Breeding of Homo-Butanol-Fermentative Strain Using Clostridium Saccharoperbutylacetonicum ATCC27021

There are some problems in acetone-butanol-ethanol fermentation, because of low n-butanol yield owing to formation of significant amount of acetone and ethanol.  We tried to breed a homo-butanol fermentative strain only to produce n-butanol as a solvent using  Clostridium saccharoperbutylacetonicum  ATCC27021.  We constructed gene disruption plasmids pNS which can insert gene fragment including recyclable antibiotics-resistance marker based on GroupII intron function,  and obtained strains that disrupted pta, ptb1, ctfB, adc, ldh1 gene which are involved in butanol fermentation using these plasmids. Disrupted strains were evaluated by cultivation. Δpta strain showed poor growth and low n-butanol yield. Δptb1 strain produced about half amount of acetone compared to wild type strain, its n-butanol yield was improved by 15%. Δptb1Δpta strain decreased in acetone formation drastically , n-butanol yield from glucose reached 85mol% of the theoretical yield. (Wild-type strain 53mol%). Δptb1ΔptaΔldh1 strain showed n-butanol yield 87mol%. Δptb1Δpta strain produced 52.5g/L(yield 84.1mol%) of n-butanol with high glucose concentration medium overlayed oleyl alcohol. (Wild type strain produced 23.6g/L (yield 41.2mol%) Metabolic analysis data of Δptb1Δpta strain indicate pyruvate and butyly-CoA concentration were significantly high and ratio of NAD/NADH was quite low. From these result, there were some redox unbalance, especially in dehydrogenation of pyruvate to acetyl-CoA and reduction of butylyl-CoA to n-butanol.