perfusion process is recognized as an attractive option for biologics production due to its high productivity. However, there are still some challenges and limitations for further process improvement due to lack of information of cell response to hydrodynamic force and nutrients. In both cases, hydrodynamics plays an important role and similar tool can be used to achieve a deeper understanding of these processes. This thesis is mainly aiming to elucidate the influence of hydrodynamic forces on microorganisms or cells in nature and during bioprocesses. In particular, shear stress in a natural environment and in a bioreactor operated in perfusion mode is studied.

    This work mainly investigates hydrodynamics in nature and bioprocess including three flow cases. The first study investigates the effect of turbulence on marine life by performing direct numerical simulations (DNS) of motile micro-organisms in isotropic homogeneous turbulence. The clustering level of micro-organisms with one preferential swimming direction (e.g. gyrotaxis) is examined. The second study uses Computation Fluid Dynamics (CFD) to simulate the fluid flow inside a Wave bioreactor bag. The phenomenon of mixing, oxygen transfer rate and shear stress in nine different operating conditions of rocking speeds and angles are discussed. In the third study, the cellular response to shear force including growth and metabolism in a cell retention device such as hollow fiber filters during a perfusion process is analyzed. Theoretical calculations and experiment validation is performed to compare two filtration modes, tangential flow filtration (TFF) or alternating tangential flow filtration (ATF). Further optimizations regarding mixing and feeding are performed in a screening scale of in a perfusion system of stirred tank bioreactor with cell separation device.

    The main findings can be summarized as that spherical gyrotaxis swimmers show significant clustering, whereas prolate swimmers remain more uniformly distributed due to their large sensitivity to the local shear. These results could explain how pure hydrodynamic effects can alter the ecology of micro-organisms for instance by varying shape and their preferential orientation (paper I). The simulations of Wave bioreactors show that the mixing and shear stress increase with the rocking angle but that increasing rocking speeds are not systematically associated with increasing mixing and shear stress. A resonance phenomenon is responsible for the fact that the lowest studied rocking speed generates the highest fluid velocity, mixing and shear stress (paper II). Theoretical velocity profile-based calculations suggested a lower shear stress for ATF by a factor 0.637 compared to TFF. This is experimentally validated by cultures of HEK (human embryonic kidney) 293 cells subjected to shear stress by a perfusion system that affects growth and metabolism using these cell separation devices (paper III). Thanks to optimization of mixing and oxygen transfer in a screening system for perfusion process, very high cell densities above 100 x 106 cells/mL of mammalian cells were achieved (paper IV).

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