Construction of a Stable Replicating Shuttle Vector for Caldicellulosiruptor species: Extending Genetic Methodologies to other Members of This Genus

University of Georgia researchers have developed a replicating shuttle vector based on a small plasmid for Caldicellulosiruptor bescii. The entire plasmid was cloned into an E. coli cloning vector. The shuttle vector enables modification of several members the genus (including C. hydrothermalis) providing them with features that are desirable to improve biomass-to-biofuel conversion. There was no evidence of DNA rearrangement during transformation and replication in C. bescii.

Biomass is a renewable resource that has shown promise to replace petroleum-based fuels while reducing greenhouse gas emissions. A key challenge towards achieving an economically viable biomass solution is that plants have built up a natural protection (or recalcitrance) against being converted to fuel. Therefore, more effort using special enzymes and microbes is needed to convert biomass into ethanol. In order to become more widely adopted, the high cost for biomass processing needs to be reduced.

A special type of bacterium, Caldicellulosiruptor bescii, has a high affinity for decomposing lignocellulosic biomass that includes agricultural residues such as rice straw, switchgrass, as well as hard- and softwoods. However, the products of C. bescii’s degradation of biomass are not effective as fuels. That is, these naturally occurring processes do not produce compounds of great economic interest. However, given its high affinity for decomposition of, and its ability to grow on, untreated biomass, C. bescii makes an attractive candidate for genetic modifications that could better enable its use in the production of biofuels and other commodity chemicals.

University of Georgia researchers have developed a genetic tool that allows for transformations of Caldi, making it a more viable organism for the degradation of recalcitrant biomass and production of biofuels and commodity chemicals. The researchers have leveraged the special properties of thermophiles, organisms that grow at relatively high temperatures, which lead to better biomass conversion. The Caldicellulosiruptor genus is the most thermophilic cellulolytic microbes known, and therefore this genus was targeted towards improving its efficiency for biomass conversion.

To improve the effectiveness of the Caldicellulosiruptor species, University of Georgia researchers constructed a replicating shuttle vector that enables advanced characterization and manipulation of genes and metabolic pathways to enhance biomass conversion. This is the first vector of its kind for members of this genus. Their technique also utilizes a native plasmid in this genus to generate a replicating vector.

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