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Mannitol is a C(6) polyol with a wide range of applications in the food and pharmaceutical industry. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in cyanobacteria proved to be genetically unstable. To stabilize mannitol production in Synechocystis sp. PCC6803, we first validate that mannitol can work as well as the native compatible solutes (e.g., sucrose and glucosyl-glycerol) in conferring salt resistance. Under this background, the mannitol production system was proven to be stable under salt stress condition in the mutants which lack sucrose and glucosyl-glycerol in their arsenal against osmotic pressure (Chapter II). However, even with this stabilized mannitol production system, its production in Synechocystis sp. PCC6803 is still very low, and therefore, it is unable to meet the requirements of industrial scale. In the following studies, we precisely control the expression level of the mannitol cassette using different promoters in chapter III and proceed to select a super mannitol producer from adaptive laboratory evolution under increasing salt pressure in Chapter IV. Both methods are used to successfully increase the mannitol production in cells. The lastly in Chapter V, we explore a new pathway of mannitol expression in Synechocystis sp. PCC6803. This thesis aims to contribute a solid theoretical basis for achieving mannitol production in Synechocystis sp. PCC6803 at a commercial scale, and possibly, inspire researchers to develop highly efficient photosynthetic cell factories for other valuable compounds with similar properties.

The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that (i) the PHB synthesis pathway becomes deregulated, and/or (ii) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve. The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232 mg L− 1, ~ 12% (w/w) of the dry biomass weight, and a productivity of 7.3 mg L− 1 d− 1; are to the best of our knowledge, the highest ever reported for PHB production directly from CO2.

The primary goal of the work presented in this thesis was to provide a unique tool that may increase our understanding of the mechanism of cytoplasmic diffusion in signal transduction (networks) in eukaryotic cells, in the form of a chimeric, light-dependent, cytoplasmic signal transduction device in the yeast Saccharomyces cerevisiae. We describe the design and functional in vitro and in vivo characterization of the intended chimera: A light-sensitive histidine protein kinase, derived from the Sln1 kinase of S. cerevisiae, translationally fused in a coiled-coil motif with the LOV domain of the stressosome component YtvA from Bacillus subtilis. In most chimeras exposure to blue light decreased the rate of autophosphorylation, but rational engineering also allowed the construction of a light-stimulated histidine protein fusion kinase. This orthogonal photo-transduction system can be used to both activate and repress gene expression in S. cerevisiae, depending on the specific promoter that is targeted. Furthermore, the device can also be used to initiate nuclear accumulation of a selected signal transduction protein with incident blue light.

Irrespective of where we come from, we all share the global responsibility of ensuring that our societies are sustainable. We have been depleting the world’s resources and filling the atmosphere with abnormal levels of CO2. But CO2 can also be used as a resource - plants and certain microbes have been doing so for billions of years. This is the foundation for the bio-based economy – reducing and eventually replacing the use of fossil fuel. Up to now, the focus in bio-based production has been on producing biofuels using plants to produce sugars, which are then used as substrate in microbial fermentation processes. But there is an alternative way - using cyanobacteria to take up CO2 from the atmosphere and directly converting it into useful products. However, the development of efficient cyanobacterial cell factories is still challenging. One typical technical hurdle is to maintain sustained productivity. This is because traditional genetic engineering strategies for microbial product formation, i.e. through introduction of heterologous pathways encoded by multiple genes with constitutive expression levels, are burdensome for cell growth. Consequently, spontaneous non-producing mutants tend to grow faster, thereby gradually taking over the population and undermining the total productivity of the culture. In general, this technical hurdle is common to any microbial system that was genetically engineered for product formation. Yet, for cyanobacteria little attention had been paid before this project commenced. Therefore, we decided to specifically study the instability of cyanobacterial direct conversion processes, aiming for conceiving novel strategies to prevent this problem.

To achieve success in setting up sustainability applications, photosynthetic organisms are required to convert solar energy with the highest efficiency. A widely proposed method to do this is to expand their effective absorption spectrum into the infrared (i.e. beyond 700 nm). Introducing an infrared-absorbing, retinal-based proton pump, as a substitute for PSI, is a promising approach to achieve this. This thesis focuses on expressing two such retinal-based pumps, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR), into Synechocystis sp. PCC 6803 and its PSI-deletion derivate. In these strains we explored the stimulatory effect of these pumps on the growth of their host. To facilitate expression of PR and GR variants with a modified chromophore, we also investigated retinal metabolism in Synechocystis. Our results show that, upon use of the psbA2 promoter, bacterial rhodopsins (PR or GR) are expressed in Synechocystis, at a level of up to 105 molecules per cell, presumably as pentamers and trimers, respectively, with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. Functional assays have revealed that PR but not GR can enhance the growth rate of Synechocystis and its ΔPSI derivative strain. Notably, the stimulatory effect is most significant in the ΔPSI strain. Moreover, our results confirm that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal, thereby making use of the SynACO enzyme (encoded by sll1541). By supplying a red-shifted retinal analogue to a sll1541-knockout mutant we could show that an infrared-absorbing PR derivative is formed in Synechocystis.

The oxygenic photoautotroph Synechocystis sp. PCC6803 is a present-day cyanobacterium, a lineage with billions of years of history. In this thesis, we explore ways to optimize the use of Synechocystis for the realization of a sustainable society, in which the carbon cycle is closed by using sunlight to produce fuel and chemical feedstock out of CO2. A deeper understanding of metabolism in Synechocystis is required to be able to reach this goal in the future. State-of-the-art photobioreactors were used to precisely control and measure parameters of growth and develop a new medium, as described in chapter 2. This chapter explores the limits of growth of Synechocystis and exposes nutrient limitations. In chapters 3 and 4, the photosynthetic apparatus itself is closely investigated with special emphasis on the role of the light-harvesting complexes typically found in cyanobacteria, revealing a remarkable flexibility in coping with varying light intensities. Because of the desire to use sunlight to fuel the assimilation of CO2, it is of paramount importance to be able to deal with the steady rhythm of day and night, as well as varying light intensities during the day. Chapters 5 to 7 cover the circadian clock of Synechocystis, an essential mechanism to time gene expression and metabolism. The clock of Synechocystis was shown to be robust and in control of large number of cellular processes, crucial for coping with the night and allowing for additional regulation to enhance production of compounds of interest. Chapter 8 discusses all findings in a broader context.

In the combustion reaction, a fuel reacts with oxygen to carbon dioxide and water. The energy liberated in this process can then be harnessed to do work, e.g. to generate electricity or drive an engine. Excessive and increasing world-wide use of fossil fuels has led to increases in atmospheric carbon dioxide levels that are predicted to cause significant climate change. In this thesis project, the cyanobacterium Synechocystis sp. PCC6803 is engineered for the conversion of water and carbon dioxide into glycerol and 2,3-butanediol. These compounds have attracted interest as (liquid) fuels and as precursors for both large scale (polymers) and small scale (specialised compounds) synthesis. The conversion process is driven by light energy. As such, it represents the reversion of the combustion reaction referred to above. Furthermore it is shown that an artificial pathway from carbon dioxide to a product does not have to be confined to a single organism. Indeed, when mixtures of different organisms are used, their unique metabolic makeup can be harnessed to drive the synthesis of compounds that would be infeasible in just a single cell type. To this end, a mixture of Synechocystis and a heterotrophic organism such as Escherichia coli is shown to be able to convert carbon dioxide into optically active (i.e. enantiomerically pure) forms of 2,3-butanediol. Lastly, a protein with an until now unknown function is demonstrated to be required for the degradation of the osmoprotectant glucosylglycerol.