The three tricarboxylate synthase activities of Corynebacterium glutamicum and increase of l-lysine synthesis

Radmacher, Eva; Eggeling, Lothar

doi:10.1007/s00253-007-1105-7

Cited by 35 publications

(37 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Given that PrpR was the name given to the 54 -dependent regulator of the prp operon of enterobacteria (18,19), Masiewicz et al (27) used the same nomenclature for Rv1129c, despite its dissimilarity in amino acid sequence to PrpR. This name was also later used for describing Cg0800 of C. glutamicum (29,55). Because PrpR/LrpG (Rv1129c) from M. tuberculosis and PrpR (Cg0800) from C. glutamicum are members of the MccR class of the ScfR family, we recommend adjusting the previously mixed and misleading nomenclature (Fig.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

Carter

Alber

2015

J Bacteriol

View full text Add to dashboard Cite

Propionyl coenzyme A (propionyl-CoA Rhodobacter sphaeroides belongs to the purple nonsulfur bacteria, a physiologically related group of phototrophic bacteria known for their metabolic versatility (1). Given their natural abundance in anaerobic portions of stagnant water, purple nonsulfur bacteria are adept at using light as a source of energy during anaerobic growth while acquiring carbon from fermentation products, such as propionate. Despite the abundance of propionate in its natural environment, R. sphaeroides was originally classified uniquely among the purple nonsulfur bacteria to be unable to use propionate as a carbon source for growth (1). A recent edition of Bergey's Manual of Systematic Bacteriology, however, recognizes that the type strain, R. sphaeroides 2.4.1, does grow with propionate (2).As a growth substrate, propionate is directly activated to propionyl coenzyme A (propionyl-CoA). Propionyl-CoA is also an intermediate of pathways responsible for the degradation of endogenously derived or exogenously supplied branched-chain amino acids as well as branched-chain and odd-numbered-chain fatty acids (3-6). In addition, propionylCoA is formed during the metabolism of 3-hydroxypropionate and during acetyl-CoA assimilation via the ethylmalonyl-CoA pathway (see Fig. S1 in the supplemental material) (7,8). The ethylmalonyl-CoA pathway substitutes for the glyoxylate bypass in many bacteria and is required for replenishing intermediates of the citric acid cycle for growth with substrates that enter central carbon metabolism at the level of acetyl-CoA (9).Propionyl-CoA is an inhibitor of several key metabolic enzymes, and therefore, the metabolism of propionyl-CoA needs to

show abstract

Section: Discussionmentioning

confidence: 99%

Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

Carter

Alber

2015

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Optimization of cellular oxaloacetate concentrations seems to be crucial, especially for improving L-lysine production. This possibility was proposed by Menkel et al (29) and was indicated by overexpression of the pyruvate carboxylase gene (40), inactivation of PEP carboxykinase (46), inactivation of citrate and methylcitrate synthases (45), and disruption of the malate:quinone oxidoreductase gene (31). However, there have not been many studies addressing the role of oxaloacetate decarboxylase (ODx), an enzyme that has high levels of activity in different C. glutamicum strains (21) and catalyzes the irreversible decarboxylation of oxaloacetate (25), at this key branch point.…”

mentioning

confidence: 99%

Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum

Klaffl

Eikmanns

2010

J Bacteriol

View full text Add to dashboard Cite

Soluble, divalent cation-dependent oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of oxaloacetate to pyruvate and CO 2 . Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 ؎ 1 kDa and that the enzyme is composed of two ϳ29-kDa homodimeric subunits and has a K m for oxaloacetate of 1.4 mM and a V max of 201 mol of oxaloacetate converted per min per mg of protein, resulting in a k cat of 104 s ؊1 . Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an L-lysineproducing strain of C. glutamicum led to accumulation of less L-lysine. However, inactivation of the odx gene did not improve L-lysine production under the conditions tested.Corynebacterium glutamicum is a respirative, Gram-positive soil bacterium that is well suited to industrial amino acid production of, e.g., L-glutamate, L-lysine, and L-valine (4,28). This organism possesses a rather complex phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (Fig. 1) compared to model organisms, such as Escherichia coli and Bacillus subtilis (49). Due to the importance of this node for supply of precursors for amino acid synthesis and due to the fact that all enzymes of this node show significant activity in glucose-grown cells (13,17,20,22,42,52), much attention has been focused on identifying targets for metabolic engineering (5,18,24,26,36,39,41,46,47,(56)(57)(58). Optimization of cellular oxaloacetate concentrations seems to be crucial, especially for improving L-lysine production. This possibility was proposed by Menkel et al. (29) and was indicated by overexpression of the pyruvate carboxylase gene (40), inactivation of PEP carboxykinase (46), inactivation of citrate and methylcitrate synthases (45), and disruption of the malate:quinone oxidoreductase gene (31). However, there have not been many studies addressing the role of oxaloacetate decarboxylase (ODx), an enzyme that has high levels of activity in different C. glutami...

show abstract

“…Later, two methylcitrate synthases were identified, PrpC1 (cg0798) and PrpC2 (cg0762), which also can catalyze the formation of citrate from oxaloacetate and acetylCoA (47). Their activity usually is not sufficient to substitute for the one of GltA, but enhanced prp gene expression due to the inactivation of the repressor PrpR allows for the partial complementation of a ⌬gltA mutant (47).…”

mentioning

confidence: 99%

“…The relevance of individual TCA cycle enzymes in C. glutamicum has been explored with the help of mutants in which the corresponding genes were disrupted or deleted, such as strains lacking citrate synthase (13,47), isocitrate dehydrogenase (12), 2-oxoglutarate dehydrogenase (25), malate-quinone oxidoreductase (41), and malate dehydrogenase (40,41). In the case of citrate synthase, which catalyzes the first, irreversible step of the TCA cycle, this approach revealed the presence of alternative enzymes which can at least partially take over the function of the deleted protein.…”

mentioning

confidence: 99%

“…The resulting cell extract was kept on ice until it was used for the assay, which was based on the reaction of coenzyme A with 5,5Ј-dithiobis(2-nitrobenzoic acid). The absorbance of the resulting yellow 2-nitro-5-thiobenzoate dianion was measured spectrophotometrically at 412 nm as described previously (47). Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Pierce, Bonn, Germany) with bovine serum albumin as the standard.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Deletion of the Aconitase Gene in Corynebacterium glutamicum Causes Strong Selection Pressure for Secondary Mutations Inactivating Citrate Synthase

et al. 2011

View full text Add to dashboard Cite

The aconitase gene acn of Corynebacterium glutamicum is regulated by four transcriptional regulators, indicating that the synthesis of this enzyme is carefully controlled. To understand the causes for this elaborate regulation, the properties of the ⌬acn-1 deletion mutant were analyzed in detail. The mutant was glutamate auxotrophic in glucose minimal medium, showed a strong growth defect, and secreted large amounts of acetate. None of these phenotypes could be complemented by plasmid-encoded aconitase, suggesting the presence of a secondary mutation. In fact, a point mutation within the gltA gene encoding citrate synthase was identified that caused the instability of the protein and an almost complete lack of its enzymatic activity. Subsequently, 27 further, independent ⌬acn clones were isolated, and 15 of them were found to contain distinct mutations in gltA, causing the loss of citrate synthase activity. A similar result was observed for mutants lacking the isocitrate dehydrogenase gene icd. In this case, 8 of 24 ⌬icd clones contained additional mutations in gltA. Indirect evidence was obtained that elevated intracellular citrate concentrations could be the cause of this selection pressure. Accordingly, the careful control of aconitase synthesis might have evolved due to the necessity to avoid inhibitory cytoplasmic citrate levels on the one hand and to prevent the excessive synthesis of an oxygen-sensitive protein requiring both iron and sulfur on the other hand.For most organisms, the tricarboxylic acid (TCA) cycle, also known as the Krebs cycle, is a key metabolic pathway. In its catabolic function, acetyl-coenzyme A (CoA), a product of the degradation of sugars, fatty acids, amino acids, and other carbon sources, is oxidized to CO 2 , thereby generating reducing equivalents (NADH and reduced quinones) for respiration and ATP by substrate-level phosphorylation. In its anabolic function, the TCA cycle produces 2-oxoglutarate and oxaloacetate as precursors of the glutamate family and the aspartate family of amino acids, respectively, in addition to other important intermediates, such as succinyl-CoA. Moreover, in many bacteria, NADPH is produced for biosynthetic purposes by the isocitrate dehydrogenase reaction.The TCA cycle has been studied quite intensively in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for large-scale industrial amino acid production, with the major products being the flavor enhancer L-glutamate (as monosodium salt) and the feed additive L-lysine, both of which are derived from intermediates of the TCA cycle. Many enzymes have been characterized, for example, citrate synthase (13), aconitase (4), isocitrate dehydrogenase (12), the 2-oxoglutarate dehydrogenase complex (25, 43, 59), fumarase (19), succinate dehydrogenase (34), malate-menaquinone oxidoreductase, and malate dehydrogenase (18,40,41). In addition, a complex regulatory network for the control of TCA cycle activity has been elucidated (for a review, see reference 7) that includes a variety of transcripti...

show abstract

The three tricarboxylate synthase activities of Corynebacterium glutamicum and increase of l-lysine synthesis

Cited by 35 publications

References 27 publications

Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum

Deletion of the Aconitase Gene in Corynebacterium glutamicum Causes Strong Selection Pressure for Secondary Mutations Inactivating Citrate Synthase

Contact Info

Product

Resources

About