Light-Driven [FeFe] Hydrogenase Based H₂ Production in E. coli: A Model Reaction for Exploring E. coli Based Semiartificial Photosynthetic Systems

Abstract

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 (H 2 ) gas production as a model system for semiartificial photosynthesis. The employed whole-cell photocatalytic 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.

Elucidating Electron Transfer Kinetics and Optimizing System Performance for Escherichia coli-Based Semi-Artificial H2 Production

“…The protocol and concentrations of the different components were adapted from our previous design-of-experiment study on E. coli-based photocatalytic hydrogen evolution systems. 19 Control samples with E. coli cells containing an inactive apo-form of CrHydA1 were analyzed in parallel. The samples (1 mL sample volume in 9 mL vials, estimated path length 5 mm) were prepared under an inert gas atmosphere (nitrogen or argon gas) and exposed to continuous white light irradiation (4000 lx, main emission 400−650 nm, spectrum, see Figure S.I.…”

“…As previously reported, 10,19 the whole-cell system using EY as the PS does produce hydrogen upon white light irradiation, and the photocatalytic system significantly outperforms the fermentative production in darkness. 22,23 However, the hydrogen production does not last longer than 24 h. One of the main reasons for the short-lived activity of the system is the decomposition of EY upon photoreduction by TEOA, apparent from a color change of the assay mix from pink to almost colorless.…”

“…10,19 Furthermore, different (sacrificial) electron donors are added to the system. Typical examples of such electron donors found in the literature are cysteine, 8,17,20 triethanolamine (TEOA), 9,19 bis-trismethane, 11 and ascorbic acid. 13,21 While a system consisting of a whole-cell biocatalyst, an artificial PS, and an electron donor is technically a complete photocatalytic system, many reports also include a mediator, e.g., methyl viologen 9 or reduced graphene oxide, 21 which facilitates the efficient transport of electrons from the photoactive system to the enzyme.…”

“…9,11,14 A variety of artificial PSs have been studied for whole-cell catalysis, e.g., inorganic materials such as TiO 2 nanoparticles, 15,16 metal oxynitrides, 9 CdS quantum dots, 11,17,18 or organic dyes such as Eosin Y (EY). 10,19 Furthermore, different (sacrificial) electron donors are added to the system. Typical examples of such electron donors found in the literature are cysteine, 8,17,20 triethanolamine (TEOA), 9,19 bis-trismethane, 11 and ascorbic acid.…”

“…10,12,19 We could further spectroscopically show that this hydrogen production activity indeed stems from the photocatalytically driven [FeFe] hydrogenase enzyme inside the cells. 19 Following our earlier design-of-experiment screening for the ideal catalytic conditions, we herein present studies focusing on the photochemistry aspects of the system. The factors determining the photocatalytic performance are identified using in vitro and in vivo steady-state and transient UV−vis absorption spectroscopy.…”