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  • 1.
    Andersson, Sofia
    et al.
    KTH, Skolan för bioteknologi (BIO), Miljömikrobiologi.
    Dalhammar, Gunnel
    KTH, Skolan för bioteknologi (BIO), Miljömikrobiologi.
    Land, Carl Johan
    KTH, Skolan för bioteknologi (BIO), Miljömikrobiologi.
    Kuttuva Rajarao, Gunaratna
    KTH, Skolan för bioteknologi (BIO), Miljömikrobiologi.
    Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans2009Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 82, nr 3, s. 535-543Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3-37%), nucleic acids (9-50%), and carbohydrates (3-21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.

  • 2. Bostrom, M.
    et al.
    Larsson, Gen
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Process design for recombinant protein production based on the promoter, P-malK2004Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 66, nr 2, s. 200-208Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    P-malK is induced through activation of MalT, by the formation of maltotriose and cyclic adenosine monophosphate ( cAMP). The possibility to influence endogenous inducer levels is used to vary the production rates in specifically designed production protocols. Induction based on a batch process protocol on maltose gives low production rates, as the result of a lack of cAMP, which is shown to be of major importance to fully induce this promoter. Two mechanisms are thus used to influence the levels of maltotriose and/or cAMP formation: ( 1) catabolite derepression achieved from low glucose concentration and ( 2) catabolite derepression/inducer exclusion from diauxic growth on glucose/maltose. Fed-batch processes based on limited amounts of glucose result in product accumulation of up to 10% of the total protein. Depending on the feed of limiting glucose, different production profiles are developed. The initial increase in the production rate is due to maltotriose formation from endogenous glycogen degradation while, later in the process, production can be further supported by elevated levels of cAMP, provided the feed rate is sufficiently low. The introduction of maltose after a preceding fed-batch process on glucose can be efficiently used to produce maltotriose in combination with cAMP formation in the event of catabolite derepression. This leads to higher production rates and a further increase in product accumulation of up to 30% of the total protein. The diauxic growth phase resulting from the shift in carbon source can be shortened and even avoided by the design of the preceding feed-rate of glucose. It is postulated that proper design of the inoculum and initial phases of production can reduce basal levels of product formation. With this promoter, the production rate can be as high as 65 units mg(-1) h(-1) and the time to reach a maximal production rate can be designed to take up to 8 h. Furthermore, the duration of the production rate can be as long as 7 h.

  • 3.
    Boström, Maria
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Markland, Katrin
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Sandén, Anna Maria
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Hedhammar, My
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO), Centra, Centrum för Bioprocessteknik, CBioPT.
    Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli2005Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 68, nr 1, s. 82-90Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

  • 4.
    Elegir, Graziano
    et al.
    Stazione Sperimentale Carta Cartoni e Paste per Carta.
    Bussini, Daniele
    Stazione Sperimentale Carta Cartoni e Paste per Carta.
    Antonsson, Stefan
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Träkemi och massateknologi.
    Lindström, Mikael E.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Träkemi och massateknologi.
    Zoia, Luca
    Univ Milan, Dipartimento Sci Ambiente & Terr.
    Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor2007Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 77, nr 4, s. 809-817Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2'azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.

  • 5. Eriksson, M.
    et al.
    Dalhammar, Gunnel
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Borg-Karlson, Anna-Karin
    KTH, Tidigare Institutioner                               , Kemi.
    Biological degradation of selected hydrocarbons in an old PAH/creosote contaminated soil from a gas work site2000Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 53, nr 5, s. 619-626Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An old PAH/creosote contaminated soil (total similar to 300 mu g PAH/g soil) from a former gas work site in Stockholm, Sweden, has been treated at 20 degrees C with the addition of various nutrients and inoculated with bacteria (isolated from the soil) to enhance the degradation of selected hydrocarbons. Microcosm studies showed that the soil consisted of two contaminant fractions: one available, easily degraded fraction and a strongly sorbed, recalcitrant one. The bioavailable fraction, monitored by headspace solid phase microextraction, contained aromatics with up to three rings, and these were degraded within 20 days down to non-detectable levels (ng PAH/g soil) by both the indigenous bacteria and the externally inoculated samples. The nutrient additives were: a minimal medium (Bushnell-Haas), nitrate, nitrite, potting soil (Anglamark, Sweden), sterile water and aeration with Bushnell-Haas medium. After 30 days treatment most of the sorbed fractions were still present in the soil. Stirring or mechanical mixing of the soil slurries had the greatest effect on degradation, indicating that the substances were too strongly sorbed for the microorganisms. When stirring the choice of nutrient seemed less important. For the non-stirred samples the addition of nitrate with the bacterial inoculum showed the best degradation, compared to the other non-stirred samples. At the end of the experiments, accumulations of metabolites/degradation products, such as 9H-fluorenone, 4-hydroxy-9H-fluorenone, 9,10-phenanthrenedione and 4H-cyclopenta[def]phenanthrenedione were detected. The metabolite 4-hydroxy-9H-fluorenone increased by several orders of magnitude during the biological treatments. Microbial activity in the soil was measured by oxygen consumption and carbon dioxide production.

  • 6. Ghebremichael, K. A.
    et al.
    Gunaratna, K. R.
    Dalhammar, Gunnel
    KTH, Skolan för bioteknologi (BIO), Miljömikrobiologi.
    Single-step ion exchange purification of the coagulant protein from Moringa oleifera seed2006Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 70, nr 5, s. 526-532Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The coagulant protein from Moringa oleifera (MO) seed was purified using a single-step batch ion exchange (IEX) method. Adsorption and elution parameters were optimized. Impact of the purification on the reduction of organic and nutrient release to the water was studied. The matrix was equilibrated using ammonium acetate buffer, and the optimum ionic strength of NaCl for elution was 0.6 M. The time for adsorption equilibrium was between 90 and 120 min. Maximum adsorption capacity of the matrix, estimated with the Langmuir model, was 68 mg protein/g adsorbent. The purified protein does not release organic and nutrient loads to the water, which are the main concerns of the crude extract. This work suggests that a readily scalable single-step IEX purification method can be used to produce the coagulant protein and it can be carried out with locally available facilities. This will promote the use of MO in large water treatment plants and other industries.

  • 7.
    Hassan, Noor
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Geiger, Barbara
    Gandini, Rosaria
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Patel, Bharat K. C.
    Kittl, Roman
    Haltrich, Dietmar
    Nguyen, Thu-Ha
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Tan, Tien Chye
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis2016Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, nr 8, s. 3533-3543Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining beta-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a beta-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k(cat), but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k(cat)). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with similar to 10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

  • 8.
    Hassan, Noor
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Nguyen, Thu-Ha
    Intanon, Montira
    Kori, Lokesh D.
    Patel, Bharat K. C.
    Haltrich, Dietmar
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO).
    Tan, Tien Chye
    KTH, Skolan för bioteknologi (BIO).
    Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis2015Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 99, nr 4, s. 1731-1744Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific beta-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-D-Galp-(1 -> 3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

  • 9.
    Karlsson, S.
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Polymerteknologi.
    Banhidi, Z.G.
    Albertsson, A-C
    Identification and characterization of alkali-tolerant clostridia isolated from biodeteriorated casein-containing building materials1988Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 28, nr 3, s. 305-310Artikkel i tidsskrift (Fagfellevurdert)
  • 10.
    Kroll, Jens
    et al.
    Westfälische Wilhelms-Universität Münster, Germany.
    Klinter, Stefan
    Westfälische Wilhelms-Universität Münster, Germany.
    Steinbüchel, Alexander
    Westfälische Wilhelms-Universität Münster, Germany.
    A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium2011Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 89, nr 3, s. 593-604Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Introduction: Hitherto the production of the biopolymer cyanophycin (CGP) using recombinant Escherichia coli strains and cheap mineral salts medium yielded only trace amounts of CGP (<0.5%, w/w) of the cell dry matter (CDM). This was probably due to the instability of the plasmids encoding the cyanophycin synthetase. Material and methods: In this study, we developed an anabolism-based media-dependent plasmid addiction system (PAS) to enhance plasmid stability, and we established a process based on a modified mineral salts medium yielding a CGP content of 42% (w/w) at the maximum without the addition of amino acids to the medium for the first time. This PAS is based on different lysine biosynthesis pathways and consists of two components: (1) a knockout of the chromosomal dapE disrupts the native succinylase pathway in E. coli and (2) the complementation by the plasmid-encoded artificial aminotransferase pathway mediated by the dapL gene from Synechocystis sp. PCC 6308, which allows the synthesis of the essential lysine precursor L,L-2,6-diaminopimelate. In addition, this plasmid also harbors cphAC595S, an engineered cyanophycin synthetase gene responsible for CGP production. Results: Cultivation experiments in Erlenmeyer flask and also in bioreactors in mineral salts medium without antibiotics revealed an at least 4.5-fold enhanced production of CGP in comparison to control cultivations without PAS. Discussion: Fermentation experiments with culture volume of up to 400 l yielded a maximum of 18% CGP (w/w) and a final cell density of 15.2 g CDM/l. Lactose was used constantly as an effective inducer and carbon source. Thus, we present a convenient option to produce CGP with E. coli at a technical scale without the need to add antibiotics or amino acids using the mineral salts medium designed in this study.

  • 11.
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO).
    Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli2002Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 60, s. 654-664Artikkel i tidsskrift (Fagfellevurdert)
  • 12.
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO).
    Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress induced promoters uspA and uspB2003Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 61, s. 495-501Artikkel i tidsskrift (Fagfellevurdert)
  • 13.
    Leta, Seyoum
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Assefa, F.
    Gumaelius, Lena
    KTH, Tidigare Institutioner, Bioteknologi.
    Dalhammar, Gunnel
    KTH, Tidigare Institutioner, Bioteknologi.
    Biological nitrogen and organic matter removal from tannery wastewater in pilot plant operations in Ethiopia2004Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 66, nr 3, s. 333-339Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The objective of this study was to set-up a pilot plant and to evaluate its effectiveness for biological nitrogen and organic matter removal from tannery wastewater in Ethiopia. A pilot wastewater treatment plant consisting of a predenitrification-nitrification process was constructed and operated for 6 months. This was fed with a raw tannery wastewater obtained from the Modjo Tannery located 70 km south of the capital, Addis Ababa. Up to 98% total nitrogen and chemical oxygen demand, and 95% ammonium nitrogen removal efficiencies were achieved in the system. The average effluent ammonium nitrogen ranged from 8.4 mg l(-1) to 86.0 mg l(-1), whereas the average effluent for nitrate nitrogen ranged from 2.9 mg l(-1) to 4.4 mg l(-1). The average values of denitrification and nitrification rates determined by nitrate and ammonium uptake rates (NUR and AUR) were 8.0 mg NO3-N [g volatile suspended solids (VSS)](-1) h(-1) and 5.4 mg NH4-N (g VSS)(-1) h(-1), respectively, demonstrating that the treatment processes of the pilot plant were effective. Further studies of the effect of chromium III on AUR showed 50% inhibition at a concentration of 85 mg l(-1), indicating that this metal was not causing process inhibition during performance operations. Thus, the predenitrification-nitrification process was found to be efficient for simultaneous removal of nitrogen and organic substrates from tannery wastewaters.

  • 14.
    Lindskog, Eva
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Svensson, Ingrid
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells2006Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 71, nr 4, s. 444-449Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

  • 15.
    Löfblom, John
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Rosenstein, Ralf
    Nguyen, Minh-Thu
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Götz, Friedrich
    Staphylococcus carnosus: from starter culture to protein engineering platform2017Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, nr 23-24, s. 8293-8307Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and "food grade" species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

  • 16. Mergulhao, F. J. M.
    et al.
    Monteiro, G. A.
    Larsson, Gen
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Sanden, A. M.
    Farewell, A.
    Nystrom, T.
    Cabral, J. M. S.
    Taipa, M. A.
    Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB2003Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 61, nr 06-maj, s. 495-501Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (1560 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.

  • 17.
    Nielsen, Jens
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Chalmers, Sweden.
    Archer, John
    Essack, Magbubah
    Bajic, Vladimir B.
    Gojobori, Takashi
    Mijakovic, Ivan
    Building a bio-based industry in the Middle East through harnessing the potential of the Red Sea biodiversity2017Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, nr 12, s. 4837-4851Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    The incentive for developing microbial cell factories for production of fuels and chemicals comes from the ability of microbes to deliver these valuable compounds at a reduced cost and with a smaller environmental impact compared to the analogous chemical synthesis. Another crucial advantage of microbes is their great biological diversity, which offers a much larger "catalog" of molecules than the one obtainable by chemical synthesis. Adaptation to different environments is one of the important drives behind microbial diversity. We argue that the Red Sea, which is a rather unique marine niche, represents a remarkable source of biodiversity that can be geared towards economical and sustainable bioproduction processes in the local area and can be competitive in the international bio-based economy. Recent bioprospecting studies, conducted by the King Abdullah University of Science and Technology, have established important leads on the Red Sea biological potential, with newly isolated strains of Bacilli and Cyanobacteria. We argue that these two groups of local organisms are currently most promising in terms of developing cell factories, due to their ability to operate in saline conditions, thus reducing the cost of desalination and sterilization. The ability of Cyanobacteria to perform photosynthesis can be fully exploited in this particular environment with one of the highest levels of irradiation on the planet. We highlight the importance of new experimental and in silico methodologies needed to overcome the hurdles of developing efficient cell factories from the Red Sea isolates.

  • 18.
    Perez-Zabaleta, Mariel
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Guevara-Martínez, Mónica
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Gustavsson, Martin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Quillaguamán, Jorge
    Larsson, Gen
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    van Maris, Antonius J. A.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH).
    Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation2019Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 103, nr 14, s. 5627-5636Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.

  • 19. Pisanelli, I.
    et al.
    Kujawa, M.
    Gschnitzer, D.
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. University of Natural Resources and Applied Life Sciences (BOKU), Vienna, Austria .
    Seiboth, B.
    Peterbauer, C.
    Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme2010Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 86, nr 2, s. 599-606Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pyranose dehydrogenase (PDH) is a flavin-dependant sugar oxidoreductase found in the family Agaricaceae, basidiomycetes that degrade lignocellulose-rich forest litter, and is catalytically related to the fungal enzymes pyranose 2-oxidase and cellobiose dehydrogenase. It has broad substrate specificity and displays similar activities with most sugar constituents of lignocellulose including disaccharides and oligosaccharides, a number of (substituted) quinones, and metal ions are suitable electron acceptors rather than molecular oxygen. In contrast to pyranose 2-oxidase and cellobiose dehydrogenase, which oxidize regioselectively at C-2 and C-1, respectively, PDH is capable of oxidation on C-1 to C-4 as well as double oxidations, depending on the nature of the substrate. This makes it a very interesting enzyme for biocatalytic applications, as many of the reaction products are otherwise unaccessible by chemical or enzymatic means. PDH was characterized in detail in a limited number of fungi, and the first encoding genes were isolated only recently. We report here, for the first time, the heterologous expression of one of these genes, encoding the major PDH protein in Agaricus meleagris, in the filamentous fungi Aspergillus nidulans, and Aspergillus niger.

  • 20. Sanden, B.
    et al.
    Dalhammar, Gunnel
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Application of an amperometric immunosensor for the enumeration of Nitrobacter in activated sludge2000Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 54, nr 3, s. 413-417Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments.

  • 21. Shokri, Atefeh
    et al.
    Veide, Andres
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Larsson, Gen
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    RelA1 gene control of Escherichia coli lipid structure and cell performace during glucose limited fed-batch conditions2006Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 73, nr 2, s. 464-473Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids. Opposite to the wild type, the cardiolipin-depleted membrane is more fragile with respect to sonication and osmotic chock, at severe limitation, and results in extensive foaming during the process. Protein leakage and cell lysis is, however, lower in the mutant most likely due to the increased amounts of saturated fatty acids, which might be a possible strategy to overcome the reduced amounts of membrane-strengthening cardiolipin. The membrane potential of the outer surface is negative, however less negative for the mutant. This was supported by aqueous two-phase extraction experiments which, furthermore indicated a difference in outer surface hydrofobicity. These findings suggest that the relA1 gene has a defined, but ppGpp-independent, role in cells with a slowly decreasing metabolism of glucose to control the membrane morphology.

  • 22.
    Steffen-Munsberg, Fabian
    et al.
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Greifswald University.
    Matzel, Philipp
    Sowa, Miriam A.
    Berglund, Per
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Bornscheuer, Uwe T.
    Höhne, Matthias
    Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases2016Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, s. 4511-4521Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme’s function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information implied that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved ‘flipping’ arginine, which enables dual substrate recognition by its side chain flexibility in other ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases.

  • 23.
    Svensson, Ingrid
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Calles, Karin
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Lindskog, Eva
    KTH, Skolan för bioteknologi (BIO).
    Henriksson, Hongbin
    KTH, Skolan för bioteknologi (BIO).
    Eriksson, Ulrika
    KTH, Skolan för bioteknologi (BIO).
    Häggström, Lena
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells2005Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 69, nr 1, s. 92-98Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

  • 24.
    Svensson, Marie
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Svensson, Ingrid
    KTH, Skolan för bioteknologi (BIO).
    Enfors, Sven-Olof
    KTH, Skolan för bioteknologi (BIO).
    Osmotic stability of the cell membrane of Escherichia coli from a temperature-limited fed-batch process2005Inngår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 67, nr 3, s. 345-350Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The temperature-limited fed-batch (TLFB) process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. In Escherichia coli cultures, it has been proven to prevent an extensive release of endotoxins, i.e. lipopolysaccharides, that occurs in the glucose-limited fed-batch (GLFB) processes at specific growth rates below 0.1 h(-1). The TLFB and the GLFB process were compared to each other when applied to produce the periplasmic, constitutively expressed, enzyme beta-lactamase. The extraction of the enzyme was performed by osmotic shock. A higher production of beta-lactamase was achieved with the TLFB technique while no difference in the endotoxin release was found during the extraction procedure. Furthermore, it was found that growth at declining temperature, generated by the TLFB technique, gradually stabilizes the cytoplasmic membrane, resulting in a significantly increased product quality in the extract from the TLFB cultures in the osmotic shock treatment.

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