Metabolic and Process Engineering of the Siderophore Yersiniabactin for Advanced Characterization and Application
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In the first chapter heterologous biosynthesis is introduced as a means to produce novel compounds with therapeutic value. Flux balance analysis is introduced as a way to computationally predict the effect of metabolic engineering on production of compounds of interest. Chelators are discussed in detail due to their importance for the treatment of metal overload. Sideromycins which are siderophores with antibiotic moieties are discussed as new generation antibiotics with great potential against MDR strains. Chelating compounds and siderophore research is important for expanding our field in regards to finding new drugs to treat metal overload and fight MDR bacteria. This dissertation targets the siderophore Yersiniabactin in an effort to increase production and to better understand it’s metal binding properties.In the second chapter the production Yersiniabactin and an anthranilate analog are greatly increased using a Plackett Burman design of experiments to enhance the cell production media. Different carbon and amino acid sources were examined as a function of cell growth and compound production.In the third chapter flux balance analysis was used to determine gene deletions, over-expressions and other medium components that could increase the production of Yersiniabactin along with other natural products. Medium components were also screened using the same Plackett and Burman design of experiments methodology which was implemented in Matlab. The result from this work identified many gene deletions, over-expressions and medium components which can increase the production of important small molecules such as Yersiniabactin, 6-dEB, Erythromycin and SAG.In the fourth chapter metals were systematically tested and screened in an effort to determine what other metals Ybt forms a complex with. Ybt-metal complexes were determined by using LC-MS. The metal removal properties of Ybt were explored by adsorbing Ybt onto XAD-16N resin which was then was subsequently used for metal removal tests. The degree of metal removal was quantified by determining the max amount of metal removal Qmax (mg Ybt/g adsorbent). This was accomplished by modeling the metal adsorption by the Langmuir and Redlich-Peterson isotherms.In the fifth chapter Ybt was immobilized on XAD-16 resin on a packed bed column and used for metal removal of Mg2+, Cu2+ and Ni2+ from wastewater. Process characteristics of the metal removal were examined by analyzing column capacity and process models such as the Thomas and Dose Response model.