Cell‐free synthesis and maturation of [FeFe] hydrogenases

Abstract

[FeFe] hydrogenases catalyze the reversible reduction of protons to molecular hydrogen (Adams (1990); Biochim Biophys Acta 1020(2): 115-145) and are of significant interest for the biological production of hydrogen fuel. They are complex proteins with active sites containing iron, sulfur, and carbon monoxide and cyanide ligands (Peters et al. (1998); Science 282(5395): 1853-1858). Maturation enzymes for [FeFe] hydrogenases have been identified (Posewitz et al. (2004); J Biol Chem 279(24): 25711-25720), but complete mechanisms have not yet been elucidated. The study of [FeFe] hydrogenases has been impeded by the lack of an easily manipulated expression/activation system capable of producing these complex and extremely oxygen-sensitive enzymes. Here we show the first expression of functional [FeFe] hydrogenases in an Escherichia coli-based cell-free transcription/translation system. We have produced and matured both algal and bacterial hydrogenases using E. coli cell extracts containing the HydG, HydE, and HydF proteins from Shewanella oneidensis. The current system produces approximately 22 microg/mL of active protein, constituting approximately 44% of the total protein produced. Active protein yield is greatly enhanced by pre-incubation of the maturation enzyme-containing extract with inorganic iron and sulfur for reconstitution of the [Fe-S] clusters in HydG, HydE, and HydF. The absence of cell walls permits direct addition of cofactors and substrates, enabling rapid production of active protein and providing control over the maturation conditions. These new capabilities will enhance the investigation of complex proteins requiring helper proteins for maturation and move us closer to the development of improved hydrogenases for biological production of hydrogen as a clean, renewable alternative fuel.

Cell-free protein synthesis: Applications come of age

“…Thus, extract preparation is simple and inexpensive. Second, E. coli based systems generally achieve the highest protein yields, from hundreds of micrograms per milliliter to milligrams per milliliter in a batch reaction, depending on the protein of interest ( e.g ., 1.7 mg mL -1 chloramphenicol acetyl transferase (Kim et al , 2011), 0.7 mg mL -1 human granulocyte-macrophage colony-stimulating factor (Zawada et al , 2011), and 0.022 mg mL -1 FeFe hydrogenase (Boyer et al , 2008)). Third, the reaction cost of the E. coli system is the lowest.…”

Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage

“…These attributes make cell-free technology an excellent platform for high-throughput recombinant expression and synthetic biology technologies (1, 48). Exploiting the benefits of CFPS, myriad proteins have been produced, such as virus-like particles (9, 10), proteins containing unnatural amino acids (1113), cytotoxic proteins (9), and a variety of biocatalytic enzymes (14, 15). …”

Cell-free synthetic biology: Thinking outside the cell

“…These efforts have opened the way to commercial production of protein therapeutics (Kim and Swartz, 2004) personalized medicines (Kanter et al, 2007), vaccines (Bundy et al, 2008) and classes of proteins that are difficult to produce in vivo , like hydrogenases (Boyer et al, 2008). A flow diagram of how CECF systems are commonly processed and utilized is shown in Figure 3.…”