Investigating the clostridium botulinum neurotoxin production process using a genome-scale metabolic network enhanced surrogate system.

Clostridium botulinum (C. botulinum) produces a neurotoxin which can be used in a clinical environment to treat diseases and disorders characterised by muscle hypertension or spasm. However, previous research has mostly focused on the biochemical mode of action of the toxin and the disease it manifests. In order to increase our understanding of the process further, this study aimed to investigate the metabolism of various biomarkers, thought to be correlated with neurotoxin biosynthesis. The objective was to increase our understanding of the metabolism which drives the production of C. botulinum toxin using a Genome Scale Metabolic Network (GSMN) enhanced surrogate system. A linear correlation was established between the accumulation of intracellular Poly β hydroxybutyrate (PHB) and neurotoxin in silico (R2 = 0.988). This correlation was confirmed by chemostat experiments in C. sporogenes demonstrating that increased supply of gaseous carbon dioxide (CO2) to the culture results in increased accumulation of PHB and in silico neurotoxin in C. botulinum. Experiments revealed the correlation is a result of modulation of carbon flux partitioning between glycolysis and the TCA cycle, ultimately increasing the availability of carbon for storage as PHB. Phosphate limitation and supplementation with Homoserine and other oxaloacetate derived amino acids, gave rise to increased PHB, owing to reduced activity and/or demand of the TCA cycle increasing the availability of acetyl CoA, the energy storage polymer’s precursor. Altering the growth medium to decrease TCA activity also resulted in decreased flagellin biosynthesis. The results of this study can be used to design a C. botulinum production process based on experimentally proven correlations and pathway analysis to yield a process which promotes neurotoxin biosynthesis over competing pathways, such as flagellin biosynthesis