No abstract

IL 60208-31 20, USA lntegrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in CIostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

No abstract

Expression of a pyruvate decarboxylase (Pdc) pathway in metabolically versatile thermophilic bacteria could create novel ethanologenic organisms, but no suitable thermostable Pdc is available. We have demonstrated that Pdc from Zymomonas mobilis can be expressed in an active form in Geobacillus thermoglucosidasius at up to 52 degrees C, while expression of Pdc polypeptides up to 54 degrees C was evident from Western blotting. By using an unstable lactate dehydrogenase (ldh) mutant of G. thermoglucosidasius, indirect evidence of Pdc activity in vivo was also obtained.

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.