Genetic manipulation of acid and solvent formation in Clostridium acetobutylicum ATCC 824

Abstract

The genes coding for enzymes involved in butanol or butyrate formation were subcloned into a novel Escherichia coli-Clostridium acetobutylicum shuttle vector constructed from pIMP1 and a chloramphenicol acetyl transferase gene. The resulting replicative plasmids, referred to as pTHAAD (aldehyde/alcohol dehydrogenase) and pTHBUT (butyrate operon), were used to complement C. acetobutylicum mutant strains, in which genes encoding aldehyde/alcohol dehydrogenase (aad) or butyrate kinase (buk) had been inactivated by recombination with Em r constructs. Complementation of strain PJC4BK (buk mutant) with pTHBUT restored butyrate kinase activity and butyrate production during exponential growth. Complementation of strain PJC4AAD (aad mutant) with pTHAAD restored NAD(H)-dependent butanol dehydrogenase activity, NAD(H)-dependent butyraldehyde dehydrogenase activity and butanol production during solventogenic growth. The development of an alternative selectable marker makes it is possible to overexpress genes, via replicative plasmids, in mutant strains that lack specific enzyme activities, thereby expanding the number of possible genetic manipulations that can be performed in C. acetobutylicum.

Intracellular Butyryl Phosphate and Acetyl Phosphate Concentrations in Clostridium acetobutylicum and Their Implications for Solvent Formation

“…The WT and the pta mutant had initial BuP peaks during the transition phase corresponding to initiation of solvent production. The initial BuP peak in the pta mutant was delayed compared to that in the WT, corresponding well with the delayed solvent production which has been noted previously (11). Both the WT and the pta mutant had a second BuP peak, before the levels finally fell to nearly zero at the end of the fermentations.…”

Electrotransformation of Clostridium acetobutylicum ATCC 824 using high-voltage radio frequency modulated square pulses

“…© 2000 The Society for Applied Microbiology application of molecular genetics to this group of bacteria (Durre 1998;Green and Bennett 1998). The preparation of genetic constructs for gene manipulations in clostridia is generally carried out in Escherichia coli as an intermediate host.…”

Biobutanol from Renewable Agricultural and Lignocellulose Resources and Its Perspectives as Alternative of Liquid Fuels

“…Reduced production of the AcrA and/or AcrB proteins of the complex confers increased butanol tolerance [42]. Green and Bennett subcloned the genes coding for enzymes involved in butanol or butyrate formation into a novel Escherichia coli-Clostridium acetobutylicum shuttle vector constructed from pIMP1 and a chloramphenicol acetyl transferase gene [43]. The resulting replicative plasmids, referred to as pTHAAD (aldehyde/alcohol dehydrogenase) and pTHBUT (butyrate operon), were used to complement C. acetobutylicum mutant strains, in which genes encoding aldehyde/alcohol dehydrogenase (aad) or butyrate kinase (buk) had been inactivated by recombination with Emr constructs.…”

“…Complementation of strain PJC4BK (buk mutant) with pTHBUT restored butyrate kinase activity and butyrate production during exponential growth. Complementation of strain PJC4AAD (aad mutant) with pTHAAD restored NAD(H)-dependent butanol dehydrogenase activity, NAD(H)-dependent butyraldehyde dehydrogenase activity and butanol production during solventogenic growth [43]. Shen and Liao constructed an Escherichia coli strain that produces 1-butanol and 1-propanol from glucose [44].…”