Metabolic engineering of a non‐allosteric citrate synthase in an Escherichia coli citrate synthase mutant

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Abstract
This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high‐resolution 13C NMR. The oxidation of [1,2,3‐13C]propionate to glutamate via the TCA cycle was measured in wild‐type (WT) and a citrate synthase mutant (CS−) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non‐allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C‐2, C‐3, and C‐4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C‐4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10‐fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C‐4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure(TCACALC) and yielded an acetate/succinyl‐CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3‐13C] propionate or [2‐13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non‐allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.