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Light-driven sustainable biocatalysis training network

Periodic Reporting for period 2 - PhotoBioCat (Light-driven sustainable biocatalysis training network)

Reporting period: 2020-01-01 to 2021-12-31

Pharmaceutical and chemical products present in everyday life are currently obtained from waste- and energy-intensive processes. Alternative routes that overcome this challenge should offer a cleaner and long-lasting solution. PhotoBioCat aimed at developing efficient and sustainable light-driven enzyme-catalyzed chemical processes to a commercial industrial level. The PhotoBioCat trained 12 early-stage researchers (ESRs) in cutting edge research projects on (i) the development of chemical and biological catalytic systems for the synthetic use of visible light for the production of pharmaceuticals, bulk-, fine- and specialty chemicals, (ii) up-scaling and optimization of commercially relevant processes together with industry, (iii) defining a road-map for the industrial implementation of photobiocatalysis. A double degree program allowed in-depth training in two complementary disciplines, further strengthened by a transferable skills training with strong industry participation. A consortium of seven universities and eight industrial partners provided a fruitful environment to foster complementary expertise in chemical synthesis, biocatalysis, synthetic biology and process engineering. PhotoBioCat thus explored (a) the catalytic application of photosynthetic microorganisms and their optimization by modern synthetic biology techniques; (b) the coupling of enzymes to light-harvesting molecules in vitro; and (c) the optimization of photobiocatalytic processes to technical scale; accompanied by specially designed training of the ESRs and dissemination & communication of the results to the scientific community and public.
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.
The work led to a significant process both on photosynthesis-driven enzyme reactions and enzymes coupled to photocatalytic reactions. Most importantly, the highly interdisciplinary approach allowed the development of new reactions, the increase of efficiency by metabolic engineering and the up-scale in new photobioreactors. It could be shown that the approach allows for significant savings in sacrificial cosubstrates, which will greatly improve the atom efficiency of redox biocatalysis. The results of PhotoBioCat are thus expected to contribute to promote biocatalysis as an environmentally-friendly method for the chemical and pharmaceutical industry. An important aspect of PhotoBioCat was the intensive interaction with the private sector, which is crucial to convert inventions to innovations. The interaction with large enterprises and small high-tech companies during the secondments of the PhD students were used for the creation of expertise on photoreactors and photobiocatalytic reactions and provided them with scientific and transferable skills required to work successfully in a highly dynamic, interdisciplinary and international field. The first phase of PhotoBioCat produced success cases how enzymes can be coupled to light-harvesting systems as electron donors for redox biocatalysis. In the second phase, the highly interdisciplinary collaboration was used to increase our understanding how enzyme and light-harvesting systems can be combined in cellular and cell-free systems, and our knowledge on the key parameters for an optimization of these reactions. The results represent an important step towards market implementation of this highly sustainable catalytic technology.
The value of training in the field of photobiocatalysis was demonstrated by the fact that all ESRs immediately found employment in industry and academia after the conclusion of their research projects.
PhotoBioCat Consortium