The ongoing research in Karin Stensjö Group

The projects carried out in the Stensjö group, concerns the regulatory and metabolic activities behind robustness in cyanobacteria. We define robustness as the ability to adapt to a changing environment. This knowledge is then used in metabolic engineering approaches to design more robust cyanobacteria for large-scale cultivation for manufacturing of valuable compounds. Our main interest is biofuels production from solar energy and water. We aim to uncover the fundamental activities of importance for how organisms sense and respond to environmental conditions such as light intensity, and nutrient levels, that alter energetic fluxes and thus the oxidation-reduction (redox) milieu, of their cells. The ability to control redox homeostasis and the level of reactive oxygen species (ROS) has enormous impact for the fitness of life. In our research we often use a systems biology approach with quantitative shot-gun proteomics as our main tool to discover processes and proteins of importance regarding robustness. Results from this project will be of importance to understand the regulation of cellular processes in order to achieve successful metabolic engineering and/or cultivation of cyanobacteria for production of biomass and biofuels. Since cyanobacteria use CO₂ (together with H₂O) as the raw material for biofuels production the use of microalgae for production of biofuels will efficiently help to reduce CO₂ emission. 

• Protein profiling of filamentous N2 fixing cyanobacteria for H2 production.
• Redox regulation in multicellular cyanobacteria – a proteomics approach.
• The relationship between oxidative stress and cyanotoxin production in cyanobacteria.
• Characterization of proteins involved in stress tolerance in cyanobacteria: Structural and functional characterization of ferritin-like proteins in Nostoc punctiforme.

The overall goal of this project is to explore the potential of cyanobacteria to become efficient producers of H₂ as a future energy carrier. Cyanobacteria are prokaryotes, which are able to use solar energy and H₂O for H₂ production. We are mapping the metabolic processes involved in hydrogen metabolism, with focus to identify and remove bottlenecks in the production. Many different approaches are needed so as to explore the full potential of cyanobacteria as future H2 producers. Our strategy is to use qualitative and quantitative high throughput proteomics for exploring the dynamic changes that takes place in the cells during different growth conditions, and to determine the reprogramming of metabolism when hydrogen metabolism is modified. As model organisms N₂fixing filamentous cyanobacteria with fully sequenced and annotated genomes are used, which is a prerequisite for large-scale proteomic work. Shot-gun (non-gel based) as well as gel based proteomics are used. To be able to characterize changes in expression of proteins under different conditions, in specific cell types as well as in engineered strains of cyanobacteria, we use iTRAQ (peptide tagging-method) together with “state of the art”, mass spectrometry. 

In order for an organism to be able to adapt to its surroundings it must employ numerous strategies to dynamically sense and translate changes in its environment, and hence to adjust the relevant processes within the cell. Redox control is one such strategy. The objective of this research project is to investigate different aspects of redox control in cyanobacteria. We take a proteomic approach and focuses are on proteins acting as redox transmitters such as thioredoxins, and proteins, which are targets for redox such as transcriptional regulators and enzymes. Multicellular cyanobacteria of the genus Nostoc are used to investigate the difference in redox regulation in cells with O₂ evolving photosynthesis and in non-photosynthetic cells specialized in fixation of atmospheric nitrogen, the heterocysts. Knowledge gained from this research will give insight in how cyanobacteria and other photosynthetic organisms adapts to changing environmental conditions and stress. With the characterized proteome of N₂ fixing (with heterocysts) and non N₂ fixing (without heterocysts) filamentous cyanobacteria Nostoc sp. PCC 7120 and Nostoc punctiforme ATCC 29133 we have developed robust experimental and statistical methods for our work, and we have shown the usefulness of quantitative shotgun proteomics in cyanobacterial research. The overall aim is to control the redox balance and reactive oxygen species (ROS) concentrations on a system level and thereby increase the robustness of cyanobacteria for future large-scale cultivation. And finally answer the questions of: How does the multicellular cyanobacteria coordinate the redox regulatory activities in the different cells and exchange information in the form of redox intermediates to maintain overall redox homeostasis to combat oxidative stress? Could we increase the ability of cyanobacteria to tolerate environmental changes?

Can we increase the ability of cyanobacteria to tolerate environmental changes and therefore limit the toxin production during cyanobacterial blooms? We strongly believe that the system focused investigations of ROS tolerance in cyanobacteria will be of importance for the understanding of the impact of ROS to cyanotoxin production and physiology of cyanobacterial strains during the bloom cycle. The study will provide new knowledge and tools for cyanotoxin control and bloom management. Furthermore this awareness will also be beneficial for the nontoxic blue-green algae yield that could be directly used as biomass for food, feed and fuels.

In this project we use different genetic and biochemical strategies to investigate the role of ferritin-like proteins in redox control, iron homeostasis and regulation of ROS levels. The ferritin-like proteins in Nostoc include one putative bacterioferritin and four Dps proteins. Dps are strongly connected to DNA protection, iron storage as well as detoxification of H₂O₂ within bacterial cells combating oxidative stress.

The first aim of this part of the project will be to study the response of N. punctiforme to oxidative stress by utilizing a proteomics approach. More precisely, the proteomic profile of deletion and overexpression strains will be studied using quantitative gel-based (LC-MS) methodology in cells cultivated under “normal” growth conditions as well as in the presence of reactive oxygen species (ROS) and under high light. This work will provide an understanding of overall cellular response to stress conditions and more specifically the role of the ferritin-like proteins such as Dps proteins and bacterioferritins in this process. The results will be of importance for future systems metabolic engineering efforts to design robust strains of cyanobacteria for large scale cultivation for production of biofuels.