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

Abstract

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...

Sigma factors and promoters in Corynebacterium glutamicum

“…Sequences of the selected presumed A -dependent promoters localized by mapping the TSPs are shown in Fig. 3 (Arndt and Eikmanns, 2007;Auchter et al, 2009;Beckers et al, 2001;Bellmann et al, 2001;Börmann et al, 1992;Brinkrolf et al, 2008;Brune et al, 2006Brune et al, , 2007Eikmanns et al, 1994;Engels and Wendisch, 2007;Engels et al, 2008;Follettie et al, 1993;Frunzke et al, 2008;Georgi et al, 2008;Gerstmeir et al, 2003;Han et al, 1990Han et al, , 2007Han et al, , 2008Hansmeier et al, 2006;Inui et al, 2007;Jakoby et al, 2000;Jungwirth et al, 2008;Kennerknecht et al, 2002;Klaffl and Eikmanns, 2010;Koch et al, 2005;Krug et al, 2005;Letek et al, 2007;Marcel et al, 1990;Mateos et al, 1994;Merkens et al, 2005;Möker et al, 2004;Morbach et al, 2000;Nishimura et al, 2007;Nolden et al, 2001;Okibe et al, 2009;Pátek et al, 1998;Pátek et al, 2003b;Peoples et al, 1988;Peters-Wendisch et al, 1998;…”

Phosphotransferase System-Independent Glucose Utilization in Corynebacterium glutamicum by Inositol Permeases and Glucokinases

“…Oxaloacetate utilized for L-lysine synthesis can be regenerated by PEP carboxylase (encoded by ppc) (13) or pyruvate carboxylase (pyc) (48). In terms of gluconeogenesis, oxaloacetate can be converted to either pyruvate by oxaloacetate decarboxylase (odx) (32) or to PEP by PEP carboxykinase (pck) (28). The irreversible conversion of PEP to pyruvate is catalyzed either by pyruvate kinase (pyk) with the formation of ATP (24) or by enzyme I (EI) of the PEP-dependent phosphotransferase system (PTS) in the course of uptake and the phosphorylation of PTS substrates (41).…”

Identification of FAH Domain-containing Protein 1 (FAHD1) as Oxaloacetate Decarboxylase

“…This can be explained by the fact that these enzymes are part of highly specialized metabolic pathways, involving chemical compounds that higher organisms are unable to convert and utilize for their metabolism (5). One recently identified prokaryotic member of the FAH superfamily found in Corynebacterium glutamicum, referred to as Cg1458, was characterized as a novel soluble oxaloacetate decarboxylase (ODx) (7,8); however, eukaryotic ODx enzymes were not identified so far.…”