Browsing by Author "Rodriguez-Moya, Maria"

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    Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture
    (Public Library of Science, 2014) Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodriguez-Moya, Maria; Gonzalez, Ramon; Altamirano, Claudia; Bioengineering; Chemical and Biomolecular Engineering
    Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h21) and low dilution rate (0.012 h21) at two cultivation temperatures (37 and 33ﾰC) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33ﾰC. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.
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    Systems biology approaches in metabolic engineering: hasrnessing the potential of proteomics to understand and engineer metabolism
    (2015-04-24) Rodriguez-Moya, Maria; Gonzalez, Ramon; Segatori, Laura; San, Ka-Yiu
    The genomic revolution has powered the development of numerous genetic sequencing tools, which have contributed to unveiling the genomes of different life forms. This boom has impacted the development of techniques to efficiently manipulate organisms through metabolic engineering and synthetic biology, and of systems biology tools for the analysis of cellular systems via functional genomics. Among functional genomics, proteomics provides valuable insights on the entire set of proteins, which are the main components of the physiological pathways in the cell. In this study, three different systems were analyzed via proteomic profiling to understand metabolism and identify targets for engineering. First, the toxic response of Escherichia coli to short-chain fatty acids was assessed in order to improve tolerance and productivity of these molecules for industrial applications. Nine proteins were identified as being differentially expressed under octanoic acid stress. Subsequent studies of deletion and overexpression mutants for the selected proteins led to identification of OmpF, an outer membrane porin, as having the largest effect on fatty acid tolerance. OmpF was proposed as a transporter of fatty acids across the cell membrane, which can facilitate its import when present in the extracellular medium, therefore causing a decrease in intracellular pH. Utilization of glycerol by E. coli under anaerobic conditions was also studied in order to gain insights on the system level effect of this unconventional but advantageous feedstock with potential to be used in chemical and fuel production. In this study, wild-type E. coli was compared to the adhE deficient mutant under glycerol-fermenting and non glycerol-fermenting states. Twenty-six differentially expressed proteins were identified as being involved in known metabolic routes for glycerol utilization, while others suggested that amino acid metabolism, and phosphate and magnesium levels play key roles in glycerol fermentation. Lastly, the effects of mild hypothermia on the proteome of CHO cells were also studied with the goal of understanding metabolic state changes in response to a lower culture temperature. Eight differentially expressed proteins were identified suggesting that secretion and redox processes are the main systems affected by mild hypothermia. The studies presented in this thesis validated the potential of proteomics as a tool for characterizing metabolic systems and identifying targets for the engineering and development of viable biofactories.