Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement

Biotechnology for Biofuels

Daehwan Chung¹  Joel Farkas¹  Janet Westpheling¹ 

[1] The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

关键词: Targeted deletion;    M.CbeI;    CbeI;    Restriction-modification enzymes;    Biomass conversion;    Caldicellulosiruptor species;   

Others  :  798023
DOI  :  10.1186/1754-6834-6-82

 received in 2013-04-01, accepted in 2013-05-21,  发布年份 2013

【 摘 要 】

Background

Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass.

Results

Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus.

Conclusion

Deletion of the cbeI gene removes a substantial barrier to routine DNA transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of restriction-modification activities in members of this genus suggests a way forward to eliminating restriction as a barrier to DNA transformation and efficient genetic manipulation of this important group of hyperthermophiles.

【 授权许可】

   
2013 Chung et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

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