In the Paris Agreement from 2015, nations agreed to limit the effects of global warming to well below 2°C. To be able to reach those goals, cheap, abundant and carbon neutral energy alternatives needs to be developed. The microorganisms that several billion years ago oxygenated the atmosphere; cyanobacteria, might hold the key for creating those energy technologies. Due to their capacity for photosynthesis, metabolic engineering of cyanobacteria can reroute the carbon dioxide they fix from the atmosphere into valuable products, thereby converting them into solar powered cell factories. Of the many products bacteria can be engineered to make, the production of terpenoids has gained increasing attention for their attractive properties as fuels, pharmaceuticals, fragrances and food additives. In this thesis, I detail the work I have done on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 for terpenoid production. By deleting an enzyme that converts squalene into hopanoids, we could create a strain that accumulates squalene, a molecule with uses as a fuel or chemical feedstock. In another study, we integrated two terpene synthases from the traditional medical plant Coleus forskohlii, into the genome of Synechocystis. Expression of those genes led to the formation of manoyl oxide, a precursor to the pharmaceutically active compound forskolin. Production of manoyl oxide in Synechocystis was further enhanced by engineering in two additional genes from C. forskohlii that boosted the flux to the product. To learn how to increase the production of squalene, manoyl oxide or any other terpenoid, we conducted a detailed investigation of each step in the MEP biosynthesis pathway, which creates the two common building blocks for all terpenoids. Each enzymatic step in the pathway was overexpressed, and increased flux was assayed by using isoprene as a reporter and several potential targets for overexpression were identified. The final part of this thesis details the characterization of native, inducible promoters and ribosomal binding sites in Synechocystis.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-308099 |
| Date | January 2016 |
| Creators | Englund, Elias |
| Publisher | Uppsala universitet, Molekylär biomimetik, Uppsala |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 ; 1455 |
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