Engineering Thermophilic Bacteria for Efficient Fermentation of Plant Biomass
Higher temperatures make plant biomass more accessible for processing, so thermophilic bacteria, which are active at higher temperatures than other bacteria, are promising candidates for biofuel production systems. To take full advantage of their potential in consolidated bioprocessing, efficient genetic tools are needed to metabolically engineer the thermophile. Researchers at the U.S. Department of Energy’s BioEnergy Science Center have been developing a series of genetic tools to manipulate Caldicellulosiruptor bescii. C. bescii is one of the most promising thermophiles for deconstructing and fermenting lignocellulose from nonfood plants. New research demonstrates a gene replacement strategy used to delete the lactate dehydrogenase gene from C. bescii. Because the plasmid contains a gene for which there is both positive and negative selection, it is possible to select first for recombination of the deleted ldh gene and then for loss of the plasmid sequences. This method allows clean genetic insertions and deletions, leaving no residual genetic material so that the method can be used repeatedly for adding and subtracting genes for metabolic engineering. The C. bescii strain containing the ldh gene deletion exhibited the expected metabolism changes, namely the engineered strain no longer produced lactate and had increased acetate and H2 production. This gene replacement demonstration paves the way for further genetic manipulation of C. bescii to produce desired biofuel fermentation products directly from plant biomass.
Cha, M., D. Chung, J. G. Elkins, A. M. Guss, and J. Westpheling. 2013. “Metabolic Engineering of Caldicellulosiruptor bescii Yields Increased Hydrogen Production from Lignocellulosic Biomass,” Biotechnology for Biofuels 6, 85. DOI: 10.1186/1754-6834-6-85.