Synthetic In Vivo Activation of Heterologous [FeFe]-Hydrogenase in Engineered Cyanobacteria

Wegelius, A., Uppsala University
Khanna, N., Uppsala University
Lindblad, P., Uppsala University
Esmieu, C., Uppsala University
Berggren, G., Uppsala University
Hydrogenases are a broad class of enzymes that catalyze reversible formation of hydrogen. Among hydrogenases, the [FeFe]-hydrogenase (HydA) is the most active hydrogen producer (1). The active site of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster connected to a [2Fe] subcluster via a cysteine. In nature, biosynthesis of the [2Fe] subcluster requires three additional hydrogenase specific maturases, HydE, HydF and HydG, which assemble the subcluster and transfer it to the apo-HydA protein, already containing the pre-assembled [4Fe-4S] cluster. This maturation process is not fully characterised and has long been an inconvenience for heterologous expression of HydA in cyanobacteria. Synthetic chemistry has been used to create precise mimics of the [2Fe] subcluster (2) and these clusters have been used to successfully activate the apo-HydA enzyme in vitro (3,4) and recently also in vivo in cells of Escherichia coli (5). In our present work, we have successfully activated an apo-HydA from Chlamydomonas reinhardtii, heterologously expressed in the unicellular cyanobacterium Synechocystis PCC 6803, using a synthetic [2Fe] subcluster mimic. This was achieved using a novel combination of cyanobacterial synthetic biology and synthetic chemistry and is, to the best of our knowledge, the first example of synthetic maturation of a metalloenzyme in vivoin cyanobacteria. The usage of an engineered, hydrogenase free, cyanobacterial background strain (6) offers a unique opportunity to elucidate the activity of the synthetically activated hydrogenase without interference from native hydrogen metabolism. The activated cells exhibit hydrogen production from HydA under a number of different conditions, including both light and darkness, with the highest production recorded under organic nitrogen deprivation. This provides interesting and valuable insight to the usage of metabolically engineered cyanobacteria as platforms for hydrogen production. Also, it provides a convenient new tool to evaluate recombinant [FeFe]-hydrogenases expressed in metabolically engineered cyanobacteria in the absence of the still incompletely characterized maturases.

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