BACKGROUND: 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. RESULTS: 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 × 10(2) cfu/μg DNA after step-by-step optimization and an efficiency of 1.6 × 10(2) 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 × 10(2) cfu/μg DNA. CONCLUSIONS: 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.

• Klíčová slova
• Butanol, Clostridium, Conjugation, Dam, Dcm, Electroporation, Methylation, Sonoporation, Transformation,
• Publikační typ
• časopisecké články MeSH

Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.

• MeSH
• biomasa MeSH
• Clostridium účinky léků   růst a vývoj   metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• mastné kyseliny analýza   MeSH
• membránové lipidy chemie   MeSH
• n-butanol metabolismus   farmakologie   MeSH
• nenasycené mastné kyseliny analýza   MeSH
• plasmalogeny analýza   MeSH
• techniky vsádkové kultivace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mastné kyseliny MeSH
• membránové lipidy MeSH
• n-butanol MeSH
• nenasycené mastné kyseliny MeSH
• plasmalogeny MeSH