Abstract
The main objective of this study was the heterologous production of the monoterpenoid β-phellandrene by Synechocystis transformants. The cyanobacterium Synechocystis 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.Through genetic and metabolic engineering, Synechocystis transformants with the ability to produce β-phellandrene, when grown under photoautotrophic conditions without affecting their biomass production as well as photosynthetic activity, were generated. Various fusion constructs of the codon optimized gene of phellandrene synthase (PHLS) along with the gene of geranyl diphosphate synthase (GPPS) with the highly-expressed endogenous cpcB and cpcA genes, encoding the phycocyanin β and α-subunits respectively, were constructed and incorporate ...
The main objective of this study was the heterologous production of the monoterpenoid β-phellandrene by Synechocystis transformants. The cyanobacterium Synechocystis 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.Through genetic and metabolic engineering, Synechocystis transformants with the ability to produce β-phellandrene, when grown under photoautotrophic conditions without affecting their biomass production as well as photosynthetic activity, were generated. Various fusion constructs of the codon optimized gene of phellandrene synthase (PHLS) along with the gene of geranyl diphosphate synthase (GPPS) with the highly-expressed endogenous cpcB and cpcA genes, encoding the phycocyanin β and α-subunits respectively, were constructed and incorporated into the genomic DNA of Synechocystis. These constructs were expressed under the native cpc operon promoter. Findings of this study indicate that the heterologous expression of GPPS, did enhance the production of β-phellandrene. However, the utilization of a strong promoter (cpc) in combination with the cpcB as a leader sequence seemed not to be sufficient for PHLS protein overexpression levels, when the rest of the cpc operon genes were not present. The cpcB.PHLS fusion protein leads to the heterologous production of β-phellandrene in Synechocystis transformants. On the contrary, the cpcA.PHLS fusion protein was found to lead to the heterologous production of a mixture of terpenoid isomers (δ-3-carene, camphene and α-pinene).In the second part of this thesis, different growth conditions for Synechocystis transformants were examined. Conditions such as higher salinity, alkalinity, CO2 concentration, and light intensity were tested. It was shown that higher alkalinity and salinity did not inhibit the ability of Synechocystis transformants to produce β-phellandrene. Thus, Synechocystis transformant under these conditions could be further exploited for the production of high commercial value products. Furthermore, throughout this study, it has been shown that increased biomass production did not necessarily lead to increased β phellandrene production.Immobilized Synechocystis transformants in calcium alginate beads were found to be able to produce β-phellandrene in significantly larger quantities, compared to free cells, remaining metabolically active for a period of twelve days. These systems provide a renewable perspective for their future application in larger scale bioreactors.The main conclusion of this thesis is that through genetic and metabolic engineering, Synechocystis are capable of heterologous production of a product with high demand and cost in industry by using light, water and CO2. Heterologous β-phellandrene production is just an example that opens new ways for the production of other similarly structured terpenoids with multiple application. The flexibility of these microorganisms to adapt to various growth conditions without affecting their ability to produce β-phellandrene, offers many advantages in their exploitation on an industrial scale to produce such products. The approaches presented in this thesis are renewable, environmentally friendly and economically viable alternatives for the mass production of organic molecules of high industrial value.
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