Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map.

In Escherichia coli, a single operon encodes the metabolic and regulatory enzymes of the glyoxylate bypass. The metabolic enzymes, isocitrate lyase and malate synthase, are expressed from aceA and aceB, and the regulatory enzyme, isocitrate dehydrogenase kinase/phosphatase, is expressed from aceK. We cloned this operon and determined its functional map by deletion analysis. The order of the genes in this operon is aceB-aceA-aceK, with aceB proximal to the promoter, consistent with the results of previous experiments using genetic techniques. The promoter was identified by S1 nuclease mapping, and its nucleotide sequence was determined. Isocitrate lyase and malate synthase were readily identified by autoradiography after the products of the operon clone were labeled by the maxicell procedure and then resolved by electrophoresis. In contrast, isocitrate dehydrogenase kinase/phosphatase, expressed from the same plasmid, was undetectable. This observation is consistent with a striking downshift in expression between aceA and aceK.     

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.     

The amrG1 gene is involved in the activation of acetate in Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic, Gram-positive microorganism, well known as a pro-ducer of several amino acids. Amino acid products are used on a large scale for food industry flavouring, feed additive, pharmaceutical and cosmetic purpose[1,2]. The organism is able to grow not only on glucose, fructose and lactose, but also on acetate, lactate as its sole carbon source. The growth on acetate requires its activation to acetyl-CoA. In C. glutamicum, acetate is activated in a two-step …     

Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585.

The glyoxylate cycle comprising isocitrate lyase (ICL) and malate synthase (MS) is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, which encodes malate synthase has been previously cloned from Streptomyces clavuligerus NRRL 3585 and characterized. In this study, the aceA gene, encoding ICL from S. clavuligerus NRRL 3585, was obtained via genome walking experiments and PCR. The fully sequenced open reading frame encodes 436 amino acids with a deduced M(r) of 47.5 kDa, consistent with the observed M(r) (49-67.5 kDa) of most ICL enzymes reported so far. The cloned aceA gene was expressed in Escherichia coli BL21(lambdaDE3) cells, from which ICL was purified as a His-tagged product and its functionality demonstrated. Furthermore, the relationship between the carbon sources, growth and ICL activity in S. clavuligerus were investigated. Rapid growth was observed when the cells were cultured on 0.5% (w/v) glycerol, while delayed growth was observed when cells were grown on 0.5% (w/v) acetate. However, in both cases, high levels of ICL activity coincided with a cessation of growth, suggesting a late physiological role played by ICL in the natural host, S. clavuligerus.     

Ethanol catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.