Caldicellulosiruptor bescii grows optimally at 78°C [172.4 ˚F] and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C₅ and C₆ sugars primarily to hydrogen gas, lactate, acetate and CO₂, and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) towards highly reduced products such as biofuels.

During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely-used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, herein termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents approximately 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary 43 redox metabolism of this cellulolytic microorganism.

—Scott et al.

While cellulosic biomass offers advantages as a feedstock for fuel and chemical production, its big challenge is its high resistance to enzymatic degradation. To date, most efforts to convert it to useful chemicals have involved energetically expensive pretreatment.

Avoiding pretreatment would boost commercial viability. To this end, the investigators, members of the Department of Energy’s BioEnergy Science Center (BESC), have been focusing on Caldicellulosiruptor species (the name of the genus means “hot cellulose-breakers,”), which inhabit volcanic hot springs around the world.

While the putative novel tungstoenzyme looked fairly promising, Adams is quick to assert that a likely sequence does not constitute proof of function.

In fact, “I would have predicted that the tungsten-processing system of C. bescii probably used molybdenum rather than tungsten,” Adams said. (The two metals have similar properties, but molybdenum is frequently used by bacteria, most notably to break the bonds of atmospheric nitrogen, enabling biological nitrogen fixation.) The investigators engineered C. bescii to produce a known tungstoenzyme from another organism. “That enzyme was active, proving that C. bescii is capable of synthesizing tungstoenzymes,” said Adams.

The investigators then grew C. bescii under a variety of conditions, including directly on cellulose and plant biomass, and found that it always produced the enzyme, which the investigators dubbed XOR, at high cellular concentrations under all growth conditions. They also tried unsuccessfully to grow “knock-out” mutants lacking a functional XOR gene. That result suggested, but does not prove, that the enzyme is necessary for growth, said Adams.

Although its role within the cell is as yet unknown, the conservation of XOR and its associated polyferredoxin across the genus Caldicellulosiruptor, together with its high expression level in C. bescii (~2% of the cytoplasmic protein), suggest that this novel tungstoenzyme serves an important role in the primary metabolism of these cellulolytic species. Determining its function will likely have an important impact on future metabolic engineering studies of C. bescii, and studies to elucidate the substrate(s) utilized by XOR are underway.

—Scott et al.

That knowledge, Adams added, would make it possible to metabolically engineer C. bescii to produce fuels and other useful chemicals from such feedstocks.

Resources

• Israel M. Scott, Gabe M. Rubinstein, Gina L. Lipscomb, Mirko Basen, Gerrit J. Schut, Amanda M. Rhaesa, W. Andrew Lancaster, Farris L. Poole II, Robert M. Kelly, and Michael W. W. Adams (2015) “A New Class of Tungsten-Containing Oxidoreductase in the Genus of the Plant Biomass-Degrading, Thermophilic Bacteria Caldicellulosiruptor” Appl. Environ. Microbiol doi: 10.1128/AEM.01634-15