Browsing by Subject "Metabolic Engineering"

Now showing 1 - 3 of 3
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    Engineering nonphosphorylative metabolism for the biosynthesis of sustainable chemicals
    (2016-12) Jambunathan, Pooja
    Lignocellulosic biomass is one of the largest sources of organic carbon on Earth with the potential to replace fossil fuels for the production of transportation fuels and chemicals. The two biggest challenges facing biosynthesis is the limited natural metabolic capacity of microorganisms and the effective utilization of lignocellulosic biomass. To overcome the first obstacle, over the past several decades researchers have successfully expanded the natural metabolic pathways of microorganisms to allow biosynthesis of a wide array of compounds with applications as advanced biofuels, industrial chemicals, and pharmaceuticals. Most industrial fermentations convert glucose, the major sugar present in biomass, into a value added chemical but are unable to utilize pentose sugars which make up ~30% of a typical biomass feedstock. To improve the overall economics of fermentation process, it is important to ensure that all major sugars present in the feedstock are efficiently converted to target chemicals. This work addresses both these challenges by establishing a novel alterative pathway called nonphosphorylative pathway in Escherichia coli which enables the utilization of underutilized pentose sugars such as D-xylose and L-arabinose using fewer steps and with higher theoretical yields than conventional glycolysis and pentose phosphate pathways (PPP). This nonphosphorylative pathway can convert D-xylose and L-arabinose to 2-ketoglutarate (2-KG), an important TCA cycle intermediate, using less than 6 steps. A growth selection platform based on 2-ketoglutarate (2-KG) auxotrophy was designed in E. coli to confirm the functionality of nonphosphorylative metabolism in host organism. The growth selection platform was also used to mine nonphosphorylative gene clusters from other organisms with improved activity. The pathway was then expanded to allow biosynthesis of two commercially important chemicals, 1,4-butanediol (BDO) and γ-aminobutyric acid (GABA). To improve production titers and yields of the process, protein engineering was used to reduce by-product formation and metabolic engineering was used to eliminate competing pathways and increase carbon flux towards the target compound. Furthermore, to improve uptake of pentoses by E. coli, pentose transporter was overexpressed to allow better carbon utilization. This nonphosphorylative metabolism serves as an efficient platform for biosynthesis and can be extended to produce a variety of compounds derived from TCA cycle including, but not limited to, L-glutamate, mesaconate, 5-aminolevulinic acid, and glutaconate. While the nonphosphorylative pathway has been successfully used for conversion of simple pentose sugars into important chemicals like BDO and GABA, the breakdown of biomass into these pentoses is the bigger challenge. This work also briefly addresses this challenge by comparing different acid hydrolysis treatment conditions to breakdown arabinoxylans in wheat bran into sugars - glucose, D-xylose, and L-arabinose - which can then be used in fermentation via nonphosphorylative metabolism.
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    Kinetic Analysis of the Catabolism of Tabersonine in Catharanthus roseus Leaf Protein Extracts
    (2018-08) Evans, Matthew
    Vinblastine and vincristine are valuable chemotherapy compounds produced by Catharanthus roseus. However, they are produced in small quantities in the plant because of their cytotoxicity and spatial separation of the metabolic pathways of the two monomeric molecules, vindoline and catharanthine, that condense to form them. There have been extensive attempts to metabolically engineer greater production of these molecules, but the specialized cell types needed for the terminal steps of vindoline biosynthesis from tabersonine and the high regulation of the pathway in the plant have made use of a homologous host difficult. Recently, all metabolic steps of the pathway have been elucidated, but there remains difficulty in culturing a heterologous host to express all metabolic steps. There is almost no knowledge of the catabolism of these alkaloids in the plant, and this knowledge could be used in future metabolic engineering attempts. Leaf protein extracts were incubated with tabersonine to attempt to find possible catabolic products, and initial rate experiments were used to calculate kinetic parameters of a possible catabolic enzyme. Results showed no formation of previously known or unknown metabolites. After performing kinetic studies, evidence was found of an allosteric enzyme acting on tabersonine with a vm of 5.4 ± 1.9 μM min-1, a Km " of 16,000 ± 14,000 μM, and a Hill coefficient of 2.0 ± 0.2.
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    Strain optimization through theoretical and experimental tools.
    (2010-08) Unrean, Pornkamol
    In this dissertation, metabolic network analysis based on elementary mode analysis (EMA), metabolic control analysis (MCA) and thermodynamic analysis of pathways are applied to quantitatively analyze cell metabolism and to engineer a cell for improved performance. By applying EMA, we acquire knowledge of all the pathways of a cell's metabolism. The pathway information permits the systematic implementation of cell manipulation to develop a strain with a desired phenotype. The rational strain improvement is achieved by limiting the cell's functionality to only efficient pathways through gene knockout mutations. This way, the functional space of the designed strain is minimized to a set of pathways that only support the efficient production of the desired product. The EMA approach has been implemented for enhanced synthesis of carotenoid in E. coli and ethanol in T. saccharolyticum. Metabolic control analysis and thermodynamic analysis of pathways are employed to examine changes in metabolic pathways within a cell during metabolic evolution. The evolution approach is utilized to select for a mutant of the designed strain that shows a further improvement in product synthesis or strain robustness. The approach is demonstrated in E. coli for enhanced carotenoid production, improved ethanol production in the presence of inhibitors, and in T. saccharolyticum for increased ethanol productivity. MCA is used as a guiding tool to identify a controlling step in the pathways, while thermodynamic analysis is used to determine changes in the distribution of pathway flux during evolution. The fermentation process is optimized to enhance production efficiency of the products. A controlled fed-batch fermentation process is designed and conducted to produce a high titer of carotenoid. The process of immobilized mixed cells of two substrate-selective strains of E. coli that allow for an efficient conversion of mixed sugars into ethanol at a high yield and a high productivity is also designed and implemented.