Date of Award
Doctor of Philosophy (PhD)
Our earth is covered with lots of extreme environments where only extremophiles can survive. These environments include the glaciers, volcanoes, deep sea, radiation polluted area and deserts and are usually found with extreme temperatures, salinity, pressure and radiation. Extremophile enzymes are found in these extremophile organisms and are with excellent properties like high thermostability. These extreme properties enable extremophile enzymes to save the energy and time cost in industrial production. Therefore, extremophile enzymes have been used in food processing, paper bleaching, detergent and biosensors. Extremophile enzymes could be classified roughly into psychrophilic, mesophilic and thermophilic enzymes based on their different temperature adaptation. Psychrophlic enzymes are believed to function when temperature is below 20oC. In the opposite, thermophilic enzymes tend to be functional when temperature is higher than 45oC. Enzymes exhibiting optimum temperature between 20oC and 45oC are considered as mesophilic. The temperature adaptation of extremophile enzymes limit their application in industry because their activities will be hampered when temperature is outside their working range. Engineering the extremophile enzymes to make them robust and improve the robust enzyme production can help conquer the temperature limitation in industry use and save cost in production. In our work, we first focused on the engineering of a thermophilic lipase to make robust enzymes, then we target on the non-conventional yeast Yarrowia lipolytica to explore the approaches to improve the robust enzyme production.
In this work, we aim to improve the cold activity of thermophilic lipase to make robust enzyme by studying the cold adaptation and active site flexibility. In addition to study the activity change over temperatures, MD simulation was also employed to analyze the relation between enzyme structure and activity change. We found temperature adaptation of enzymes are complicated and affected by substrate binding pocket and enzyme structure fluctuation. Meanwhile, the cold activity was engineered without sacrificing the thermostability, which corroborates the hypothesis that the cold activity and thermostability are not exclusive. More importantly, charged residues were found to form allosteric pathways to regulate the fluctuation of enzyme structure. Our work indicates that allostery cannot be neglected when modify the enzyme temperature adaptation.
Finally, for a better production of robust enzymes, the secretory pathway in Yarrowia lipolytica was studied. As one of the non-conventional oleaginous yeasts, Yarrowia lipolytica is becoming more and more popular due to its unique features such as excellent secretory capacity and wide range of hydrophobic substrate utilization. Previous studies to improve the protein production in Y. lipolytica mainly focused on expression level and process optimization. In our work, several key enzymes were either overexpressed or knocked out to tune the enzyme secretion level. Our combined strategy in the secretory pathway engineering lead to 50 folds improvement in the secretion capacity.
Wang, Weigao, "Engineering of Robust Enzymes and Their Production in the Non-Conventional Oleaginous Yeast Yarrowia lipolytica" (2021). All Dissertations. 2837.