| 1. |
- Adlercreutz, Patrick
(författare)
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Comparison of lipases and glycoside hydrolases as catalysts in synthesis reactions
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 101:2, s. 513-519
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Forskningsöversikt (refereegranskat)abstract
- Lipases and glycoside hydrolases have large similarities concerning reaction mechanisms. Acyl-enzyme intermediates are formed during lipase-catalyzed reactions and in an analogous way, retaining glycoside hydrolases form glycosyl-enzyme intermediates during catalysis. In both cases, the covalent enzyme intermediates can react with water or other nucleophiles containing hydroxyl groups. Simple alcohols are accepted as nucleophiles by both types of enzymes. Lipases are used very successfully in synthesis applications due to their efficiency in catalyzing reversed hydrolysis and transesterification reactions. On the other hand, synthesis applications of glycoside hydrolases are much less developed. Here, important similarities and differences between the enzyme groups are reviewed and approaches to reach high synthesis yields are discussed. Useful strategies include the use of low-water media, high nucleophile concentrations, as well as protein engineering to modify the selectivity of the enzymes. The transglycosylases, hydrolases which naturally catalyze mainly transfer reactions, are of special interest and might be useful guides for engineering of other hydrolases.
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| 2. |
- Antonopoulou, Io, et al.
(författare)
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Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application
- 2016
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 100:15, s. 6519-6543
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Tidskriftsartikel (refereegranskat)abstract
- Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry
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| 3. |
- Antonopoulou, Io, et al.
(författare)
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Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 101:8, s. 3213-3226
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Tidskriftsartikel (refereegranskat)abstract
- Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.
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| 4. |
- Bjerketorp, Joakim, et al.
(författare)
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Formulation and stabilization of an Arthrobacter strain with good storage stability and 4-chlorophenol-degradation activity for bioremediation
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102:4, s. 2031-2040
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Tidskriftsartikel (refereegranskat)abstract
- Chlorophenols are widespread and of environmental concern due to their toxic and carcinogenic properties. Development of less costly and less technically challenging remediation methods are needed; therefore, we developed a formulation based on micronized vermiculite that, when air-dried, resulted in a granular product containing the 4-chlorophenol (4-CP)-degrading Gram-positive bacterium Arthrobacter chlorophenolicus A6. This formulation and stabilization method yielded survival rates of about 60% that remained stable in storage for at least 3 months at 4 °C. The 4-CP degradation by the formulated and desiccated A. chlorophenolicus A6 cells was compared to that of freshly grown cells in controlled-environment soil microcosms. The stabilized cells degraded 4-CP equally efficient as freshly grown cells in two different set-ups using both hygienized and non-treated soils. The desiccated microbial product was successfully employed in an outdoor pot trial showing its effectiveness under more realistic environmental conditions. No significant phytoremediation effects on 4-CP degradation were observed in the outdoor pot experiment. The 4-CP degradation kinetics from both the microcosms and the outdoor pot trial were used to generate a predictive model of 4-CP biodegradation potentially useful for larger-scale operations, enabling better bioremediation set-ups and saving of resources. This study also opens up the possibility of formulating and stabilizing also other Arthrobacter strains possessing different desirable pollutant-degrading capabilities.
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| 5. |
- Brandenburg, Jule, et al.
(författare)
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Bioethanol and lipid production from the enzymatic hydrolysate of wheat straw after furfural extraction
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102, s. 6269-6277
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Tidskriftsartikel (refereegranskat)abstract
- This study investigates biofuel production from wheat straw hydrolysate, from which furfural was extracted using a patented method developed at the Latvian State Institute of Wood Chemistry. The solid remainder after furfural extraction, corresponding to 67.6% of the wheat straw dry matter, contained 69.9% cellulose of which 4% was decomposed during the furfural extraction and 26.3% lignin. Enzymatic hydrolysis released 44% of the glucose monomers in the cellulose. The resulting hydrolysate contained mainly glucose and very little amount of acetic acid. Xylose was not detectable. Consequently, the undiluted hydrolysate did not inhibit growth of yeast strains belonging to Saccharomyces cerevisiae, Lipomyces starkeyi, and Rhodotorula babjevae. In the fermentations, average final ethanol concentrations of 23.85 g/l were obtained, corresponding to a yield of 0.53 g ethanol per g released glucose. L. starkeyi generated lipids with a rate of 0.08 g/h and a yield of 0.09 g per g consumed glucose. R. babjevae produced lipids with a rate of 0.18 g/h and a yield of 0.17 per g consumed glucose. In both yeasts, desaturation increased during cultivation. Remarkably, the R. babjevae strain used in this study produced considerable amounts of heptadecenoic, alpha,- and gamma-linolenic acid.
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| 6. |
- Brink, Daniel P., et al.
(författare)
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Mapping the diversity of microbial lignin catabolism : experiences from the eLignin database
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; , s. 3979-4002
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Forskningsöversikt (refereegranskat)abstract
- Lignin is a heterogeneous aromatic biopolymer and a major constituent of lignocellulosic biomass, such as wood and agricultural residues. Despite the high amount of aromatic carbon present, the severe recalcitrance of the lignin macromolecule makes it difficult to convert into value-added products. In nature, lignin and lignin-derived aromatic compounds are catabolized by a consortia of microbes specialized at breaking down the natural lignin and its constituents. In an attempt to bridge the gap between the fundamental knowledge on microbial lignin catabolism, and the recently emerging field of applied biotechnology for lignin biovalorization, we have developed the eLignin Microbial Database (www.elignindatabase.com), an openly available database that indexes data from the lignin bibliome, such as microorganisms, aromatic substrates, and metabolic pathways. In the present contribution, we introduce the eLignin database, use its dataset to map the reported ecological and biochemical diversity of the lignin microbial niches, and discuss the findings.
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| 7. |
- da Costa, BLV, et al.
(författare)
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Anaerobiosis revisited: growth of Saccharomyces cerevisiae under extremely low oxygen availability.
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 102:5, s. 2101-2116
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Forskningsöversikt (refereegranskat)abstract
- The budding yeast Saccharomyces cerevisiae plays an important role in biotechnological applications, ranging from fuel ethanol to recombinant protein production. It is also a model organism for studies on cell physiology and genetic regulation. Its ability to grow under anaerobic conditions is of interest in many industrial applications. Unlike industrial bioreactors with their low surface area relative to volume, ensuring a complete anaerobic atmosphere during microbial cultivations in the laboratory is rather difficult. Tiny amounts of O2 that enter the system can vastly influence product yields and microbial physiology. A common procedure in the laboratory is to sparge the culture vessel with ultrapure N2 gas; together with the use of butyl rubber stoppers and norprene tubing, O2 diffusion into the system can be strongly minimized. With insights from some studies conducted in our laboratory, we explore the question 'how anaerobic is anaerobiosis?'. We briefly discuss the role of O2 in non-respiratory pathways in S. cerevisiae and provide a systematic survey of the attempts made thus far to cultivate yeast under anaerobic conditions. We conclude that very few data exist on the physiology of S. cerevisiae under anaerobiosis in the absence of the anaerobic growth factors ergosterol and unsaturated fatty acids. Anaerobicity should be treated as a relative condition since complete anaerobiosis is hardly achievable in the laboratory. Ideally, researchers should provide all the details of their anaerobic set-up, to ensure reproducibility of results among different laboratories.
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| 8. |
- Eberlein, Christian, et al.
(författare)
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Immediate response mechanisms of Gram-negative solvent-tolerant bacteria to cope with environmental stress : cis-trans isomerization of unsaturated fatty acids and outer membrane vesicle secretion
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102:6, s. 2583-2593
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Forskningsöversikt (refereegranskat)abstract
- Bacteria have evolved an array of adaptive mechanisms enabling them to survive and grow in the presence of different environmental stresses. These mechanisms include either modifications of the membrane or changes in the overall energy status, cell morphology, and cell surface properties. Long-term adaptations are dependent on transcriptional regulation, the induction of anabolic pathways, and cell growth. However, to survive sudden environmental changes, bacterial short-term responses are essential to keep the cells alive after the occurrence of an environmental stress factor such as heat shock or the presence of toxic organic solvents. Thus far, two main short-term responses are known. On the one hand, a fast isomerization of cis into trans unsaturated fatty leads to a quick rigidification of the cell membrane, a mechanism known in some genera of Gram-negative bacteria. On the other hand, a fast, effective, and ubiquitously present countermeasure is the release of outer membrane vesicles (OMVs) from the cell surface leading to a rapid increase in cell surface hydrophobicity and finally to the formation of cell aggregates and biofilms. These immediate response mechanisms just allow the bacteria to stay physiologically active and to employ long-term responses to assure viability upon changing environmental conditions. Here, we provide insight into the two aforementioned rapid adaptive mechanisms affecting ultimately the cell envelope of Gram-negative bacteria.
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| 9. |
- Encarnacao, Joao Crispim, et al.
(författare)
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Detecting ligand interactions in real time on living bacterial cells
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : SPRINGER. - 0175-7598 .- 1432-0614. ; 102:9, s. 4193-4201
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Tidskriftsartikel (refereegranskat)abstract
- Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracerA (R), originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria. This required the development of new immobilization methods for bacteria, since LigandTracer depends on cells being firmly attached to a Petri dish. The evaluated Escherichia coli CJ236 and BL21 as well as Staphylococcus carnosus TM300 strains were immobilized to plastic Petri dishes using antibody capture, allowing us to depict kinetic binding traces of fluorescently labeled antibodies directed against surface-displayed bacterial proteins for as long as 10-15 h. Interaction parameters, such as the affinity and kinetic constants, could be estimated with high precision (coefficient of variation 9-44%) and the bacteria stayed viable for at least 16 h. The other tested attachment protocols were inferior to the antibody capture approach. Our attachment protocol is generic and could potentially also be applied to other assays and purposes.
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| 10. |
- Froslev Nielsen, Jens Christian, 1987, et al.
(författare)
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Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 101:22, s. 8237-8248
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Tidskriftsartikel (refereegranskat)abstract
- The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO2 generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have investigated the impact of industrial AFA treatments on the physiology and transcriptome of the industrial ethanol strain Saccharomyces cerevisiae CAT-1. The investigated AFA included industrially used AFA acquired from Brazilian ethanol plants and commercially available AFA commonly used in the fermentation literature. In batch fermentations, it was shown that industrial AFA compromised growth rates and glucose uptake rates, while commercial AFA had no effect in concentrations relevant for defoaming purposes. Industrial AFA were further tested in laboratory scale simulations of the Brazilian ethanol production process and proved to decrease cell viability compared to the control, and the effects were intensified with increasing AFA concentrations and exposure time. Transcriptome analysis showed that AFA treatments induced additional stress responses in yeast cells compared to the control, shown by an up-regulation of stress-specific genes and a down-regulation of lipid biosynthesis, especially ergosterol. By documenting the detrimental effects associated with chemical AFA, we highlight the importance of developing innocuous systems for foam control in industrial fermentation processes.
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| 11. |
- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; , s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.
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| 12. |
- Gutiérrez, Alicia, et al.
(författare)
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Replenishment and mobilization of intracellular nitrogen pools decouples wine yeast nitrogen uptake from growth.
- 2016
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Ingår i: Applied microbiology and biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 100:7, s. 3255-65
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Tidskriftsartikel (refereegranskat)abstract
- Wine yeast capacity to take up nitrogen from the environment and catabolize it to support population growth, fermentation, and aroma production is critical to wine production. Under nitrogen restriction, yeast nitrogen uptake is believed to be intimately coupled to reproduction with nitrogen catabolite repression (NCR) suggested mediating this link. We provide a time- and strain-resolved view of nitrogen uptake, population growth, and NCR activity in wine yeasts. Nitrogen uptake was found to be decoupled from growth due to early assimilated nitrogen being used to replenish intracellular nitrogen pools rather than being channeled directly into reproduction. Internally accumulated nitrogen was later mobilized to support substantial population expansion after external nitrogen was depleted. On good nitrogen sources, the decoupling between nitrogen uptake and growth correlated well with relaxation of NCR repression, raising the potential that the latter may be triggered by intracellular build-up of nitrogen. No link between NCR activity and nitrogen assimilation or growth on poor nitrogen sources was found. The decoupling between nitrogen uptake and growth and its influence on NCR activity is of relevance for both wine production and our general understanding of nitrogen use.
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| 13. |
- Hao, Xiuli, et al.
(författare)
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Survival in amoeba-a major selection pressure on the presence of bacterial copper and zinc resistance determinants? Identification of a "copper pathogenicity island"
- 2015
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 99:14, s. 5817-5824
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Forskningsöversikt (refereegranskat)abstract
- The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the "copper pathogenicity island" identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs.
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| 14. |
- Hassan, Noor, et al.
(författare)
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Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis
- 2015
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 99:4, s. 1731-1744
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 15. |
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| 16. |
- Hassan, Noor, et al.
(författare)
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Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis
- 2016
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 100:8, s. 3533-3543
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 17. |
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| 18. |
- Hultberg, M., et al.
(författare)
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Fungi-based treatment of brewery wastewater-biomass production and nutrient reduction
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Verlag (Germany). - 0175-7598 .- 1432-0614. ; 101:11, s. 4791-4798
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Tidskriftsartikel (refereegranskat)abstract
- The beer-brewing process produces high amounts of nutrient-rich wastewater, and the increasing number of microbreweries worldwide has created a need for innovative solutions to deal with this waste. In the present study, fungal biomass production and the removal of organic carbon, phosphorus and nitrogen from synthetic brewery wastewater were studied. Different filamentous fungi with a record of safe use were screened for growth, and Trametes versicolor, Pleurotus ostreatus and Trichoderma harzianum were selected for further work. The highest biomass production, 1.78 ± 0.31 g L(-1) of dry weight, was observed when P. ostreatus was used for the treatment, while T. harzianum demonstrated the best capability for removing nutrients. The maximum reduction of chemical oxygen demand, 89% of the initial value, was observed with this species. In the removal of total nitrogen and phosphorus, no significant difference was observed between the species, while removal of ammonium varied between the strains. The maximum reduction of ammonium, 66.1% of the initial value, was also found in the T. harzianum treatment. It can be concluded that all treatments provided significant reductions in all water-quality parameters after 3 days of growth and that the utilisation of filamentous fungi to treat brewery wastewater, linked to a deliberate strategy to use the biomass produced, has future potential in a bio-based society.
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| 19. |
- Hultberg, Malin, et al.
(författare)
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Fungi-based treatment of brewery wastewater—biomass production and nutrient reduction
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 101, s. 4791-4798
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Tidskriftsartikel (refereegranskat)abstract
- The beer-brewing process produces high amounts of nutrient-rich wastewater, and the increasing number of microbreweries worldwide has created a need for innovative solutions to deal with this waste. In the present study, fungal biomass production and the removal of organic carbon, phosphorus and nitrogen from synthetic brewery wastewater were studied. Different filamentous fungi with a record of safe use were screened for growth, and Trametes versicolor, Pleurotus ostreatus and Trichoderma harzianum were selected for further work. The highest biomass production, 1.78 +/- 0.31 g L-1 of dry weight, was observed when P. ostreatus was used for the treatment, while T. harzianum demonstrated the best capability for removing nutrients. The maximum reduction of chemical oxygen demand, 89% of the initial value, was observed with this species. In the removal of total nitrogen and phosphorus, no significant difference was observed between the species, while removal of ammonium varied between the strains. The maximum reduction of ammonium, 66.1% of the initial value, was also found in the T. harzianum treatment. It can be concluded that all treatments provided significant reductions in all water-quality parameters after 3 days of growth and that the utilisation of filamentous fungi to treat brewery wastewater, linked to a deliberate strategy to use the biomass produced, has future potential in a bio-based society.
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| 20. |
- Hüttner, Silvia, 1984, et al.
(författare)
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Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 101:13, s. 5301-5311
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Tidskriftsartikel (refereegranskat)abstract
- The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)d-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 degrees C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.
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| 21. |
- Kaldalu, Niilo, et al.
(författare)
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Persisters - as elusive as ever
- 2016
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 100:15, s. 6545-6553
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Forskningsöversikt (refereegranskat)abstract
- Persisters-a drug-tolerant sub-population in an isogenic bacterial culture-have been featured throughout the last decade due to their important role in recurrent bacterial infections. Numerous investigations detail the mechanisms responsible for the formation of persisters and suggest exciting strategies for their eradication. In this review, we argue that the very term "persistence" is currently used to describe a large and heterogeneous set of physiological phenomena that are functions of bacterial species, strains, growth conditions, and antibiotics used in the experiments. We caution against the oversimplification of the mechanisms of persistence and urge for a more rigorous validation of the applicability of these mechanisms in each case.
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| 22. |
- Katsimpouras, Constantinos, et al.
(författare)
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A thermostable GH26 endo-β-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation
- 2016
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 100:19, s. 8385-8397
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Tidskriftsartikel (refereegranskat)abstract
- The endomannanase gene em26a from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 26, was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative endomannanase, dubbed MtMan26A, was purified to homogeneity (60 kDa) and subsequently characterized. The optimum pH and temperature for the enzymatic activity of MtMan26A were 6.0 and 60 °C, respectively. MtMan26A showed high specific activity against konjac glucomannan and carob galactomannan, while it also exhibited high thermal stability with a half-life of 14.4 h at 60 °C. Thermostability is of great importance, especially in industrial processes where harsh conditions are employed. With the aim of better understanding its structure–function relationships, a homology model of MtMan26A was constructed, based on the crystallographic structure of a close homologue. Finally, the addition of MtMan26A as a supplement to the commercial enzyme mixture Celluclast® 1.5 L and Novozyme® 188 resulted in enhanced enzymatic hydrolysis of pretreated beechwood sawdust, improving the release of total reducing sugars and glucose by 13 and 12 %, respectively.
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| 23. |
- Krustok, Ivo, 1987-, et al.
(författare)
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Effect of lake water on algal biomass and microbial community structure in municipal wastewater based lab-scale photobioreactors
- 2015
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 99:21, s. 6537-6549
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Tidskriftsartikel (refereegranskat)abstract
- Photobioreactors are a novel environmental technology that can produce biofuels with the simultaneous removal of nutrients and pollutants from wastewaters. The aim of this study was to evaluate the effect of the lake water addition to the production of algal biomass, and phylogenetic and functional structure of the algal and bacterial communities in the lab-scale bioreactors treating municipal wastewater.The lake water addition has significant benefit to the overall algal biomass growth and nutrient reduction in the reactors with wastewater and lake water (ratio 70/30 v/v). The metagenome based survey showed that the most abundant algal phylum in these reactors was Chlorophyta with Scenedesmus being the most prominent genus. The most abundant bacterial phyla were Proteobacteria and Bacteroidetes with most dominant families being Sphingobacteriaceae, Cytophagaceae, Flavobacteriaceae, Comamonadaceae, Planctomycetaceae, Nocardiaceae and Nostocaceae. These photobioreactors were also effective in reducing the overall amount of pathogens in wastewater compared to reactors with wastewater/tap water mixture. Functional analysis of the photobioreactor metagenomes revealed an increase in relative abundance genes related to photosynthesis, synthesis of vitamins important for auxotrophic algae, and decrease in virulence and nitrogen metabolism subsystems in lake water reactors.
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| 24. |
- Lundemo, Pontus, et al.
(författare)
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Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 101:3, s. 1121-1131
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Tidskriftsartikel (refereegranskat)abstract
- Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 β-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (rs/rh) from 0.33 to 1.45–2.71. Further increase in rs/rh was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5–10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.
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| 25. |
- Löfblom, John, et al.
(författare)
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Staphylococcus carnosus : from starter culture to protein engineering platform
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 101:23-24, s. 8293-8307
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 26. |
- Maciejewska, B, et al.
(författare)
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Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 101:2, s. 673-684
-
Tidskriftsartikel (refereegranskat)abstract
- Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus BKp15virus^ within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between L-alanine and D-glutamic acid.
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| 27. |
- Mathew, Sindhu, et al.
(författare)
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Xylo- and arabinoxylooligosaccharides from wheat bran by endoxylanases, utilisation by probiotic bacteria, and structural studies of the enzymes
- 2018
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102:7, s. 3105-3120
-
Tidskriftsartikel (refereegranskat)abstract
- Xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) were produced from the insoluble arabinoxylan fraction of pretreated wheat bran by endoxylanases. The glycoside hydrolase (GH) family 10 xylanases GsXyn10A from Geobacillus stearothermophilus and RmXyn10A-CM from Rhodothermus marinus produced the AXOS A3X, A2XX and A2 + 3XX in addition to XOS. RmXyn10A-CM also produced XA2 + 3XX due to its non-conserved aglycone region accommodating additional arabinose substitutions in subsite +2. The GH11 enzymes, Pentopan from Thermomyces lanuginosus and NpXyn11A from Neocallimastix patriciarum had minor structural differences affecting hydrogen bonds in subsites −3 and +3, with similar hydrolysis profiles producing XA3XX as major AXOS and minor amounts of XA2XX but different ratios of X3/X2. In vitro analysis of the prebiotic properties of (A)XOS produced by Pentopan revealed nearly complete uptake of X2 and X3 by the probiotic bacteria Lactobacillus brevis and Bifidobacterium adolescentis. In contrast to previous reports, the GH43 arabinofuranosidase BaAXHd-3 from B. adolescentis cleaved α-1,3-linked arabinose on some single substituted AXOS.
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| 28. |
- Moreno, David, 1986, et al.
(författare)
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Evolutionary engineered Candida intermedia exhibits improved xylose utilization and robustness to lignocellulose-derived inhibitors and ethanol
- 2019
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 103:3, s. 1405-1416
-
Tidskriftsartikel (refereegranskat)abstract
- The development of robust microorganisms that can efficiently ferment both glucose and xylose represents one of the major challenges in achieving a cost-effective lignocellulosic bioethanol production. Candida intermedia is a non-conventional, xylose-utilizing yeast species with a high-capacity xylose transport system. The natural ability of C. intermedia to produce ethanol from xylose makes it attractive as a non-GMO alternative for lignocellulosic biomass conversion in biorefineries. We have evaluated the fermentation capacity and the tolerance to lignocellulose-derived inhibitors and the end product, ethanol, of the C. intermedia strain CBS 141442 isolated from steam-exploded wheat straw hydrolysate. In a mixed sugar fermentation medium, C. intermedia CBS 141442 co-fermented glucose and xylose, although with a preference for glucose over xylose. The strain was clearly more sensitive to inhibitors and ethanol when consuming xylose than glucose. C. intermedia CBS 141442 was also subjected to evolutionary engineering with the aim of increasing its tolerance to inhibitors and ethanol, and thus improving its fermentation capacity under harsh conditions. The resulting evolved population was able to ferment a 50% (v/v) steam-exploded wheat straw hydrolysate (which was completely inhibitory to the parental strain), improving the sugar consumption and the final ethanol concentration. The evolved population also exhibited a better tolerance to ethanol when growing in a xylose medium supplemented with 35.5 g/L ethanol. These results highlight the potential of C. intermedia CBS 141442 to become a robust yeast for the conversion of lignocellulose to ethanol.
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| 29. |
- Morrill, Johan, et al.
(författare)
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β-Mannanase-catalyzed synthesis of alkyl mannooligosides
- 2018
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102:12, s. 5149-5163
-
Tidskriftsartikel (refereegranskat)abstract
- β-Mannanases catalyze the conversion and modification of β-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), β-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable β-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 β-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate β-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three β-mannanases using methanol or 1-hexanol as acceptor. Among the three β-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted β-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using β-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable β-mannans.
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| 30. |
- Nielsen, Jens, et al.
(författare)
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Building a bio-based industry in the Middle East through harnessing the potential of the Red Sea biodiversity
- 2017
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 101:12, s. 4837-4851
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 31. |
- Nordberg Karlsson, Eva, et al.
(författare)
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Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties
- 2018
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102:21, s. 9081-9088
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Forskningsöversikt (refereegranskat)abstract
- Xylan has a main chain consisting of β-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the − 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the − 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects.
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| 32. |
- Passoth, Volkmar, et al.
(författare)
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Biofuel production from straw hydrolysates: current achievements and perspectives
- 2019
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 103, s. 5105-5116
-
Forskningsöversikt (refereegranskat)abstract
- Straw is an agricultural residue of the production of e.g. cereals, rapeseed or sunflowers. It includes dried stalks, leaves, and empty ears and corncobs, which are separated from the grains during harvest. Straw is a promising lignocellulosic feedstock with a beneficial greenhouse gas balance for the production of biofuels and chemicals. Like all lignocellulosic materials, straw is recalcitrant and requires thermochemical and enzymatic pretreatment to enable access to the three major biopolymers of strawthe polysaccharides cellulose and hemicellulose and the polyaromatic compound lignin. Straw is used for commercial ethanol and biogas production. Considerable research has also been conducted to produce biobutanol, biodiesel and biochemicals from this raw material, but more research is required to establish them on a commercial scale. The major hindrance for launching industrial biofuel and chemicals' production from straw is the high cost necessitated by pretreatment of the material. Improvements of microbial strains, production and extraction technologies, as well as co-production of high-value compounds represent ways of establishing straw as feedstock for the production of biofuels, chemicals and food.
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| 33. |
- Paul, Catherine, et al.
(författare)
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A GH57 4-α-glucanotransferase of hyperthermophilic origin with potential for alkyl glycoside production.
- 2015
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 99:17, s. 7101-7113
-
Tidskriftsartikel (refereegranskat)abstract
- 4-α-Glucanotransferase (GTase) enzymes (EC 2.4.1.25) modulate the size of α-glucans by cleaving and reforming α-1,4 glycosidic bonds in α-glucans, an essential process in starch and glycogen metabolism in plants and microorganisms. The glycoside hydrolase family 57 enzyme (GTase57) studied in the current work catalyzes both disproportionation and cyclization reactions. Amylose was converted into cyclic amylose (with a minimum size of 17 glucose monomers) as well as to a spectrum of maltodextrins, but in contrast to glycoside hydrolase family 13 cyclodextrin glucanotransferases (CGTases), no production of cyclodextrins (C6-C8) was observed. GTase57 also effectively produced alkyl-glycosides with long α-glucan chains from dodecyl-β-D-maltoside and starch, demonstrating the potential of the enzyme to produce novel variants of surfactants. Importantly, the GTase57 has excellent thermostability with a maximal activity at 95 °C and an activity half-life of 150 min at 90 °C which is highly advantageous in this manufacturing process suggesting that enzymes from this relatively uncharacterized family, GH57, can be powerful biocatalysts for the production of large head group glucosides from soluble starch.
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| 34. |
- Perez-Zabaleta, Mariel, 1987-, et al.
(författare)
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Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
- 2019
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 103:14, s. 5627-5636
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Tidskriftsartikel (refereegranskat)abstract
- Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.
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| 35. |
- Ravi, Krithika, et al.
(författare)
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Conversion of lignin model compounds by Pseudomonas putida KT2440 and isolates from compost
- 2017
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 101:12, s. 5059-5070
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Tidskriftsartikel (refereegranskat)abstract
- Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p-coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p-coumarate, and 4-hydroxybenzoate were considerably higher (0.26–0.27 h−1) than those on ferulate and vanillate (0.21 and 0.22 h−1), as were the uptake rates.There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted in to vanillate at a rate of 4.87 mmol (gCDW h)−1 after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growthwhen mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules.
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| 36. |
- Schifferdecker, Anna, et al.
(författare)
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Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.
- 2016
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598.
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Tidskriftsartikel (refereegranskat)abstract
- Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.
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| 37. |
- Shi, Tian Qiong, et al.
(författare)
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Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system
- 2018
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 102:22, s. 9541-9548
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Forskningsöversikt (refereegranskat)abstract
- © 2018, The Author(s). The oleaginous yeast Yarrowia lipolytica is widely used for the production of both bulk and fine chemicals, including organic acids, fatty acid-derived biofuels and chemicals, polyunsaturated fatty acids, single-cell proteins, terpenoids, and other valuable products. Consequently, it is becoming increasingly popular for metabolic engineering applications. Multiple gene manipulation tools including URA blast, Cre/LoxP, and transcription activator-like effector nucleases (TALENs) have been developed for metabolic engineering in Y. lipolytica. However, the low efficiency and time-consuming procedures involved in these methods hamper further research. The emergence of the CRISPR/Cas system offers a potential solution for these problems due to its high efficiency, ease of operation, and time savings, which can significantly accelerate the genomic engineering of Y. lipolytica. In this review, we summarize the research progress on the development of CRISPR/Cas systems for Y. lipolytica, including Cas9 proteins and sgRNA expression strategies, as well as gene knock-out/knock-in and repression/activation applications. Finally, the most promising and tantalizing future prospects in this area are highlighted.
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| 38. |
- Speda, Jutta, et al.
(författare)
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Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state
- 2016
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 100:18, s. 7989-8002
-
Tidskriftsartikel (refereegranskat)abstract
- Novel enzymes that are stable in diverse conditions are intensively sought because they offer major potential advantages in industrial biotechnology, and microorganisms in extreme environments are key sources of such enzymes. However, most potentially valuable enzymes are currently inaccessible due to the pure culturing problem of microorganisms. Novel metagenomic and metaproteomic techniques that circumvent the need for pure cultures have theoretically provided possibilities to identify all genes and all proteins in microbial communities, but these techniques have not been widely used to directly identify specific enzymes because they generate vast amounts of extraneous data. In a first step towards developing a metaproteomic approach to pinpoint targeted extracellular hydrolytic enzymes of choice in microbial communities, we have generated and analyzed the necessary conditions for such an approach by the use of a methanogenic microbial community maintained on a chemically defined medium. The results show that a metabolic steady state of the microbial community could be reached, at which the expression of the targeted hydrolytic enzymes were suppressed, and that upon enzyme induction a distinct increase in the targeted enzyme expression was obtained. Furthermore, no cross talk in expression was detected between the two focal types of enzyme activities under their respective inductive conditions. Thus, the described approach should be useful to generate ideal samples, collected before and after selective induction, in controlled microbial communities to clearly discriminate between constituently expressed proteins and extracellular hydrolytic enzymes that are specifically induced, thereby reducing the analysis to only those proteins that are distinctively up-regulated.
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| 39. |
- Steffen-Munsberg, Fabian, et al.
(författare)
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Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases
- 2016
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Berlin/Heidelberg. - 0175-7598 .- 1432-0614. ; 100, s. 4511-4521
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 40. |
- Varriale, Simona, et al.
(författare)
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Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis
- 2018
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 102:12, s. 5185-5196
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Tidskriftsartikel (refereegranskat)abstract
- The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.
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| 41. |
- Wei, Yongjun, 1986, et al.
(författare)
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Cocoa butter-like lipid production ability of non-oleaginous and oleaginous yeasts under nitrogen-limited culture conditions
- 2017
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 101:9, s. 3577-3585
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Tidskriftsartikel (refereegranskat)abstract
- Cocoa butter (CB) extracted from cocoa beans is the main raw material for chocolate production. However, growing chocolate demands and limited CB production has resulted in a shortage of CB supply. CB is mainly composed of three different kinds of triacylglycerols (TAGs), POP (C16:0-C18:1-C16:0), POS (C16:0-C18:1-C18:0), and SOS (C18:0-C18:1-C18:0). The storage lipids of yeasts, mainly TAGs, also contain relative high-level of C16 and C18 fatty acids and might be used as CB-like lipids (CBL). In this study, we cultivated six different yeasts, including one non-oleaginous yeast strain, Saccharomyces cerevisiae CEN.PK113-7D, and five oleaginous yeast strains, Trichosporon oleaginosus DSM11815, Rhodotorula graminis DSM 27356, Lipomyces starkeyi DSM 70296, Rhodosporidium toruloides DSM 70398, and Yarrowia lipolytica CBS 6124, in nitrogen-limited medium and compared their CBL production ability. Under the same growth conditions, we found that TAGs were the main lipids in all six yeasts and that T. oleaginosus can produce more TAGs than the other five yeasts. Less than 3% of the total TAGs were identified as potential SOS in the six yeasts. However, T. oleaginosus produced 27.8% potential POP and POS at levels of 378 mg TAGs/g dry cell weight, hinting that this yeast may have potential as a CBL production host after further metabolic engineering in future.
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| 42. |
- Wei, Yongjun, 1986, et al.
(författare)
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Identification of genes involved in shea butter biosynthesis from Vitellaria paradoxa fruits through transcriptomics and functional heterologous expression
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 103:9, s. 3727-3736
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Tidskriftsartikel (refereegranskat)abstract
- Shea tree (Vitellaria paradoxa) is one economically important plant species that mainly distributes in West Africa. Shea butter extracted from shea fruit kernels can be used as valuable products in the food and cosmetic industries. The most valuable composition in shea butter was one kind of triacylglycerol (TAG), 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0). However, shea butter production is limited and little is known about the genetic information of shea tree. In this study, we tried to reveal genetic information of shea tree and identified shea TAG biosynthetic genes for future shea butter production in yeast cell factories. First, we measured lipid content, lipid composition, and TAG composition of seven shea fruits at different ripe stages. Then, we performed transcriptome analysis on two shea fruits containing obviously different levels of SOS and revealed a list of TAG biosynthetic genes potentially involved in TAG biosynthesis. In total, 4 glycerol-3-phosphate acyltransferase (GPAT) genes, 8 lysophospholipid acyltransferase (LPAT) genes, and 11 diacylglycerol acyltransferase (DGAT) genes in TAG biosynthetic pathway were predicted from the assembled transcriptome and 14 of them were cloned from shea fruit cDNA. Furthermore, the heterologous expression of these 14 potential GPAT, LPAT, and DGAT genes in Saccharomyces cerevisiae changed yeast fatty acid and lipid profiles, suggesting that they functioned in S. cerevisiae. Moreover, two shea DGAT genes, VpDGAT1 and VpDGAT7, were identified as functional DGATs in shea tree, showing they might be useful for shea butter (SOS) production in yeast cell factories.
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| 43. |
- Wiebe, M. G., et al.
(författare)
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A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae
- 2015
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 99:22, s. 9439-47
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Tidskriftsartikel (refereegranskat)abstract
- An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.
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| 44. |
- Wilen, Britt-Marie, 1966, et al.
(författare)
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The mechanisms of granulation of activated sludge in wastewater treatment, its optimization, and impact on effluent quality
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102:12, s. 5005-5020
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Tidskriftsartikel (refereegranskat)abstract
- Granular activated sludge has gained increasing interest due to its potential in treating wastewater in a compact and efficient way. It is well-established that activated sludge can form granules under certain environmental conditions such as batch-wise operation with feast-famine feeding, high hydrodynamic shear forces, and short settling time which select for dense microbial aggregates. Aerobic granules with stable structure and functionality have been obtained with a range of different wastewaters seeded with different sources of sludge at different operational conditions, but the microbial communities developed differed substantially. In spite of this, granule instability occurs. In this review, the available literature on the mechanisms involved in granulation and how it affects the effluent quality is assessed with special attention given to the microbial interactions involved. To be able to optimize the process further, more knowledge is needed regarding the influence of microbial communities and their metabolism on granule stability and functionality. Studies performed at conditions similar to full-scale such as fluctuation in organic loading rate, hydrodynamic conditions, temperature, incoming particles, and feed water microorganisms need further investigations.
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| 45. |
- Xiros, Charilaos, et al.
(författare)
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A cellulolytic fungal biofilm enhances the consolidated bioconversion of cellulose to short chain fatty acids by the rumen microbiome
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 103:8, s. 3355-3365
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Tidskriftsartikel (refereegranskat)abstract
- The ability of the multispecies biofilm membrane reactors (MBM reactors) to provide distinguished niches for aerobic and anaerobic microbes at the same time was used for the investigation of the consolidated bioprocessing of cellulose to short chain fatty acids (SCFAs). A consortium based consolidated bioprocess (CBP) was designed. The rumen microbiome was used as the converting microbial consortium, co-cultivated with selected individual aerobic fungi which formed a biofilm on the tubular membrane flushed with oxygen. The beneficial effect of the fungal biofilm on the process yields and productivities was attributed to the enhanced cellulolytic activities compared with those achieved by the rumen microbiome alone. At 30 °C, the MBM system with Trichoderma reesei biofilm reached a concentration 39% higher (7.3 g/L SCFAs), than the rumen microbiome alone (5.1 g/L) using 15 g/L crystalline cellulose as the substrate. Fermentation temperature was crucial especially for the composition of the short chain fatty acids produced. The temperature increase resulted in shorter fatty acids produced. While a mixture of acetic, propionic, butyric, and caproic acids was produced at 30 °C with Trichoderma reesei biofilm, butyric and caproic acids were not detected during the fermentations at 37.5 °C carried out with Coprinopsis cinerea as the biofilm forming fungus. Apart from the presence of the fungal biofilm, no parameter studied had a significant impact on the total yield of organic acids produced, which reached 0.47 g of total SCFAs per g of cellulose (at 30 °C and at pH 6, with rumen inoculum to total volume ratio equal to 0.372).
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| 46. |
- Yu, Dawei, et al.
(författare)
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Multiple effects of trace elements on methanogenesis in a two-phase anaerobic membrane bioreactor treating starch wastewater.
- 2016
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 100:15, s. 6631-6642
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Tidskriftsartikel (refereegranskat)abstract
- For enhancing anaerobic membrane bioreactor (AnMBR) treating food processing wastewater due to speed-limited methanogenesis step, multiple effects of trace element (TE) supplementation on methanogenesis of a two-phase AnMBR were firstly investigated in batch tests. TE supplementation included individual element, combination and recovery of Fe, Ni, Co, Cu and Zn supplementation. Multiple effects of TE supplementation were highest stimulated by 22.4 ± 5.6 % (TE313) for chemical oxygen demand (COD) removal, 43.1 ± 12.5 % (TE303) for specific methanogenic activity (SMA) and 13.9 ± 3.7 % (TE405) for biomass growth, respectively, although only 7.5 ± 0.6 % (TE106) for methane production. Dosage of TEs played a critical role in methane production, COD removal and biomass growth of the AnMBR's methanogenesis. Low dosages of TE supplementation improved the COD removal and slightly stimulated the COD bioconverting to methane and biomass, but their specific methanation activities were inhibited in the initial rapid methanogenesis stage. Several methanation functional species were increased in abundance like Methanosarcina and Methanoculleus.
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