Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum.

Metabolic flux analysis was used to investigate the roles of the acid formation pathways in Clostridium acetobutylicum. The acid formation pathways were revealed to serve different roles in wildtype fermentations than previously expected. Specifically, enzymes known to catalyze butyrate formation were found to uptake butyrate without concomitant production of acetone. This role was further corroborated by flux analysis of a recombinant strain overexpressing the butyrate formation enzymes. Analysis of wildtype fermentation data also revealed an important role for the acetate formation enzymes, namely the cycling of carbon between acetate and acetylCoA during the stationary phase. Next, metabolic flux analysis was used to compare the patterns of activity in two butyrate kinase deficient strains of C. acetobutylicum. The strain developed by gene inactivation, PJC4BK, exhibited a shift in acid formation fluxes toward acetate while the strain developed by antisense RNA strategies, 824(pRD4), did not exhibit such a shift. However, both strains exhibited altered solvent formation patterns. PJC4BK exhibited a strong transient enhancement of solvent formation fluxes. In contrast, 824(pRD4) exhibited relatively lower levels of solvent formation fluxes, although fluxes were sustained over a longer period of time.

[1]  E. Papoutsakis,et al.  Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents , 1988, Applied and environmental microbiology.

[2]  Host-plasmid interactions in recombinant strains of Clostridium acetobutylicum ATCC 824 , 1994 .

[3]  Eva R. Kashket,et al.  Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum , 1986, Applied and environmental microbiology.

[4]  E. Papoutsakis,et al.  The effect of pH on nitrogen supply, cell lysis, and solvent production in fermentations of Clostridium acetobutylicum , 1985, Biotechnology and bioengineering.

[5]  E. Papoutsakis,et al.  Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. , 1996, Microbiology.

[6]  E. Papoutsakis,et al.  Solventogenesis in Clostridium acetobutylicum fermentations related to carboxylic acid and proton concentrations , 1988, Biotechnology and bioengineering.

[7]  H. Blaschek,et al.  Effect of Acetate on Molecular and Physiological Aspects of Clostridium beijerinckii NCIMB 8052 Solvent Production and Strain Degeneration , 1999, Applied and Environmental Microbiology.

[8]  E. Papoutsakis,et al.  Studies of Recombinant Clostridium acetobutylicum with Increased Dosages of Butyrate Formation Genesa , 1994, Annals of the New York Academy of Sciences.

[9]  Eleftherios T. Papoutsakis,et al.  Antisense RNA Strategies for Metabolic Engineering of Clostridium acetobutylicum , 1999, Applied and Environmental Microbiology.

[10]  G. Stephanopoulos,et al.  Network rigidity and metabolic engineering in metabolite overproduction , 1991, Science.

[11]  E. Papoutsakis.  Equations and calculations for fermentations of butyric acid bacteria , 1984, Biotechnology and bioengineering.

[12]  D. T. Jones,et al.  Acetone-butanol fermentation revisited. , 1986, Microbiological reviews.

[13]  E. Papoutsakis,et al.  Stoichiometric modeling of Clostridium acetobutylicum fermentations with non-linear constraints. , 1999, Journal of biotechnology.