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Identifier

000441204

Title

Βελτιστοποίηση των συνθηκών παραγωγής του μονοτερπενίου β-φελλανδρενίου από το γενετικά τροποποιημένο κυανοβακτήριο Synechocystis sp. PCC 6803

Alternative Title

Optimal conditions for the production of the monoterpene β-phellandrene by the genetically modified cyanobacterium Synechocystis sp. PCC 6803

Author

Χρυσουλάκη, Ελένη Ε.

Thesis advisor

Γανωτάκης, Δημήτριος

Reviewer

Παυλίδης, Ιωάννης
Τσιώτης, Γεώργιος

Abstract

The main objective of this study was the finding of optimal conditions in order to increase the heterologous production of the monoterpene β-phellandrene by Synechocystis sp. PCC 6803 transformants. This cyanobacterium is a model organism, in which the “photosynthesis to fuel” approach has been successfully applied. Thus, it is a suitable microorganism for biotechnological exploitation and production of a large number of products, by using only sunlight, water and carbon dioxide (CO₂) as raw materials. Previous laboratory studies have extensively studied the growth, photosynthetic activity and production of β-phellandrene by mutant strains of the cyanobacterium, under conditions of high salinity or even under conditions that used only 100% seawater and the experiment lasted ten days with measurement values taken every two days. Both their growth and production were successful. Therefore, in the first chapter of this work, the wild type of cyanobacterium was studied under various conditions of enriched seawater, which is abundant on the planet. The aim was to find the most suitable growth condition of the strain in order to then study this condition in the mutant strains and what is the effect on the production of the desired product. The conditions that stood out from those studied were those with enriched seawater with all the components of BG11 and HEPES / NaOH as buffer with pH 7.5 and enriched seawater with some components of BG11 with the same buffer. Under these conditions the growth simulates to a very good extent the corresponding growth of the WT strain in the control condition-BG11. The same results were seen in photosynthetic activity. Therefore, in order to have a fairly economical method in the rest of our study, as a condition to be applied to mutant strains was chosen, the one with enriched seawater with some components present in the nutrient medium BG11. In the next chapter, the mutants Δcpc+cpcB.PHLS+cpcA.GPPS and Δcpc+cpcB.PHLS+cpc(-cpcA) were studied for growth, photosynthetic activity and production of β-phellandrene in the optimal condition of seawater with some components of BG11. The experiments lasted ten days and the measurements were performed every second day. The results showed great similarity to the control condition in terms of growth and photosynthetic activity. The yield of β-phellandrene seemed to be lower than the control condition-BG11, but the cells are still able to produce it successfully. Finally, in the last chapter the constructs Δcpc+cpcB.PHLS+cpc(-cpcA) and Δcpc+cpcB.PHLS+cpcA.GPPS+cpc were immobilized in alginate polymeric spheres in the condition with enriched seawater for 12 days and β-phellandrene production was measured, always compared to the production of the same construct in free cells under the same condition. A previous study in the laboratory showed that immobilization of the cells of the construct Δcpc+cpcB.PHLS+cpc(-cpcA) gives higher production of β-phellandrene compared to the free cells in the nutrient medium BG11, but also in 100% seawater. Immobilization of cells in enriched seawater seems to have lower yields than free cells. These two strains studied remain metabolically active for the period of twelve days of studied and the production of β-- phellandrene is possible.

Language

Greek

Subject

Biofuels

Cpc operon

Cpc οπερόνιο

Cyanobacteria

Fusion proteins

Geranyl diphosphate synthase (GPPS)

Metabolic engineering

Phellandrene synthase (PHLS)

Photosynthesis