Pyruvate formation and sugar metabolism in an amino acid-producing bacterium, Brevibacterium flavum.

Abstract

A Brevibacteriumflavum mutant lacking pyruvate kinase, No. 70, grew on glucose, fructose and sucrose as well as the original wild strain did, but was unable to grow on ribose or gluconate unless pyruvate was added. Mutants that required pyruvate for growth on ribose were derived directly from the wild strain. Manyof them were completely or partially defective in pyruvate kinase activity. These pyruvate kinase mutants were also unable to grow on gluconate. A phosphoenolpy-ruvate (PEP) : sugar phosphotransferase system (PTS) was found in B. flavum, which catalyzed the formation ofpyruvate and sugar phosphate from PEPand sugar. The system required Mg2+ , acted on glucose, fructose, mannose, glucosamine and 2-deoxyglucose, and existed in the cells grown on any of the carbon sources tested. Cells grown on fructose, mannitol and sucrose, however, exhibited higher PTS activities on fructose than those grown on others. Glucose PTSactivity was about 20-fold stronger than that of glucokinase. Other sugar metabolic enzymes, inducible mannitol dehydrogenase, gluconokinase, ribokinase and maltase, as well as constitutive invertase were also detected. Oxaloacetate decarboxylase and malic enzyme, which also catalyzed the pyruvate formation, were found in B. flavum, but the latter activity was very low in cells grown on glucose. The levels of these enzymesin pyruvate kinase mutants unable to grow on ribose or gluconate derived from the wild strain were almost identical to those in the wild-type strain. Since phosphoenolpyruvate (PEP) is a branch-point intermediate in the sugar metabolism for amino acid biosynthesis, studies on enzymes catalyzing PEP metabolism seem interesting, especially for an amino acid-producing bacterium, Brevibacterium flavum. Studies on PEP carboxylase1} which catalyzes the formation of oxaloacetate, an intermediate in amino acid biosynthesis, and on pyruvate kinase (PK)2) which catalyzes the first step of degradation of PEP to CO2 were reported previously. Pyruvate produced from PEP via the PK reaction is not only oxidized to yield CO2but also used for the biosynthesis of various essential cell constituents. The growth on glucose of mutants lacking PK, however, was substantially identical to that of the wild-type strain, suggesting that enzymesother than PKmight serve to form sufficient pyruvate for growth. The present study revealed that the PEP : sugar phosphotransferase system (PTS) as well as PK significantly contribute to the pyruvate formation during growth on glucose, whereas only PK is responsible for the formation during growth on sugars which are not metabolized via the PTS reaction. MATERIALS AND METHODS Chemicals. PEP, ADPand ATP were purchased from Sigma Chemical Co., dithiothreitol from Calbiochem, and NAD, NADHand NADPfrom Boehringer Mannheim GmbH.All the enzymes used for enzyme assays and for the metabolite determinations were obtained from the latter company. Abbreviations: PK, pyruvate kinase; PTS, phosphotransferase system; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; FBP, fr...

Metabolic Analysis of Glutamate Production by Corynebacterium glutamicum

“…Glyceraldehyde-3-phosphate dehydrogenase was assayed according to the method of Crow and Wittenberger (1979) with the following reaction mixture: NAD (1 mM), sodium arsenate (5 mM), cysteine HCl (5 mM), triethanolamine buffer (125 mM, pH 7.8), dl-glyceraldehyde-3-P (4 mM), and diluted enzyme extract. Malic enzyme was assayed by the method described by Mori and Shiio (1987) with an optimized reaction mixture consisting of phosphate buffer (100 mM, pH 7.8), MgCl 2 (5 mM), NADP + (0.6 mM), and malate (40 mM) as the substrate. Assays were initiated by the addition of malate.…”

Growth Recovery on Glucose under Aerobic Conditions of an <i>Escherichia coli</i> Strain Carrying a Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Deletion by Inactivating <i>arcA</i> and Overexpressing the Genes Coding for Glucokinase and Galactose Permease

“…Several groups have modified the PEP node to increase the availability of this molecule for the synthesis of aromatic compounds [Frost and Lievense, 1984;Miller et al, 1987;Mori and Shio, 1987;Flores et al, 1996;Gosset et al, 1996;Yi et al, 2002]. One of these strategies includes the elimination of PTS which consumes half of the PEP produced during glycolysis for glucose internalization [Flores et al, 1996;Postma et al, 1996;Saier, 2002;Gosset, 2005].…”

Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes inEscherichia coli

“…It is evident that if we are interested in optimizing carbon flow for the production of certain metabolites with maximal yields, this basic scheme needs to be modified. Accordingly, for the production of several aromatic compounds (Frost and Draths, 1995), the modification of the phosphoenolpyruvate node (PEP) has been the focus of several groups (Gubler et al, 1994;Miller et al, 1987;Mori and Shiio, 1987;Patnaik and Liao, 1994;Sano and Ito, 1987). Flux distribution at the PEP node is tightly regulated by allosteric regulation of the enzymes involved in this node.…”