A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture

Abstract

Background: Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible.

Results: We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation.

Conclusions: Incorporating gene regulation into the model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required.

Figure 1

A schematic view of the joint metabolic and gene regulation network model of AB fermentation in C. acetobutylicum. Acids are outlined in red and solvents in blue. The AB fermentation is characterized by a bi-phase metabolism. Following glycolysis the cells produce either the acids acetate (A) and butyrate (B) at a high pH, or the solvents acetone (An) and butanol (Bn) at a low pH while ethanol (En) is made in both phases but at a relatively low level. A characteristic reaction in this metabolism is the conversion of acetate (or butyrate) and acetoacetyl-CoA into acetyl-CoA (or butyryl-CoA) and acetoacetate, this being the first step in the formation of solvents from acids. We reduced the metabolic network published in [10] to ten reactions R_i found in Table 1. For reactions R₃, R₅, R₆, R₇, and R₁₀ we include gene regulation for the enzymes which are involved in the production of the solvents.

Figure 2

Comparison of our model (solid lines) with the data of the dynamic shift chemostat experiments (dots). Figure 2(a) shows results for the 'forward' dynamic shift experiment. The two repetitions of this 'forward' dynamic shift experiment are shown in Figure 2(b) and Figure 2(c). The cells produce mainly acetate and butyrate when grown at a pH value of 5.7. During the transition phase C. acetobutylicum switches its metabolism (as a function of the external pH) towards the generation of the solvents acetone and butanol at a pH of 4.5. Ethanol is produced during acidogenesis and solventogenesis at approximately the same levels. In Figure 2(d) we demonstrate the comparison of the model and the data for the 'reverse' dynamic shift experiment.

Figure 3

Steady-state curves of butanol (Bn) for varying production of (a) Adc (acetoacetate decarboxylase), (b) and (c) the CtfA/B (CoA-transferase), (d) AdhE (alcohol aldehyde dehydrogenase), (e) BdhA and/or BdhB (butanol dehydrogenases) and (f) ThlA (thiolase). In (c) we have altered the axes of (b) in order to be able to see clearly the effect of downregulation of ctfA/B.