Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism

Abstract

The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO2, and H2 as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.

Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites

“…HydG is responsible for synthesizing a di‐iron precursor to the H‐cluster active site of the FeFe hydrogenase (Kuchenreuther et al., ). As the presence or absence of HydG protein can effectively regulate assembly of functional FeFe hydrogenase by modulating correct active site assembly (Biswas et al., ), and the FeFe hydrogenase is a main hydrogen generation route in C. bescii (Cha et al., ), its regulation by Rex is consistent and expected. The regulon of this CopG transcription factor remains unknown and unpredicted.…”

“…These redox systems are plastic and subject to modulation through genetic modifications or by altering growth conditions. Eliminating lactate production in C. bescii increased overall hydrogen production (Cha, Chung, Elkins, Guss, & Westpheling, ), while eliminating the NiFe membrane‐bound hydrogenase decreased ethanol yield in a strain expressing an exogenous bifunctional AdhE (Cha, Chung, & Westpheling, ). Another method of redox modulation by Caldicellulosiruptor is demonstrated by the closely related bacterium Caldicellulosiruptor saccharolyticus.…”

Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms

“…With a genetic system now in place, gene ‘knockouts’ in C. bescii can be strategies to understand Caldicellulosiruptor metabolism and physiology. For instance, an uncharacterized [Ni‐Fe] hydrogenase maturation gene cluster ( hypABFCDE —Athe_1088‐Athe_1093) was deleted from the aforementioned, modified ethanol‐producing C. bescii strain containing adhE from C. thermocellum . The resulting strain produced 20% less H 2 than its parent, yet its H 2 yield per mol of cellobiose increased 63% (3.58 versus 2.19 mol H 2 /mol cellobiose).…”

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

“…Deletion of the lactate dehydrogenase gene (ldh) in C. bescii eliminated lactate production and increased acetate and hydrogen production by 21% and 34%, respectively, compared to the parent strain (13). The deletion of the maturation genes required for the nickel-iron hydrogenase showed that this enzyme was not responsible for the majority of the hydrogen production by C. bescii (14). The addition of a bifunctional alcohol dehydrogenase gene (adhE) from Clostridium thermocellum, resulting in strain JWCB032, allowed for the production of ethanol from plant biomass at 65°C (15), and production at 75°C was obtained by expressing the genes encoding AdhE and AdhB from Thermoanaerobacter pseudethanolicus 39E, although the ethanol yield was much lower (16).…”