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Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetateglucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By 13 Clabeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. ) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.In their natural environments microorganisms often encounter situations when not a single carbon source but mixtures of carbon and energy sources are present. Under such conditions, bacteria often utilize one carbon source preferentially, with the further carbon source(s) being consumed only, when the preferred one is exhausted. As already shown by Monod (29), the preferred carbon source in general supports the best growth rate and/or growth yield, and the successive utilization of the substrates is often represented by a biphasic growth behavior (29). The classical example of this phenomenon is the diauxic growth of Escherichia coli on glucose plus lactose, and the study of the underlying principles initiated the era of research on gene regulation. On the other hand, by analysis of growth and carbon source consumption, it was shown that, e.g., Leuconostoc oenos cometabolizes glucose with citrate or fructose (38). Also, E. coli cometabolizes hexoses under carbon limitation conditions (reviewed in reference 20). Several other bacteria use two carbon sources in parallel (reviewed in reference 16); among these is Corynebacterium glutamicum, a gram-positive bacterium known for its ability to excrete amino acids. C. glutamicum grows aerobically on a variety of carbohydrates and organic acids as carbon sources (23). The organism cometabolizes glucose and fructose, glucose and lactate, and glucose and pyruvate (6, 7), whereas it shows diauxic growth on glucose-glutamate mixtures (21). The carbon sources glucose and acetate have been shown to serve as substrates for amino acid production by C. glutamicum (17). There is considerable knowledge about the enzymes and genes involved in acetate and glucose metabolism as well as their regulation (35,36,52,53), whereas neither growth on acetate-glucose mixtures nor metabolite fluxes during growth on acetate have been studied in detail.The utilization of acetate involves its uptake ...

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of L-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y P/S ) of 0.60 ؎ 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y P/S to 0.77 ؎ 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y P/S , indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH؉H ؉ to NADPH؉H ؉ . In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ⌬aceE ⌬pqo ⌬ilvE ⌬ldhA ⌬mdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h ؊1 , and showed an overall Y P/S of about 0.48 mol per mol of glucose in the production phase.

In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.