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Identifier

000429148

Title

Βελτιστοποίηση της παραγωγής β-φελλανδρενίου από ένα γενετικά τροποποιημένο κυανοβακτήριο

Alternative Title

Optimization of the production of β-phellandrene by a cyanobacterium genetically modified

Author

Ψυχογυιού, Μαρία Ελένη N.

Thesis advisor

Γανωτάκης, Δημήτριος

Reviewer

Παυλίδης, Ιωάννης
Τσιώτης, Γεώργιος

Abstract

In the present study the improvement of the terpenoid β-phellandrene production by the cyanobacterium Synechocystis sp PCC 6803 was studied. The heterologous expression of β-phellandrene synthase by this microorganism was achieved in previous studies after modifying the cpc operon. This operon expresses phycocyanin, a pigment localized on the photosynthetic antena of the microorganism, facilitating the capturing of the light energy in the low sea level were the organism is existing and lower yield of sun radiation is approaching. Different constructs of Synechocystis have been designed containg the gene of β-phellandrene synthase in their genome. In this study, the increase of the yield of β-phellandrene was achieved by studying a new construct Δcpc+cpcB.PHLS+cpcA.GPPS+cpc. The difference between this construct and the previous studied is that all genes of cpc operon located in Wild Type have been maintained, fusing the gene of the β-phellandrene synthase and the synthase of its procursor in the biosynthatic pathway of endogenous terpenoids, geranyl diphosphate, with the genes expressing the two subunits of phycocyanin. The presence of all genes was necessary for the overexpression of the two synthases and led to higher production of the terpenoid. Furthermore, the organism follows similar photosynthetic activity and growth to the wild type. In the second chapter, the word “improvement” refers to the development of a more cost effective and environmental friendly method for β-phellandrene production. Previous studies have been conducted on the construct Δcpc+cpcB.PHLS+cpcA.GPPS under high salt concentration including growth, photosynthetic activity and production, simulating the conditions in sea water, which is available in huge amounts in the planet compared to fresh water. In addition, same parameters were examined in alkaline environment which prevents the growth of a plethora of microorganisms and can be used as a possible method to prevent contaminations. The new construct was studied in same conditions, yet giving lower production of β-phellandrene. Moreover, both Δcpc+cpcB.PHLS+cpcA.GPPS and Δcpc+cpcB.PHLS+cpcA.GPPS +cpc constructs were examined in sea water. After cultivation in 100% sea water β-phellandrene production by both constructs was achieved. In the last chapter, Δcpc+cpcB.PHLS+cpc(-cpcA) construct was immobilised in alginic polymeric spheres in 100% sea water and the system was studied for the ability to produce β-phellandrene in 12 experimental days, using free cells in 100% sea water as control. Previous studies in the laboratory showed that cell immobilisation concluded in the production of higher amounts of β-phellandrene compared to BG-11 free cells cultures. The immobilised cells in sea water produced similar yield observed in immobilised cells in BG-11, in contrast to free cells that seem to be affected in growth and the yield of β-phellandrene production by the presence of sea water.

Language

Greek

Subject

Biofuels

Cpc opero

Cpc οπερόνιο

Cyanobacteria

Fused genes

Geranyl diphosphate synthase (GPPS)

Monoterpenes

Photosynthesis

Phycocyanin