UGA team develops method for genetic engineering of Caldicellulosiruptor thermophilic bacteria; another pathway for efficient conversion of biomass to fuels and chemicals
24 August 2012
|The UGA team reports a method for modifying the extremely thermophilic, cellulose-degrading C.bescii. Source: ORNL. Click to enlarge.|
Researchers at the University of Georgia, who are also members of Department of Energy’s BioEnergy Science Center (BESC), have developed a method for the genetic manipulation of members of bacterial genus Caldicellulosiruptor, a group of anaerobic thermophiles with optimum growth temperatures between 65 °C and 78 °C (149–172 °F). (Earlier post.)
In a paper in the open-access journal PLoS ONE, the team reports the first example of DNA transformation of a member of this genus, C. bescii. Their efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination provide the basis for rapid and efficient methods of genetic manipulation.
In July, a team of researchers at BESC from North Carolina State University, Oak Ridge National Laboratory and the University of Georgia reported the analysis of the genomes of eight species of Caldicellulosiruptor and identified key proteins for the deconstruction of plant biomass into fermentable sugars.
Current methods for the use of lignocellulosic biomass as a substrate for microbial conversion to products of interest rely on pretreatment of the biomass with acids, alkali or organic solvents, often at high temperature and the addition of hydrolytic enzymes that partially digest the plant cell walls. Enzymatic pretreatment is particularly expensive and often prohibitive for the production of low value commodity products from biomass. Thermophilic microorganisms offer special advantages for biomass conversion, in part, because they offer the potential to decrease hydrolysis times by several-fold with the same cellulase loading or to decrease cellulase loading by several fold at constant hydrolysis times. Organisms that can use complex biomass as substrate reduce the need for pretreatment and enzymatic hydrolysis and, therefore, the cost of the process.
Caldicellulosiruptor species have the ability to utilize non-pretreated biomass including both low-lignin napier and Bermuda grasses as well as high–lignin switchgrass and a hardwood, popular, for growth. Members of this genus are the most thermophilic of all known organisms capable of using non-pretreated cellulosic biomass. The sequences of eight Caldicellulosiruptor genomes have been published and reveal enzymes likely to be important in lignocellulose utilization. In addition, microarray analysis of cells grown on various substrates implicates specific genes and gene clusters in biomass degradation.—Chung et al.
While there are many interesting microorganisms that do interesting and important chemistry, the ability to manipulate them genetically is essential to making them useful, the authors note.
Janet Westpheling, a microbial geneticist in the department of genetics in the UGA Franklin College of Arts and Sciences and a scientist of BESC and her colleagues used enzymes unique to Caldicellulosiruptor to overcome a defense mechanism the bacteria use to protect themselves from invading viruses.
The novelty of their discovery is a previously unknown methyltransferase, an enzyme that transfers a methyl group from a donor to an acceptor apparently unique to this species, capable of modifying the DNA, protecting it and allowing transformation.
We had clues that restriction (degradation) of DNA by the organisms themselves was a barrier to DNA transformation, but this is the first time it has been shown to be an absolute barrier.—Janet Westpheling
Paul Gilna, the director of the BioEnergy Science Center headquartered at Oak Ridge National Laboratory, noted that the work is a real breakthrough for BESC that will open the way to develop further the Caldicellulosiruptor bacteria as a new platform for the efficient conversion of lignocellulosic biomass into fuels.
This work was supported by the Department of Energy Office of Biological and Environmental Research BioEnergy Science Center.
BESC is one of three DOE Bioenergy Research Centers established by the DOE’s Office of Science in 2007. The centers support multidisciplinary, multi-institutional research teams pursuing the fundamental scientific breakthroughs needed to make production of cellulosic biofuels, or biofuels from nonfood plant fiber, cost-effective on a national scale. The three centers are coordinated at ORNL, Lawrence Berkeley National Laboratory and the University of Wisconsin-Madison in partnership with Michigan State University.
Chung D, Farkas J, Huddleston JR, Olivar E, Westpheling J (2012) Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725. PLoS ONE 7(8): e43844. doi: 10.1371/journal.pone.0043844
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