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Ferredoxin (Fd) is a small metalloprotein holding one or two Fe-S clusters in its inner shell. Like many other metalloproteins, Fd is redox active and involved in electron transfer during cellular metabolism. The electrons from reduced Fd are mostly used to regenerate NADPH under physiological conditions. Increasing number of attempts have been reported, however, where Fd delivers electrons to enable biosynthesis of valuable compounds. Various compounds ranging from H 2 to vitamin D 3 have been synthesized successfully using electrons mediated by Fd molecules. In this review, we provide an overview of the engineering studies utilizing Fd for biosynthesis of targeted molecules. The emphasis is on the role and activity of Fd as well as the methods used to improve the rate of electron transfer. Both microbial and electrochemical biosynthesis technologies are described and compared with respect to productivity and the compound being produced. In addition to the ferredoxins from the microbial organisms, artificially designed de novo types are described, highlighting the potential of the emerging computational methods used in metabolic and protein engineering. We believe that the recent advances in utilization of Fd for biosynthesis can result in breakthrough innovation across the biotechnology industry., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

[FeFe] hydrogenases catalyze rapid H 2 production but are highly O 2 -sensitive. Developing O 2 -tolerant enzymes is needed for sustainable H 2 production technologies, but the lack of a quantitative and predictive assay for O 2 tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O 2 sensitivity by using an assay employing ferredoxin NADP + reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H 2 production in an O 2 -containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction solutions. The second assay validates the more convenient NADPH-driven assay, which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O 2 However, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O 2 tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15-amino acid linker but not for a longer linker. We suggest that this precise, solution phase assay for [FeFe] hydrogenase O 2 sensitivity and the insights we provide constitute an important advance toward the discovery of the O 2 -tolerant [FeFe] hydrogenases required for photosynthetic, biological H 2 production., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hydrogenases, ferredoxins, and ferredoxin-NADP+ reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H2 production technologies. A high-throughput H2 production assay device (H2PAD) is presented that enables simultaneous evaluation of 96 individual H2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H2PAD senses the chemo-optical response of Pd/WO3 thin films to the H2 produced. H2PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH-driven H2 production rates. H2PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems. [ABSTRACT FROM AUTHOR]