Carbon‐flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose

Abstract

Growth of Corynebacterium glutamicum on fructose was significantly less than that obtained on glucose, despite similar rates of substrate uptake. This was in part due to the production of overflow metabolites (dihydroxyacetone and lactate) but also to the increased production of CO 2 during growth on fructose. These differences in carbon-metabolite accumulation are indicative of a different pattern of carbon-flux distribution through the central metabolic pathways. Growth on glucose has been previously shown to involve a high flux (Ͼ 50% of total glucose consumption) via the pentose pathway to generate anabolic reducing equivalents. NMR analysis of carbon-isotope distribution patterns of the glutamate pool after growth on 1-13 C-or 6- 13C-enriched fructose indicates that the contribution of the pentose pathway is significantly diminished during exponential growth on fructose with glycolysis being the predominant pathway (80 % of total fructose consumption). The increased flux through glycolysis during growth on fructose is associated with an increased NADH/NAD ϩ ratio susceptible to inhibit both glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenase, and provoking the overflow of metabolites derived from the substrates of these two enzymes. The biomass yield observed experimentally is higher than can be estimated from the apparent quantity of NADPH associated with the pentose pathway and the flux through isocitrate dehydrogenase, suggesting an additional reaction yielding NADPH. This may involve a modified tricarboxylic acid cycle involving malic enzyme, expressed to significantly higher levels during growth on fructose than on glucose, and a pyruvate carboxylating anaplerotic enzyme.Keywords : Corynebacterium glutamicum; fructose metabolism; NMR analysis; NADH/NAD ϩ ratio; overflow metabolism.For several decades, Corynebacterium glutamicum and re-as to more efficiently supply the specific biosynthetic pathways with the necessary carbon precursors and coenzymes. lated species have been exploited industrially for the production of various amino acids. Improvements of the fermentation strateAlthough many enzymes of intermediary metabolism have been isolated from Brevibacterium flavum and characterised pregies employed and of the bacterial strains by genetic engineering techniques have been achieved, leading to progressively increas-dominantly by the team of Shiio [5Ϫ7], global carbon-flux models based on regulatory or energetic principles have only reing rates of production and/or yields [1]. These strategies have often been based upon overcoming the natural feedback regula-ceived attention recently. Over the last five years, various groups have examined how glucose is catabolised by the central pathtion mechanisms specific to each biosynthetic pathway [2Ϫ4] and have enabled a detailed understanding of these biochemical ways using NMR [8Ϫ10], enzymatic [11] and mathematical modelling [12,13] approaches. A general consensus opinion has sequences to be established. It is now apparent that further impr...

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

“…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). It was proposed that to meet the NADPH requirement for growth and lysine production on fructose or on fructose/glucose mixtures, malic enzyme activity has to be increased (Dominguez et al 1998).…”

“…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). It was proposed that to meet the NADPH requirement for growth and lysine production on fructose or on fructose/glucose mixtures, malic enzyme activity has to be increased (Dominguez et al 1998). As fructose is transported into the cell as fructose-1-phosphate by a PEP-dependent phosphotransferase system and enters glycolysis after phosphorylation to fructose-1,6-bisphosphate (Kotrba et al 2001;Parche et al 2001), overexpression of the fructose-1,6-bisphosphatase gene fbp (Rittmann et al 2003) was alternatively proposed to increase the PPP flux as well as lysine production on fructose (Kiefer et al 2004;Pons et al 1996).…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…The role of malic enzyme is still not completely clear. Its contribution to NADPH supply has been demonstrated for growth on fructose [32] as well as in a pyruvate kinase deficient lysine production strain [120]. Lysine production flux (%) Fig.…”

“…The major enzymes are glucose 6-phosphate dehydrogenase [28,29] and 6-phosphogluconate dehydrogenase [28,30] in the oxidative part of the PPP and the TCA cycle enzyme isocitrate dehydrogenase [31]. In selected cases, NADPH supply might involve malic enzyme [32,33]. In addition to the central catabolism, anabolic routes have also been elucidated.…”

“…From 13 C metabolic flux studies of lysine producing strains under different physiological conditions, remarkable insights into function and regulation of the NADPH metabolism of C. glutamicum could be obtained. The NADPH metabolism of C. glutamicum is highly flexible, adjusting to the physiological growth state [126], the nutrient status [13,32,111], or the genetic background [108,131]. In most cases this results in an apparent NADPH excess, pointing at so far unassigned reactions which consume NADPH.…”