A shift in the dominant phenotype governs the pH-induced metabolic switch of Clostridium acetobutylicumin phosphate-limited continuous cultures

Abstract

In response to changing extracellular pH levels, phosphate-limited continuous cultures of Clostridium acetobutylicum reversibly switches its metabolism from the dominant formation of acids to the prevalent production of solvents. Previous experimental and theoretical studies have revealed that this pH-induced metabolic switch involves a rearrangement of the intracellular transcriptomic, proteomic and metabolomic composition of the clostridial cells. However, the influence of the population dynamics on the observations reported has so far been neglected. Here, we present a method for linking the pH shift, clostridial growth and the acetone-butanol-ethanol fermentation metabolic network systematically into a model which combines the dynamics of the external pH and optical density with a metabolic model. Furthermore, the recently found antagonistic expression pattern of the aldehyde/alcohol dehydrogenases AdhE1/2 and pH-dependent enzyme activities have been included into this combined model. Our model predictions reveal that the pH-induced metabolic shift under these experimental conditions is governed by a phenotypic switch of predominantly acidogenic subpopulation towards a predominantly solventogenic subpopulation. This model-driven explanation of the pH-induced shift from acidogenesis to solventogenesis by population dynamics casts an entirely new light on the clostridial response to changing pH levels. Moreover, the results presented here underline that pH-dependent growth and pH-dependent specific enzymatic activity play a crucial role in this adaptation. In particular, the behaviour of AdhE1 and AdhE2 seems to be the key factor for the product formation of the two phenotypes, their pH-dependent growth, and thus, the pH-induced metabolic switch in C. acetobutylicum.

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

“…Additionally, the effect of several single mutations on the product formation at acidogenic and solventogenic steady state was investigated. Recently, we developed a model that combines the growth and the network of ABE fermentation of clostridial populations in continuous cultures under phosphate limitation (Millat et al 2013b). In this model, we proposed that a phenotypic switch governs the pH-induced metabolic switch under these experimental conditions.…”

“…In particular, the consideration of the population dynamics is important for the investigation presented here, because it separates the effect of changing population sizes from intracellular alterations. In the following section, this mathematical model is introduced briefly, for a more detailed description we refer to (Millat et al 2013b).…”

“…However, this activity would not be consistent with the results reported in , where the authors concluded from 13 C NMR studies, enzyme assays, and thermodynamic considerations that a CoA-transferase-mediated mechanism is more likely responsible for acid re-assimilation than a reverse action of acetate and butyrate formation pathways, acyl-CoA synthase reactions, or generation of acetyl and butyryl phosphate followed by their direct reduction to the corresponding aldehyde. Additionally, our recent theoretical analysis of the product formation rate, using the wild type ATCC 824 (COSMIC strain, see Materials and Methods), suggests that the re-assimilation of acetate is less active than that of butyrate in phosphate-limited continuous cultures (Millat et al 2013b).…”

“…As a consequence of the mutation, the acetoacetate formation and, consecutively, the formation of acetone are abolished. The experimentally measured time courses of the external pH, the optical density (OD 600 ), and the fermentation products provide the basis for a theoretical investigation of the metabolic switch in the mutant, thereby modifying our two-population model developed for the wild type (Millat et al 2013b).…”

“…The model in Millat et al (2013b) combines the pH-dependent growth of an acidogenic and a solventogenic population with a pH-dependent metabolic model of clostridial ABE fermentation. As a consequence, it distinguishes the two involved processes, acid re-assimilation and drop of population size that determine the decline of the acids after the initiation of the pH-shift.…”