Browsing by Author "Martínez, Irene"
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Item Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures(Public Library of Science, 2015) Vergara, Mauricio; Berrios, Julio; Martínez, Irene; Díaz-Barrera, Alvaro; Acevedo, Cristian; Reyes, Juan G.; Gonzalez, Ramon; Altamirano, Claudia; Bioengineering; Chemical and Biomolecular EngineeringBackground: Chinese hamster ovary (CHO) cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum. Methods: In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD) processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA) were carried out at two temperatures (37°C and 33°C) and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system) or ERAD II (Autophagosoma/Lisosomal system) pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1) and low (0.012 h-1) dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA). Results and Conclusion: Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO cells is modulated predominantly by specific growth rate, indicating that the culture temperature has a lower weighted effect within the range of conditions evaluated in this work.Item Metabolic engineering of carbon and redox flow in the production of small organic acids(Springer, 2014) Thakker, Chandresh; Martínez, Irene; Li, Wei; San, Ka-Yiu; Bennett, George N.; Bioengineering; Biosciences; Chemical and Biomolecular EngineeringThe review describes efforts toward metabolic engineering of production of organic acids. One aspect of the strategy involves the generation of an appropriate amount and type of reduced cofactor needed for the designed pathway. The ability to capture reducing power in the proper form, NADH or NADPH for the biosynthetic reactions leading to the organic acid, requires specific attention in designing the host and also depends on the feedstock used and cell energetic requirements for efficient metabolism during production. Recent work on the formation and commercial uses of a number of small mono- and diacids is discussed with redox differences, major biosynthetic precursors and engineering strategies outlined. Specific attention is given to those acids that are used in balancing cell redox or providing reduction equivalents for the cell, such as formate, which can be used in conjunction with metabolic engineering of other products to improve yields. Since a number of widely studied acids derived from oxaloacetate as an important precursor, several of these acids are covered with the general strategies and particular components summarized, including succinate, fumarate and malate. Since malate and fumarate are less reduced than succinate, the availability of reduction equivalents and level of aerobiosis are important parameters in optimizing production of these compounds in various hosts. Several other more oxidized acids are also discussed as in some cases, they may be desired products or their formation is minimized to afford higher yields of more reduced products. The placement and connections among acids in the typical central metabolic network are presented along with the use of a number of specific non-native enzymes to enhance routes to high production, where available alternative pathways and strategies are discussed. While many organic acids are derived from a few precursors within central metabolism, each organic acid has its own special requirements for high production and best compatibility with host physiology.