Butanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of its whole genome sequence. However, there is no established method for the transfer of foreign DNA into this strain; this is the next step necessary for progress in its use for butanol production. We have described functional protocols for conjugation and transformation of the bio-butanol producer C. pasteurianum NRRL B-598 by foreign plasmid DNA. We show that the use of unmethylated plasmid DNA is necessary for efficient transformation or successful conjugation. Genes encoding DNA methylation and those for restriction-modification systems and antibiotic resistance were searched for in the whole genome sequence and their homologies with other clostridial bacteria were determined. Furthermore, activity of described novel type I restriction system was proved experimentally. The described electrotransformation protocol achieved an efficiency 1.2 × 102 cfu/μg DNA after step-by-step optimization and an efficiency of 1.6 × 102 cfu/μg DNA was achieved by the sonoporation technique using a standard laboratory ultrasound bath. The highest transformation efficiency was achieved using a combination of these approaches; sono/electroporation led to an increase in transformation efficiency, to 5.3 × 102 cfu/μg DNA. Both Dam and Dcm methylations are detrimental for transformation of C. pasteurianum NRRL B-598. Methods for conjugation, electroporation, sonoporation, and a combined method for sono/electroporation were established for this strain. The methods described could be used for genetic improvement of this strain, which is suitable for bio-butanol production.

/pdf/dam-and-dcm-methylations-prevent-gene-transfer-into-50l9xjyio7.pdf

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation.

The genus Clostridium contains widely diverse species which have been shown to be associated with foodborne illness as well as solvent production (Blaschek, 1999). C. perfringens is representative of a clostridial species which is associated with food poisoning, while non-toxinogenic C. beijerinckii has important industrial and biotechnological potential because of its ability to produce acetone and butanol during fermentation of carbohydrates. The development of genetic systems for the clostridia was recently reviewed (Blaschek and White, 1995). Electroporation induced transformation has been shown to have considerable potential for introducing DNA into a wide variety of gram+ and gram− bacterial species (Dower et al., 1992) and electroporation protocols are available for previously non-transformable clostridial strains (Chen et al., 1996). However, the transformation efficiencies appear to vary greatly among different species and strains. It has been suggested that DNase and restriction enzyme activity are common barriers to successful transformation of the clostridia. The cell wall may also represent a barrier to DNA uptake in C. perfringens (Kim and Blaschek, 1989). As we learn more about mechanisms and factors involved in transforming various bacteria, protocols for overcoming these obstacles will likely emerge. Electroporation protocols have been developed for the clostridia which are based on using polyethylene glycol (PEG) in the shocking menstruum. During electroporation-induced transformation, PEG may limit cytoplasmic leakage and, via exclusion of the aqueous phase, help to push the plasmid DNA into the cell and consequently increase cell viability and transformation efficiency.

Clostridium in Biotechnology and Food Technology

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Clostridium in Biotechnology and Food Technology