Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon

Abstract

Acetohydroxy acid synthase (AHAS) and isomeroreductase (IR) catalyze subsequent reactions in the flux of metabolites towards isoleucine, valine, leucine, and pantothenate. A 4,705-bp DNA fragment from Corynebacterium glutamicum known to code for AHAS and IR was sequenced and analyzed by Northern (RNA blot) analysis. As in other bacteria, the AHAS of this gram-positive organism is encoded by two genes, ilvB and ilvN. Gene disruption verified that these genes encode the single AHAS activity in C. glutamicum. The start of ilvB was determined by amino-terminal sequencing of a fusion peptide. By Northern analysis of the ilvBNC cluster, three in vivo transcripts of 3.9, 2.3, and 1.1 kb were identified, corresponding to ilvBNC, ilvNC, and ilvC messages, respectively. The ilvC transcript (encoding IR) was by far the most abundant one. With a clone from which the ilvB upstream regions had been deleted, only the ilvNC and ilvC transcripts were synthesized, and with a clone from which the ilvN upstream regions had been deleted, only the smallest ilvC transcript was formed. It is therefore concluded that in the ilv operon of C. glutamicum, three promoters are active. The amounts of the ilvBNC and ilvNC transcripts increased in response to the addition of a-ketobutyrate to the growth medium. This was correlated to an increase in specific AHAS activity, whereas IR activity was not increased because of the relatively large amount of the ilvC transcript present under all conditions assayed.Therefore, the steady-state level of the ilvBNC and ilvNC messages contributes significantly to the total activity of the single AHAS. The ilvC transcript of this operon, however, is regulated independently and present in a large excess, which is in accord with the constant IR activities determined.Within the highly branched pathway of isoleucine synthesis, acetohydroxy acid synthase (AHAS) is unique because it naturally uses different substrates, allowing the ultimate synthesis of isoleucine (Ile), valine (Val), leucine (Leu), and pantothenate in parallel reactions. Consequently, AHAS activity is complexly regulated at various levels. Up to five isoenzymes exist in enterobacteria, of which three have been extensively studied. AHAS I (ilvBN) is feedback sensitive to Val; its expression is controlled by Val and Leu but also by cyclic AMP (8). AHAS II (ilvGM) is controlled by Val-, Leu-, and Ile-mediated attenuation. ilvGM is embedded in the ilvGMEDA operon, which, together with the downstream ilvYC genes, codes for all enzymes needed for Ile synthesis (28). AHAS III (ilvIH) is feedback sensitive to Val, and its expression is controlled by interaction of the Leu-Lrp protein complex with regulatory regions (32). In addition to these mechanisms for regulating total AHAS activity, each enzyme has a different specificity towards its two possible substrate mixtures, again influencing the total flux through this one reaction (1).In contrast to this plurality of sophisticated mechanisms for enterobacteria, only limited information is available for g...

Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

“…Biochemical characterization of IlvBN from M. tuberculosis and M. avium showed that the recombinant enzyme was sensitive to valine, and that the AHAS activity could be inhibited by sulfonylureas (Zohar et al, 2003;Choi et al, 2005), thus making it a functional equivalent of the type II enzyme of enterobacteria. Although Grampositive bacteria possess a single species of AHAS, mostly classified as IlvBN type (Keilhauer et al, 1993;Porat et al, 2004), the presence of multiple genes coding for the catalytic subunits of AHAS in M. tuberculosis points towards the possible existence of multiple AHAS isozymes in this organism.…”

“…The biochemical characterization of recombinant AHAS from Bacillus stearothermophilus, which has been designated IlvBN, revealed that it is a functional equivalent of the type III AHAS (IlvIH) of enterobacteria (Porat et al, 2004). Similarly, Corynebacterium glutamicum was shown to possess a single species of AHAS (IlvBN), which is responsible for the synthesis of BCAA (Keilhauer et al, 1993). Biochemical characterization of a recombinant AHAS (IlvBN) from two mycobacterial species, Mycobacterium avium and M. tuberculosis, has been reported (Zohar et al, 2003;Choi et al, 2005).…”

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

“…For analysing lysine or glutamate production, a brain-heart infusion preculture was inoculated from a fresh Luria-Bertani (LB) plate and cultivated overnight. After washing cells in CGXII medium (as described by Keilhauer et al 1993, but with 30 mg/l 3,4-dihydroxybenzoic acid as iron chelator) without carbon source, the main culture with CGXII medium was inoculated to an OD 600 of 1. For lysine production, 10% (w/v) glucose, 10% (w/v) fructose, 10% (w/v) sucrose, or 5% (w/v) glucose plus 5% (w/v) fructose were used as carbon source, whereas for glutamate production, 4% (w/v) glucose, 4% (w/v) fructose, 4% (w/v) sucrose or 2% (w/v) glucose + 2% (w/v) fructose were used.…”

Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production

“…C. glutamicum strains and plasmids used in this work are listed in Table 1. For analyzing growth and lysine production, a first preculture was grown in brain heart infusion medium with 2% (wt/vol) glucose for 8 h and an aliquot of cells was transferred either to CGXII minimal medium (15) containing 4% (wt/vol) glucose or to modified CGX minimal medium (32) with 10% (wt/vol) glucose to give an optical density at 600 nm (OD 600 ) of 1. The CGXII medium was supplemented with 30 mg/liter 3,4-dihydroxybenzoic acid as iron chelator and, if appropriate, with 0.3 g/liter leucine.…”