Work on cyanobacterial whole-cell biocatalysts focused on the use of electrons from photosynthetic water splitting for heterologous redox reactions. For this, the photosynthetic electron transport first reduces ferredoxin, which then via the ferredoxin-NADP reductase reduces NADPH. The results of PhotoBioCat significantly expanded the scope of the method towards novel applications. The expression of the gene of a cyanobacterial CYP450 monooxygenase established a new reaction for the selective hydroxylation of steroids. This system directly uses ferredoxin, which is reduced by the photosystem I. Screening of a panel of bacterial Baeyer-Villiger monooxygenases led to the identification of a new enzyme that showed improved activity both in the heterotroph Escherichia coli and the photoautotroph Synechocystis. Interestingly, the specific activity of the recombinant cyanobacteria was higher than those of the E. coli strain, underlining the potential of cyanobacteria as sustainable biocatalysts. Moreover, a new system for the application of cyanobacteria for the recycling of redox cofactors was developed. In this system, a mediator molecule (such as acetone) is reduced within the cell on the expense of NADPH, which in turn is regenerated with electrons from the photosynthetic electron transport. The reduced mediator then is exported from the cell to the supernatant, where it is used for redox cofactor recycling for a redox reaction in the outer volume of the reactor. This system is modular and can in principle be applied for any reaction requiring nicotinamide cofactors. After the successful increase of the diversity of reactions fueled by photosynthesis, focus shifted on an increase of the capacity of the cells to donate electrons for heterologous redox reactions, which was achieved by the deletion of competing electron sinks. This metabolic engineering approach identified several targets for improvement, and a 1.4-2 fold activity improvement could be achieved. The metabolic engineering went along with the optimization of the conditions of the photobiotransformation reaction. In this context, the up-scale of photobiocatalytic reactions is a challenge as the increasing distance between light source and photocatalyst leads to losses by absorption, and hence a lower catalyst activity. By using the principle of internal illumination in a bubble-column photobioreactor with floating LEDs, it was shown that this challenge can be overcome. The demonstration of the so far highest volumetric yield of a photobiotransformation in a scalable photobioreactor is an important milestone on the way to the industrial application of the technology. Work on photobiocatalysis in cell-free systems compared light-driven enzymes with indirect photobiocatalysis using organic and inorganic photocatalysts. The ESRs explored new reaction concepts for the coupling of highly selective enzymes to light-driven supply of redox cofactors.
Work spanned from the coupling of bacterial whole-cell biocatalysts to photocatalysts in the cell-free space to the investigation of photosynthesis in cell-free systems. Work on enzyme-photocatalyst coupled systems established a new system where the selectivity of an enzyme cascade could be controlled with the wavelength of the light. The up-scale of cascade reactions comprised of photocatalysts and enzymes identified the light-sensitivity of some of the photocatalysts as bottleneck.
An integral element of PhotoBioCat includes the public communication of the research by imparting awareness on the positive impact of biocatalysis to the environment. The ESRs actively contributed to public engagement activities, including the creation of videos that were uploaded to YouTube, social media channels and through the participation in the European Researchers’ Night. Scientific results were disseminated in the form of conference presentations and high-impact publications.
