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Frank Chaplen
Associate Professor, Biological and Ecological Engineering
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RESEARCH:
Metabolic Engineering. Metabolic engineering provides the mathematical, computational and experimental tools for analyzing and manipulating biochemical pathways and cell properties. This is a fast-evolving, new field that is highly interdisciplinary in nature and requires knowledge and techniques from biochemistry, cell and molecular biology, genetics, cell physiology, systems analysis and chemical engineering. Most industrial applications of metabolic engineering are aimed at improving cell properties and increasing metabolite yields and productivities, and, as such, it is playing a growing role in the development of economically viable bioprocesses.
Projects in metabolic engineering will involve the development of new experimental tools for analyzing biochemical pathways. Many of the experimental approaches currently utilized are system specific, invasive, or do not allow easy real-time measurements to be made of parameters of interest, such as changes in enzyme and metabolite concentrations or in enzyme activity as conditions in the extracellular environment are varied. One example of a project in metabolic engineering that is currently being tested is an analytical tool for measuring in vivo enzyme concentrations. This approach use recombinant DNA techniques to fluorescently label enzymes in metabolic pathways. Fluorescence levels from the labeled enzymes can then be utilized to provide a measure of in vivo enzyme concentrations.
Industrial Animal Cell Culture. Mammalian cell culture is an important technology for the commercial production of many high value proteins, such as cytokines, erythropoietin, interferons, tissue plasminogen activator and monoclonal antibodies. Economical processes must give high titers of product of a consistent quality while operating at high volumetric productivities for long periods of time. An important consideration for such processes is the role of toxic by-products. Mammalian cells grown in culture produce a number of toxic by-products that are known to affect cell function. Most
research has focused on ammonium and lactate, generally considered to be the major inhibitory waste products in mammalian cell culture systems. Projects in this area will focus on more fully investigating a by-product of mammalian cell culture that is much less studied than ammonium or lactate, but that may also be detrimental to both cell growth and product quality. This compound is methylglyoxal.
Methylglyoxal, an a -ketoaldehyde, is produced mainly through spontaneous phosphate elimination from glycolytic pathway intermediates. Previous studies have indicated that > 99% of the methylglyoxal in the cell may be reversibly bound to cellular structures that contain amine and sulfhydryl groups, such as proteins. In addition, increased intracellular methylglyoxal has been associated with decreased Chinese hamster ovary (CHO) cell survival in culture. Examples of ongoing efforts include chemostat studies to quantify the impact of changes in environmental conditions in the bioreactor on methylglyoxal levels in the cell, development of comprehensive flux analysis models to characterize effect of changes in free methylglyoxal levels on the concentration of reversibly bound intracellular methylglyoxal, and elucidation of the toxic effects of methylglyoxal in culture using genetically engineered CHO cells that overexpress the major detoxification enzyme for methylglyoxal, glyoxalase I.