A Recombinant 12‐His Tagged Pyrococcus furiosus Soluble [NiFe]‐Hydrogenase I Overexpressed in Thermococcus kodakarensis KOD1 Facilitates Hydrogen‐Powered in vitro NADH Regeneration

Abstract

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H2). Here, the authors expressed the four αβγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200‐fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12‐His tagged recombinant SHI (rSHI) is obtained by one‐step affinity adsorption on nickel‐charged resin. Size‐exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half‐life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN‐containing diaphorase (DI) is used to match NADP‐preferred SHI and NAD‐preferred lactate dehydrogenase (LDH). This study provides a cost‐efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.

High‐efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme

“…A T. kodakarensis ‐ E. coli (Tk‐Ec) shuttle vector pTE1 (Song et al, 2018a,b) was used to transform into T. kodakarensis TS559 (Fig. 2A).…”

“…Thermococcus kodakarensis TS559 transformants growing without agmatine were selected on ASW‐YT plates (Song et al, 2018a,b), and T. kodakarensis transformants resistant to simvastatin (Sim) were selected on ASW‐YT plates containing simvastatin (Hileman and Santangelo, 2012). The solid media were prepared by adding 1% (w/v) Gelrite ® as described previously (Song et al, 2018a,b). Further modifications of the medium for selection of transformants were as described below.…”

“…All primers used for PCR are listed in Table S3. The Tk ‐ Ec shuttle vector pTE1 was constructed as described previously (Song et al, 2018a,b). In brief, two DNA fragments were amplified from plasmid pUC19 with primers 1F/1R and from pTS543 (Santangelo et al ., 2010) with primers 2F/2R by high‐fidelity PCR.…”

“…mevinolin or simvastatin) (Hileman and Santangelo, 2012). Now, T. kodakarensis is a model archaeon for in‐depth understanding of physiology and the origin of life of archaea, the discovery of novel metabolic pathways (Santangelo et al ., 2011; Aslam et al ., 2017) and thermophilic enzymes (Elshawadfy et al ., 2014; Zhang and Tripathi, 2017; Okano et al ., 2018), and biotechnological applications for the expression of complicated enzyme complexes, such as soluble [NiFe] hydrogenase (Song et al, 2018b). Although it is naturally competent for the uptake of either linear or circular DNA (Sato et al ., 2005), its low transformation efficiency of 10 1 –10 2 transformants per μg of (circular) plasmid DNA still limits its wide adoption (Hileman and Santangelo, 2012; Straub et al ., 2018).…”

Biohydrogen production beyond the Thauer limit by precision design of artificial microbial consortia

“…The PFOR pathway is operative in Clostridiaceae during H 2 production, which occurs through the action of [NiFe]- and/or [FeFe]-hydrogenases 36 , 37 . Up to now, dark fermentative biohydrogen producing wild-type or metabolically engineered mono-cultures were not successful in improving Y (H2/S) beyond the Thauer limit 29 , 38 , 39 . Therefore, to boost Y (H2/S) , undefined microbial consortia or defined co- and multi-cultures of H 2 -producing microbes were examined in complex or defined medium 40 – 43 .…”