Identification of the aceA gene encoding isocitrate lyase required for the growth of Pseudomonas aeruginosa on acetate, acyclic terpenes and leucine

Pseudomonas aeruginosa PAO1 mutants affected in acyclic monoterpenes, n-octanol, and acetate assimilation were isolated using transposon mutagenesis. The isocitrate lyase gene (aceA) corresponding to ORF PA2634 of the PAO1 strain genome was identified in one of these mutants. The aceA gene encodes a protein that is 72% identical to the isocitrate lyase (ICL) characterized from Colwellia maris, but is less than 30% identical to their homologues from pseudomonads. The genetic arrangement of aceA suggests that it is a monocistronic gene, and no adjacent related genes were found. The ICL protein was detected as a 60-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis from cultures grown on acetate, but not in glucose-grown PAO1 cultures. Genetic complementation further confirmed that the aceA gene encodes the ICL enzyme. The ICL enzyme activity in crude extracts from cultures of the PAO1 strain was induced by acetate, citronellol and leucine, and repressed by growth on glucose or citrate. These results suggest that ICL is involved in the assimilation of acetate, acyclic monoterpenes of the citronellol family, alkanols, and leucine, in which the final intermediary acetyl-coenzyme A may be channelled to the glyoxylate shunt.     

代谢工程改造大肠杆菌提高乙醇酸产率

乙醇酸(Glycolate)是一种在工业上有多种用途的重要化合物。本研究首先在大肠杆菌MG1655(DE3)中敲除了ldh A(乳酸脱氢酶),获得菌株Mgly1,作为出发菌株。然后通过调节乙醇酸合成途径的关键酶——异柠檬酸裂解酶(ace A)、乙醛酸还原酶(ycd W)、异柠檬酸脱氢酶激酶/磷酸化酶(ace K)的表达水平,得到乙醇酸产率为0.24 g/g葡萄糖(占理论产率的28.2%)。过量表达柠檬酸合成酶(glt A),乙醇酸产率提高到0.326 g/g葡萄糖(占理论产率的38.3%)。然后在Mgly1中敲除了glc B和ace B(苹果酸合成酶),减少了乙醇酸合成的前体乙醛酸的消耗。最终获得的工程菌株Mgly335乙醇酸产率达到0.522 g/g葡萄糖(占理论产率的61.4%)。     

The regulation of NADP-linked isocitrate dehydrogenase in Aspergillus nidulans

The regulation of NADP-linked isocitrate dehydrogenase (NADP-IDH) has been studied in wild-type and mutant strains of Aspergillus nidulans. In the wild-type strain studied, the levels of NADP-IDH vary in a similar way to those of acetamidase, acetyl-CoA synthase, isocitrate lyase and malate synthase under all growth conditions used. Similarly, fac mutants, which are altered in the regulation of these enzymes of acetate utilization, are affected in NADP-IDH levels in a parallel fashion, as are cre mutants, which show altered carbon catabolite repression of this group of enzymes. Possible functions of the NADP-IDH enzyme are considered.     

Acetate-nonutilizing Mutants of Neurospora crassa II. Biochemical Deficiencies and the Roles of Certain Enzymes

The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing (acu) mutants of Neurospora crassa, both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed.     

Biochemical studies on acetate non-utilizing mutants of Aspergillus terreus IRRL 16043

The strain Aspergillus terreus IRRL 16043 can utilize glucose as well as acetate as a sole carbon source. Thirty-nine mutants were isolated from the wild-type by treatment with a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNTG) which could not utilize acetate as a sole carbon source, and were designated as acetate non-utilizing (acu). By complementation and biochemical analyses they were divided into three functional groups, acu A, acu B and acu C lacking isocitrate lyase, malate synthase and acetyl-CoA synthetase activity, respectively.     

Metabolic Role, Regulation of Synthesis, Cellular Localization, and Genetic Control of the Glyoxylate Cycle Enzymes in Neurospora crassa

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.     

Characterization of Sinorhizobium meliloti triose phosphate isomerase genes

A Tn5 mutant strain of Sinorhizobium meliloti with an insertion in tpiA (systematic identifier SMc01023), a putative triose phosphate isomerase (TPI)-encoding gene, was isolated. The tpiA mutant grew more slowly than the wild type on rhamnose and did not grow with glycerol as a sole carbon source. The genome of S. meliloti wild-type Rm1021 contains a second predicted TPI-encoding gene, tpiB (SMc01614). We have constructed mutations and confirmed that both genes encode functional TPI enzymes. tpiA appears to be constitutively expressed and provides the primary TPI activity for central metabolism. tpiB has been shown to be required for growth with erythritol. TpiB activity is induced by growth with erythritol; however, basal levels of TpiB activity present in tpiA mutants allow for growth with gluconeogenic carbon sources. Although tpiA mutants can be complemented by tpiB, tpiA cannot substitute for mutations in tpiB with respect to erythritol catabolism. Mutations in tpiA or tpiB alone do not cause symbiotic defects; however, mutations in both tpiA and tpiB caused reduced symbiotic nitrogen fixation.     

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.     

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.     

Biochemical and structural studies of malate synthase from Mycobacterium tuberculosis

Establishment or maintenance of a persistent infection by Mycobacterium tuberculosis requires the glyoxylate pathway. This is a bypass of the tricarboxylic acid cycle in which isocitrate lyase and malate synthase (GlcB) catalyze the net incorporation of carbon during growth of microorganisms on acetate or fatty acids as the primary carbon source. The glcB gene from M. tuberculosis, which encodes malate synthase, was cloned, and GlcB was expressed in Escherichia coli. The influence of media conditions on expression in M. tuberculosis indicated that this enzyme is regulated differentially to isocitrate lyase. Purified GlcB had K(m) values of 57 and 30 microm for its substrates glyoxylate and acetyl coenzyme A, respectively, and was inhibited by bromopyruvate, oxalate, and phosphoenolpyruvate. The GlcB structure was solved to 2.1-A resolution in the presence of glyoxylate and magnesium. We also report the structure of GlcB in complex with the products of the reaction, coenzyme A and malate, solved to 2.7-A resolution. Coenzyme A binds in a bent conformation, and the details of its interactions are described, together with implications on the enzyme mechanism.