CyanoFactory, a European consortium to develop technologies needed to advance cyanobacteria as chassis for production of chemicals and fuels

CyanoFactory, Design, construction and demonstration of solar biofuel production using novel (photo)synthetic cell factories, was an R&D project developed in response to the European Commission FP7-ENERGY-2012-1 call “Future Emerging Technologies” and the need for significant advances in both new science and technologies to convert solar energy into a fuel. CyanoFactory was an example of “purpose driven” research and development with identified scientific goals and creation of new technologies. The present overview highlights significant outcomes of the project, three years after its successful completion. The scientific progress of CyanoFactory involved: (i) development of a ToolBox for cyanobacterial synthetic biology; (ii) construction of DataWarehouse/Bioinformatics web-based capacities and functions; (iii) improvement of chassis growth, functionality and robustness; (iv) introduction of custom designed genetic constructs into cyanobacteria, (v) improvement of photosynthetic efficiency towards hydrogen production; (vi) biosafety mechanisms; (vii) analyses of the designed cyanobacterial cells to identify bottlenecks with suggestions on further improvements; (viii) metabolic modelling of engineered cells; (ix) development of an efficient laboratory scale photobioreactor unit; and (x) the assembly and experimental performance assessment of a larger (1350 L) outdoor flat panel photobioreactor system during two seasons. CyanoFactory - Custom design and purpose construction of microbial cells for the production of desired products using synthetic biology – aimed to go beyond conventional paths to pursue innovative and high impact goals. CyanoFactory brought together ten leading European partners (universities, research organizations and enterprises) with a common goal – to develop the future technologies in Synthetic biology and Advanced photobioreactors

Structural response of Photosystem 2 to iron deficiency: Characterization of a new Photosystem 2–IdiA complex from the cyanobacterium Thermosynechococcus elongatus BP-1

Iron deficiency triggers various processes in cyanobacterial cells of which the synthesis of an additional antenna system (IsiA) around photosystem (PS) 1 is well documented. Here we show that PS2 also undergoes prominent structural changes upon iron deficiency: Prerequisite is the isolation and purification of a PS2–IdiA complex which is exclusively synthesized under these conditions. Immunoblotting in combination with size exclusion chromatography shows that IdiA is only bound to dimeric PS2. Using single particle analysis of negatively stained specimens, IdiA can be localized in averaged electron micrographs on top of the CP43 subunit facing the cytoplasmic side in a model derived from the known 3D structure of PS2. The presence of IdiA as integral part of PS2 is the first example of a new PS2 protein being expressed under stress conditions, which is missing in highly purified PS2 complexes isolated from iron-sufficient cells.

Rational redesign of the ferredoxin-NADP⁺-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H₂-production

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]‑hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H2 production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria