Isolation and Characterization of Random Citrate Synthase Variants That Increase Acetate Generation by Escherichia...

A method to select Escherichia coli citrate synthase variants having reduced activity was developed based initially on the increased generation of acid, observed visually by prolonged color change in the pH indicator bromocresol purple. Five hundred fifty-two potential variants were isolated and each were cultured on glucose as sole carbon source. Of this library, 41 variants showed increased duration of the color change, indicating elevated acid formation. These strains were then examined in single shake flasks, and 16 demonstrated at least 2.5× greater acetate production than the wild-type citrate synthase parent strain. These 16 strains were selected for sequencing and quantitative studies. In shake flask studies, an inverse correlation was observed between maximum specific growth rate and acetate yield, with lowered growth rate being an indication of mutation severity. The wild-type citrate synthase and two variant enzymes were purified and their kinetic parameters determined. These three strains were also compared under carbon-limited, steady-state conditions at a growth rate of 0.20 h^-1, where acetate yield and specific glucose uptake were correlated with severity of mutation. The results are explained mathematically in terms of an enzyme partitioning ratio using the relative kinetics of the competing enzymes (modified citrate synthase and phosphotransacetylase). The results demonstrate how partitioning of glycolytic flux between the tricarboxylic acid and other competitive pathways to products derived from acetyl-CoA can be modulated by decreasing citrate synthase activity, a method which complements other methods including those using promoter strength and expression level.