PhD defence by Konstantinos Vavitsas

Title of thesis:
Photosynthetic production of diterpenoids in chloroplasts and cyanobacteria

Summary
Terpenoids are one of the largest classes of chemical compounds, some of them with industrial interest as nutraceuticals, biofuels, or chemical feedstocks. Diterpenoids are a large terpenoid subclass, and their chemical structure consists of a core skeleton of 20 carbon atoms. This skeleton can be further modified by cyclizing enzymes, and be decorated by the addition of chemical groups. Even though they are mainly plant-derived compounds, diterpenoid production in photosynthetic organisms is rather unexplored, with a few successful studies reported in the literature.

In this thesis, I elaborate on the potential of using plant chloroplasts and cyanobacteria as biosynthetic vessels, with a focus on diterpenoid production, and on the potential direct linking of photosynthesis to drive electron-consuming enzymes, such as the monooxygenases cytochrome P450s. I subsequently present the full localization of a diterpenoid biosynthetic pathway within the Nicotiana benthamiana chloroplast, and the protein modifications required to achieve this goal. Faster-growing and simpler photosynthetic systems, however, have a larger bioproduction potential. Therefore I examine the effects of introducing two heterologous biosynthetic pathways in the cyanobacterium Synechocystis sp. PCC 6803, using targeted metabolite analysis and computational modelling. Finally, I present a novel genetic engineering technique for the moss Physcomitrella patens, where PCR-amplified DNA fragments can be inserted into the organism and be assembled correctly by its native homologous recombination mechanisms. This theoretical and experimental work puts together the existing knowledge on terpenoid production and photosynthetic biotechnology, reveals the existing limitations and potential bottlenecks, and paves the way for future work towards the optimal use of photosynthesis to drive terpenoid production.

Supervisor
Professor Poul Erik Jensen

Assessment committee
Research Director Stéphane Lemaire, IBPC Paris
Assistant Professor Paul Hudson, KTH Stockholm
Associate Professor Yumiko Sakuragi (chair)

After the defence there will be a reception in room K117-2 and H117-3