The use of genome scale metabolic flux variability analysis for process feed formulation based on an investigation of the effects of the zwf mutation on antibiotic production in Streptomyces coelicolor

Deletion of either of the genes (zwf), coding for glucose-6-phosphate dehydrogenase isoforms in Streptomyces coelicolor, resulted in an increase in in vitro determined glucose-6-phosphate dehydrogenase activity in the surviving isoform. Mutants carrying either one of these deletions exhibited significantly increased actinorhodin biosynthesis, an effect that was not predicted by simple flux balance simulation. The flux variability distribution, obtained using experimental measured variables as constraints, was examined. The reactions whose flux variabilities were most significantly affected, as a result of the increased actinorhodin production and changes in nutrient uptake rates, included reactions from a number of different pathways. These could be connected together to form an actinorhodin-generating network. They included glutamate synthesising and catabolising reactions. Subsequent experiments, in which glutamate was fed to steady state cultures, resulted in increased actinorhodin production and the in silico model predicted the importance of the same reactions that were amplified in the zwf model. Introduction of a constitutively expressed copy of actII-orf4, the pathway-specific activator of the actinorhodin pathway genes, on the integrating plasmid pIJ8714 resulted in a switch from growth dissociated (typical secondary metabolite) to growth associated antibiotic production.