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Semi-artificial photosynthesis aims to harness the power of biocatalysis while breaking away from the limitations of Nature's photosynthetic machinery, by merging artificial light harvesters with enzyme catalysts. However, the artificial photocatalytic components are generally toxic towards the biological components. In this study, we investigate a system wherein Escherichia coli cells, heterologously expressing an [FeFe] hydrogenase, act as hydrogen evolution catalyst in combination with an artificial photosensitizer, sacrificial electron donor, and redox mediator. Previously, the use of artificial components or their reaction products was found to be toxic to E. coli cells. To overcome this challenge, we examined alternative electron donors and redox mediators, achieving turnover numbers (TON, 39.6 mu mol H-2 per 1 mL sample with OD600 = 5) and turnover frequencies (TOF, 812 nmol H-2 h(-1) per 1 mL sample with OD600 = 5) on par with previously reported high performing E. coli-based systems while greatly reducing cytotoxic effects. Transient absorption spectroscopy revealed how the choice of photosensitizer, electron donor, and redox mediator affects the observed photocatalytic TOFs. Following optimization of the redox mediator and electron donor the biocatalyst demonstrated remarkable stability throughout the experiments. We identified the availability of electrons from the electron donor as the primary limiting factor, with approximately 85% of electrons being effectively utilized for hydrogen production. Furthermore, the observed activity with different [FeFe] hydrogenases verified the broad applicability of the identified photocatalytic components to promote light-driven catalysis in bio-hybrid systems.

Both photo- and biocatalysis are well-established andintensivelystudied. The combination of these two approaches is also an emergingresearch field, commonly referred to as semi-artificial photosynthesis.Semi-artificial photosynthesis aims at combining highly efficientsynthetic light harvesters with the self-healing and potent catalyticproperties of biocatalysis. In this study, a semi-artificial photocatalyticsystem featuring Escherichia coli bacteria,which heterologously express the [FeFe] hydrogenase enzyme HydA1 fromgreen algae, is employed as a hydrogen gas production catalyst. Toprobe the influence of photochemistry on overall system performance,the E. coli whole-cell catalyst iscombined with two different photosensitizers and redox mediators.The addition of a redox mediator greatly improves the rates and longevityof the photocatalytic system, as reflected in increases of both theturn-over number (0.777 vs 10.9 & mu;mol H-2 mL(-1) OD600 (-1)) and the turn-over frequency(175 vs 334 & mu;mol H-2 mL(-1) h(-1) OD600 (-1)). The redoxmediator is found to both protect from photobleaching and enable electrontransport to the hydrogenase from an extracellular photosensitizer.However, E. coli cells are stronglyaffected by the photocatalytic system, leading to a decrease in cellintegrity and cell viability, possibly due to toxic decompositionproducts formed during the photocatalytic process. We furthermoreemployed steady-state and transient absorption spectroscopy to investigatesolution potentials and the kinetics of electron transfer processesbetween the sacrificial electron donor, photosensitizer, redox mediator,and the [FeFe] hydrogenase as the final electron acceptor. These resultsallowed us to rationalize the different activities observed in photocatalyticassays and offer a better understanding of the factors that influencethe photocatalytic performance of E. coli-based whole-cell systems.

Biohybrid technologies like semiartificial photosynthesis are attracting increased attention, as they enable the combination of highly efficient synthetic light-harvesters with the self-healing and outstanding performance of biocatalysis. However, such systems are intrinsically complex, with multiple interacting components. Herein, we explore a whole-cell photocatalytic system for hydrogen (H2) gas production as a model system for semiartificial photosynthesis. The employed whole-cell photo catalytic system is based on Escherichia coli cells heterologously expressing a highly efficient, but oxygen-sensitive, [FeFe] hydrogenase. The system is driven by the organic photosensitizer eosin Y under broad-spectrum white light illumination. The direct involvement of the [FeFe] hydrogenase in the catalytic reaction is verified spectroscopically. We also observe that E. coli provides protection against O-2 damage, underscoring the suitability of this host organism for oxygen-sensitive enzymes in the development of (photo) catalytic biohybrid systems. Moreover, the study shows how factorial experimental design combined with analysis of variance (ANOVA) can be employed to identify relevant variables, as well as their interconnectivity, on both overall catalytic performance and O-2 tolerance.