(Ondřej Prášil – MBÚ)
The success of our project requires the cultivation, manipulation and rapid analysis of thousands of mutant strains of cyanobacteria and algae grown under precisely controlled conditions. Automated phenotyping of algae and cyanobacteria is essential for rapid and efficient characterization of random and targeted mutants and for selection of highly productive strains. There are no commercially available robotic solutions that accomplish all the required tasks: cultivation, manipulation and detailed analysis at the scale expected in the project. Therefore, in this project we will develop the AlgaScreen robotic system for automated cultivation, manipulation and basic characterization of cyanobacterial/algal strains on agar or culture plates. The system will be equipped with new sensor stations and artificial intelligence to create a unique, fully automated phenotyping line. The new optical sensors will be able to detect and quantify phycobilisomes and fluorescent proteins, as well as lipid, carbohydrate and metabolic product content in the mutant cells being analysed.
(Vendula Krynická – MBÚ)
Pressure on a phototrophic cell to accumulate large amounts of bioproducts inevitably leads to conflicts in nutrient allocation, as growth and production use the same limiting resources. To solve this problem, we reprogram the cyanobacterium Synechocystis to be switchable to a genetically integrated survival program. Upon activation of this program, cells divert carbon and nitrogen (C/N) metabolites, including amino acids and bilins stored in light-harvesting antennae (phycobilisomes), to storage polymers. To prevent further investment of cells in biomass, growth will be blocked during the production phase. The basic Syn2Cell prototype, constructed in the first phase of the project, will be refined using high-throughput screening techniques as well as advances in other tasks of the project. Synechocystis tak, aby byla přepínatelná na geneticky integrovaný program přežití. Po aktivaci tohoto programu buňky přesměrují metabolity uhlíku a dusíku (C/N), včetně aminokyselin a bilinů uložených ve světlosběrných anténách, do zásobních polymerů. Aby se zabránilo dalším investicím buněk do biomasy, bude růst v produkční fázi blokován. Základní prototyp Syn2Cell, zkonstruovaný v první fázi projektu, bude zdokonalen pomocí vysoce výkonných screeningových technik a také pokrokem v dalších úkolech projektu.
(Josef Komenda – MBÚ)
Program přežití, který se aktivuje u sinic v podmínkách nedostatku živin, způsobuje degradaci fotosyntetického aparátu. Vzhledem k tomu, že modifikovaný program přežívání bude využit v buňkách Syn2Cells (úkol 1.2) k přesměrování metabolitů do bioproduktů, je zásadní zlepšit stabilitu fotosyntetického aparátu během produkční fáze. Za tímto účelem identifikujeme rozhodující proteinové faktory, které kontrolují akumulaci a kontrolu kvality obou fotosystémů. Případné odstranění, modifikace nebo zvýšená akumulace těchto faktorů by měla vést k udržení aktivní fotosyntézy i po napodobení podmínek nedostatku dusíku. Současně využijeme strategii náhodné/cílené mutageneze a specifické selekce kolonií, které si udrží vysokou fotosyntetickou aktivitu i během podmínek živinového stresu. Výsledkem kombinace těchto dvou přístupů by měl být nakonec prototyp Syn2Cell s vysokou fotosyntetickou aktivitou v produkční fázi.
(Eva Kiss – MBÚ)
This task focuses on how cyanobacteria regulate central metabolism at the post-translational level to direct metabolic fluxes to storage compounds, and how they catabolize amino acids and bilins released from their giant light-harvesting antennae. These processes are mostly unknown; however, the results suggest that tight posttranslational control of enzymes involved in central carbon and nitrogen metabolic pathways plays a key role. The main goal is to investigate the dynamics of enzyme complexes that enable the metabolic change from a rapid growth to a survival program. We identify the regulatory function of interacting protein factors as well as the importance of oligomerization and post-translational modifications of central enzymes and their protein partners. The resulting list of protein factors and specific enzyme modifications will be used for targeted mutagenesis to improve the properties of the Syn2Cell system. The results obtained will also provide a comprehensive view of the post-translational regulation of metabolite levels, which is important for overcoming potential bottlenecks in Syn2Cell metabolism.
(Roman Kouřil – UPOL)
The newly set metabolism of Syn2Cell cells will require optimal regulation of redox and ATP homeostasis to ensure sufficient energy. For this proper setup, the formation of new types of membrane protein supercomplexes and the associated reorganization of membrane microdomains will be crucial. Using confocal and electron microscopy methods, we will investigate the structural reorganization of thylakoid membranes and proteins of the photosynthetic apparatus, and through this we will identify potential genetic targets for tuning energy homeostasis. Thus, by solving this task, we will obtain the necessary data set that will allow us to adjust the energetics of the cyanobacterial cell with respect to the energy needs of the Syn2Cell cell.
(Meri Eichner – MBÚ)
There are millions of cells in every milliliter of microalgae culture. In recent years, it has become apparent that these cultures can exhibit considerable intercellular heterogeneity, meaning that the production of desired compounds varies greatly from cell to cell. To maximize bioproduct yields in Syn2Cell cultures (Task 1.2), it is necessary to: i) exclude cells that do not manifest the desired genetic modification ("revertants" and "cheaters"), and ii) select for rare spontaneous mutations with unique phenotypes ("supercells"). To this end, we will develop a high-throughput screening and selection approach combining flow cytometry, cell sorting and automated culturing and monitoring.
(Iva Mozgová – BC)
Green algae are an important source of biomass for biofuel and feed production. As eukaryotic phototrophs, they share most of the primary metabolic pathways with prokaryotic cyanobacteria; in contrast, growth, biomolecule partitioning and cell division require an additional level of coordination between the genomes of the nucleus and organelles and between their products. This makes algae the perfect eukaryotic equivalent of cyanobacteria – they share phototrophy but overcome the limitations of the prokaryotic system, such as the insolubility of biosynthetic products, problems with protein folding, or the absence of some post-translational modifications. Due to the higher complexity and underdeveloped tools of synthetic biotechnology, it is currently not possible to construct an eukaryotic equivalent of Syn2Cell. Task 1.7. focuses on understanding how evolutionary innovations in eukaryotes affect the metabolic switch between growth and production. It will reveal how epigenetic mechanisms contribute to the establishment of growth and production states and to the coordination of chloroplast and nuclear genome expression. Genetic screening will identify novel organelle signaling components that coordinate cell growth and accumulation of storage compounds. This will provide the basis for the application of strategies that allow targeted improvement of the production phase in algae.
(David Bína – JČU)
The light-harvesting complexes of cyanobacteria, phycobilisomes (PBS), represent a large and rapidly degradable source of organic carbon and nitrogen that can be directed towards the production of desired bioproducts (Task 1.2). However, for efficient photosynthesis in high cell density culture during the production phase, photosynthetic complexes (photosystems) require an external antenna system. For this purpose, we propose to develop chlorophyll- and carotenoid-based antennae that functionally resemble the light-harvesting complexes of algae and plants. This synthetic antennae, with a higher density of chromophores than PBS, will provide a steady supply of energy from a wider range of photons once the genetic program for the production phase is activated (see Task 1.2).
(Roman Sobotka – MBÚ)
Synthetic biology of cyanobacteria and algae is a rapidly developing field and more and more biological tools and other standardized genetic elements are becoming available for microalgae. However, compared to widely used bacterial or yeast systems, the current set of molecular tools is still relatively limited. Particularly noticeable is the lack of a system for robust and very strong gene expression in cyanobacteria. The low accumulation of heterogeneously expressed proteins in cyanobacteria is a persistent problem that could be however tackled by a shuttle vector with inducible high copy number (HC). We aim to convert the pCC5.2 plasmid, which is natively present in Synechocystis, into an inducible HC shuttle vector that can be maintained in E. coli. The resulting vector will be used for metabolic engineering of Synechocystis (depletion - decoys of transcription factors, task 1.2) and for bioproduction of valuable compounds such as nostatin (see task 2.5.). Synechocystis, na indukovatelný HC shuttle vektor, který lze udržovat i v E. coli. Výsledný vektor bude využitý pro metabolické inženýrství Synechocystis (vysycení – decoys transkripčních faktorů, úkol 1.2) a pro bioprodukci cenných látek jako je nostatin A (viz úkol 2.5.).
(Michal Koblížek – MBÚ)
Apart from oxygenic cyanobacteria, there is another large group of phototrophic microorganisms that perform anoxygenic phototrophy using bacteriochlorophyll. Another phototrophic group are rhodopsin-containing bacteria. These are metabolically very diverse groups. Some phototrophic species have a clear biotechnological potential as they can fix atmospheric nitrogen or produce hydrogen. Some other species can produce substances that promote the growth of agricultural crops, other species produce a wide range of carotenoids with significant biotechnological potential. We therefore propose to use the robotic AlgaScreen system (see Task 1.1.) to isolate new phototrophic organisms. The selected strains will be sequenced and their genome annotated. The selected metabolic pathways can be used to construct a modified Syn2Cell cyanobacteria. We plan to use a transposon mutagenesis-based method to identify important genes and metabolic pathways. The obtained mutants will be further tested using a screening robot.
(Jan Janouškovec – MBÚ)
Bioproduction of high-value compounds is often hindered by small gaps in knowledge of basic biochemistry. An example is the biosynthesis of photosynthetic pigments such as chlorophylls and carotenoids, where the discovery of individual missing enzymes may revolutionize our ability to modify microalgal metabolism to produce valuable compounds. In this task, we will expand the catalogue of enzymes for the synthetic biology of phototrophs using state-of-the-art approaches to search for the responsible genes in genomes and functionally test them. The result will be engineered cells that express target molecules and can be adapted for use in biotechnology, medicine and the food industry.
(Pavel Hrouzek – MBÚ)
Cyanobacteria are a huge source of new chemical compounds. As prokaryotic organisms, they possess highly versatile biosynthetic mechanisms known to produce compounds with therapeutic potential, many of which are recently in various stages of clinical trials. In the proposed task, we will apply state-of-the-art screening methods to find molecules applicable in two extremely important areas of medical research. The first involves the search for quorum-sensing inhibitors with potential use in anti-viral therapy, used to control the development of antimicrobial resistance. In the second, we focus on compounds with potential use as antiproliferative agents against triple negative breast cancer cells, a disease with inadequate therapeutic coverage.
(Karolína Štěrbová – MBÚ)
The biosynthetic apparatus of cyanobacterial secondary metabolites is usually encoded by colocalized genes in so-called biosynthetic gene clusters (BGCs). Such an arrangement is advantageous for identifying the genes responsible for the biosynthesis of the compound, but also makes the BGC accessible for expression in another organism, as it usually contains all the genes required for the synthesis of the compound. In our previous studies, we have identified several BGCs, including one that encodes the antiproliferative compound nostatin A. In this task, we will focus on understanding the biosynthetic steps required for the synthesis of the studied bioactive metabolites as well as the structural motifs important for their bioactivity. Subsequently, we will prepare mutant strains of Synechocystis capable of biosynthesizing these high-value bioactive products. This biotechnological approach is of considerable benefit in the context of the molecules mentioned above, as these compounds are relatively difficult to prepare by classical organic synthesis.
