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Applied and Environmental Microbiology, February 2007, p. 861-868, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01818-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Armin Kabus, Axel Niebisch, and Michael Bott^*

Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany

Received 2 August 2006/ Accepted 19 November 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa₃ and cytochrome bd. Cytochrome aa₃ forms a supercomplex with the cytochrome bc₁ complex, which contains an unusual diheme cytochrome c₁. Both the bc₁-aa₃ supercomplex and cytochrome bd transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome bd for growth and lysine production. When cultivated in glucose minimal medium, a cydAB deletion mutant of C. glutamicum ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome bd oxidase in ATCC 13032 led to a reduction of the growth rate by 45% and of the maximal biomass by 35%, presumably as a consequence of increased electron flow through the inefficient cytochrome bd oxidase. In the L-lysine-producing C. glutamicum strain MH20-22B, deletion of the cydAB genes had only minor effects on growth rate and biomass formation, but lysine production was increased by 12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Corynebacterium glutamicum is an aerobic gram-positive soil bacterium (8) that is used for the large-scale biotechnological production of amino acids, in particular L-glutamate (17) and L-lysine (12, 16). Several studies have indicated the importance of proper oxygenation conditions under manufacturing operations to attain high amino acid yields (2, 29, 38). In addition, overexpression of the glbO gene encoding a hemoglobin-like protein was shown to improve lysine synthesis (21). Biochemical and genetic studies revealed that C. glutamicum possesses a branched respiratory chain with two terminal oxidases (for review see references 3 and 4). The reducing equivalents formed by the oxidation of substrates are initially transferred to menaquinone by several dehydrogenases, including a type II NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, and pyruvate:quinone oxidoreductase. Reoxidation of menaquinol is catalyzed either by the cytochrome bc₁ complex, which passes the electrons to the terminal oxidase cytochrome aa₃, or by cytochrome bd oxidase (Fig. 1). The cytochrome bc₁ complex is encoded by the qcrCAB genes encoding cytochrome c₁, Rieske iron-sulfur protein, and cytochrome b, respectively (22). Cytochrome c₁ is unusual in that it contains two covalently bound heme groups (22, 34) and represents the only c-type cytochrome in C. glutamicum. In accord with our suggestion that the second heme group of cytochrome c₁ takes over the function of a separate cytochrome c in electron transfer to cytochrome aa₃, we recently demonstrated that the bc₁ complex and cytochrome aa₃ oxidase form a supercomplex (23), which presumably allows a high rate of electron transfer between the two complexes. It was also shown that cytochrome aa₃ oxidase consists of four subunits encoded by the genes ctaD (subunit I), ctaC (subunit II), ctaE (subunit III), and ctaF (subunit IV) (23). The last three genes are located immediately upstream of qcrCAB, whereas ctaD is located separately 345 kb upstream of ctaC, as revealed by the genome sequence of C. glutamicum (13). The cytochrome bd oxidase consists of two subunits encoded by cydA (subunit I) and cydB (subunit II) (18), which are located upstream of cydD and cydC (Fig. 2). In Escherichia coli the last two genes are required for the formation of active cytochrome bd (11, 27) and encode an ABC transporter which was reported to catalyze the export of L-cysteine (25) and glutathione (26). Since C. glutamicum does not possess a proton- or sodium ion-pumping NADH dehydrogenase, only the cytochrome bc₁-aa₃ supercomplex and cytochrome bd oxidase couple electron transfer to the generation of an electrochemical proton gradient. Besides being required for ATP synthesis and various secondary transport processes, the electrochemical proton gradient appears to be required as a driving force for succinate dehydrogenase, which probably catalyzes a reversed electron transfer when oxidizing succinate to fumarate with menaquinone as electron acceptor (31).

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FIG. 1. Model of the branched respiratory chain of C. glutamicum. As indicated by the number of protons presumably translocated to the exterior per two electrons transferred to oxygen, the branch composed of the cytochrome bc1-aa3 supercomplex has a threefold-higher bioenergetic efficiency than the cytochrome bd branch. Since C. glutamicum does not possess a proton- or sodium ion-pumping NADH dehydrogenase, only the bc1-aa3 supercomplex and cytochrome bd oxidase couple electron transport to the generation of an electrochemical proton gradient.

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FIG. 2. Physical map of the C. glutamicum cydABDC gene cluster. The cydA and cydB genes encode subunit I and subunit II of cytochrome bd oxidase, respectively. The cydC and cydD genes encode an ABC transporter presumably required for the formation of active cytochrome bd oxidase. The sequence deleted in strains 13032cydAB and MH20-22BcydAB is indicated. The gray bars indicate the regions amplified for construction of the plasmid pK19mobsacB-cydAB.

Mutants of the C. glutamicum wild-type strain ATCC 13032 which are unable to synthesize or assemble the bc₁ complex or cytochrome aa₃ oxidase showed a severe growth defect on brain heart infusion agar plates as well as in glucose minimal medium, indicating that the bc₁-aa₃ branch of the respiratory chain is of major importance for aerobic respiration (22, 23). Cytochromes of the a, b, and c types are spectroscopically detectable under all growth conditions and in all growth phases tested hitherto. In this study, we analyzed the role of cytochrome bd oxidase for growth of C. glutamicum and lysine production. To this end, we deleted and overexpressed the cytochrome bd oxidase genes in C. glutamicum ATCC 13032 and its lysine-producing derivative MH20-22B.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and culture conditions.
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 (OD₆₀₀) 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. After overnight incubation, cells of the second preculture were harvested, washed three times with 0.9% (wt/vol) NaCl, and used for inoculation of either CGXII medium with 4% (wt/vol) glucose or CGX medium with 10% (wt/vol) glucose to an OD₆₀₀ of 1. Cultivations were done in baffled 500-ml Erlenmeyer flasks with 60 ml medium at 30°C and 150 rpm. E. coli DH5, which was used as a host for cloning, was cultivated in LB medium or on LB agar plates at 37°C. When appropriate, kanamycin was used at a concentration of 25 µg/ml (C. glutamicum) or 50 µg/ml (E. coli) and isopropyl-ß-D-thiogalactopyranoside (IPTG) was used at a concentration of 1 mM.

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TABLE 1. Strains of C. glutamicum and plasmids used in this study

DNA manipulations.
Enzymes used for DNA restriction, ligation, or dephosphorylation were obtained either from Roche Diagnostics (Mannheim, Germany) or from New England Biolabs (Frankfurt am Main, Germany). For Southern hybridization (35), 10 µg genomic DNA isolated as described previously (10) was completely digested with appropriate restriction enzymes, size fractionated on a 1% agarose gel, and transferred onto a nylon membrane (Hybond-N⁺; Amersham Pharmacia Biotech) by vacuum-supported diffusion. Labeling of the probes with digoxigenin (DIG), hybridization, washing, and detection were carried out with the DIG Chem-Link labeling and detection kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). For size determination, DIG-labeled DNA molecular weight markers II and III (Roche Diagnostics, Mannheim, Germany) were used. Plasmid DNA from E. coli or C. glutamicum was isolated with the QIAprep Spin Miniprep kit according to the manufacturer's instructions (QIAGEN, Hilden, Germany).

Construction of cydAB deletion mutants.
In-frame cydAB deletion mutants of C. glutamicum ATCC 13032 and MH20-22B were constructed as described previously (22). For this purpose, the cydA upstream region including the first six codons of cydA and the cydB downstream region including the last 12 codons of cydB were amplified with the Expand High Fidelity kit (Roche Diagnostics) using the primer pairs cydAB-1/cydAB-2 and cydAB-3/cydAB-4, respectively (Table 2). The resulting PCR products of 561 bp and 574 bp, respectively, were subsequently fused by crossover PCR to a product of 1,114 bp. After digestion with EcoRI and PstI, this fragment was cloned into pK19mobsacB (30) cut with the same restriction enzymes to yield pK19mobsacB-cydAB. DNA sequence analysis revealed that the cloned PCR product contained no unwanted mutations. Subsequently, plasmid pK19mobsacB-cydAB was transferred by electroporation (37) into the C. glutamicum strains ATCC 13032 and MH20-22B and the transformation mixture was plated on LBHIS agar (37) containing 25 µg kanamycin/ml. After selection for the first and second recombination events, genomic DNA of kanamycin-sensitive and sucrose-resistant clones was analyzed by PCR with the primers cydAB-for and cydAB-rev in order to distinguish between wild-type and cydAB clones. For Southern blot analysis of selected cydAB clones, the DIG-labeled 1.1-kb EcoRI/PstI insert of pK19mobsacB-cydAB was used as a probe.

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TABLE 2. Oligonucleotides used in this worka

Overproduction of the cytochrome bd oxidase.
In order to overproduce cytochrome bd oxidase, the cydAB genes or the cydABDC genes were cloned into the expression plasmid pEKEx2 (9), which contains an IPTG-inducible tac promoter. The cydAB genes including the cydA ribosome binding site were amplified by PCR using chromosomal C. glutamicum ATCC 13032 DNA as template and the primer pair cydAB-for/cydAB-rev. After digestion of the 2,587-bp PCR product with SacI and EcoRI, the fragment was ligated into the pEKEx2 vector cut with the same restriction enzymes, transferred into E. coli DH5, and plated on LB plates containing 50 µg kanamycin/ml. Plasmids from kanamycin-resistant clones were isolated and restricted with SacI and EcoRI, and one of the plasmids containing the cydAB fragment (pEKEx2-cydAB) was transferred into C. glutamicum ATCC 13032 and MH20-22B by electroporation. The same strategy was used for the construction of a cydABDC expression plasmid. In this case, primers cydAB-for and cydABDC-rev were used to amplify the 5,683-bp cydABDC gene cluster. The pEKEx2-cydABDC plasmid was transferred into C. glutamicum strains ATCC 13032 and MH20-22B.

Determination of glucose and L-lysine.
Glucose concentrations were determined enzymatically using hexokinase and glucose-6-phosphate dehydrogenase. Samples (1 ml) of the cultures were centrifuged for 10 min at 16,100 x g, and aliquots of the supernatant were directly used for glucose determination. NADH formation was measured spectrophotometrically at 340 nm in a microplate reader (ThermoMax; MWG, Ebersberg, Germany), and concentrations were calculated using an extinction coefficient () of 6.3 mM^–1 cm^–1. Alternatively, glucose concentrations were determined with an enzyme electrode (EBIO Compact; Eppendorf, Hamburg, Germany). Quantitative determination of L-lysine in supernatants was carried out by reversed-phase high-pressure liquid chromatography after precolumn derivatization with a mixture of o-phthaldialdehyde and ß-mercaptoethanol (Pierce Biotechnology Inc., Rockford, IL) according to reference 19 using an Agilent 1100 LC system (Agilent Technologies, Waldbronn, Germany) equipped with an octyldecylsilane Hypersil 120- x 4-mm column (CS Chromatographie Service GmbH, Langerwehe, Germany) with a 5-µm particle size and a guard cartridge (40 x 4 mm). Ornithine at a final concentration of 1 mM was used as an internal standard in each sample. Substances were eluted with a flow rate of 0.35 ml min^–1 within the first minute and 0.6 ml min^–1 for the following 15 min at 40°C with a gradient of 0.1 M sodium acetate, pH 7.2, and methanol. Fluorescence of the amino acid isoindole derivatives was measured after excitation at 230 nm at an emission wavelength of 450 nm.

Determination of growth parameters.
Growth was followed by measuring the OD₆₀₀ with an Ultrospec 500-Pro spectrophotometer (Amersham Biotech). The biomass concentration was calculated from OD₆₀₀ values using an experimentally determined correlation factor of 0.25 g (dry weight) of cells (cdw) liter^–1 for an OD₆₀₀ of 1.

Membrane isolation and determination of protein concentration.
For the isolation of cell membranes, 5 g (wet weight) cells from stationary phase was suspended in 7 ml 100 mM Tris-HCl buffer, pH 7.5, containing 1 mM of phenylmethylsulfonyl fluoride and disrupted by five passages at 207 MPa through a French press cell (SLM Aminco). Cell debris was removed by centrifugation at 8,000 x g for 15 min, and the supernatant was ultracentrifuged at 150,000 x g for 90 min. The pellet containing the membrane fraction was resuspended in 7 ml 10 mM Tris-HCl (pH 7.5), ultracentrifuged once more for 90 min at 150,000 x g, and resuspended in 1 to 2 ml of the same buffer. For redox difference spectroscopy, suspensions containing 6 mg membrane protein ml^–1 were used. Protein concentrations were determined with the Bradford protein assay (5) using bovine serum albumin as a standard.

Redox difference spectroscopy.
Dithionite-reduced-minus-ferricyanide-oxidized difference spectra of membranes and dithionite-reduced spectra of intact cells (oxidation of cytochromes in intact cells proved to be difficult) were recorded at room temperature using 5-mm-light-path cuvettes with a Jasco V560 spectrophotometer equipped with a silicon photodiode detector for turbid samples (7).

Oxygen consumption rates.
Oxygen consumption rates were measured with intact cells from stationary phase in a thermostatically controlled, magnetically stirred 2-ml chamber with a Clarke-type oxygen electrode (Rank Brothers, Cambridge, United Kingdom) at 25°C. The chamber was filled with 940 µl air-saturated 20 mM Tris-HCl buffer, pH 8.0, and 20 µl air-saturated 222 mM glucose solution. Oxygen consumption was followed with a chart recorder after adding 40 µl of a stock cell suspension in 20 mM Tris-HCl, pH 8.0, to give a final OD₆₀₀ of 5 in the chamber.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Influence of cytochrome bd oxidase absence on growth of C. glutamicum wild-type strain ATCC 13032.
In order to test the relevance of cytochrome bd oxidase for growth of the C. glutamicum wild-type strain ATCC 13032, a mutant lacking the cydAB genes for subunits I and II of cytochrome bd was constructed as described in Materials and Methods. For the deletion, codons 7 to 507 of cydA and codons 1 to 319 of cydB were replaced by a 21-bp sequence tag. The genomic structure of the mutant was verified by PCR (data not shown) and Southern blot analysis (Fig. 3). In the parental strain, two hybridizing PstI-EcoRI fragments of 1.7 kb and 4.6 kb were detected, whereas only a single hybridizing PstI-EcoRI fragment of 3.8 kb was present in the cydAB deletion mutant. Figure 4A shows a growth comparison of the parental strain and the 13032cydAB mutant in CGXII minimal medium with 4% (wt/vol) glucose. The absence of cytochrome bd oxidase had no influence during the exponential growth phase, and the mutant reached the same maximal growth rate (0.35 h^–1) as the wild type did. However, growth after the exponential phase was strongly inhibited by the cydAB deletion and the mutant reached only 60% of the maximal biomass of the wild type (10.6 versus 18.2 g cdw liter^–1). This result clearly indicates an important role for cytochrome bd oxidase under the prevailing culture conditions. Remarkably, a distinct cytochrome d-specific peak at 630 nm could be observed in the reduced spectrum of wild-type cells from stationary phase only after subtraction of the reduced spectrum of the cydAB mutant (data not shown), indicating that the concentration of cytochrome bd oxidase in the wild type is quite low. The observed growth defect of the cydAB mutant could be complemented, at least partially, by transformation with plasmid pEKEx2-cydAB. The resulting strain, when cultivated in glucose minimal medium in the presence of 1 mM IPTG, showed a maximal growth rate in the exponential phase similar to those of the wild type and the cydAB/pEKEx2 strain but reached a biomass concentration of 15.3 g cdw/liter after 28 h compared to 11.7 g cdw/liter of the cydAB/pEKEx2 strain and 17.6 g cdw/liter of the wild type (mean values of two independent experiments).

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FIG. 3. Southern blot analysis of genomic DNA from C. glutamicum ATCC 13032 (lane 1), MH20-22B (lane 2), 13032cydAB (lane 3), and MH20-22cydAB (lane 4). DNA restricted with EcoRI and PstI was separated by agarose gel electrophoresis, blotted onto a nylon membrane, denatured, and hybridized with the 1.1-kb DIG-labeled cydAB insert from pK19mobsacB-cydAB. St, DIG-labeled DNA standards II and III (Roche Diagnostics).

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FIG. 4. Influence of cytochrome bd oxidase absence (A) or overproduction (B) on growth of C. glutamicum. (A) Growth of C. glutamicum 13032 (filled squares) and 13032cydAB (open triangles) in CGXII medium with 4% (wt/vol) glucose. (B) Growth of C. glutamicum 13032/pEKEx2 (filled squares) and 13032/pEKEx2-cydABDC (open triangles) in CGXII medium with 4% (wt/vol) glucose, 25 µg/ml kanamycin, and 1 mM IPTG. The inset shows dithionite-reduced spectra of intact stationary-phase cells from 13032/pEKEx2 (gray line) and 13032/pEKEx2-cydABDC (black line). The shoulder at 552 nm and the peaks at 562 nm, 603 nm, and 630 nm, which are characteristic for cytochromes of the c, b, a, and d types, respectively, are indicated. Each growth curve shows the mean values of three cultures including standard deviations.

Influence of cytochrome bd oxidase overproduction on growth of C. glutamicum wild-type strain ATCC 13032.
In order to determine the effect of cytochrome bd oxidase overproduction on growth, the wild type was transformed with plasmid pEKEx2-cydABDC. As shown in the inset of Fig. 4B, the dithionite-reduced spectrum of stationary-phase cells of 13032/pEKEx-cydABDC grown in the presence of IPTG revealed a clear cytochrome d-specific peak at 630 nm and an increased cytochrome b-specific peak at 560 nm compared to the reference strain 13032/pEKEx2, indicating that cytochrome bd was present. The respiration rate of suspensions of stationary-phase cells supplied with 4.4 mM glucose was 107 ± 11 nmol O₂ consumed min^–1 mg protein^–1 for 13032/pEKEx2 and 140 ± 6 nmol O₂ consumed min^–1 mg protein^–1 for 13032/pEKEx2-cydABDC, indicating that the overproduced cytochrome bd oxidase was catalytically active. As shown in Fig. 4B, growth of 13032/pEKEx2-cydABDC in glucose minimal medium differed significantly from that of the reference strain. The maximal growth rate (0.19 h^–1) was reduced 2-fold and the maximal biomass (11.2 g liter^–1) by 1/3.

Influence of cytochrome bd oxidase absence on growth and lysine formation by C. glutamicum MH20-22B.
In contrast to wild-type cells, a significant part of the carbon flux in strain MH20-22B is directed towards L-lysine rather than towards biomass production (32). Therefore, it was of interest to analyze the effect of a cydAB deletion on growth of and lysine formation by this producer strain. A cydAB deletion mutant of MH20-22B was constructed as described above for ATCC 13032, and the successful deletion was confirmed again by Southern blot analysis (Fig. 3). As shown in Fig. 5, the growth behavior of MH20-22BcydAB did not differ significantly from that of MH20-22B. The cydAB mutant reached the same maximal growth rate (0.29 h^–1) and a similar maximal biomass concentration (10.8 g cdw liter^–1) as the parent strain did (10.5 g cdw liter^–1). Redox difference spectroscopy of membranes revealed a small but distinct cytochrome d-specific peak at 630 nm in MH20-22B cells that was absent in the cydAB deletion mutant (inset of Fig. 5A). The presence of cytochrome bd oxidase in MH20-22B was also confirmed by the observation that respiration of stationary-phase cells (33 ± 1 nmol O₂ consumed min^–1 mg protein^–1) was not inhibited by 10 mM cyanide, whereas that of MH20-22cydAB cells (27 ± 3 nmol O₂ consumed min^–1 mg protein^–1) was rapidly and completely inhibited (data not shown). The increased level of cytochrome bd oxidase in MH20-22B compared to its parent ATCC 13032 is likely due to increased expression of the cydAB genes (33) and appears to result from the mutagenesis and screening procedures applied during the development of this strain (32).

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FIG. 5. Influence of cytochrome bd deletion on growth, glucose consumption, and lysine production by C. glutamicum MH20-22B. (A and B) Two independent growth experiments with MH20-22B (filled squares) and MH20-22BcydAB (open triangles) in modified CGX minimal medium containing 10% glucose. (C and D) Glucose consumption and lysine formation (as lysine-HCl) of the four cultures shown in panels A and B, respectively.

Whereas the cydAB deletion in strain MH20-22B had no marked effect on growth and glucose consumption, it stimulated lysine synthesis. As shown in Fig. 5C and D, both the rate of lysine production and the lysine concentration were increased in MH20-22BcydAB. In four independent experiments, the final lysine titer was increased on average by 12%, the glucose-specific lysine yield by 14%, the biomass-specific lysine yield by 6%, and the space-time yield by 9% (Table 3).

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TABLE 3. Influence of a cydAB deletion in C. glutamicum MH20-22B on lysine production parametersa

Influence of cytochrome bd oxidase overproduction on growth and lysine formation by C. glutamicum MH20-22B.
As outlined in the Discussion, the increased lysine production by MH20-22BcydAB might be a consequence of a bioenergetically more efficient respiratory chain. Therefore, it was of interest to test whether an increased level of cytochrome bd oxidase has an effect on lysine production opposite from that of the cydAB deletion. In a first attempt, only the structural genes for cytochrome bd oxidase, cydA and cydB, were cloned into the expression plasmid pEKEx2 and transferred to MH20-22B. The resulting strain, MH20-22B/pEKEx2-cydAB, was cultivated in CGXII minimal medium with 4% (wt/vol) glucose. Neither the size of the cytochrome d peak nor biomass formation nor lysine production showed any differences compared to the reference strain MH20-22B/pEKEx2 (data not shown). This indicated that overexpression of the cydAB genes alone is not sufficient for increased synthesis of an active cytochrome bd oxidase. As it is known that synthesis of functional cytochrome bd oxidase requires the products of cydC (11) and cydD (27), we cloned the entire cydABDC gene cluster into the pEKEx2 plasmid and transferred it into MH20-22B. Dithionite-reduced spectra of stationary-phase cells revealed significantly higher levels of b- and d-type cytochromes in MH20-22B/pEKEx2-cydABDC than in the reference strain MH20-22B/pEKEx2, confirming the overproduction of cytochrome bd (Fig. 6A, inset). The oxygen consumption rates of suspensions from stationary-phase cells were found to be 37 ± 6 nmol O₂ consumed min^–1 mg protein^–1 for MH20-22B/pEKEx2 and 63 ± 21 nmol O₂ consumed min^–1 mg protein^–1 for MH20-22B/pEKEx2-cydABDC. The 1.7-fold-increased respiration rate clearly showed that overexpression of cydABDC led to an active cytochrome bd oxidase. When strains were cultivated in CGXII minimal medium with 4% (wt/vol) glucose (Fig. 6A), the growth rate (0.22 h^–1) and the maximal biomass (9.9 g cdw liter^–1) of MH20-22B/pEKEx2-cydABDC were 12% and 16% lower, respectively, than those of MH20-22B/pEKEx2 (0.25 h^–1 and 11.8 g cdw liter^–1, respectively). The lysine titer of MH20-22B/pEKEx2-cydABDC (7.1 g liter^–1) was 14% lower than that of MH20-22B/pEKEx2 (8.3 g liter^–1). Thus, overproduction of cytochrome bd oxidase had a negative impact on growth of and lysine production by MH20-22B (Fig. 6).

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FIG. 6. Influence of cytochrome bd overproduction on growth (A) and lysine production (B) by C. glutamicum MH20-22B. (A) Growth of C. glutamicum MH20-22B/pEKEx2 (filled squares) and MH20-22B/pEKEx2-cydABDC (open triangles) in CGXII medium with 4% (wt/vol) glucose, 25 µg ml–1 kanamycin, and 1 mM IPTG. The inset shows dithionite-reduced spectra of stationary-phase cells of MH20-22B/pEKEx2 (gray line) and MH20-22B/pEKEx2-cydABDC (black line). Peaks characteristic of a-, b-, c-, and d-type cytochromes are indicated. (B) L-Lysine formation by the cultures shown in panel A. Numbers given are mean values and standard deviations from three independent cultivations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The respiratory chain of C. glutamicum involves two branches for transfer of electrons to oxygen. One branch is formed by the cytochrome bc₁ complex and the terminal oxidase cytochrome aa₃ (23); the second branch is formed by cytochrome bd oxidase (18). We previously showed that mutants lacking either the bc₁ complex or cytochrome aa₃ had severe growth defects, suggesting that the bc₁-aa₃ supercomplex is of major importance for aerobic respiration (22, 23). In this study, we investigated the role of the second branch of the respiratory chain, cytochrome bd oxidase, by analyzing mutant strains which either lacked or overproduced this terminal oxidase. In the wild-type strain ATCC 13032, a cydAB deletion had no effect on growth in the exponential phase but inhibited growth thereafter, resulting in a strong reduction of the biomass formed (Fig. 4A). This shows that cytochrome bd oxidase is present and active in the wild type, although neither cells nor membranes of stationary-phase cells showed a distinct cytochrome d-specific peak in reduced or reduced-minus-oxidized spectra, respectively. The behavior of the 13032cydAB mutant could be due to low dissolved oxygen concentrations at the end of the exponential growth phase, which strongly restrict the activity of the low-affinity cytochrome aa₃ oxidase but not that of the high-affinity cytochrome bd oxidase. In Mycobacterium smegmatis, like C. glutamicum a member of the suborder Corynebacterineae (36), cytochrome bd oxidase was recently shown to be important for growth under microaerobic conditions and to be induced two- to threefold upon reduction of the O₂ partial pressure in the growth medium from 21% to 0.5% air saturation (14).

Whereas the absence of cytochrome bd had no influence on exponential-phase growth of C. glutamicum, constitutive overproduction of this terminal oxidase by the expression plasmid pEKEx2-cydABDC caused a twofold reduction of the growth rate and a 35% reduction in biomass formation. Although inhibitory effects due to the overproduction of four membrane proteins cannot be excluded, the more likely reason for the reduced growth is the difference in bioenergetic efficiency between the cytochrome bc₁-aa₃ supercomplex and cytochrome bd oxidase (6). As outlined previously (3), the former pathway should couple transport of two electrons from menaquinol to oxygen to the transfer of six protons from the cytoplasm to the outside, whereas the latter pathway can transfer only two protons to the outside per two electrons transported to oxygen (20, 28) (Fig. 1). This results in a threefold-lower P/O ratio of the bd branch than of the bc₁-aa₃ branch. Consequently, a shift of the electron flow from the bc₁-aa₃ branch to the bd branch, which is to be expected upon overproduction of an active cytochrome bd oxidase, might lead to a decreased rate of ATP synthesis by F₁F_o-ATP synthase, an increased amount of glucose required for catabolism and maintenance, and a decreased amount of glucose available for biomass formation. A rough calculation indicates that 13032/pEKEx2 uses 57% of the consumed glucose for energy generation and maintenance and 43% for biomass formation, whereas 13032/pEKEx2-cydABDC uses 72% of the glucose for catabolism and only 28% for anabolism. Assuming a Y_ATP value of 10 g (dry weight) cells/mol ATP for both strains, 13032/pEKEx2 forms 13.7 mol ATP/mol glucose, but 13032/pEKEx-cydABDC forms only 7 mol ATP/mol glucose.

Overall, the effects caused by the cydAB deletion and by cydABDC overexpression suggest that cytochrome bd functions as an emergency or rescue oxidase in C. glutamicum which permits respiration under conditions where cytochrome aa₃ activity is insufficient due to low dissolved oxygen concentrations or other reasons. The low concentration of cytochrome bd oxidase indicated by the redox spectra appears to be sufficient to fulfill this function and ensures that, whenever possible, the major portion of electrons is transferred to oxygen via the energetically favorable cytochrome bc₁-aa₃ supercomplex.

C. glutamicum MH20-22B uses a significant portion of the consumed glucose for lysine formation rather than for biomass formation. Deletion of cydAB in MH20-22B had only minor effects on growth rate, biomass formation, and glucose consumption, whereas the lysine synthesis rate and the final lysine titer were increased by 12%. Overproduction of cytochrome bd oxidase in MH20-22B led to reductions of the growth rate by 10%, of biomass production by 16 to 19%, and of lysine synthesis by 14%. As discussed above, these effects might be explained by a reduced P/O ratio due to an increased electron flux through the inefficient bd branch. The positive effect of cytochrome bd absence on lysine formation, on the other hand, can be explained by an increased P/O ratio in MH20-22BcydAB, which results in a reduced glucose demand for energy generation and maintenance and an increased availability of glucose for lysine synthesis. A comparable stimulation of lysine production was also observed for a cydAB deletion mutant of C. glutamicum DM1730 (obtained from Degussa AG), a "minimal mutation strain" (pyc-P458S, hom-V59A, lysC-T311I, zwf-A243T) that was derived from ATCC 13032 by "genome breeding" (24) (data not shown). The results show that modulation of the respiratory chain composition in favor of an increased bioenergetic efficiency (P/O ratio) can be used as a metabolic engineering strategy to improve the performance of microbial producer strains. The conclusion derived here for lysine-producing strains of C. glutamicum was previously also suggested by Zamboni et al. (39), who showed that deletion of cytochrome bd oxidase improved riboflavin production by Bacillus subtilis (39).

 
Financial support by R&D Feed Additives of Degussa AG is gratefully acknowledged.

 
* Corresponding author. Mailing address: Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany. Phone: 49 2461 615515. Fax: 49 2461 612710. E-mail: m.bott{at}fz-juelich.de.

Published ahead of print on 1 December 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, February 2007, p. 861-868, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01818-06
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