Investigating Metabolite Profile and Relative Flux Ratio Under Growth Phase of Ralstonia Eutropha H16

To achieve high yield of biochemical synthesis, understanding overall metabolism, such as the concentrations of metabolites or fluxes among metabolites of microbe is important. However, even with intensively studied microbe strains, it is difficult to estimate in-vivo concentration of metabolites or determine active pathways in specific phase due to low stability of metabolites. Advanced analysis instruments, e.g. NMR, GC/MS, LC/TOFMS with proper and strain-optimized sampling methodology have enabled us to determine intracellular metabolite pool sizes and active metabolic pathways. To compare metabolic differences according to carbon sources, Ralstonia eutropha H16, a natural PHB producer, was cultivated under 2 different carbon sources and intracellular metabolite samples were analyzed by LC/TOFMS. To obtain reliable in-vivo metabolite concentration data, we compared cell harvesting methods and compared cell extraction methods. Results showed that rapid filtration provided more consistent values of metabolite concentrations. On the other hand, R. eutropha H16 underwent serious metabolite leakage during methanol quenching possibly by cell membrane damage. This damage resulted in detecting smaller amount of intracellular metabolites and relatively inconsistent metabolite concentrations among samples. Between the two extraction methods, boiling ethanol method showed a little higher-efficiency. We can speculate that chloroform/methanol/water extraction needs more modification to improve the efficiency. The optimal quenching and extraction methods were successfully applied to monitoring metabolite profiling of R. eutropha H16 grown with fructose or acetate. To determine relative flux ratio among ED, EMP and PP pathway of R. eutropha H16 under exponential growth phase, 1-13C labeled fructose was fed and proteins were harvest at 3 different time points to confirm stable flux ratio. Relative flux ratio was calculated by analyzing ion fragments of proteiongenic amino acids using GC/MS.