Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing

Abstract

To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of 'H-detected 13C nuclear magnetic resonance spectroscopy t o follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [l-'3Clglucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments i n carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology t o C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 73.7%. Due t o the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

“…This implies the in vivo activity of NADPH consuming reactions. Similar findings were reported for the lysine producing strain C. glutamicum MH 20±22 B [6]. Possible candidates pointed out by the authors were in vivo NADPH regeneration by an oxidase, malic enzyme or alternative reactions, which have not been identified yet.…”

“…13 C tracer experiments with MALDI-TOF MS (this paper), NMR [6], and metabolite balancing [17] lead to similar conclusions concerning the metabolism of C. glutamicum during lysine production. This supports the consistency of the applied techniques.…”

“…From the experimentally determined biomass yield and from the biomass composition of C. glutamicum previously Table 2. Precursor demand for biomass formation calculated from the experimentally determined yield coefficient Y X/S 0.0599 g´(mmol glucose) 21 and from biomass composition of C. glutamicum previously estimated [6] determined by Marx et al [6], the demand for the 12 biomass precursors was calculated for the phase of maximum lysine production ( Fig. 1.…”

“…A disadvantage of the NMR approach by Marx et al [6] is the enormous experimental effort, the insensitivity of NMR and the requirement of steady-state continuous cultures, which impedes its use as a broadly applied method for metabolic flux analysis. Moreover, nongrowing batch cultures cannot be analyzed by such a technique.…”

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

“…In principle, NADPH can be synthesized in C. glutamicum by the enzymes of the oxidative pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (Moritz et al 2000), by isocitrate dehydrogenase (Eikmanns et al 1995) and by malic enzyme (Gourdon et al 2000). Carbon flux analysis revealed that during growth on glucose, NADPH for lysine biosynthesis is provided by the PPP, whereas isocitrate dehydrogenase and malic enzyme are of minor importance (Marx et al 1996(Marx et al , 1997(Marx et al , 1999. During growth of C. glutamicum on fructose or on fructose/glucose mixtures, a low carbon flux through the oxidative PPP was demonstrated (Dominguez et al 1998;Kiefer et al 2004;Pons et al 1996).…”