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.

Shigellae are pathogenic bacteria that cause the disease shigellosis. Two methods for studying secreted effectors encoded by this pathogen’s virulence plasmid are described. First, protein microarrays were used to identify substrates of an E3 ubiquitin ligase called IpaH7.8. Second, a deletion collection containing mutants for every gene on the virulence plasmid was used in two screens: one to identify mutants that elicit atypical levels of Interleukin-8 (IL-8) from U937 cells, and one to identify mutants that bind the dye Congo red abnormally. Although protein microarrays were an ineffective tool, the deletion collection proved valuable. Most mutants were less effective at sequestering Congo red than wild-type S. flexneri, although this ability was enhanced in several mutants. Four mutants, ?ospB, ?orf186, ?mxiH and ?mxiK, elicited higher levels of IL-8 from U937 cells than wild type S. flexneri. These results validate the use of the deletion collection as a tool for studying bacterial pathogenesis.

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Philosophiae Doctor - PhD / Starch is a major carbohydrate reserve in many plants, providing energy during heterotrophic growth and it is contained in large amounts in staple foods such as potatoes, wheat, maize, rice, sorghum and cassava. Apart from being a major product for use in the food industry, starch is also attracting interest from the biofuels industry as a source of bioethanol. This study reports on the development of genetic tools aimed at increasing starch production in sorghum (Sorghum bicolor L Moench), a crop of key agronomic importance worldwide by exploiting a new discovery of a transcription factor gene that regulates starch accumulation in Arabidopsis thaliana namely LEAFY COTYLEDON I (LECl). Ectopic over expression of this gene in arabidopsis has previously been shown to induce a massive hyper accumulation of starch in vegetative tissues. Therefore, we set out to investigate the function of its orthologous gene counterpart in sorghum with the aim of manipulating starch yield directly. Deduced protein sequence analyses showed that the putative sorghum LEAFY COTYLEDON I gene (SbLEC1) cloned in this study shares an overall high amino acid sequence identity (70 %) with the arabidopsis LEC 1, while the functional central B domain shows an even higher percentage sequence identity (91 %) with the same region of arabidopsis LEC 1. The putative SbLEC1 protein shares 14 out of the 16, signature ammo acids characteristic of the Central B Domain with arabidopsis. Furthermore, the putative SbLEC1 protein was also shown to share a significantly high sequence identity (> 80 %) with other well-characterized LEC1 protein sequences from organisms such as maize, rice, rapeseed as well as other organisms documented in the NCB I database. Similarly, much of the sequence similarity lies within the functional central B domain compared to any other region. Gene expression profiling using semi-quantitative PCR showed that SbLEC1 transcripts accumulated in developing seeds as well as in embryogenic calli tissue and no SbLEC 1 transcripts were detectable in leaf, root or sheath tissue. In order to confirm that the identified transcription factor is a functional ortholog, the full cDNA encoding putative SbLEC 1 transcription factor was identified, isolated and cloned from the sweet sorghum MN 1812 genotype. Plant transformation gene constructs based on the pCAMBIA1305.2 binary vector harbouring the transcription factor gene under the control of different promoter sequences were then assembled and immobilized into Agrobacterium tumefaciens strain LBA4404 in preparation for sorghum and arabidopsis transformation. Transient GUS expression studies showed that the five SbLEC1 gene constructs developed in this study were successfully transformed into arabidopsis (Ws ecotype) and sorghum (variety MN1812) callus and cell suspension cultures. The transformed tissues thus represent essential tools that are useful to evaluate the effect of over expressing the putative SbLEC1 protein. Transient GUS expression assays also further revealed differences in efficiency among promoters in driving transgene expression. Transient GUS activity was highest for the maize ubiquitin promoter (MUbi1), followed by the sorghum LEC1 promoter (SLECP), the arabidopsis LEC1 promoter (ALECP) and lastly the maize alcohol dehydrogenase promoter (MAdh1). The ability of the putative SbLEC 1 gene to complement the arabidopsis lecI mutation was also investigated and our findings were not conclusive as they only revealed partial complementation. A detailed comparison of SbLECI full cDNA sequences isolated and cloned from twenty-eight different F2 population plants from different sorghum varieties revealed the existence of sequence variation within the SbLEC 1 gene, which appeared to be allelic. The allelic variation was further shown to affect the amino acid composition of the putative SbLEC 1 protein. Heterologous protein expression studies of the SbLECI gene using an E. coli system showed that the predicted 29.16 kDa putative SbLEC 1 protein could be expressed in vitro both as an development of an efficient tissue culture protocol is a prerequisite for plant genetic engineering, this study also reports on the evaluation of thirteen sorghum genotypes from different genetic backgrounds for their in vitro culture response. A tissue culture protocol for three previously unexplored sorghum genotypes namely Agricol white, AS4 and MNI812 was established. The effect of plant genotype, explant and medium composition on in vitro culture response was highly significant (95 % Cl) in this study. Taken together, the findings in our study demonstrate efforts to draw a baseline foundation for the development of molecular technologies that can be used to increase starch production in sweet sorghum as a water efficient and sustainable feedstock for biofuel production.