Amino Acid Aminotransferases in an Amino Acid-producing Bacterium,<i>Brevibacterium flavum</i>

Shiio, Isamu; Mori, Michiko; Ozaki, Hachiro

doi:10.1080/00021369.1982.10865552

Cited by 5 publications

(10 citation statements)

References 5 publications

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“…Although overexpression of the odx gene led to accumulation of less L-lysine, inactivation of the odx gene did not improve L-lysine production, which disproved the proposal based on the results of Klapa et al (24). Analysis of the kinetic properties revealed that the K m for oxaloacetate (1.4 mM) was higher than those of other enzymes catalyzing oxaloacetate conversion, such as citrate synthase, aspartate aminotransferase, and malate dehydrogenase (K m values, 0.0015 mM, 0.11 mM, and 0.25 mM, respectively) (15,21,54). The low affinity of oxaloacetate decarboxylase for oxaloacetate, a compound that occurs only at low intracellular concentrations but is involved in a multitude of reactions, is one possible explanation for the absence of a positive effect of the odx deletion on L-lysine production.…”

Section: Discussionmentioning

confidence: 37%

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

Klaffl

Eikmanns

2010

J Bacteriol

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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...

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Section: Discussionmentioning

confidence: 37%

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

Klaffl

Eikmanns

2010

J Bacteriol

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“…(2) were approximated by the respective K m values (Stryer, 1988). Shiio et al (1982) found nearly identical maximum forward and reverse activities of C. glutamicum aspartate aminotransferase. Hence, the measured specific activity was taken as the value of both v AAT, max(f ) and v AAT, max(r) .…”

Section: Estimated Intracellular Oxaloacetate Concentrationmentioning

confidence: 74%

“…However, due to lysine production and growth a substantial net flux was catalyzed by aspartate aminotransferase. Hence, a more accurate estimation of oxaloacetate concentration needs to consider the kinetics of the enzyme, which follow a Ping-Pong Bi-Bi mechanism (Segel, 1975;Shiio et al, 1982):…”

Section: Estimated Intracellular Oxaloacetate Concentrationmentioning

confidence: 99%

“…K m... are the Michaelis constants for the respective metabolites, and K d.... are dissociation constants. For the C. glutamicum enzyme, K mOAA, AAT is 0.11 mM, and K mGlu, AAT is 24 mM (Shiio et al, 1982). Michaelis constants for aspartate and a-ketoglutarate are not available for C. glutamicum, therefore K m values of the Escherichia coli enzyme (K maKG, AAT =0.071 mM, K mAsp, AAT =4.4 mM; Mavrides and Orr, 1975) were used as approximations.…”

Section: Estimated Intracellular Oxaloacetate Concentrationmentioning

confidence: 99%

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Metabolic Consequences of Altered PhosphoenolpyruvateCarboxykinase Activity in Corynebacterium glutamicum Reveal Anaplerotic Regulation Mechanisms in Vivo

Petersen

Mack

Graaf

et al. 2001

Metabolic Engineering

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“…Strange that we rarely think of l -glutamate as parameter we should investigate. Under normal circumstances, l -glutamate is enough to supply the amino for l -lysine biosynthesis, but it’s inevitably encountered some special circumstances, such as the strain with attenuation of TCA cycle [ 14 ]. Moreover, l -lysine biosynthesis is closely related to the biosynthesis of l -glutamate.…”

Section: Introductionmentioning

confidence: 99%

Rational modification of tricarboxylic acid cycle for improving l-lysine production in Corynebacterium glutamicum

Wu²,

Gao³

et al. 2018

Microb Cell Fact

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BackgroundOxaloacetate (OAA) and l-glutamate are essential precursors for the biosynthesis of l-lysine. Reasonable control of all potentially rate-limiting steps, including the precursors supply rate, is of vital importance to maximize the efficiency of l-lysine fermentation process.ResultsIn this paper, we have rationally engineered the tricarboxylic acid (TCA) cycle that increased the carbon yield (from 36.18 to 59.65%), final titer (from 14.47 ± 0.41 to 23.86 ± 2.16 g L−1) and productivity (from 0.30 to 0.50 g L−1 h−1) of l-lysine by Corynebacterium glutamicum in shake-flask fermentation because of improving the OAA and l-glutamate availability. To do this, the phosphoenolpyruvate–pyruvate–oxaloacetate (PEP–pyruvate–OAA) node’s genes ppc and pyc were inserted in the genes pck and odx loci, the P1 promoter of the TCA cycle’s gene gltA was deleted, and the nature promoter of glutamate dehydrogenase-coding gene gdh was replaced by Ptac-M promoter that resulted in the final engineered strain C. glutamicum JL-69Ptac-M gdh. Furthermore, the suitable addition of biotin accelerates the l-lysine production in strain JL-69Ptac-M gdh because it elastically adjusts the carbon flux for cell growth and precursor supply. The final strain JL-69Ptac-M gdh could produce 181.5 ± 11.74 g L−1 of l-lysine with a productivity of 3.78 g L−1 h−1 and maximal specific production rate (qLys, max.) of 0.73 ± 0.16 g g−1 h−1 in fed-batch culture during adding 2.4 mg L−1 biotin with four times.ConclusionsOur results reveal that sufficient biomass, OAA and l-glutamate are equally important in the development of l-lysine high-yielding strain, and it is the first time to verify that fed-batch biotin plays a positive role in improving l-lysine production.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0958-z) contains supplementary material, which is available to authorized users.

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Amino Acid Aminotransferases in an Amino Acid-producing Bacterium,Brevibacterium flavum

Cited by 5 publications

References 5 publications

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

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

Metabolic Consequences of Altered PhosphoenolpyruvateCarboxykinase Activity in Corynebacterium glutamicum Reveal Anaplerotic Regulation Mechanisms in Vivo

Rational modification of tricarboxylic acid cycle for improving l-lysine production in Corynebacterium glutamicum

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