Improving proteomic methods and investigating H2 production in Synechocystis sp. PCC6803

The annual EU consumption of energy is approaching 3 Terawatt.hr−1, but the majority of this is powered by fossil fuel. Burning of fossil fuels has produced a global catastrophe, climate change, and carbon-free replacement technologies are urgently required to prevent this from becoming worse in the coming years. The CyanoFactory consortium worked to optimise the organism Synechocystis sp. PCC6803 (herein Synechocystis) to produce industrially relevant levels of bio-hydrogen as one such potential solution. This thesis discusses aspects of this ambitious project, focusing on understanding and optimising the internal protein network of the organism to engineer a functional and efficient system. Synechocystis is a model cyanobacteria – and so has a significant body of research associated with it compared with other cyanobacteria, but is nowhere near as well studied as the other major model organisms such as E. coli or S. cerevisiae – particularly in protein-level studies, although this is changing with time. Whole-proteome studies are highly advanced in medical applications, however bioengineering using proteomics still lags behind studies which directly measure individual proteins, metabolic outputs, or nucleic acid studies. A number of proposals emerge from the literature as the most effective way to move forward, part of which is filling the gaps in the literature for Synechocystis and production strains in general. The major improvement missing from this field is the broad-spectrum inclusion of broadly applicable bioengineering techniques, such as synthetic biology, being integrated with whole-proteome studies, rather than just focusing on individual pathways. This gap is likely to be filled in the near future, with the recent improvements to proteomic technologies and the increasing popularity of the methodology – which has seen a sharp increase since the start of 2015. The current gap between the medical studies and production strains provides an opportunity to test a variety of different approaches, that look more at general whole-cell level responses rather than targeted observations. These gaps in knowledge are assessed herein, and new methods for analysing Synechocystis specifically are proposed. These proposals cover both alterations to the practical protocol, including physically lysing cells based on meta-analysis of the literature with experimental verification, more accurate methods of determining protein levels – which are generally complicated by coloured compounds found in cyanobacteria; and computational protocols for improving the quantity, quality and relevance of the data obtained, including better observation of low-abundance proteins in a complex background, assessment and recommendations for expanding the number of different samples that can be measured simultaneously, and simpler tools for identifying broad-sweeping changes, where metabolic-network derived investigations are unsuitable. Isobaric tags are popular methods for analysing the relative quantity of proteins observed in a cell-wide sample, however there are different technologies for this method. The two most popular tag-based quantification technologies – iTRAQ and TMT – are directly compared, to determine which method is more suitable for analyses in Synechocystis. The study was focused on Synechocystis, however the observations are also more generally applicable to other investigations. To perform this study, a modelled assessment of the ‘proteomic background’ of Synechocystis was carried out, providing an impression of the internal proteome distribution – a valuable set of information for carrying out more accurate engineering of the internal mechanisms with technologies, such as Synthetic Biology. The study found that whilst TMT tags generally produced more quantifications, the iTRAQ tags were more accurate over a greater range – however to take advantage of this would require a larger number of repeated injections of the iTRAQ samples, producing a relatively inflated cost for better quality data. Combining these tools, a direct assessment was carried out of the systemic changes that occur in Synechocystis under hydrogen-producing conditions, along with an assessment of a media proposed for optimised H2 production. This experiment first carried out with the methods used more widely at the start of this analysis, and the second was conducted afterwards, utilising many of the methodological improvements proposed in this thesis. Ultimately, an increase in data quantity and quality was observed. As hydrogen production is a response to a change of conditions, the pathway-level assessment of the proteome changes show a concordant switch between 2 very clear states under the experimental conditions used. This suggests that finding a way to produce hydrogen directly – under normal growth conditions in light – will be extremely challenging as it fundamentally competes with the growth and function of the organism; however an integrated approach, merging the production of high-value side products during the day, coupled with hydrogen production at night for generating power to run the bioreactor system, has a much greater chance of success. A decision on which products should be targeted to make the system economically viable will dictate further analysis of the data. The major conclusions of this work show that the suggested improvements are beneficial to proteomic studies in Synechocystis, producing an improvement to quantity, quality and accessibility of proteomic data. These observations have been applied to hydrogen production systems, demonstrating that whilst bio-hydrogen is unlikely to be the white knight that will save the world from climate change, it can be integrated into large-scale production systems to improve energy efficiency – where the energy saved can reduce costs and power-inputs required from carbon-based fuels. The methods suggested here, whilst ultimately adding little to the assessment of H2 production, have huge potential when integrated into future project focused on the production of more economically viable complex organic molecules or fine chemicals.