Cyano-GenApp

Cyanobacteria are exceptional microbes, which are (like plants) capable of using light as an energy source via photosynthesis. Due to their properties they are interesting candidates for manufacturing processes in industry. However, they are poorly established as cell factories as they are challenging in handling and genetic manipulation. One central goal of this project is to develop tools for facilitating genetic manipulation of cyanobacteria. A very popular strategy of genetic manipulation is CRISPR/Cas9. This method is beneficial when compared to other strategies as it is more efficient and does not require the use of a genetic marker for selection of mutant strains. The system is based on a protein (Cas9) which cuts the DNA at target positions specified by a guide RNA, thus finally allowing integration of foreign DNA. CRISPR/Cas9 is established in several organisms, however in cyanobacteria the application of this method is restricted by toxic effects of the Cas9 protein. As an approach to overcome this problem, the usability of Cas proteins natively occurring in cyanobacteria is tested in course of this project. This promises to bring a reduction of toxic effects and an increase in the mutation efficiency. Another obstacle to genetic manipulation of cyanobacteria is the genetic organisation of these organisms. In contrast to most other bacteria, cyanobacteria possess multiple copies of their genome, which hampers the generation of genetically pure mutant strains. Hence, for this project, cultivation conditions resulting in a reduction of the genome copy number are applied. The combination of this strategy and a highly efficient engineering method such as CRISPR/Cas promise to allow the generation of genetically pure mutant strains in one single step and without making use of a selection marker. This would be a milestone in genetic manipulation of cyanobacteria and expand the option s for their final application. Another, separate part of the project Cyano-GenApp is the production of hydrogen in cyanobacteria. Hydrogen is a good alternative to fossil fuels and thus a promising renewable energy perspective. Cyanobacteria possess a specific enzyme for hydrogen production, the so termed Hox hydrogenase. This enzyme is highly efficient, yet it is sensitive to oxygen produced during photosynthesis in the presence of light. Thus, cyanobacteria can only produce hydrogen in the absence of light (at night) - conditions, which in turn allow only poor growth of cyanobacterial cells. A totally new approach to address this issue is to replace the native Hox hydrogenase by a hydrogenase with more beneficial properties. This more beneficial hydrogenase will be created by fusion of two hydrogenases, namely the native cyanobacterial hydrogenase and an oxygen-stable hydrogenase obtained from a knallgas bacterium. This strategy bears the potential hydrogen production in cyanobacteria under optimized growth conditions, at best in an industrial scale.