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- Bäcklund, Emma
(författare)
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Impact of glucose uptake rate on recombinant protein production in Escherichia coli
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures. The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.
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| 4. |
- Immerstrand, Tina, et al.
(författare)
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Characterization of the properties of Pediococcus parvulus for probiotic or protective culture use.
- 2010
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Ingår i: Journal of Food Protection. - 0362-028X. ; 73:5, s. 960-966
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Tidskriftsartikel (refereegranskat)abstract
- Pediococcus parvulus 2.6 (previously Pediococcus damnosus 2.6, here confirmed as P. parvulus by 16S DNA sequencing) displayed antibacterial activity toward several bacterial species, including isolates found as contaminants in oats, herein genetically identified as Bacillus cereus. No inhibition of Listeria monocytogenes was found under the conditions used. Antibacterial activity was retrieved after ammonium sulfate or acetone precipitation showed it to be peptide mediated. P. parvulus 2.6 has previously shown good technological properties in oat-based products. This, together with the currently found inhibition of food spoilage microorganisms like B. cereus, makes it suitable as a food protective culture. Survival trials of P. parvulus 2.6 at conditions mimicking the gastrointestinal tract were prompted by previously found cholesterol-lowering effects in humans after consumption of oat products cofermented by using P. parvulus 2.6 and Bifidobacterium spp. Viability was measured with in vitro, gutlike simulations at 37 degrees C. High survival was shown under two of three conditions (gastric juice, bile, and small intestine juice), defined as main obstacles of the gastrointestinal tract. The critical step was bile exposure. At a concentration of 20%, viability was low, but 0.3% bile (mean concentration in the intestine) did not have a major influence on growth. Viability of P. parvulus 2.6 was significantly decreased in gastric juice at pH 1.5 (with pepsin), but it was not significantly affected at pH 2.5, and was also improved at a lower pH in 20% oat milk. Viability was judged sufficient for colonization at gutlike conditions, qualifying the strain for further probiotic studies.
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| 5. |
- Latifian, Maryam, et al.
(författare)
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Nitrogen and Phosphorus Removal from Urine by Sequential Struvite Formation and Recycling Process
- 2014
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Ingår i: Clean - Soil, Air, Water. - : Wiley. - 1863-0650. ; 42:8, s. 1157-1161
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Tidskriftsartikel (refereegranskat)abstract
- Recovering nutrients from urine as solid minerals such as struvite can eliminate many hurdles related to the use of liquid urine as fertilizer in the current urine separation systems. The focus of this study has been to increase nitrogen and phosphorus removal efficiencies by developing a struvite formation and recycling process. Struvite crystallization was carried out in urine samples by addition of MgO where 92% of phosphorus as orthophosphate was recovered. In order to optimize the use of calcined struvite in the struvite recycling step, four calcination treatments were tested and calcination at 200 degrees C for 3 h was chosen for further experiments. To remove the remaining ammonia present in urine a struvite recycling step was used subsequently. Through sequential struvite formation and struvite recycling process up to 90% of ammonium nitrogen was successfully removed.
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| 6. |
- 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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| 7. |
- Lindström, Cecilia, et al.
(författare)
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Evaluation of commercial microbial hydrocolloids concerning their effects on plasma lipids and caecal formation of SCFA in mice
- 2012
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Ingår i: Food Hydrocolloids. - : Elsevier BV. - 0268-005X. ; 28:2, s. 367-372
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Tidskriftsartikel (refereegranskat)abstract
- Exopolysaccharides (EPS) are excreted by microorganisms into the surrounding environment and have been shown to have various physiological effects and are commonly used as food additives due to their rheological properties. Four commercially available microbial EPS with different polymeric structures and composition were tested in LDL receptor knock-out mice to investigate their effect on blood cholesterol, lipoproteins and caecal formation of SCFA. After four weeks on a Western diet supplemented with 4% EPS there were significant increases in caecal content and caecal tissue weight for the EPS groups compared to the control. The total pool of caecal short chain fatty acids was increased when mice were fed scleroglucan, xanthan and dextran. There were no differences in plasma cholesterol levels on the experimental diets compared to the control. Plasma triglycerides did not differ between groups. The results indicate that EPS supplementation to a Western diet may help in maintaining a healthy intestinal environment. (C) 2012 Elsevier Ltd. All rights reserved.
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| 8. |
- Lindström, Cecilia, et al.
(författare)
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Oral administration of live exopolysaccharide-producing Pediococcus parvulus, but not purified exopolysaccharide suppressed Enterobacteriaceae without affecting bacterial diversity in caecum of mice.
- 2013
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Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 79:16, s. 5030-5037
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Tidskriftsartikel (refereegranskat)abstract
- Growing evidence indicates that the gut microbiota could have an important role in the development of diet and lifestyle induced diseases. It has been shown that modulation of the gut microbiota by means of probiotics and prebiotics could improve host health. An oat based product fermented by the exopolysaccharide (EPS)-producing Pediococcus parvulus 2.6 has been reported to have a bifidogenic effect. To find out whether the effect could be attributed to the EPS or the bacterium, mice were fed a diet supplemented with 2 % purified EPS or 10(8) CFU/g of live P. parvulus 2.6 for six weeks. Both supplementations altered the gut microbiota composition but in different directions. Purified EPS not only significantly lowered the microbial diversity (P < 0.001) but decreased the bifidobacterial population (P = 0.01). In contrast, the live EPS-producing P. parvulus 2.6 antagonized Enterobacteriaceae without disturbing the homeostasis of the caecal microbiota.
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