PhD Defence Krutika Bavishi: A synthetic biology approach to study the dhurrin pathway in Sorghum bicolor
It is with great delight we announce the PhD defence of Krutika Bavishi.
All are welcome!
Title: A synthetic biology approach to study the dhurrin pathway in Sorghum bicolor
Abstract: The Cytochrome P450 family of enzymes constitutes the largest family of proteins in plants. These enzymes orchestrate the production of a large array of secondary metabolites that enable the plants to cope with environmental stresses. These natural products are also industrially valuable, providing colors, fragrances, pharmaceuticals etc. However, the ability to conduct proficient metabolic engineering and fully exploit their commercial potential requires a detailed understanding of the properties of its constituent enzymes and organization of the P450-centric pathways. A simple and well-characterized system is a pre-requisite to investigate these fundamental details.
The dhurrin pathway was selected as the model pathway and its key membrane appended enzymes- CYP79A1, CYP71E1 and its NADPH P450 oxidoreductase (POR) were expressed, purified and reconstituted in biomimetic membrane systems to study them in aqueous solutions. These building blocks were mixed-and-matched with a synthetic biology toolkit consisting of a multitude of bio-chemical/physical techniques. The application of a novel detergent-free SMALP technology to sorghum microsomes (native membranes) allowed identification of the protein and lipid components that constitute the dhurrin ‘metabolon’ (Paper I). Furthermore, the redox properties and electrocatalytic potential of CYP79A1, CYP71E1 and POR reconstituted in nanodiscs were studied by direct electrochemistry (Paper II). Detailed insights about enzyme conformational dynamics that govern their plasticity can be provided by single molecule methods that provide direct evidences of molecular heterogeneity (Review, Paper III). POR, under freely diffusing conditions was studied by single molecule FRET (smFRET) to directly observe and quantify a distribution of multiple conformational subpopulations and its regulation by ionic strength and lipid bilayer (Paper IV). Moreover, the effect of choice of membrane scaffold protein (MSP) in nanodisc based neutron reflection experiments was investigated (Paper V). smFRET analyses of surface-immobilized POR by TIRF microscopy enabled the acquisition of fluorescence trajectories that represent its real time conformational dynamics (Chapter 1). Functionalized amphipathic surfactants ‘amphipols’ have been employed to solubilize CYP79A1 and CYP71E1 aiming to circumvent the classical utilization of detergents for electrochemistry (Chapter 2).
The insights gained from these studies would facilitate an enhanced fundamental understanding of P450-containing systems that are pivotal to all living systems. This would translate into designing of rational strategies for their commercial and biosensing applications.
Reception afterwards in the 117 meeting rooms
Supervisors:
Prof. Dr. Birger Lindberg Møller
Dr. Tomas Laursen
Co-supervisors:
Assoc. Prof. Nikos S. Hatzakis
Dr. Jean-Etienne Bassard
Assessment committee:
Prof. Thomas Günther Pomorski (Chair)
Prof. Dr. Rita Bernhardt
Senior Researcher Dr. Ilia Denisov