Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids

Abstract

13C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct 13C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal 13C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using 13C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs). Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the 13C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution.

Metabolic engineering of isopropyl alcohol‐producing Escherichia coli strains with 13C‐metabolic flux analysis

“…Bacterial cells were cultured in 100 ml of the M9 medium containing 50 μg/ml ampicillin and 13 C‐labeled glucose in a shake flask (500 ml) at 30°C with reciprocal shaking at 120 rpm in triplicate (Okahashi, Kajihata, Furusawa, & Shimizu, ). [1‐ 13 C]glucose (98–99%) and [U‐ 13 C]glucose (99%) were purchased from Cambridge Isotope Laboratories (Andover, MA).…”

“…The supernatant was filtered and stored at −30°C until analysis. Concentrations of ethanol, acetone, and IPA as well as glucose, lactate, formate, and acetate in the supernatant were determined by GC and high performance liquid chromatography as described previously (Okahashi et al, , ). The concentrations of pyruvate, citrate, α‐ketoglutarate, succinate, fumarate, and malate in the media were quantified by GC‐MS.…”

“…Concentrations of ethanol, acetone, and IPA as well as glucose, lactate, formate, and acetate in the supernatant were determined by GC and high performance liquid chromatography as described previously (Okahashi et al, 2014. The concentrations of pyruvate, citrate, α-ketoglutarate, succinate, fumarate, and malate in the media were quantified by GC-MS. A 50 μl of the medium was mixed with 10 μl of 5 mM ribitol solution, and the mixture was evaporated to dryness on a Speed Vac (Thermo Scientific, Tokyo, Japan).…”

“…For 13 C-MFA in growth phase, 13 C-labeling of tert-butyldimethylsilylproteinogenic amino acids was measured by GC-MS (Agilent 7890A GC and 5975C Mass Selective Detector; Agilent Technologies, Santa Clara, CA) using the previously described method (Okahashi et al, 2014). For 13 C-MFA in nitrogen-starved non-growth phase, intracellular metabolites such as organic acids and sugar phosphates were extracted from the E. coli cells whose 13 C-labeling patterns were determined by GC-MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) by the method described previously with slight modifications (Nishino, Okahashi, Matsuda, & Shimizu, 2015;Okahashi et al, 2014Okahashi et al, , 2015. The culture broth (3 ml) was rapidly mixed with 12 ml of a 60% (v/v) aqueous solution of methanol (containing 10 mM ammonium acetate) precooled to −80°C (Rühl, Rupp, et al, 2012).…”

Metabolic characterization of cultured mammalian cells by mass balance analysis, tracer labeling experiments and computer-aided simulations

“…The 13 C-labeling of intracellular aspartate was analyzed by GC-MS using a method previously described (32). The effect of naturally occurring isotopes was removed from the raw mass spectra by a previously reported method (16).…”