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- Anfelt, Josefine
(författare)
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Metabolic engineering strategies to increase n-butanol production from cyanobacteria
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of sustainable replacements for fossil fuels has been spurred by concerns over global warming effects. Biofuels are typically produced through fermentation of edible crops, or forest or agricultural residues requiring cost-intensive pretreatment. An alternative is to use photosynthetic cyanobacteria to directly convert CO2 and sunlight into fuel. In this thesis, the cyanobacterium Synechocystis sp. PCC 6803 was genetically engineered to produce the biofuel n-butanol. Several metabolic engineering strategies were explored with the aim to increase butanol titers and tolerance.In papers I-II, different driving forces for n-butanol production were evaluated. Expression of a phosphoketolase increased acetyl-CoA levels and subsequently butanol titers. Attempts to increase the NADH pool further improved titers to 100 mg/L in four days.In paper III, enzymes were co-localized onto a scaffold to aid intermediate channeling. The scaffold was tested on a farnesene and polyhydroxybutyrate (PHB) pathway in yeast and in E. coli, respectively, and could be extended to cyanobacteria. Enzyme co-localization increased farnesene titers by 120%. Additionally, fusion of scaffold-recognizing proteins to the enzymes improved farnesene and PHB production by 20% and 300%, respectively, even in the absence of scaffold.In paper IV, the gene repression technology CRISPRi was implemented in Synechocystis to enable parallel repression of multiple genes. CRISPRi allowed 50-95% repression of four genes simultaneously. The method will be valuable for repression of competing pathways to butanol synthesis.Butanol becomes toxic at high concentrations, impeding growth and thus limiting titers. In papers V-VI, butanol tolerance was increased by overexpressing a heat shock protein or a stress-related sigma factor.Taken together, this thesis demonstrates several strategies to improve butanol production from cyanobacteria. The strategies could ultimately be combined to increase titers further.
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| 2. |
- Pallas, Anna, 1977-
(författare)
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The Effect of Boron in Metal Borides and BN – A Theoretical Approach
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Boron, B, has many interesting electronic and structural properties, which makes it an ideal material for technical and industrial needs. The different binary materials that have been in focus in the present thesis, do all include B; metal diborides (MB2), cubic boron nitride (c-BN) forming an interface with diamond, and various phases of BN [cubic (c-BN), hexagonal (h-BN), wurtzitic (w-BN), and rhombohedral (r-BN)]. Density Functional Theory (DFT) methods have been used in studying structural geometries, energetical stabilities, electronic properties, and surface reactivity.A structural and electronic comparison has been made for various MB2 compounds in planar and puckered structural forms. The resulting MB2 structure was found to correlate to the degree of electron transfer from the metal atom to B. A transfer of more than one electron was observed to induce a planar B structure. This is to be compared with the planar MgB2 structure, for which an electron transfer of two electrons was observed.The initial nucleation of c-BN onto a diamond substrate has also been focused in the present thesis. This step has experimentally been found to be critical for a phase-pure c-BN thin film growth to occur. The evolution of an interfacial diamond//BN structure was investigated, with the purpose to simulate a layer-by-layer growth of c-BN. The obtained results were found to strongly support the experimental findings, in that there is a need for an extra energy in order to avoid non-cubic phases in the closest vicinity to the substrate. However, the simulations showed that it is possible to diminish this need of extra energy by completely terminating the surface by species like H or F. These calculations also showed that terminated diamond//BN generally show a stronger interfacial bond energy, thereby improving the adhesion to the diamond substrate. The importance with surface termination was not found crucial for thicker BN adlayers.A combined effect of doping and surface termination was investigated for the various BN allotropes, (using O, C, and Si). The electron induced in c-BN by the O (or C) dopant was observed to move towards the surface B atoms, and thereby creating a more reactive surface. For the upper surface N atoms, doping was observed to create a less reactive N surface. The Si dopants did only show a positive effect on surface reactivity at the B surface sites on both h-BN (001) and r-BN (001) surfaces.
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| 3. |
- Rodrigues, João S.
(författare)
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Engineering Synechocystis sp. PCC 6803 for microbial terpenoid production
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- As part of the efforts to develop a carbon-neutral economy, photosynthetic microorganisms such as cyanobacteria have been extensively studied as solar-powered cell factories for the production of organic compounds with industrial interest, using the atmospheric CO2 as carbon source.Among other metabolites, cyanobacteria produce terpenoids, a diverse group of natural products that play pivotal roles in the cell. These metabolites have industrial relevance as fragrances, colorants and biofuel precursors. The research here presented focused on the production of terpenoids in the model cyanobacterium Synechocystis sp. PCC 6803, with special emphasis on two compounds: bisabolene and isoprene. Bisabolene is a 15-carbon-atom molecule that can be used as feedstock for chemical synthesis and for biofuel generation. In my first two projects, I demonstrated that heterologous expression of the bisabolene synthase is enough to complete the biosynthetic pathway, and cultivation of bisabolene-producing Synechocystis strains in high-density conditions significantly benefits microbial production. Furthermore, overexpression of key enzymes from the native metabolism increased precursor supply. The best producing strain reached a bisabolene titre of 180 mg L-1 after 8 days of cultivation.Isoprene is a small volatile terpenoid with high relevance to industry, and it plays an important role in thermotolerance in several plants. In order to map competitive pathways to terpenoid production in Synechocystis, I utilized a synthetic biology tool for gene knockdown of several targets and evaluated their influence on isoprene production. Six candidate genes were identified as potential targets for further modifications, to improve reallocation of the cellular resources to terpenoid production. In another study, I analysed how the production of isoprene in Synechocystis can affect the metabolism and cell physiology, and how isoprene production can be improved by optimizing cultivation conditions, such as light quality, light intensity and temperature. Cultivation under violet light or at higher temperatures improved significantly isoprene production. I also observed that isoprene itself modifies the physiology of the cell, contributing to a reduction in size and growth rate, and confers additional thermotolerance to Synechocystis.In my last project, I participated in a collaborative work where a hybrid photobiological-photochemical approach was applied to convert photosynthetically-derived isoprene into jet fuels using light as energy source. I cultivated isoprene-producing cyanobacteria in closed vials and designed a capturing system to trap the produced isoprene. Subsequent photochemical dimerization rendered a mixture of products that, upon minor chemical modifications, fulfilled the requirements to be used as jet fuels.
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