Developing cyanobacteria as a microbial factory for the production of novel compounds

Fossil fuels are a finite resource and sustainable alternative methods of producing fuel molecules and the numerous chemical products derived from fossil fuels need to be found. Genetically engineered cyanobacteria represent one promising ‘green’ platform for the production of biofuels and high-value chemicals. As photosynthetic prokaryotes, cyanobacteria are able to fix atmospheric carbon dioxide using sunlight and channel that carbon into organic products. Manipulation of these biochemical pathways should allow the hyper-accumulation of certain products or the synthesis of novel compounds. Compared to other phototrophic platforms (plants and eukaryotic algae), cyanobacteria are much easier to grow and easier to manipulate genetically. Nevertheless, the current genetic tools to engineer cyanobacteria are not as advanced as those developed for E. coli and S. cerevisiae, and there is a need for new technologies in this area. The work presented in this thesis therefore aimed to develop novel tools for cyanobacteria and demonstrate their use as a platform for the production of new compounds. The research focussed on the model organism Synechocystis sp. PCC 6803 – the availability of a sequenced and annotated genome and the ability to integrate DNA via natural transformation at a specific locus by homologous recombination making it ideal for genetic engineering. A new method was developed in Synechocystis for creating transformants that no longer contain an antibiotic resistance marker, and improvements to transcription and translation of transgenes investigated. The expression of GFP and limonene synthase were tested under various promoters, and several native and nonnative integenic regions, containing ribosomal binding sites, were functionally tested in a two-gene operon containing GFP as a reporter gene. The production of limonene and (S)- styrene oxide, a chiral compound used as a building block for pharmaceuticals, was attempted in Synechocystis using the newly developed tools