| 1. |
- Lindström, Cecilia, et al.
(författare)
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Effects of Pediococcus parvulus 2.6 and its exopolysaccharide on plasma cholesterol levels and inflammatory markers in mice
- 2012
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 2:66
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Tidskriftsartikel (refereegranskat)abstract
- Intake of dietary fibres may reduce the prevalence of physiological risk factors of the metabolic syndrome, such as high plasma lipid levels and low-grade inflammatory state. Dietary fibres are usually of plant origin however microbial exopolysaccharides (EPSs) have analogue structures that could potentially exert similar physiological effects. Pediococcus parvulus 2.6 (Pd 2.6) excretes a ropy EPS and has previously shown probiotic potential. The aim of this work was to evaluate physiological effects of Pd 2.6 and its EPS in vivo. The live Pd 2.6 (both the ropy and non-ropy isogenic variant) and its purified EPS were fed to hypercholesterolemic LDL-receptor deficient mice for 6 weeks to investigate their effects on cholesterol levels and the inflammatory tone of the animals. Both variants of Pd 2.6 survived passage through the mouse gut fulfilling an important criterion of probiotics. The ability to produce EPS was conferring an advantage to survival (faecal recovery of 3.7 (1.9-8.7) vs. 0.21 (0.14-0.34) *108 CFU, P < 0.001, median and 25th and 75th percentiles). The ropy Pd 2.6 decreased the levels of soluble vascular cell adhesion molecule-1 compared to the EPS alone (591 ± 14 vs. 646 ± 13 ng/ml, P < 0.05). An increase in liver weight in mice fed the purified EPS was observed, but with no change in liver lipids. No changes in blood lipids were detected in any group. Further the EPS induced growth of the caecal tissue and increased the amount of caecal content showing bulking properties like that of a dietary fibre.
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| 2. |
- Olofsson, Kim, et al.
(författare)
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A mutated xylose reductase increases bioethanol production more than a glucose/xylose facilitator in simultaneous fermentation and co-fermentation of wheat straw.
- 2011
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 1:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: Genetically engineered Saccharomyces cerevisiae strains are able to ferment xylose present in lignocellulosic biomass. However, better xylose fermenting strains are required to reach complete xylose uptake in simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic hydrolyzates. In the current study, haploid Saccharomyces cerevisiae strains expressing a heterologous xylose pathway including either the native xylose reductase (XR) from P. stipitis, a mutated variant of XR (mXR) with altered co-factor preference, a glucose/xylose facilitator (Gxf1) from Candida intermedia or both mXR and Gxf1 were assessed in SSCF of acid-pretreated non-detoxified wheat straw. The xylose conversion in SSCF was doubled with the S. cerevisiae strain expressing mXR compared to the isogenic strain expressing the native XR, converting 76% and 38%, respectively. The xylitol yield was less than half using mXR in comparison with the native variant. As a result of this, the ethanol yield increased from 0.33 to 0.39 g g-1 when the native XR was replaced by mXR. In contrast, the expression of Gxf1 only slightly increased the xylose uptake, and did not increase the ethanol production. The results suggest that ethanolic xylose fermentation under SSCF conditions is controlled primarily by the XR activity and to a much lesser extent by xylose transport.
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| 3. |
- Sánchez I Nogué, Violeta, et al.
(författare)
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Isolation and characterization of a resident tolerant Saccharomyces cerevisiae strain from a spent sulfite liquor fermentation plant
- 2012
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 2:1, s. 68-
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Tidskriftsartikel (refereegranskat)abstract
- Spent Sulfite Liquor (SSL) from wood pulping facilities is a sugar rich effluent that can be used as feedstock for ethanol production. However, depending on the pulping process conditions, the release of monosaccharides also generates a range of compounds that negatively affect microbial fermentation. In the present study, we investigated whether endogenous yeasts in SSL-based ethanol plant could represent a source of Saccharomyces cerevisiae strains with a naturally acquired tolerance towards this inhibitory environment. Two isolation processes were performed, before and after the re-inoculation of the plant with a commercial baker’s yeast strain. The isolates were clustered by DNA fingerprinting and a recurrent Saccharomyces cerevisiae strain, different from the inoculated commercial baker’s yeast strain, was isolated. The strain, named TMB3720, flocculated heavily and presented high furaldehyde reductase activity. During fermentation of undiluted SSL, TMB3720 displayed a 4-fold higher ethanol production rate and 1.8-fold higher ethanol yield as compared to the commercial baker’s yeast. Another non-Saccharomyces cerevisiae species, identified as the pentose utilizing Pichia galeiformis, was also recovered in the last tanks of the process where the hexose to pentose sugar ratio and the inhibitory pressure are expected to be the lowest.
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| 4. |
- Björn, Camilla, et al.
(författare)
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Anti-infectious and anti-inflammatory effects of peptide fragments sequentially derived from the antimicrobial peptide centrocin 1 isolated from the green sea urchin, Strongylocentrotus droebachiensis
- 2012
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial resistance against antibiotic treatment has become a major threat to public health. Antimicrobial peptides (AMPs) have emerged as promising alternative agents for treatment of infectious diseases. This study characterizes novel synthetic peptides sequentially derived from the AMP centrocin 1, isolated from the green sea urchin, for their applicability as anti-infective agents. The microbicidal effect of centrocin 1 heavy chain (CEN1 HC-Br), its debrominated analogue (CEN1 HC), the C-terminal truncated variants of both peptides, i.e. CEN1 HC-Br (1–20) and CEN1 HC (1–20), as well as the cysteine to serine substituted equivalent CEN1 HC (Ser) was evaluated using minimal microbicidal concentration assay. The anti-inflammatory properties were assessed by measuring the inhibition of secretion of pro-inflammatory cytokines. All the peptides tested exhibited marked microbicidal and anti-inflammatory properties. No difference in efficacy was seen comparing CEN1 HC-Br and CEN1 HC, while the brominated variant had higher cytotoxicity. C-terminal truncation of both peptides reduced salt-tolerability of the microbicidal effect as well as anti-inflammatory actions. Also, serine substitution of cysteine residue decreased the microbicidal effect. Thus, from the peptide variants tested, CEN1 HC showed the best efficacy and safety profile. Further, CEN1 HC significantly reduced bacterial counts in two different animal models of infected wounds, while Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance against this peptide under continued selection pressure. In summary, CEN1 HC appears a promising new antimicrobial agent, and clinical studies are warranted to evaluate the applicability of this AMP for local treatment of infections in man.
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| 5. |
- Kocharin, Kanokarn, 1978, et al.
(författare)
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Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae
- 2012
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- Through metabolic engineering microorganisms can be engineered to produce new products and further produce these with higher yield and productivities. Here, we expressed the bacterial polyhydroxybutyrate (PHB) pathway in the yeast Saccharomyces cerevisiae and we further evaluated the effect of engineering the formation of acetyl coenzyme A (acetyl-CoA), an intermediate of the central carbon metabolism and precursor of the PHB pathway, on heterologous PHB production by yeast. We engineered the acetyl-CoA metabolism by co-transformation of a plasmid containing genes for native S. cerevisiae alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA acetyltransferase (ERG10) and a Salmonella enterica acetyl-CoA synthetase variant (acsL641P), resulting in acetoacetyl-CoA overproduction, together with a plasmid containing the PHB pathway genes coding for acetyl-CoA acetyltransferase (phaA), NADPH-linked acetoacetyl-CoA reductase (phaB) and poly(3-hydroxybutyrate) polymerase (phaC) from Ralstonia eutropha H16. Introduction of the acetyl-CoA plasmid together with the PHB plasmid, improved the productivity of PHB more than 16 times compared to the reference strain used in this study, as well as it reduced the specific product formation of side products.
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| 6. |
- Kocharin, Kanokarn, 1978, et al.
(författare)
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Specific growth rate and substrate dependent polyhydroxybutyrate production in Saccharomyces cerevisiae
- 2013
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 3:18, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- Production of the biopolymer polyhydroxybutyrate (PHB) in Saccharomyces cerevisiae starts at the end of exponential phase particularly when the specific growth rate is decreased due to the depletion of glucose in the medium. The specific growth rate and the type of carbon source (fermentable/non-fermentable) have been known to influence the cell physiology and hence affect the fermentability of S. cerevisiae. The production of PHB utilizes cytosolic acetyl-CoA as a precursor and the S. cerevisiae employed in this study is therefore a strain with the enhanced cytosolic acetyl-CoA supply. Growth and PHB production at different specific growth rates were evaluated on glucose, ethanol and a mixture of glucose and ethanol as carbon source. Ethanol as carbon source yielded a higher PHB production compared to glucose since it can be directly used for cytosolic acetyl-CoA production and hence serves as a precursor for PHB production. However, this carbon source results in lower biomass yield and hence it was found that to ensure both biomass formation and PHB production a mixture of glucose and ethanol was optimal, and this resulted in the highest volumetric productivity of PHB, 8.23 mg/L · h-1, at a dilution rate of 0.1 h-1.
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| 7. |
- Adeboye, Peter, 1982, et al.
(författare)
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The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulosic hydrolysates
- 2014
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4:46, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the severity of the inhibitory effects of 13 phenolic compounds usually found in spruce hydrolysates (4-hydroxy-3-methoxycinnamaldehyde, homovanilyl alcohol, vanillin, syringic acid, vanillic acid, gallic acid, dihydroferulic acid, p-coumaric acid, hydroquinone, ferulic acid, homovanillic acid, 4-hydroxybenzoic acid and vanillylidenacetone). The effects of the selected compounds on cell growth, biomass yield and ethanol yield were studied and the toxic concentration threshold was defined for each compound. Using Ethanol Red, the popular industrial strain of Saccharomyces cerevisiae, we found the most toxic compound to be 4-hydroxy-3-methoxycinnamaldehyde which inhibited growth at a concentration of 1.8 mM. We also observed that toxicity did not generally follow a trend based on the aldehyde, acid, ketone or alcohol classification of phenolic compounds, but rather that other structural properties such as additional functional groups attached to the compound may determine its toxicity. Three distinctive growth patterns that effectively clustered all the compounds involved in the screening into three categories. We suggest that the compounds have different cellular targets, and that. We suggest that the compounds have different cellular targets and inhibitory mechanisms in the cells, also compounds who share similar pattern on cell growth may have similar inhibitory effect and mechanisms of inhibition.
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| 8. |
- Bao, Jichen, 1988, et al.
(författare)
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Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast
- 2018
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein anterograde trafficking from the endoplasmic reticulum to the Golgi apparatus by the moderate overexpression of SEC16 could increase recombinant protein secretion in S. cerevisiae. In this study, the retrograde trafficking in a strain with moderate overexpression of SEC16 was engineered by overexpression of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of α-amylase but did not induce production of reactive oxygen species. An expanded ER membrane was detected in both the GCS1 and GLO3 overexpressio n strains. Physiological characterizations during batch fermentation showed that GLO3 overexpression had better effect on recombinant protein secretion than GCS1 overexpression. Additionally, the GLO3 overexpression strain had higher secretion of two other recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-α-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast.
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| 9. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae
- 2016
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could largely prevent this breakdown. An evaluation of in vivo function of a subset of phosphoketolases was conducted by monitoring acetate levels during growth, confirming that candidates showing high activity in vitro indeed showed increased acetate accumulation, but expression also decreased cellular fitness. The study shows that expression of several bacterial phosphoketolase candidates in S. cerevisiae can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds.
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| 10. |
- Bisagni, Serena, et al.
(författare)
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Cloning, expression and characterization of a versatile Baeyer-Villiger monooxygenase from Dietzia sp. D5.
- 2014
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- A novel BVMO encoding gene was identified from a draft genome sequence of a newly isolated strain of Dietzia. Analysis of the protein sequence revealed that it belongs to a group of BVMOs whose most characterized member is cyclopentadecanone monooxygenase (CPDMO). The gene was PCR amplified, cloned and successfully expressed in E. coli. The expressed recombinant enzyme was purified using metal affinity chromatography. Characterization of the purified enzyme revealed that it has a broad substrate scope and oxidized different compounds including substituted and unsubstituted alicyclic, bicyclic-, aliphatic-ketones, ketones with an aromatic moiety, and sulfides. The highest activities were measured for 2- and 3-methylcyclohexanone, phenylacetone, bicyclo-[3.2.0]-hept-2-en-6-one and menthone. The enzyme was optimally active at pH 7.5 and 35°C, a temperature at which its half-life was about 20 hours. The stability studies have shown that this enzyme is more stable than all other reported BVMOs except the phenylacetone monooxygenase from the thermophilic organism Thermobifida fusca.
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| 11. |
- Bonzom, Cyrielle, 1987, et al.
(författare)
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Glycosylation influences activity, stability and immobilization of the feruloyl esterase 1a from Myceliophthora thermophila
- 2019
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host.
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| 12. |
- Dines Knudsen, Jan, et al.
(författare)
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NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level.
- 2014
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4
-
Tidskriftsartikel (refereegranskat)abstract
- A reporter system was constructed to measure perturbations in the NADH/NAD(+) co-factor balance in yeast, by using the green fluorescent protein gene under the control of the GPD2 promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD(+) via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.
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| 13. |
- Fellahi, Soltana, et al.
(författare)
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Identification of two new keratinolytic proteases from a Bacillus pumilus strain using protein analysis and gene sequencing
- 2016
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- The Bacillus strain (CCUG 66887) has a high capacity to excrete keratinase with the ability to degrade both alpha- and beta keratin. In this study we aimed to show the characteristics of the keratinolytic protease and to identify its gene by using liquid chromatography-electrospray ionization tandem mass spectrometry methods (nanoHPLC-ESI-MS/MS) followed by Mascot data base search. The results showed that the enzyme in fact consists of two different keratinases, both with a molecular mass of 38 kDa. Further, DNA sequencing generated the open reading frame (ORF) of one of the genes (Ker1), and de novo genome sequencing identified the ORF of the second gene (Ker2). The two keratinase genes contain 1153 base pairs each and have a gene similarity of 67 %. In addition, the Bacillus strain was classified as Bacillus pumilus and its genes were annotated in the GeneBank at NCBI (accession: CP011109.1). Amino acid sequences alignment with known B. pumilus proteases indicated that the two keratinases of B. pumilus strain C4 are subtilisin-like serine proteases belonging to the Protease S8 family. Taken together, these result suggest the two keratinases as promising candidates for enzymatic processing of keratinous wastes in waste refinery.[on SciFinder (R)]
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| 14. |
- Fernando, Dinesh, et al.
(författare)
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Comparison of traditional field retting and Phlebia radiata Cel 26 retting of hemp fibres for fibre-reinforced composites
- 2017
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Classical field retting and controlled fungal retting of hemp using Phlebia radiata Cel 26 (a mutant with low cellulose degrading ability) were compared with pure pectinase treatment with regard to mechanical properties of the produced fibre/epoxy composites. For field retting a classification of the microbial evolution (by gene sequencing) and enzyme profiles were conducted. By phylogenetic frequency mapping, different types of fungi, many belonging to the Ascomycota phylum were found on the fibres during the first 2 weeks of field retting, and thereafter, different types of bacteria, notably Proteobacteria, also proliferated on the field retted fibres. Extracts from field retted fibres exhibited high glucanase activities, while extracts from P. radiata Cel 26 retted fibres showed high polygalacturonase and laccase activities. As a result, fungal retting gave a significantly higher glucan content in the fibres than field retting (77 vs. 67%) and caused a higher removal of pectin as indicated by lower galacturonan content of fibres (1.6%) after fibres were retted for 20 days with P. radiata Cel 26 compared to a galacturonan content of 3.6% for field retted fibres. Effective fibre stiffness increased slightly after retting with P. radiata Cel 26 from 65 to 67 GPa, while it decreased after field retting to 52 GPa. Effective fibre strength could not be determined similarly due to variations in fibre fracture strain and fibre-matrix adhesion. A maximum composite strength with 50 vol% fibres of 307 MPa was obtained using P. radiata Cel 26 compared to 248 MPa with field retting.
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| 15. |
- García-Hidalgo, Javier, et al.
(författare)
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Identification of the two-component guaiacol demethylase system from Rhodococcus rhodochrous and expression in Pseudomonas putida EM42 for guaiacol assimilation
- 2019
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- A diversity of softwood lignin depolymerization processes yield guaiacol as the main low molecular weight product. This key aromatic compound can be utilized as a carbon source by several microbial species, most of which are Gram positive bacteria. Microbial degradation of guaiacol is known to proceed initially via demethylation to catechol, and this reaction is catalyzed by cytochrome P450 monooxygenases. These enzymes typically require a set of redox partner proteins, whose number and identities were not described until very recently in the case of guaiacol. In this work we identified two proteins involved in guaiacol demethylation by the actinomycete Rhodococcus rhodochrous. Additionally, we constructed four different polycistronic operons carrying combinations of putative redox partners of this guaiacol demethylation system in an inducible expression plasmid that was introduced into the Gram negative host Pseudomonas putida EM42, and the guaiacol consumption dynamics of each resulting strain were analyzed. All the polycistronic operons, expressing a cytochrome P450 together with a putative ferredoxin reductase from R. rhodochrous and putative ferredoxins from R. rhodochrous or Amycolatopsis ATCC 39116 enabled P. putida EM42 to metabolize and grow on guaiacol as the sole carbon source.
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| 16. |
- Hernández, Armando, et al.
(författare)
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Impact of the fermentation parameters pH and temperature on stress resilience of Lactobacillus reuteri DSM 17938
- 2019
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- This study was undertaken to investigate the impact of culture pH (4.5–6.5) and temperature (32–37 °C) on the stress resilience of Lactobacillus reuteri DSM 17938 during freeze-drying and post freeze-drying exposure to low pH (pH 2)and bile salts. Response-surface methodology analysis revealed that freeze-drying survival rates Ncells after drying/Ncells before drying*100 were linearly related to pH with the highest survival rate of 80% when cells were cultured at pH 6.5 and the lowest was 40% when cells were cultured at pH 4.5. The analysis further revealed that within the chosen temperature range the culture temperature did not significantly affect the freeze-drying survival rate. However, fermentation at pH 4.5 led to better survival rates when rehydrated cells were exposed to low pH shock or bile salts. Thus, the effect of pH onfreeze-drying survival was in contrast to effects on low pH and bile salts stress tolerance. The rationale behind this irreconcilability is based on the responses being dissimilar and are not tuned to each other. Culturing strain DSM17938 at pH values higher than 5.5 could be a useful option to improve the survivability and increase viable cell numbers in the final freeze-dried product. However, the dissimilar responses for the process- and application parameterstested here suggest that an optimal compromise has to be found in order to obtain the most functional probiotic product possible.
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| 17. |
- Hernandez-Cortes, G., et al.
(författare)
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Improvement on the productivity of continuous tequila fermentation by Saccharomyces cerevisiae of Agave tequilana juice with supplementation of yeast extract and aeration
- 2016
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Agave (Agave tequilana Weber var. azul) fermentations are traditionally carried out employing batch systems in the process of tequila manufacturing; nevertheless, continuous cultures could be an attractive technological alternative to increase productivity and efficiency of sugar to ethanol conversion. However, agave juice (used as a culture medium) has nutritional deficiencies that limit the implementation of yeast continuous fermentations, resulting in high residual sugars and low fermentative rates. In this work, fermentations of agave juice using Saccharomyces cerevisiae were put into operation to prove the necessity of supplementing yeast extract, in order to alleviate nutritional deficiencies of agave juice. Furthermore, continuous fermentations were performed at two different aeration flow rates, and feeding sterilized and non-sterilized media. The obtained fermented musts were subsequently distilled to obtain tequila and the preference level was compared against two commercial tequilas, according to a sensorial analysis. The supplementation of agave juice with air and yeast extract augmented the fermentative capacity of S. cerevisiae S1 and the ethanol productivities, compared to those continuous fermentations non supplemented. In fact, aeration improved ethanol production from 37 to 40 g L-1, reducing sugars consumption from 73 to 88 g L-1 and ethanol productivity from 3.0 to 3.2 g (Lh)(-1), for non-aerated and aerated (at 0.02 vvm) cultures, respectively. Supplementation of yeast extract allowed an increase in specific growth rate and dilution rates (0.12 h(-1), compared to 0.08 h(-1) of non-supplemented cultures), ethanol production (47 g L-1), reducing sugars consumption (93 g L-1) and ethanol productivity [5.6 g (Lh)(-1)] were reached. Additionally, the effect of feeding sterilized or non-sterilized medium to the continuous cultures was compared, finding no significant differences between both types of cultures. The overall effect of adding yeast extract and air to the continuous fermentations resulted in 88 % increase in ethanol productivity. For all cultures, pH was not controlled, reaching low pH values (from 2.6 to 3). This feature suggested a reduced probability of contamination for prolonged continuous cultures and explained why no significant differences were found between continuous cultures fed with sterilized or non-sterilized media. Concentrations of volatile compounds quantified in the distillates (tequila) were in the allowed ranges established by the Mexican regulation of tequila (NOM-006-SCFI-2012, Norma Oficial Mexicana: Bebidas alcoholicas-Tequila-specificaciones, 2012). The preference level of the distillates was similar to that of two well-known commercial tequilas. The results suggested the possibility of implementing this innovative technology on an industrial scale, attaining high productivities and using non-sterilized agave juice.
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| 18. |
- Hidayat, B.J., et al.
(författare)
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The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images
- 2015
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 5:1, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- © 2015, Hidayat et al. Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.
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| 19. |
- Kaczmarzyk, Danuta, et al.
(författare)
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Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria
- 2016
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Ingår i: AMB Express. - : BioMed Central (BMC). - 2191-0855. ; 6:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10-C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystis aas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids.
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| 20. |
- Konzock, Oliver, 1991, et al.
(författare)
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Cinnamic acid and p-coumaric acid are metabolized to 4-hydroxybenzoic acid by Yarrowia lipolytica
- 2023
-
Ingår i: AMB Express. - 2191-0855. ; 13:1
-
Tidskriftsartikel (refereegranskat)abstract
- Yarrowia lipolytica has been explored as a potential production host for flavonoid synthesis due to its high tolerance to aromatic acids and ability to supply malonyl-CoA. However, little is known about its ability to consume the precursors cinnamic and p-coumaric acid. In this study, we demonstrate that Y. lipolytica can consume these precursors through multiple pathways that are partially dependent on the cultivation medium. By monitoring the aromatic acid concentrations over time, we found that cinnamic acid is converted to p-coumaric acid. We identified potential proteins with a trans-cinnamate 4-monooxygenase activity in Y. lipolytica and constructed a collection of 15 knock-out strains to identify the genes responsible for the reaction. We identified YALI1_B28430g as the gene encoding for a protein that converts cinnamic acid to p-coumaric acid (designated as TCM1). By comparing different media compositions we found that complex media components (casamino acids and yeast extract) induce this pathway. Additionally, we discover the conversion of p-coumaric acid to 4-hydroxybenzoic acid. Our findings provide new insight into the metabolic capabilities of Y. lipolytica and hold great potential for the future development of improved strains for flavonoid production.
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| 21. |
- Nair, Ramkumar, 1988-, et al.
(författare)
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Mycelial pellet formation by edible ascomycete filamentous fungi, Neurospora intermedia
- 2016
-
Ingår i: AMB Express. - : Springer Berlin/Heidelberg. - 2191-0855. ; 6:31
-
Tidskriftsartikel (refereegranskat)abstract
- Pellet formation of filamentous fungi in submerged culture is an imperative topic of fermentation research. In this study, we report for the first time the growth of filamentous ascomycete fungus,Neurospora intermedia in its mycelial pellet form. In submerged culture, the growth morphology of the fungus was successfully manipulated into growing as pellets by modifying various cultivation conditions. Factors such as pH (2.0–10.0), agitation rate (100–150 rpm), carbon source (glucose, arabinose, sucrose, and galactose), the presence of additive agents (glycerol and calcium chloride) and trace metals were investigated for their effect on the pellet formation. Of the various factors screened, uniform pellets were formed only at pH range 3.0–4.0, signifying it as the most influential factor for N. intermedia pellet formation. The average pellet size ranged from 2.38 ± 0.12 to 2.86 ± 0.38 mm. The pellet formation remained unaffected by the inoculum type used and its size showed an inverse correlation with the agitation rate of the culture. Efficient glucose utilization was observed with fungal pellets, as opposed to the freely suspended mycelium, proving its viability for fast- fermentation processes. Scale up of the pelletization process was also carried out in bench-scale airlift and bubble column reactors (4.5 L).
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| 22. |
- Narayanan, Venkatachalam, et al.
(författare)
-
Adaptation to low pH and lignocellulosic inhibitors resulting in ethanolic fermentation and growth of Saccharomyces cerevisiae
- 2016
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- Lignocellulosic bioethanol from renewable feedstocks using Saccharomyces cerevisiae is a promising alternative tofossil fuels owing to environmental challenges. S. cerevisiae is frequently challenged by bacterial contamination anda combination of lignocellulosic inhibitors formed during the pre-treatment, in terms of growth, ethanol yield andproductivity. We investigated the phenotypic robustness of a brewing yeast strain TMB3500 and its ability to adapt tolow pH thereby preventing bacterial contamination along with lignocellulosic inhibitors by short-term adaptation andadaptive lab evolution (ALE). The short-term adaptation strategy was used to investigate the inherent ability of strainTMB3500 to activate a robust phenotype involving pre-culturing yeast cells in defined medium with lignocellulosicinhibitors at pH 5.0 until late exponential phase prior to inoculating them in defined media with the same inhibitorcocktail at pH 3.7. Adapted cells were able to grow aerobically, ferment anaerobically (glucose exhaustion by 19 ± 5 hto yield 0.45 ± 0.01 g ethanol g glucose−1) and portray significant detoxification of inhibitors at pH 3.7, when comparedto non-adapted cells. ALE was performed to investigate whether a stable strain could be developed to growand ferment at low pH with lignocellulosic inhibitors in a continuous suspension culture. Though a robust populationwas obtained after 3600 h with an ability to grow and ferment at pH 3.7 with inhibitors, inhibitor robustness was notstable as indicated by the characterisation of the evolved culture possibly due to phenotypic plasticity. With furtherresearch, this short-term adaptation and low pH strategy could be successfully applied in lignocellulosic ethanolplants to prevent bacterial contamination.
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| 23. |
- Navarrete Roman, Clara, 1981, et al.
(författare)
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Enhanced ethanol production and reduced glycerol formation in fps1∆ mutants of Saccharomyces cerevisiae engineered for improved redox balancing
- 2014
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4:1, s. 1-8
-
Tidskriftsartikel (refereegranskat)abstract
- Ethanol is by volume the largest fermentation product. During ethanol production by Saccharomyces cerevisiae about 4-5% of the carbon source is lost to glycerol production. Different approaches have been proposed for improving the ethanol yield while reducing glycerol production. Here we studied the effect of reducing glycerol export/formation through deletion of the aquaglyceroporin gene FPS1 together with expressing gapN encoding NADP+-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans and overexpressing the ATP-NADH kinase gene UTR1 from S. cerevisiae. This strategy will allow reducing the redox balance problem observed when the glycerol pathway is blocked, and hereby improve ethanol production. We found that our strategy enabled increasing the ethanol yield by 4.6% in the case of the best producing strain, compared to the reference strain, without any major effect on the specific growth rate.
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| 24. |
- Nunes, Diogo Jp, et al.
(författare)
-
Effect of nitrogen availability on the poly-3-d-hydroxybutyrate accumulation by engineered Saccharomyces cerevisiae
- 2017
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 7:1
-
Tidskriftsartikel (refereegranskat)abstract
- Poly-3-d-hydroxybutyrate (or PHB) is a polyester which can be used in the production of biodegradable plastics from renewable resources. It is naturally produced by several bacteria as a response to nutrient starvation in the excess of a carbon source. The yeast Saccharomyces cerevisiae could be an alternative production host as it offers good inhibitor tolerance towards weak acids and phenolic compounds and does not depolymerize the produced PHB. As nitrogen limitation is known to boost the accumulation of PHB in bacteria, the present study aimed at investigating the effect of nitrogen availability on PHB accumulation in two recombinant S. cerevisiae strains harboring different xylose consuming and PHB producing pathways: TMB4443 expressing an NADPH-dependent acetoacetyl-CoA reductase and a wild-type S. stipitis XR with preferential use of NADPH and TMB4425 which expresses an NADH-dependent acetoacetyl-CoA reductase and a mutated XR with a balanced affinity for NADPH/NADH. TMB4443 accumulated most PHB under aerobic conditions and with glucose as sole carbon source, whereas the highest PHB concentrations were obtained with TMB4425 under anaerobic conditions and xylose as carbon source. In both cases, the highest PHB contents were obtained with high availability of nitrogen. The major impact of nitrogen availability was observed in TMB4425, where a 2.7-fold increase in PHB content was obtained. In contrast to what was observed in natural PHB-producing bacteria, nitrogen deficiency did not improve PHB accumulation in S. cerevisiae. Instead the excess available carbon from xylose was shunted into glycogen, indicating a significant gluconeogenic activity on xylose.
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| 25. |
- Palmqvist, Benny, et al.
(författare)
-
Combining the effects of process design and pH for improved xylose conversion in high solid ethanol production from Arundo donax.
- 2014
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4
-
Tidskriftsartikel (refereegranskat)abstract
- The impact of pH coupled to process design for the conversion of the energy crop Arundo donax to ethanol was assessed in the present study under industrially relevant solids loadings. Two main process strategies were investigated, i.e. the traditional simultaneous saccharification and co-fermentation (SSCF) and a HYBRID design, where a long high temperature enzymatic hydrolysis step was carried out prior to continued low temperature SSCF, keeping the same total reaction time. Since acetic acid was identified as the major inhibitor in the slurry, the scenarios were investigated under different fermentation pH in order to alleviate the inhibitory effect on, in particular, xylose conversion. The results show that, regardless of fermentation pH, a higher glucan conversion could be achieved with the HYBRID approach compared to SSCF. Furthermore, it was found that increasing the pH from 5.0 to 5.5 for the fermentation phase had a large positive effect on xylose consumption for both process designs, although the SSCF design was more favored. With the high sugar concentrations available at the start of fermentation during the HYBRID design, the ethanol yield was reduced in favor of cell growth and glycerol production. This finding was confirmed in shake flask fermentations where an increase in pH enhanced both glucose and xylose consumption, but also cell growth and cell yield with the overall effect being a reduced ethanol yield. In conclusion this resulted in similar overall ethanol yields at the different pH values for the HYBRID design, despite the improved xylose uptake, whereas a significant increase in overall ethanol yield was found with the SSCF design.
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| 26. |
- Raghavendran, Vijayendran, 1976, et al.
(författare)
-
A comparative study of the enzymatic hydrolysis of batch organosolv-pretreated birch and spruce biomass
- 2018
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- A shift towards a sustainable and green society is vital to reduce the negative effects of climate change associated with increased CO2emissions. Lignocellulosic biomass is both renewable and abundant, but is recalcitrant to deconstruction. Among the methods of pretreatment available, organosolv (OS) delignifies cellulose efficiently, significantly improving its digestibility by enzymes. We have assessed the hydrolysability of the cellulose-rich solid fractions from OS-pretreated spruce and birch at 2% w/v loading (dry matter). Almost complete saccharification of birch was possible with 80 mg enzyme preparation/gsolids(12 FPU/gsolids), while the saccharification yield for spruce was only 70%, even when applying 60 FPU/gsolids. As the cellulose content is enriched by OS, the yield of glucose was higher than in their steam-exploded counterparts. The hydrolysate was a transparent liquid due to the absence of phenolics and was also free from inhibitors. OS pretreatment holds potential for use in a large-scale, closed-loop biorefinery producing fuels from the cellulose fraction and platform chemicals from the hemicellulose and lignin fractions respectively.
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| 27. |
- Raghavendran, Vijayendran, 1976, et al.
(författare)
-
The protective role of intracellular glutathione in Saccharomyces cerevisiae during lignocellulosic ethanol production
- 2020
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- To enhance the competitiveness of industrial lignocellulose ethanol production, robust enzymes and cell factories are vital. Lignocellulose derived streams contain a cocktail of inhibitors that drain the cell of its redox power and ATP, leading to a decrease in overall ethanol productivity. Many studies have attempted to address this issue, and we have shown that increasing the glutathione (GSH) content in yeasts confers tolerance towards lignocellulose inhibitors, subsequently increasing the ethanol titres. However, GSH levels in yeast are limited by feedback inhibition of GSH biosynthesis. Multidomain and dual functional enzymes exist in several bacterial genera and they catalyse the GSH biosynthesis in a single step without the feedback inhibition. To test if even higher intracellular glutathione levels could be achieved and if this might lead to increased tolerance, we overexpressed the genes from two bacterial genera and assessed the recombinants in simultaneous saccharification and fermentation (SSF) with steam pretreated spruce hydrolysate containing 10% solids. Although overexpressing the heterologous genes led to a sixfold increase in maximum glutathione content (18 µmol gdrycellmass−1) compared to the control strain, this only led to a threefold increase in final ethanol titres (8.5 g L− 1). As our work does not conclusively indicate the cause-effect of increased GSH levels towards ethanol titres, we cautiously conclude that there is a limit to cellular fitness that could be accomplished via increased levels of glutathione.
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| 28. |
- Ravi, Krithika, et al.
(författare)
-
Biological conversion of aromatic monolignol compounds by a Pseudomonas isolate from sediments of the Baltic Sea
- 2018
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- Bacterial strains were isolated from the sediments of the Baltic Sea using ferulic acid, guaiacol or a lignin-rich softwood waste stream as substrate. In total nine isolates were obtained, five on ferulic acid, two on guaiacol and two on a lignin-rich softwood stream as a carbon source. Three of the isolates were found to be Pseudomonas sp. based on 16S rRNA sequencing. Among them, isolate 9.1, which showed the fastest growth in defined M9 medium, was tentatively identified as a Pseudomonas deceptionensis strain based on the gyrB sequencing. The growth of isolate 9.1 was further examined on six selected lignin model compounds (ferulate, p-coumarate, benzoate, syringate, vanillin and guaiacol) from different upper funneling aromatic pathways and was found able to grow on four out of these six compounds. No growth was detected on syringate and guaiacol. The highest specific growth and uptake rates were observed for benzoate (0.3 h−1 and 4.2 mmol gCDW −1 h−1) whereas the lowest were for the compounds from the coniferyl branch. Interestingly, several pathway intermediates were excreted during batch growth. Vanillyl alcohol was found to be excreted during growth on vanillin. Several other intermediates like cis,cis-muconate, catechol, vanillate and 4-hydroxybenzoate from the known bacterial catabolic pathways were excreted during growth on the model compounds.
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| 29. |
- Rönnander, Jonas, et al.
(författare)
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Biotransformation of vanillin into vanillyl alcohol by a novel strain of Cystobasidium laryngis isolated from decaying wood
- 2018
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- Vanillin is an aromatic aldehyde found as a component of lignocellulosic material, and in the cured pods of orchidaceae plants. Like other phenolic substances, vanillin has antimicrobial activity and can be extracted from lignin either by a thermo-chemical process or through microbial degradation. Vanillin, can serve as a model monomer in biodegradation studies of lignin. In the present study, a yeast isolated from decaying wood on the Faroe Islands, was identified as Cystobasidium laryngis strain FMYD002, based on internal transcribed spacer sequence analysis. It demonstrated the ability to convert vanillin to vanillyl alcohol, as detected by ultra-high performance liquid chromatography–quadrupole-time-of-flight. Structural analysis of vanillyl alcohol was carried out by using gas chromatography–mass spectrometry and 1H NMR spectroscopy, and further verified by synthesis. The reduction of vanillin to vanillyl alcohol has been documented for only a few species of fungi. However, to our knowledge, this biotransformation has not yet been reported for basidiomycetous yeast species, nor for any representative of the subphylum Pucciniomycotina. The biotransformation capability of the present strain might prove useful in the industrial utilisation of lignocellulosic residues.
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| 30. |
- Sandström, Anders, et al.
(författare)
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Engineering of Saccharomyces cerevisiae for the production of poly-3-d-hydroxybutyrate from xylose.
- 2015
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Poly-3-d-hydroxybutyrate (PHB) is a promising biopolymer naturally produced by several bacterial species. In the present study, the robust baker's yeast Saccharomyces cerevisiae was engineered to produce PHB from xylose, the main pentose found in lignocellulosic biomass. The PHB pathway genes from the well-characterized PHB producer Cupriavidus necator were introduced in recombinant S. cerevisiae strains already capable of pentose utilization by introduction of the fungal genes for xylose utilization from the yeast Scheffersomyces stipitis. PHB production from xylose was successfully demonstrated in shake-flasks experiments, with PHB yield of 1.17 ± 0.18 mg PHB g(-1) xylose. Under well-controlled fully aerobic conditions, a titer of 101.7 mg PHB L(-1) was reached within 48 hours, with a PHB yield of 1.99 ± 0.15 mg PHB g(-1) xylose, thereby demonstrating the potential of this host for PHB production from lignocellulose.
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| 31. |
- Shibasaki, Seiji, et al.
(författare)
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Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function
- 2014
-
Ingår i: AMB Express. - : Springer Berlin/Heidelberg. - 2191-0855. ; 4:82
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.
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| 32. |
- Sjöberg, Gustav, et al.
(författare)
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Characterization of volatile fatty-acid utilization in Escherichia coli aiming for robust valorisation of food residues
- 2020
-
Ingår i: AMB Express. - : Springer Nature. - 2191-0855. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Valorisation of food residues would greatly benefit from development of robust processes that create added value compared to current feed- and biogas applications. Recent advances in membrane-bioreactor-based open mixed microbial cultures, enable robust conversion of fluctuating streams of food residues to a mixture of volatile fatty acids (VFAs). In this study, such a mixed stream of VFAs was investigated as a substrate for Escherichia coli, a well-studied organism suitable for application in further conversion of the acids into compounds of higher value, and/or that are easier to separate from the aqueous medium. E. coli was cultured in batch on a VFA-rich anaerobic digest of food residues, tolerating up to 40 mM of total VFAs without any reduction in growth rate. In carbon-limited chemostats of E. coli W3110 ΔFadR on a simulated VFA mixture, the straight-chain VFAs (C2-C6) in the mixture were readily consumed simultaneously. At a dilution rate of 0.1 h−1, mainly acetic-, propionic- and caproic acid were consumed, while consumption of all the provided acids were observed at 0.05 h−1. Interestingly, also the branched isovaleric acid was consumed through a hitherto unknown mechanism. In total, up to 80% of the carbon from the supplied VFAs was consumed by the cells, and approximately 2.7% was excreted as nucleotide precursors in the medium. These results suggest that VFAs derived from food residues are a promising substrate for E. coli.
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| 33. |
- Stagge, Stefan, et al.
(författare)
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Identification of benzoquinones in pretreated lignocellulosic feedstocks and inhibitory effects on yeast
- 2015
-
Ingår i: AMB Express. - : BioMed Central (BMC). - 2191-0855. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Pretreatment of lignocellulosic biomass under acidic conditions gives rise to by-products that inhibit fermenting microorganisms. An analytical procedure for identification of p-benzoquinone (BQ) and 2,6-dimethoxybenzoquinone (DMBQ) in pretreated biomass was developed, and the inhibitory effects of BQ and DMBQ on the yeast Saccharomyces cerevisiae were assessed. The benzoquinones were analyzed using ultra-high performance liquid chromatographyelectrospray ionization-triple quadrupole-mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Pretreatment liquids examined with regard to the presence of BQ and DMBQ originated from six different lignocellulosic feedstocks covering agricultural residues, hardwood, and softwood, and were produced through impregnation with sulfuric acid or sulfur dioxide at varying pretreatment temperature (165-204 degrees C) and residence time (6-20 min). BQ was detected in all six pretreatment liquids in concentrations ranging up to 6 mg/l, while DMBQ was detected in four pretreatment liquids in concentrations ranging up to 0.5 mg/l. The result indicates that benzoquinones are ubiquitous as by-products of acid pretreatment of lignocellulose, regardless of feedstock and pretreatment conditions. Fermentation experiments with BQ and DMBQ covered the concentration ranges 2 mg/l to 1 g/l and 20 mg/l to 1 g/l, respectively. Even the lowest BQ concentration tested (2 mg/l) was strongly inhibitory to yeast, while 20 mg/l DMBQ gave a slight negative effect on ethanol formation. This work shows that benzoquinones should be regarded as potent and widespread inhibitors in lignocellulosic hydrolysates, and that they warrant attention besides more well-studied inhibitory substances, such as aliphatic carboxylic acids, phenols, and furan aldehydes.
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| 34. |
- Szabo, Enikö Barbara, 1985, et al.
(författare)
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Comparison of the bacterial community composition in the granular and the suspended phase of sequencing batch reactors
- 2017
-
Ingår i: Amb Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Granulation of activated sludge is an increasingly important area within the field of wastewater treatment. Granulation is usually achieved by high hydraulic selection pressure, which results in the wash-out of slow settling particles. The effect of the harsh wash-out conditions on the granular sludge ecosystem is not yet fully understood, but different bacterial groups may be affected to varying degrees. In this study, we used high-throughput amplicon sequencing to follow the community composition in granular sludge reactors for 12 weeks, both in the granular phase and the suspended phase (effluent). The microbiome of the washed out biomass was similar but not identical to the microbiome of the granular biomass. Certain taxa (e.g. Flavobacterium spp. and Bdellovibrio spp.) had significantly (p < 0.05) higher relative abundance in the granules compared to the effluent. Fluorescence in situ hybridization images indicated that these taxa were mainly located in the interior of granules and therefore protected from erosion. Other taxa (e.g. Meganema sp. and Zooglea sp.) had significantly lower relative abundance in the granules compared to the effluent, and appeared to be mainly located on the surface of granules and therefore subject to erosion. Despite being washed out, these taxa were among the most abundant members of the granular sludge communities and were likely growing fast in the reactors. The ratio between relative abundance in the granular biomass and in the effluent did not predict temporal variation of the taxa in the reactors, but it did appear to predict the spatial location of the taxa in the granules.
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| 35. |
- Vahed, M., et al.
(författare)
-
Molecular dynamics simulation and experimental study of the surface-display of SPA protein via Lpp-OmpA system for screening of IgG
- 2020
-
Ingår i: AMB Express. - : Springer Nature. - 2191-0855. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Staphylococcal protein A (SpA) is a major virulence factor of Staphylococcus aureus. S. aureus is able to escape detection by the immune system by the surface display of protein A. The SpA protein is broadly used to purify immunoglobulin G (IgG) antibodies. This study investigates the fusion ability of Lpp′-OmpA (46–159) to anchor and display five replicate domains of protein A with 295 residues length (SpA295) of S. aureus on the surface of Escherichia coli to develop a novel bioadsorbent. First, the binding between Lpp’-OmpA-SPA295 and IgGFc and the three-dimensional structure was investigated using molecular dynamics simulation. Then high IgG recovery from human serum by the surface-displayed system of Lpp′-OmpA-SPA295 performed experimentally. In silico analysis was demonstrated the binding potential of SPA295 to IgG after expression on LPP-OmpA surface. Surface-engineered E. coli displaying SpA protein and IgG-binding assay with SDS-PAGE analysis exhibited high potential of the expressed complex on the E. coli surface for IgG capture from human serum which is applicable to conventional immune precipitation.
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| 36. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Nutrient-supplemented propagation of Saccharomyces cerevisiae improves its lignocellulose fermentation ability
- 2020
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Propagation conditions have been shown to be of considerable importance for the fermentation ability of Saccharomyces cerevisiae. The limited tolerance of yeast to inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. We have investigated the hypothesis that the addition of nutrients during propagation leads to yeast cultures with improved ability to subsequently ferment lignocellulosic materials. This hypothesis was tested with and without short-term adaptation to wheat straw or corn stover hydrolysates during propagation of the yeast. The study was performed using the industrial xylose-fermenting S. cerevisiae strain CR01. Adding a mixture of pyridoxine, thiamine, and biotin to unadapted propagation cultures improved cell growth and ethanol yields during fermentation in wheat straw hydrolysate from 0.04 g g−1 to 0.19 g g−1 and in corn stover hydrolysate from 0.02 g g−1 to 0.08 g g−1. The combination of short–term adaptation and supplementation with the vitamin mixture during propagation led to ethanol yields of 0.43 g g−1 in wheat straw hydrolysate fermentation and 0.41 g g−1 in corn stover hydrolysate fermentation. These ethanol yields were improved compared to ethanol yields from cultures that were solely short-term adapted (0.37 and 0.33 g g−1). Supplementing the propagation medium with nutrients in combination with short-term adaptation was thus demonstrated to be a promising strategy to improve the efficiency of industrial lignocellulosic fermentation.
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| 37. |
- Wallace, Valeria, et al.
(författare)
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Re-assessment of YAP1 and MCR1 contributions to inhibitor tolerance in robust engineered Saccharomyces cerevisiae fermenting undetoxified lignocellulosic hydrolysate
- 2014
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4:1, s. 56-12
-
Tidskriftsartikel (refereegranskat)abstract
- Development of robust yeast strains that can efficiently ferment lignocellulose-based feedstocks is one of the requirements for achieving economically feasible bioethanol production processes. With this goal, several genes have been identified as promising candidates to confer improved tolerance to S. cerevisiae. In most of the cases, however, the evaluation of the genetic modification was performed only in laboratory strains, that is, in strains that are known to be quite sensitive to various types of stresses. In the present study, we evaluated the effects of overexpressing genes encoding the transcription factor (YAP1) and the mitochondrial NADH-cytochrome b5 reductase (MCR1), either alone or in combination, in an already robust and xylose-consuming industrial strain of S. cerevisiae and evaluated the effect during the fermentation of undiluted and undetoxified spruce hydrolysate. Overexpression of either gene resulted in faster hexose catabolism, but no cumulative effect was observed with the simultaneous overexpression. The improved phenotype of MCR1 overexpression appeared to be related, at least in part, to a faster furaldehyde reduction capacity, indicating that this reductase may have a wider substrate range than previously reported. Unexpectedly a decreased xylose fermentation rate was also observed in YAP1 overexpressing strains and possible reasons behind this phenotype are discussed.
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| 38. |
- Wasserstrom, Lisa, et al.
(författare)
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Exploring d-xylose oxidation in Saccharomyces cerevisiae through the Weimberg pathway
- 2018
-
Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- Engineering of the yeast Saccharomyces cerevisiae towards efficient d-xylose assimilation has been a major focus over the last decades since d-xylose is the second most abundant sugar in nature, and its conversion into products could significantly improve process economy in biomass-based processes. Up to now, two different metabolic routes have been introduced via genetic engineering, consisting of either the isomerization or the oxido-reduction of d-xylose to d-xylulose that is further connected to the pentose phosphate pathway and glycolysis. In the present study, cytosolic d-xylose oxidation was investigated instead, through the introduction of the Weimberg pathway from Caulobacter crescentus in S. cerevisiae. This pathway consists of five reaction steps that connect d-xylose to the TCA cycle intermediate α-ketoglutarate. The corresponding genes could be expressed in S. cerevisiae, but no growth was observed on d-xylose indicating that not all the enzymes were functionally active. The accumulation of the Weimberg intermediate d-xylonate suggested that the dehydration step(s) might be limiting, blocking further conversion into α-ketoglutarate. Although four alternative dehydratases both of bacterial and archaeon origins were evaluated, d-xylonate accumulation still occurred. A better understanding of the mechanisms associated with the activity of dehydratases, both at a bacterial and yeast level, appears essential to obtain a fully functional Weimberg pathway in S. cerevisiae.
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| 39. |
- Wei, Yongjun, 1986, et al.
(författare)
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Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes
- 2017
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0–C18:1–C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0–C18:1–C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1–C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis. © 2017, The Author(s).
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