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 H 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.
Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, β = 98.21, γ = 90°.
Due to energetic considerations, microalgae are becoming more and more popular for the production of biomass in general and the production of high value products in special applications. Major benefits of these organisms are the use of sunlight as a free energy source and the fixation of CO 2 by incorporation into reduced carbon compounds. Both can contribute to reducing atmospheric CO 2 (a "climate killer" gas) and also can be regarded as energy-rich storage compounds for periods of energy shortage. There are, however, also alternative strategies for converting sun energy directly into biofuels, i.e. high energy compounds, which are energetically and economically highly attractive: The classical route of energy storage via carbon compounds is extremely inefficient, ranging in most cases (due to metabolic constrains) from below 1 % and only in exceptional cases up to about 5 % efficiency , which is in contrast to the high efficiency of the primary (light-) reactions of photosynthesis [2,3]. For this reason, biofuel production has to be coupled as closely as possible to the light reactions of photosynthesis, combined with a re-routing of electrons which minimizes the steps of biofuel production and also the loss of electrons due to carbon fixation [4][5][6]. The attractive overall strategy is to receive the required electrons from the most abundant and cheapest compound available on earth -water -and to transfer them directly to the biofuel. The most useful and most direct-to-produce biofuel would be hydrogen [6], as it is a high energy compound that releases its energy by reaction with oxygen, producing as the only "waste" product again the starting compound water. The energy of this reaction can be used directly -for instance in a fuel cell -or can be easily stored for a longer time. By transferring the electrons primarily to protons instead of reducing CO 2 , this process is completely environmentally acceptable. In contrast, the release of energy stored in reduced carbon compounds inevitably leads to the production of CO 2 -besides the high energy loss due to the many metabolic steps involved.As this is a new concept which is against the principles of nature -it does not secure survival of the cell by storing energy but rather "wastes" the collected sun energy by setting free the high energy compound hydrogen -the metabolism of such a cell has to be modified in many individual steps. This is also a challenge from the bioenergetic point of view, since the amount of sun energy collected by a photosynthetic cell that can be harnessed for mankind is completely unknown. Such an "exploita-
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