Genome‐scale model for Clostridium acetobutylicum: Part I. Metabolic network resolution and analysis

Abstract

A genome-scale metabolic network reconstruction for Clostridium acetobutylicum (ATCC 824) was carried out using a new semi-automated reverse engineering algorithm. The network consists of 422 intracellular metabolites involved in 552 reactions and includes 80 membrane transport reactions. The metabolic network illustrates the reliance of clostridia on the urea cycle, intracellular L-glutamate solute pools, and the acetylornithine transaminase for amino acid biosynthesis from the 2-oxoglutarate precursor. The semi-automated reverse engineering algorithm identified discrepancies in reaction network databases that are major obstacles for fully automated network-building algorithms. The proposed semi-automated approach allowed for the conservation of unique clostridial metabolic pathways, such as an incomplete TCA cycle. A thermodynamic analysis was used to determine the physiological conditions under which proposed pathways (e.g., reverse partial TCA cycle and reverse arginine biosynthesis pathway) are feasible. The reconstructed metabolic network was used to create a genome-scale model that correctly characterized the butyrate kinase knock-out and the asolventogenic M5 pSOL1 megaplasmid degenerate strains. Systematic gene knock-out simulations were performed to identify a set of genes encoding clostridial enzymes essential for growth in silico.

Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

“…Despite this long history, there are still key pathways of primary metabolism in C. acetobutylicum that remain unresolved. In particular, as is common for most anaerobic bacteria, the tricarboxylic acid (TCA) cycle remains illdefined (14,23,28).C. acetobutylicum is capable of growing on minimal medium (i.e., using glucose as the sole carbon source) (20), and it therefore must be able to synthesize ␣-ketoglutarate, the carbon skeleton of the glutamate family of amino acids.…”

“…Despite this long history, there are still key pathways of primary metabolism in C. acetobutylicum that remain unresolved. In particular, as is common for most anaerobic bacteria, the tricarboxylic acid (TCA) cycle remains illdefined (14,23,28).…”

“…In one case, it was proposed that the TCA cycle functions in the reductive (counterclockwise) direction to produce ␣-ketoglutarate (14). In the second, it was hypothesized that glutamate is synthesized from ornithine by running the arginine biosynthesis pathway in reverse, bypassing the need for the TCA cycle (28). With the exception of the TCA cycle, the other core metabolic pathways, e.g., of glucose catabolism and amino acid and nucleotide biosyntheses, appear based on sequence homology to be complete and analogous to those in more well-studied bacteria, such as Escherichia coli (23).…”

The Role of PerR in O2-Affected Gene Expression ofClostridium acetobutylicum

“…Especially the latter one has regained attention lately as an attractive biofuel (14). Thus, metabolic engineering and in silico modeling of clostridial metabolism are ranked highly among recent efforts to increase productivity (39,53,61,64). Furthermore, the sequenced genome and the design of microarrays for global-scale transcriptional analysis of C. acetobutylicum have substantially improved the understanding of solvent formation and allowed a detailed view on its unique life cycle (34,50,54).…”

The putative influence of the agr operon upon survival mechanisms used by Clostridium acetobutylicum

“…In a similar vein, models of sporulation in Gram-positive bacteria predominantly concern B. subtilis, with three in particular incorporating genetic regulation of the initiation network [9,20,21] using methods similar to those that we shall exploit here. Mathematical modelling of C. acetobutylicum has concentrated on the metabolic interactions involved in the fermentation process and have successfully represented the cells either during acidogenesis or in solventogenesis [22,23,24,25,26]. However, the nature of the existing models means that the switch between the two phases has to be imposed either by an artificial switch, or by an alteration in the parameter set.…”