Elsevier

Journal of Biotechnology

Volume 104, Issues 1–3, 4 September 2003, Pages 129-153
Journal of Biotechnology

The respiratory chain of Corynebacterium glutamicum

https://doi.org/10.1016/S0168-1656(03)00144-5Get rights and content

Abstract

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, d-lactate dehydrogenase, l-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and l-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc1 complex to the aa3-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c1 to the CuA center in subunit II of cytochrome aa3. Rather, cytochrome c1 contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc1 complex and cytochrome aa3 oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc1aa3 branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F1F0-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.

Introduction

Corynebacterium glutamicum was isolated by Kinoshita and coworkers in the mid 1950s as a biotin-auxotrophic bacterium that excretes large amounts of l-glutamate when growing aerobically under biotin limitation with glucose as carbon source (Kinoshita et al., 1957). It belongs to the Gram-positive bacteria with a high G+C content of the chromosomal DNA (53.8% in the case of C. glutamicum) and, more specifically, to a group of bacteria including the genera Corynebacterium, Mycobacterium, Nocardia and Rhodococcus which contain mycolic acids (α-alkyl-β-hydroxy fatty acids) in the cell wall. After the discovery that C. glutamicum is suitable for the conversion of sugars and organic acids to l-glutamate, mutants were isolated that were able to excrete also other amino acids, in particular l-lysine. Nowadays, both l-glutamate, which is mainly used as a flavor enhancer, and l-lysine, mainly used as a feed additive, are produced industrially almost exclusively with C. glutamicum at a scale of 1×106 and 0.5×106 tons per year, respectively (Ikeda, 2003, Kimura, 2003, Pfefferle et al., 2003).

In the past, the common practice to obtain amino acid overproducing strains was by mutagenesis and selection. Within the last decade, however, metabolic engineering, i.e. the improvement of enzymatic, transport and regulatory functions of the cell with the application of recombinant DNA technology (Bailey, 1991), was established as an alternative that allows the rational construction of production strains. Metabolic engineering includes the deletion, overexpression or timed expression of selected genes or the replacement of a wild-type enzyme with one having defined altered properties. In contrast to strain construction by mutagenesis and selection, metabolic engineering requires at least some basic knowledge on the metabolism of the organism investigated and in particular tools for genetic manipulation.

Due to the importance of C. glutamicum for amino acid production, most studies on this organism focused on amino acid biosynthesis, on precursor supply by central metabolic pathways and anaplerotic reactions, on carbon and nitrogen fluxes and on transport processes relevant for amino acid production (for review, see Eikmanns et al., 1993, Sahm et al., 1995, Sahm et al., 2000, Eggeling et al., 1997, Eggeling and Sahm, 2001, Burkovski and Krämer, 2002). In recent years, additional topics received attention, driven (i) by the establishment of the C. glutamicum genome sequence, (ii) by the realization that construction of the “optimal” production strain requires knowledge on the physiology and biochemistry of the organism as a whole rather than only of selected parts, and (iii) by the possibility to use C. glutamicum as a non-pathogenic model organism to study features in common with pathogenic corynebacteria and mycobacteria, e.g. C. diphtheriae and Mycobacterium tuberculosis. Respiration was one of the topics receiving attention, resulting in a continuation of the studies made in the 1970s and 1980s (Kanzaki et al., 1973, Kanzaki et al., 1974, Sugiyama et al., 1973a, Sugiyama et al., 1973b, Trutko et al., 1982, Kawahara et al., 1988). This review summarizes the current knowledge on the C. glutamicum respiratory chain, including relevant information from the genome sequence on aspects that have not yet been studied experimentally.

Section snippets

The early studies on respiration in C. glutamicum

First interest into the respiratory chain of C. glutamicum was probably initiated by the finding that copper ions were necessary for the maximal production of l-glutamate from acetate by an oleic acid-requiring strain of C. glutamicum designated as Brevibacterium thiogenitalis. A similar effect was observed for succinate and fumarate, but not for glucose, lactate, ethanol and n-paraffin. The decreased glutamate formation in the absence of copper ions correlated with an increased oxidation of

Enzymes reducing menaquinol

In this and the following sections, the individual components of the C. glutamicum respiratory chain are described. Fig. 1 gives an overview on these components, whereas Fig. 2 shows more details of their subunit composition, localization, cofactors and involvement in the generation of a proton-motive force.

Enzymes oxidizing menaquinol

Reduced-minus-oxidized difference spectra obtained with intact cells, cell extracts and membranes of C. glutamicum revealed peaks at 600, 560 and 550 nm, indicating the presence of a-, b- and c-type cytochromes, respectively (Sugiyama et al., 1973a, Sugiyama et al., 1973b, Trutko et al., 1982). In the CO-reduced-minus-reduced difference spectrum a peak at 427 nm and a trough at 443 nm were found, typical for a cytochrome aa3-type terminal oxidase (Trutko et al., 1982). Isolated membranes

Evidences for an additional terminal oxidase activity

Genome analysis revealed that cytochrome aa3 and cytochrome bd are the only terminal oxidases present in C. glutamicum. However, some experimental results indicated the presence of an additional terminal oxidase activity whose molecular nature has not yet been elucidated: (i) The cyanide-resistant respiration of C. glutamicum measured by Trutko et al., 1982, Matsushita et al., 1998 was not accompanied by the appearance of cytochrome d in redox difference spectra. (ii) Kusumoto et al. (2000)

F1F0-ATP synthase

F1F0-ATP synthase is essential for ATP generation by oxidative phosphorylation. The genes encoding the eight subunits of this enzyme are organized as in most other bacteria in the atpIBEFHAGDC operon (Cgl1205–1213), which includes the atpI gene of unknown function. The promoter-proximal genes atpB, atpE and atpF encode the three components of the membrane-integral F0 part, i.e. the a-, c- and b-subunit, respectively. The promoter-distal genes atpH, atpA, atpG, atpD and atpC encode the five

Bioenergetic considerations

According to our present knowledge, the C. glutamicum respiratory chain using oxygen as terminal electron acceptor involves three enzymes which couple electron transfer to the generation of an electrochemical proton gradient across the cytoplasmic membrane, i.e. the cytochrome bc1 complex, cytochrome aa3 oxidase and cytochrome bd oxidase. According to generally accepted values (Nicholls and Ferguson, 1992), the number of protons formally transported across the membrane per electron (H+/e) is

MK biosynthesis

MK was identified as the only isoprenoid quinone present in C. glutamicum, with dihydromenaquinone-8 (i.e. MK containing eight isoprene units with one double bond hydrogenated) and MK-9 as minor components and dihydromenaquinone-9 as major component (Kanzaki et al., 1974, Collins et al., 1977, Collins et al., 1979). Genome analysis led to the identification of all genes required for the synthesis of MK from chorismate, an intermediate in the biosynthesis of aromatic amino acids (Fig. 6). These

Heme biosynthesis and cytochrome c maturation

Heme biosynthesis in C. glutamicum (Fig. 7) occurs via the C5 pathway that uses glutamate as the substrate for the synthesis of δ-aminolevulinic acid. The genes for heme biosynthesis (reviewed in O'Brian and Thöny-Meyer, 2002) in C. glutamicum are arranged in one large cluster (Cgl0429–0442, Fig. 8) except for the hemA, hemC, hemN and hemH genes encoding glutamyl-tRNA reductase (EC 1.2.1.-, Cgl0417), porphobilinogen deaminase (EC 4.3.1.8, Cgl0418), coproporphyrinogen III dehydrogenase (EC

Biotechnological aspects

C. glutamicum is an aerobic respiratory organism and thus synthesizes most of its ATP by oxidative phosphorylation. As shown by the effects of copper (Kanzaki et al., 1973, Sugiyama et al., 1973b), cyanide (Trutko et al., 1982) or a defective F1F0-ATP synthase (Sekine et al., 2001), interferences with the system of oxidative phosphorylation can have a strong influence on metabolism in general and on amino acid production in particular. Further support for this finding comes from a number of

Concluding remarks

The recent experimental studies in several laboratories and the information obtained by the genome sequence have established a good picture of the aerobic respiratory chain of C. glutamicum. It involves several dehydrogenases, MK, a cytochrome bc1aa3 supercomplex and cytochrome bd menaquinol oxidase. Cytochrome c1 represents the only c-type cytochrome of C. glutamicum and is unusual in that it contains two covalently bound heme groups, both of which are essential for assembly, stability and

Acknowledgements

Support of the studies on Corynebacterium glutamicum in the laboratory of M.B. by the EU project “VALPAN”, the BMBF “Genomik” program (Genome research on bacteria relevant for agriculture, environment and biotechnology; cluster IV: Corynebacteria), the BMBF “Proteomics” program (New methods for proteome analysis: application and combination with metabolome analysis using Corynebacterium glutamicum as an example), the Degussa AG, and the DFG-Graduiertenkolleg “Molekulare Physiologie: Stoff- und

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