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- Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinasePublication . Calado, Cecília; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm−3 to 10 g dm−3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7‐fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9‐fold. After implementation of a fed‐batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8‐, 2.9‐, 5.3‐ and 6.4‐fold, respectively, in relation to the previously‐mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins.
- Development of scalable processes to produce therapeutic mesenchymal stromal cell-derived extracellular vesicles and their characterizationPublication . Cunha, Raquel M. S.; Vargas, Elga; Pires, Filipa; Calado, Cecília; Cabral, Joaquim M.S.; Silva, Cláudia; Fernandes, AnaIn summary, this study contributes to the establishment of a scalable process for MSC-EV production.
- Impact of the human mesenchymal stem cells donor on conditional medium compositionPublication . Pereira, Maria João Canha; Ramalhete, Luís; Aleixo, Sandra; da Silva, Cláudia L.; Cabral, Joaquim M.S.; Calado, Cecília; Fernandes, AnaExosomes produced by Mesenchymal Stem Cells (MSCs) can represent a very appealing strategy for cell-free therapies. However, to achieve this reality it is necessary to further understand the process associated to the MSC culture when conditioned to produce exosomes. In the present work, it was evaluated how different MSC obtained from different donors may affect the conditioned media composition and how this can be influenced by the conditioned media type (DMEM versus Xeno-Free medium, XF). The molecular fingerprint of the conditioned media composition was obtained by mid-infrared (MIR) spectroscopy, as this technique reflects fundamental vibrations of a high diversity of functional chemical groups present in biological samples. It was observed by principal component analysis of the second derivative spectra of conditioned media that the media chemical composition depends more from the MSCs donor than the conditioning days. Diverse spectral regions, characteristic of defined chemical groups, enabled to discriminate the chemical composition of the media according to the MSC donor. All of this was observed in both types of media (DMEM and XF). This work is a step forward to understand how different MSC donors and conditioned media may affect the exosomes characteristics.
- Optimisation of culture conditions and characterisation of cutinase produced by recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Taipa, M. Ângela; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The optimum culture dissolved oxygen concentration and culture pH for the production of cutinase from Fusarium solani pisi by the recombinant Saccharomyces cerevisiae SU50 strain was investigated. Dissolved oxygen concentrations in the fermentation culture of 5, 30 and 60% of air saturation were evaluated. A high cutinase production, specific cutinase activity and the highest cutinase yield on biomass and the highest specific cutinase production rate were obtained with a 5% of air saturation, which could have impact on process economics. Furthermore, at a low dissolved oxygen concentration, the specific growth rate, specific cutinase production rate, cutinase yield on biomass, cutinase activity and specific cutinase activity were increased when the pH control was changed from 5.25 to 6.25. The cutinase, accumulated in the yeast culture presents two glycosilated isoforms with molecular weights of 22.8 and 24.9 kDa measured by SDS-PAGE. Furthermore, cutinase in clarified culture samples presents a linear relationship between estereolytic and lypolitic activity and a high stability at room temperature.
- Scaling-up the manufacturing of well-characterized mesenchymal stromal cell-derived extracellular vesicles for biomedical applicationsPublication . Fernandes, Ana; Rosa, Sara; Silva, Ricardo; Cunha, Raquel M. S.; Fuzeta, Miguel de Almeida; Calado, Cecília; Carvalho, Carla; Cabral, Joaquim M.S.; Azevedo, Ana; Silva, CláudiaThe platform established herein could be applied to the production of well-characterized SPC-EVs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as EVs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer.
- Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiaePublication . Calado, Cecília; Monteiro, Sandra M. S.; Cabral, Joaquim M.S.; Fonseca, Luís P. P.The importance of controlling the expression of heterologous cutinase in a recombinant Saccharomyces cerevisiae SU50 strain was investigated. Maximum specific growth rate and the biomass yield increased 1.91 and 1.16 fold, respectively, when cutinase production was induced by galactose in a pre-fermentation step. However, only 19% of specific cell activity was obtained in comparison to other fermentations following a pre-fermentation step without induction of cutinase expression. Thus, the pre-fermentation step was performed using a selective medium not containing galactose, and the fermentation was performed with a cheaper and complex non-selective medium containing galactose. Under these conditions, and with the aim of maximising the specific cutinase activity, a pre-fermentation with low volume and high density of viable cells must be used. However, due to the low pre-fermentation volume, low yeast cell concentrations and low specific cell activities were obtained after 96 h of fermentation. Otherwise, when the aim was to maximise cutinase yield and productivity, a pre-fermentation volume of 10% (v/v) in relation to fermentation and in the exponential growth phase with a cell concentration between 1.1 and 1.8 g dcw/l should be used. A higher pre-fermentation volume, such as 20% (v/v), would still be economical in the case of a pre-fermentation with low cell density or low cell viability.
- Design and operation of a fully controlled platform for the production and purification of well-defined mesenchymal stromal cell (MSC)-derived extracellular vesiclesPublication . Fernandes, Ana; Rosa, Sara; Silva, Ricardo; Cunha, Raquel; Fuzeta, Miguel Almeida; Calado, Cecília; Carvalho, Carla; Cabral, Joaquim M.S.; Azevedo, AnaCurrently, the production of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC) is performed in laborious and time-consuming static culture systems using serum-containing media, and their purification is through ultracentrifugation based methods. The whole process lacks on resolution, selectivity and capacity. Additionally, several differences were observed, in terms of the cargo of EVs, between EVs isolated from culture supernatants of MSC expanded under different culture conditions, stressing the importance of controlling all culture process parameters. A robust and indepth process to manufacture MSC-EVs may provide a new therapeutic paradigm for cell-free MSC-based therapies.
- Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strainPublication . Calado, Cecília; Almeida, Claúdio; Cabral, Joaquim M.S.; Fonseca, Luís P. P.Saccharomyces cerevisiae SU50 strain was cultivated with different concentrations of glucose and galactose with the aim of increasing cutinase activity, cutinase yield on the carbon source, and bioreactor productivity. Cultivations in shake flasks with galactose as the sole carbon source, with sugar concentrations between 10 and 40 g/l, exhibited growth-associated cutinase production and a constant specificity of cutinase secretion. Furthermore, as the galactose concentration increased to values higher than 15 g/l, a progressively higher maximum specific galactose consumption rate and a consequent higher alcoholic fermentation occurred, resulting in progressively lower biomass yields on the carbon source and cutinase yields on biomass. Using high glucose and galactose concentrations in a well-aerated bioreactor resulted in a high biomass productivity (0.5 g dcw/l/h), a high cutinase yield on biomass (21.5 U/mg dew), a final high cutinase secretion efficiency (97%), and plasmid stability (99%). Based on these studies, a two phase fed-batch cultivation strategy was developed. A batch phase with high glucose and galactose concentrations, followed by a fedbatch with a constant feed rate with galactose as the sole carbon source in order to minimize the repression of the GAL 7 promoter, were established. The feed rate was estimated to maintain a pre-determined concentration of galactose (20 g/l) on the culture medium in order to maximize the efficiency of cutinase secretion and minimize the galactose alcoholic fermentation. By this cultivation strategy, enhancements of 3.6-fold in cutinase activity, 1.2-fold in cutinase yield on the carbon source, and 8.7-fold culture productivity were obtained in relation to a batch cultivation performed in shake flasks with 20 g/l of galactose.
- Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspectsPublication . Ferreira, Bruno S.; Calado, Cecília; Keulen, Frederik van; Fonseca, Luís P. P.; Cabral, Joaquim M.S.; R., Da Fonseca M. M.Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell—required for the induction of cutinase production—and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.
- A flow injection analysis system for on‐line monitoring of cutinase activity at outlet of an expanded bed adsorption column almost in real timePublication . Almeida, Cláudio F.; Calado, Cecília; Bernardino, Susana A.; Cabral, Joaquim M.S.; Fonseca, Luís P. P.Expanded bed adsorption (EBA) was used to recover, concentrate and purify Fusarium solani pisi cutinase, secreted by a recombinant Saccharomyces cerevisiae strain, directly from a whole fermentation culture. A flow injection analysis (FIA) system for monitoring Fusarium solani pisi cutinase based on microencapsulation of p‐nitrophenylbutyrate (p‐NPB) in a micellar system (sodium cholate, tetrahydrofuran, phosphate buffer) was developed for monitoring this target enzyme at the outlet tube of the EBA column. Slight differences in yeast cultivation conditions during cutinase production may influence the fermentation performance, which affects directly the adsorption of cutinase during the loading step and consequently the efficiency of the EBA process. This effect can be especially relevant when it is necessary to stop the application of feedstock to the EBA column when the outlet concentration (A) of the desired product is lower than 5% of the feed concentration (Ao). Excellent correlations between the FIA system and the off‐line analytical method for monitoring cutinase activity during the different EBA steps were obtained. Additionally, the blocking/fouling of the sample injector and tubes of the FIA system initially observed were eliminated due to the excellent surfactant properties of the sodium cholate contained in the phosphate buffer and used to dilute the enzyme samples. This FIA system was shown to be a powerful analytical tool for monitoring cutinase activity almost in real time (45–60 s), maximizing enzyme adsorption while minimizing product loss and consequently maximizing the recovery yield of the product. Copyright © 2006 Society of Chemical Industry