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Impact of glucose feed rate on productivity and recombinant protein quality in Escherichia coliSandén, Anna Maria January 2005 (has links)
<p>The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate. </p><p>The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, q<sub>p</sub>, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins.</p><p>By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation, </p><p>In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied.</p><p>In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the P<sub>malK </sub>production system.</p><p>A novel fed-batch process based on the promoters for the universal stress proteins A and B (P<sub>uspA</sub>, P<sub>uspB</sub>) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects.</p>
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Impact of glucose feed rate on productivity and recombinant protein quality in Escherichia coliSandén, Anna Maria January 2005 (has links)
The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate. The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, qp, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins. By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation, In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied. In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the PmalK production system. A novel fed-batch process based on the promoters for the universal stress proteins A and B (PuspA, PuspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects. / <p>QC 20101008</p>
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Deammonification Process Kinetics and Inhibition EvaluationMusabyimana, Martin 12 November 2008 (has links)
A number of innovative nitrogen removal technologies have been developed to address the treatment challenges caused by stringent regulations and increasing chemical and energy cost. A major contributing factor to these challenges is the liquid stream originating from the process of dewatering anaerobically digested solids. This liquid, also knows as centrate, reject water or sludge liquor, can cause an increase of up to 25% in ammonia loading. The recently discovered anaerobic ammonia oxidation (anammox) process is a major breakthrough for treatment of these streams as it has the potential to remove up to 85% of nitrogen load without external carbon source addition. The anammox process is combined with another process that oxidizes half of the ammonia to nitrite (nitritation) in a separate reactor such as in the SHARON process, or in the same reactor such as in the DEaMmONification (DEMON) process. Despite intensive laboratory research for the last 10 years to fully understand these processes, there is still a high level of skepticism surrounding the implementation of full-scale systems. The reason for this skepticism could be due to frequent failures observed in the lab scale systems as well as reported slow bacterial growth. We think that this technology might be used more effectively in the future if process kinetics, inhibition and toxicity can be better understood.
This work focused on the DEMON process with a goal to understand the kinetics and inhibition of the system as a whole and the anammox process in particular. A DEMON pilot study was undertaken at the Alexandria Sanitation Authority (ASA) and had several study participants, including ASA, the District of Columbia Water and Sewer Authority (DCWASA), CH2M Hill Inc., Envirosim Ltd, the University of Innsbruck and Virginia Tech. We investigated the growth rate of anammox bacteria within a quasi-optimal environment. Laboratory-scale experiments were conducted to assess anaerobic ammonia oxidation inhibition by nitrite as well as aerobic ammonia oxidation inhibition by compounds present in the DEMON reactor feed, such as a defoaming agent, a sludge conditioning polymer, and residual iron from phosphorus removal practices.
The study revealed that the DEMON process can be efficiently controlled to limit nitrite accumulation capable of causing process inhibition. The target ammonium loading rate of 0.5 kg/m3/d was reached, and no upset was noticed for a loading up to 0.80 kg/m3/d with an HRT of 1.7 days. The ammonia removal efficiency reached an average of 76% while total nitrogen removal efficiency had an average of 52%. Most of the process upsets were caused by aerobic ammonia oxidation failure rather than anammox inhibition. Failure in ammonia oxidation affected pH control, a variable which is at the center of the DEMON process control logic. The pilot study is summarized in Chapter 3 of this Dissertation.
The low anammox maximum specific growth rate (µmax,An) as well as nitrite inhibition are historically reported to be the major process challenges according to the literature, but the degree to which each contributes to process problems differs widely in the literature. In this study, we estimated µmax,An by using the high F:M protocol commonly used for nitrifying populations. We also studied the effect of both short term and sustained nitrite exposure on anammox activity. In this study, µmax,An was estimated to be 0.017 h-1. The study results also suggest that anammox bacteria can tolerate a spike of nitrite-N at concentrations as high as 400 mg/L as long as this concentration is not sustained. Sustained concentrations above 50 mg/L caused a gradual loss of activity over the long term.
Finally, the inhibition of aerobic ammonia oxidizing bacteria (AerAOB) observed in the DEMON reactor was investigated using laboratory experiments and is reported in Chapter 6. AerAOB inhibition was, in most cases, the main reason for process upset. Compounds that were suspected to be the cause of the inhibition were tested. The study noticed that a defoaming agent, polymer and ferrous iron had some inhibiting properties at the concentrations tested. / Ph. D.
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Microalgal growth and lipid production : trends, multiple regression models, and validation in a photobioreactorGuha Roy, Aimee January 2014 (has links)
Algae are a promising new source of oil for biodiesel. They are aquatic organisms that do not require cropland, and they can produce many useful side-products for bioenergy, aquaculture, and nutraceutical production. To be cost-effective, algae need high and reliable oil productivities; however, there is still a great deal to learn about the effects of culturing conditions on algae growth rates and lipid production. These culturing conditions include light intensity, gas flow, use of CO<sub>2</sub>, and culture volume. An extensive database of published research on algae growth rates and lipid contents under a wide variety of environmental conditions was prepared. By graphing data from 116 publications on 132 microalgae species, several key trends were identified relating to culturing parameters and algae biomass and lipid production. In addition, data from 131 publications on 128 microalgae species were graphed to look at presence of flagella, nutrient limitation, lipid productivity, and productivity tradeoffs. Moreover, cell size information was gathered for 146 species. The interactions between culture variables are complex, so it is difficult to quantify the degree to which each culture variable affects algae growth rates and lipid production. Therefore, several multivariate analyses were performed to generate a set of general and simple predictive models to assess specific growth rates, maximum lipid contents, and volumetric lipid productivities. These models were used to determine which culture parameters were significant predictors of algae growth rates and lipid production, and the contribution of each environmental parameter was quantified. In addition to models for algae in general, genera specific models were prepared for Chlorella, Isochrysis, Nannochloropsis, Phaeodactylum, and Tetraselmis. These models show high predictive capabilities, and they greatly extend the range of species-specific multiple regression models available. Furthermore, one Tetraselmis model was validated using Tetraselmis impellucida growth experiments in a large novel photobioreactor.
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Interação entre Cylindrospermopsis raciborskii e Microcystis aeruginosa: implicações no crescimento de culturas e na produção de microcistinas / Interaction between Cylindrospermopsis raciborskii and Microcystis aeruginosa: implications in cultures growth and microcystins productionSantos, Paulo Vagner dos 18 June 2009 (has links)
Cianobactérias desempenham importante papel ecológico em diversos ecossistemas terrestres e aquáticos. Contudo, tornam-se problema de saúde pública quando biossintetizam compostos secundários tóxicos, denominados cianotoxinas, as quais podem atuar sobre diversos organismos. Sistemas aquáticos impactados favorecem o desenvolvimento destes microrganismos, que podem tornar-se dominantes em função dos diversos mecanismos que lhes conferem vantagens adaptativas em relação aos demais grupos fitoplanctônicos. Muitas vezes, elas apresentam-se sob forma de florescimento, cuja toxicidade varia de acordo com espécies e cepas que o compõem. Apesar das cianobactérias Cylindrospermopsis raciborskii e Microcystis aeruginosa formarem florações, terem potencial tóxico, serem competitivas e estarem presentes em diversos reservatórios, é desconhecido o efeito da interação entre elas na produção de microcistina. Para esclarecer esta questão, nesta pesquisa, foi estudada a interação entre cepas de C. raciborskii e M. aeruginosa visando: caracterizar o cultivo das espécies em monocultura e cultura mista; verificar se o caráter tóxico das cepas favoreceu a competição; mensurar a microcistina de M. aeruginosa e avaliar o efeito da interação sobre esta variável. Em salas climatizadas de cultivo (22ºC, fotoperíodo 12h, 60 \'mü\'mol photon/\'M POT.2\'.s) experimentos de interação foram realizados em culturas batch compostas por três tratamentos: (1) monocultura de M. aeruginosa, (2) monocultura de C. raciborskii, (3) cultura mista de M. aeruginosa e C. raciborskii. Foram realizadas análises da densidade e biovolume dos organismos, da velocidade específica de crescimento (\'mü\') e tempo de duplicação (Td) e da concentração de microcistinas, clorofila-a e nutrientes. A interação reduziu em até 22% a \'mü\' das linhagens estudadas. Tanto C. raciborskii quanto M. aeruginosa apresentaram vantagem competitiva em pelo menos um dos ensaios e, no caso de linhagem tóxica de M. aeruginosa, verificou-se que o seu caráter tóxico favoreceu a competição da espécie com linhagem atóxica de C. raciborskii. Além disso, M. aeruginosa elevou em até 53% a síntese de microcistina ao interagir com C. raciborskii. Este processo poderia se relacionar a três principais mecanismos: competição por interferência, produção de alelopáticos e comunicação química. Estudos de balanços genéticos dos florescimentos bem como da possível comunicação química existente entre cianobactérias são ferramentas investigativas importantes que poderão ajudar no aprimoramento da prevenção e manejo de florações tóxicas. / Cyanobacteria play important ecological role in many terrestrial and aquatic ecosystems. However, they become a public health problem when synthesize secondary toxic compounds, known as cyanotoxins, which may act on several organisms. Impacted aquatic systems favor their growth and these microorganisms can become dominant due to mechanisms that confer them adaptive advantages in relation to other phytoplanktonic groups. Frequently, cyanobacteria can form water blooms, whose toxicity varies according to their species and strains composition. Although the cyanobacteria Cylindrospermopsis raciborskii and Microcystis aeruginosa can form blooms, have toxic potential, are competitive and present in several reservoirs, it is unknown the interaction effect between them in the production of microcystins. To solve this question, the interaction among strains of C. raciborskii and M. aeruginosa was studied to: characterize the culture of the species in monoculture and mixed culture; check whether the character of toxic strains favored the competition; measure microcystin concentration produced by M. aeruginosa and evaluate interaction effect on this variable. In climatized growth room (22°C, 12h photoperiod, 60 \'mü\'mol photon/\'M POT.2\'.s) four interaction experiments composed by three treatments were performed in batch cultures: (1) monoculture of M. aeruginosa, (2) monoculture of C. raciborskii, (3) mixed culture of M. aeruginosa and C. raciborskii. Density and biovolume, specific growth rate (\'mü\'), doubling time (Td) and concentration of microcystin, chlorophyll-a and nutrients analyses were performed. The interaction reduced up to 22% the of studied strains. Both C. raciborskii and M. aeruginosa presented competitive advantage in at least one of the tests. In the case of toxic strain of M. aeruginosa, it was found that its toxic character favored the competition with non-toxic strain of C. raciborskii. Furthermore, M. aeruginosa increased by 53% the synthesis of microcystin while interacting with C. raciborskii. This process could be related to three main mechanisms: interference competition, communication and production of allelopathic chemicals. Genetical balance studies of blooms as the possible chemical communication existing among cyanobacteria are important investigative tools that might help prevention and management improvement of toxic blooms.
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Interação entre Cylindrospermopsis raciborskii e Microcystis aeruginosa: implicações no crescimento de culturas e na produção de microcistinas / Interaction between Cylindrospermopsis raciborskii and Microcystis aeruginosa: implications in cultures growth and microcystins productionPaulo Vagner dos Santos 18 June 2009 (has links)
Cianobactérias desempenham importante papel ecológico em diversos ecossistemas terrestres e aquáticos. Contudo, tornam-se problema de saúde pública quando biossintetizam compostos secundários tóxicos, denominados cianotoxinas, as quais podem atuar sobre diversos organismos. Sistemas aquáticos impactados favorecem o desenvolvimento destes microrganismos, que podem tornar-se dominantes em função dos diversos mecanismos que lhes conferem vantagens adaptativas em relação aos demais grupos fitoplanctônicos. Muitas vezes, elas apresentam-se sob forma de florescimento, cuja toxicidade varia de acordo com espécies e cepas que o compõem. Apesar das cianobactérias Cylindrospermopsis raciborskii e Microcystis aeruginosa formarem florações, terem potencial tóxico, serem competitivas e estarem presentes em diversos reservatórios, é desconhecido o efeito da interação entre elas na produção de microcistina. Para esclarecer esta questão, nesta pesquisa, foi estudada a interação entre cepas de C. raciborskii e M. aeruginosa visando: caracterizar o cultivo das espécies em monocultura e cultura mista; verificar se o caráter tóxico das cepas favoreceu a competição; mensurar a microcistina de M. aeruginosa e avaliar o efeito da interação sobre esta variável. Em salas climatizadas de cultivo (22ºC, fotoperíodo 12h, 60 \'mü\'mol photon/\'M POT.2\'.s) experimentos de interação foram realizados em culturas batch compostas por três tratamentos: (1) monocultura de M. aeruginosa, (2) monocultura de C. raciborskii, (3) cultura mista de M. aeruginosa e C. raciborskii. Foram realizadas análises da densidade e biovolume dos organismos, da velocidade específica de crescimento (\'mü\') e tempo de duplicação (Td) e da concentração de microcistinas, clorofila-a e nutrientes. A interação reduziu em até 22% a \'mü\' das linhagens estudadas. Tanto C. raciborskii quanto M. aeruginosa apresentaram vantagem competitiva em pelo menos um dos ensaios e, no caso de linhagem tóxica de M. aeruginosa, verificou-se que o seu caráter tóxico favoreceu a competição da espécie com linhagem atóxica de C. raciborskii. Além disso, M. aeruginosa elevou em até 53% a síntese de microcistina ao interagir com C. raciborskii. Este processo poderia se relacionar a três principais mecanismos: competição por interferência, produção de alelopáticos e comunicação química. Estudos de balanços genéticos dos florescimentos bem como da possível comunicação química existente entre cianobactérias são ferramentas investigativas importantes que poderão ajudar no aprimoramento da prevenção e manejo de florações tóxicas. / Cyanobacteria play important ecological role in many terrestrial and aquatic ecosystems. However, they become a public health problem when synthesize secondary toxic compounds, known as cyanotoxins, which may act on several organisms. Impacted aquatic systems favor their growth and these microorganisms can become dominant due to mechanisms that confer them adaptive advantages in relation to other phytoplanktonic groups. Frequently, cyanobacteria can form water blooms, whose toxicity varies according to their species and strains composition. Although the cyanobacteria Cylindrospermopsis raciborskii and Microcystis aeruginosa can form blooms, have toxic potential, are competitive and present in several reservoirs, it is unknown the interaction effect between them in the production of microcystins. To solve this question, the interaction among strains of C. raciborskii and M. aeruginosa was studied to: characterize the culture of the species in monoculture and mixed culture; check whether the character of toxic strains favored the competition; measure microcystin concentration produced by M. aeruginosa and evaluate interaction effect on this variable. In climatized growth room (22°C, 12h photoperiod, 60 \'mü\'mol photon/\'M POT.2\'.s) four interaction experiments composed by three treatments were performed in batch cultures: (1) monoculture of M. aeruginosa, (2) monoculture of C. raciborskii, (3) mixed culture of M. aeruginosa and C. raciborskii. Density and biovolume, specific growth rate (\'mü\'), doubling time (Td) and concentration of microcystin, chlorophyll-a and nutrients analyses were performed. The interaction reduced up to 22% the of studied strains. Both C. raciborskii and M. aeruginosa presented competitive advantage in at least one of the tests. In the case of toxic strain of M. aeruginosa, it was found that its toxic character favored the competition with non-toxic strain of C. raciborskii. Furthermore, M. aeruginosa increased by 53% the synthesis of microcystin while interacting with C. raciborskii. This process could be related to three main mechanisms: interference competition, communication and production of allelopathic chemicals. Genetical balance studies of blooms as the possible chemical communication existing among cyanobacteria are important investigative tools that might help prevention and management improvement of toxic blooms.
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Effects of microcarrier concentration, agitation rate, and serum concentration on the specific growth rate of mouse L cells in batch culturesNorcio, Lawrence P. January 1995 (has links)
No description available.
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Superexpressão de CDC48 e HSP104 na levedura Saccharomyces cerevisiae. / Overexpression of CDC48 e HSP104 in the yeast Saccharomyces cerevisiae.Franco, Letícia Veloso Ribeiro 19 December 2016 (has links)
Este trabalho iniciou-se com o objetivo de superexpressar proteínas com atividade ATPase, como tentativa de alterar a conservação de energia livre na levedura S. cerevisiae, de maneira a aumentar o rendimento da fermentação alcoólica. Para isso, duas ATPases nativas de S. cerevisiae, as chaperonas codificadas pelos genes HSP104 e CDC48, foram superexpressas, individualmente, sob o controle de quatro promotores de diferentes forças, provocando diferentes gastos energéticos na levedura. Entretanto, não foi possível obter aumento no rendimento em etanol. Em seguida, foi feito um estudo que visou comparar essas linhagens em situação de estresse térmico, ácido ou osmótico, tipicamente encontrados no processo brasileiro de produção de etanol. A 40 °C, uma linhagem superexpressando CDC48 apresentou velocidade específica máxima de crescimento 17 % maior que a linhagem de referência, indicando maior tolerância ao estresse térmico. Finalmente, avaliou-se Hsp104 e Cdc48 em um contexto fisiológico no qual as atividades dessas proteínas pudessem ser mais requeridas. Como as chaperonas moleculares são conhecidas por agirem como primeira linha de defesa contra a formação de proteínas incorretamente enoveladas e agregados proteicos, estudaram-se a morfologia e a fisiologia da superexpressão de HSP104 e CDC48 em linhagens com desarranjo no controle de qualidade de proteínas intracelulares, causado por mutações no proteassomo 20S. A superexpressão de CDC48 ou HSP104 reverteu em parte a morfologia alterada de alguns desses mutantes de proteassomo. / The initial goal of this work was to overexpress proteins with ATPase activity in Saccharomyces cerevisiae, as an attempt to alter the conservation of free energy in this yeast, in order to increase alcoholic fermentation yield. Therefore, two native S. cerevisiae ATPases, the chaperones encoded by HSP104 and CDC48, were individually overexpressed under the control of four promoters with different strengths, in order to provoke different levels of energy expenditure. Increments in the ethanol yield could not be observed in any of the constructed strains. Subsequently, a study was carried out to compare these mutant strains with reference strains under heat, acid or osmotic stress, which are typically found in the industrial fuel ethanol production in Brazil. At 40 oC a strain overexpressing CDC48 displayed a maximum specific growth rate 17 % higher than that of the reference strain, indicating a greater tolerance to heat stress. Finally, Hsp104 and Cdc48 were evaluated in a physiological context in which the activity of these proteins would be required in a higher level. Since molecular chaperones are known to act as the first defense line against the formation of misfolded proteins and aggregates, the physiological and morphological effects of HSP104 or CDC48 overexpression were analyzed in strains with protein quality control disarrangements caused by mutations in proteasome 20S. The overexpression of either CDC48 or HSP104 partially reversed the altered morphology of some of these proteasome mutants.
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Impact of glucose uptake rate on recombinant protein production in Escherichia coliBäcklund, Emma January 2011 (has links)
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. / QC 20110608
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Analytical tools for monitoring and control of fermentation processesSundström, Heléne January 2007 (has links)
The overall objective of this work has been to adopt new developments and techniques in the area of measurement, modelling and control of fermentation processes. Flow cytometry and software sensors are techniques which were considered ready for application and the focus was set on developing tools for research aiming at understanding the relationship between measured variables and process quality parameters. In this study fed-batch cultivations have been performed with two different strains of Escherichia coli (E.coli) K12 W3110 with and without a gene for the recombinant protein promegapoietin. Inclusion body formation was followed during the process with flow cytometric detection by labelling the inclusion bodies with first an antibody against the protein promegapoietin and then a second fluorescent anti-antibody. The approach to label inclusion bodies directly in disintegrated and diluted cell slurry could be adopted as a method to follow protein production during the process, although the labelling procedure with incubation times and washings was somewhat time-consuming (1.5 h). The labelling of inclusion bodies inside the cells to follow protein production was feasible to perform, although an unexplained decrease in the relative fluorescence intensity occurred late in process. However, it is difficult to translate this qualitative measurement into a quantitative one, since a quantitative protein analysis should give data proportional to the volume, while the labelling of the spheric inclusion bodies gives a signal corresponding to the area of the body, and calibration is not possible. The methods were shown to be useful for monitoring inclusion body formation, but it seems difficult to get quantitative information from the analysis. Population heterogeneity analysis was performed, by using flow cytometry, on a cell population, which lost 80-90% viability according to viable count analysis. It was possible to show that the apparent cell death was due to cells incapable of dividing on agar plates after induction. These cells continued to produce the induced recombinant protein. It was shown that almost all cells in the population (≈97%) contained PMP, and furthermore total protein analysis of the medium indicated that only about 1% of the population had lysed. This confirms that the "non-viable" cells according to viable count by cfu analysis produced product. The software sensors XNH3 and µNH3, which utilises base titration data to estimate biomass and specific growth rate was shown to correlate well with the off-line analyses during cultivation of E. coli W3110 using minimal medium. In rich medium the µNH3 sensor was shown to give a signal that may be used as a fingerprint of the process, at least from the time of induction. The software sensor KLaC* was shown to respond to foaming in culture that probably was caused by increased air bubble dispersion. The RO/S coefficient, which describes the oxygen to substrate consumption, was shown to give a distinct response to stress caused by lowered pH and addition of the inducing agent IPTG. The software sensor for biomass was applied to a highly automated 6-unit multi-bioreactor system intended for fast process development. In this way also specific rates of substrate and oxygen consumption became available without manual sampling. / QC 20100819
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