PhD defence by Cecilie Cetti Hansen

We are happy to announce the PhD defence by Cecilie Cetti Hansen!

Title: Plant Specialized Metabolism: Biosynthesis, Evolution and Plasticity

Supervisors:
Assistant professor Elizabeth H. J. Neilson
Professor Birger Lindberg Møller

Assessment committee:
Senior researcher Hugues Renault, University of Strasbourg (France),
Professor Thomas Vogt, Leibniz Institute of Plant Biochemistry (Germany),
Associate professor Nanna Bjarnholt (chair), University of Copenhagen

Summary:
Plants produce an impressive number of structurally diverse metabolites. Cytochrome P450 enzymes (CYPs) and UDP-glycosyltransferases (UGTs) catalyze key reactions in many different metabolite pathways including formation of phytohormones, membrane sterols, signaling molecules, pigments, and chemical defense compounds. The important functions of these different metabolites ultimately allow plants to thrive, adapt and reproduce in their environment. This thesis presents new findings and insight into the evolution, organization and regulation of biosynthetic pathways mediated by CYP, UGT and cytochrome P450 reductase (POR) enzymes, the latter of which donate electrons necessary for CYP catalysis.

The thesis comprises of seven chapters which are organized into three overall themes that each addresses the complexity of metabolite biosynthesis on different scales. The first part addresses the dynamic evolution of specialized metabolites, using the biosynthesis of cyanogenic glucosides as a model pathway. It is demonstrated that the biosynthetic orchestration in Eucalyptus trees and the fern Phlebodium aureum consists of additional enzymes compared to pathways previously characterized in other cyanogenic plant species. The formation of cyanogenic glucosides is typically catalyzed by two multifunctional CYPs (a CYP79 and a CYP71) and a UGT85. In contrast to the multifunctional CYP71, Eucalyptus has recruited a CYP706 and a CYP71 that act sequentially for formation of the cyanohydrin from the oxime intermediate produced by the CYP79. Another intriguing difference was revealed by comparison of cyanogenic glucoside biosynthesis between two distinct phylogenetic groups within the Eucalyptus genus. This showed that some Eucalyptus species utilizes a UGT85, whereas other species have recruited a UGT87 for glucosylation of the cyanohydrin to form the cyanogenic glucoside. Cyanogenic glucoside production in P. aureum was shown to include a CYP981, belonging to a fern-specific CYP family that constitutes the largest family of the fern CYPome. These results expand the collection cyanogenic glucoside CYP and UGT pathway members to include the CYP706, CYP981 and UGT87 families.

The second research theme expands upon part one and provides critical insights on the evolution and plasticity of metabolic pathways involving CYPs and POR. Specifically, CYPomes from the entire plant kingdom are summarized revealing dynamic evolution of this enzyme family. Based on an extensive literature study, a database on functionally characterized published plant CYPs was established: the Plant Cytochrome P450 Database (https://erda.dk/public/vgrid/PlantP450/). The second part further discusses how CYPs and POR enzymes may organize into multienzyme complexes thereby forming a metabolic highway. Moreover, it is shown that certain small metabolites may help tune POR-CYP interactions by changing the specificity of POR towards certain CYP enzymes.

The final theme presents a combinatorial approach involving CYPs and PORs for optimized microbial production of steviol, the precursor to the sweet non-calorie steviol glucosides. This work tested a small collection of CYPs and PORs and identified the combination and gene copy numbers resulting in increased steviol yields.

Collectively, this thesis expands our understanding of the remarkable evolution shaping the metabolic landscape of plants, how plants fine-tune their metabolic plasticity, and ways to apply CYPs and POR in a bio-based production system.

All are welcome for the defence and for the reception afterwards!!!