- Original article
- Open Access
Fixation of CO2 in Clostridium cellulovorans analyzed by 13C-isotopomer-based target metabolomics
© Shinohara et al.; licensee Springer. 2013
Received: 25 September 2013
Accepted: 5 October 2013
Published: 9 October 2013
Clostridium cellulovorans has been one of promising microorganisms to use biomass efficiently; however the basic metabolic pathways have not been completely known. We carried out 13C-isotopomer-based target metabolome analysis, or carbohydrate conversion process analysis, for more profound understanding of metabolic pathways of the bacterium. Our findings that pyruvate + oxaloacetate, fumarate, and malate inside and outside cells exhibited 13C incorporation suggest that C. cellulovorans exactly fixed CO2 and partly operated the TCA cycle in a reductive manner. Accompanied with CO2 fixation, the microorganism was also found to produce and secrete lactate. Overall, our study demonstrates that a part of C. cellulovorans metabolic pathways related to glycolysis and the TCA cycle are involved in CO2 fixation.
C. cellulovorans, an anaerobic mesophilic bacterium, can degrade and assimilate not only various kinds of carbohydrates (including cellulose, xylan, pectin, cellobiose, glucose, fructose, galactose, and mannose) and but also actual biomass (rice straw and corn waste) (Tamaru et al. 2010b), and whose whole genome was recently sequenced for the first time by our group (Tamaru et al. 2010a). This wide spectrum of degradation depends on extracellular multi-protein complexes called cellulosomes in several cellulosome-producing Clostridium species reported; however, most of the researches focus on the cellulosome itself. In order to use Clostridium species for practical applications, it is important to elucidate the basic biology of these bacteria, especially their metabolic processes that are highly associated with the conversion of carbohydrates to final products.
C. cellulovorans has been suggested to have a CO2 fixation pathway, because of its ability to grow under a higher concentration of ‘100%’ CO2 compared to other Clostridium species (an atmosphere of 20% CO2 (C. cellulovorans); 5% CO2 (C. acetobutylicum and C. kluyveri); 10% CO2 (C. thermocellum and C. difficile) (Sleat et al. 1984; Amador-Noguez et al. 2010; Waller et al. 2013; Saujet et al. 2011; Thauer et al. 1968). Previously, a few studies have characterized the metabolic pathway of C. kluyveri and C. acetobutylicum (Jungermann et al. 1970; Amador-Noguez et al. 2010). In the genome analysis of C. cellulovorans (Tamaru et al. 2010a), the genes of 2 important CO2 fixation enzymes, namely pyruvate:ferredoxin oxidoreductase (PFOR) and phosphoenolpyruvic acid (PEP) carboxylase (PEPC) were annotated. Notably, PFOR of glycolysis and PEPC of the TCA cycle are both in the node of main metabolic pathways in C. cellulovorans. Therefore, the study of CO2 fixation by metabolome analysis would help to clarify the complete metabolic pathway of C. cellulovorans. In particular, 13C-labeling studies of metabolic products are useful for understanding the in vivo metabolism since 13-carbon isotope can distinguish fluxes through different pathways when these fluxes result in different positional isotopic enrichments in metabolic intermediates (Ratcliffe and Shachar-Hill 2006; McKinlay et al. 2007).
Materials and methods
Cultivation conditions and growth rate analysis
C. cellulovorans 743B (ATCC 35296) was grown anaerobically at 37°C in an atmosphere of ‘100%’ CO2 unless otherwise noted. Liquid cultivation media contained the following reagents: 0.45 g/l KH2PO4 · H2O, 0.45 g/l K2HPO4, 0.9 g/l NaCl, 0.3675 g/l NH4Cl, 0.1575 g/l MgCl2 · 6H2O, 0.12 g/l CaCl2 · 2H2O, 5.2 mg/l Na2-EDTA, 1.5 mg/l FeCl2 · 4H2O, 0.942 mg/l CoCl2 · 6H2O, 0.85 mg/l MnCl2 · 4H2O, 0.07 mg/l ZnCl2 · 6H2O, 0.062 mg/l H3BO4, 0.036 mg/l Na2MoO4 · 2H2O, 0.024 mg/l NiCl2 · 6H2O, 0.017 mg/l CuCl2 · 6H2O, 5g/l NaHCO3, 4 g/l Bacto™ Yeast Extract (Becton and Dickinson Company), 3 g/l glucose, and 1 g/l L-cysteine. For labeling experiments, NaHCO3 and glucose were replaced by NaH13CO3 and [U-13C]-glucose, respectively (both 99% purity; Cambridge Isotope Laboratories, Andover, MA).
Quenching and extraction of intracellular metabolites
Quenching and metabolite extraction were carried out as previously described (Winder et al. 2008), with some modifications. In brief, culture broths were injected rapidly into 4 volumes of 60% aqueous methanol solution (−40°C) for quenching. Supernatants after centrifugation at 3000 × g at −9°C for 10 min for quenching were removed rapidly, and washed with 1 ml of 60% aqueous methanol (−40°C), followed by centrifugation at 3000 × g at −9°C for 10 min. Subsequently, supernatants were thoroughly removed, and cell pellets were frozen in liquid nitrogen and kept at −80°C until the following extraction procedures. Cell pellets were suspended in 500 μl of 100% methanol (−40°C), frozen in liquid nitrogen, and allowed to thaw on dry ice. After, the freeze-thaw cycle was performed 3 times in total, the suspensions were centrifuged at 16000 × g, at −9°C, for 5 min. Supernatants were retained and stored on dry ice, and another aliquot (500 μl) of 100% methanol (−40°C) was added to each pellet. The procedure was repeated twice, and the second aliquot of methanol was combined with the first one.
Target metabolites detected by GC/MS
Retention time (min)
Pyruvate + OAA
GC/MS analysis and data processing
Derivatized metabolites were analyzed using GCMS-QP2010 Ultra (Shimadzu, Kyoto, Japan) equipped with a 30 m × 0.25 mm i.d. fused silica capillary column coated with 0.25-μm CP-SIL 8 CB low bleed (Agilent Technologies, Santa Clara, CA). Aliquots (1 μl) were injected in the split mode (25/1, supernatant analysis; 5/1, intracellular analysis) at 230°C, using helium as carrier gas at a flow rate of 1.12 ml/min. The column temperature was held at 80°C for 2 min isothermally, raised to 130°C (4°C/min) and then to 330°C (25°C/min), and maintained for 6 min isothermally. The interface and MS source temperatures were 250°C and 200°C, respectively, and the ion voltage was 1 kV. Data were collected by GCMS solution (Shimadzu), and identified metabolites are shown in Table 1. Mass isotopomer distributions were corrected for natural isotope abundance as previously described (Nanchen et al. 2007). The GC/MS analysis was performed on 3 biological replicates of each sample.
CO2incorporation into C. cellulovorans metabolites
Glucose metabolism into metabolic pathway intermediates
Lactate secretion accompanied with CO2 fixation
Our findings also indicate that little citrate and succinate was produced from glucose (Figure 3). Isocitrate dehydrogenase, which operates downstream of citrate in the TCA cycle and operates in an oxidative manner with NAD(P)+, could not be used. It is known that citrate is produced from glutamate in some organisms. The metabolic information of C. acetobutylicum (Amador-Noguez et al. 2010) also suggested that C. cellulovorans could use amino acids (glutamate/glutamine) to make other metabolites. If C. cellulovorans produces citrate from glutamate, redox balance would be better maintained because glutamate dehydrogenase or glutamate synthase uses NADP+ and isocitrate dehydrogenase uses NADPH. In particular, when C. cellulovorans lives under a reductive condition, such a pathway is more reasonable than the pathway of citrate production from acetyl-CoA in an oxidative manner. To examine this hypothesis in the future investigation, it will be a promising approach to study how 13C atoms are incorporated into metabolites when using media containing 13C-labeled glutamate. Some other amino acids may be needed to maintain the metabolic pathway in C. cellulovorans, because the bacterium cannot be cultivated in media without yeast extract, which has glutamate (Sleat et al. 1984).
As mentioned above, we speculate that C. cellulovorans could use the mechanism to maintain redox balance, because the oxidizability (the ability to oxygenate other metabolites) is valuable for the condition which was absent from O2. C. cellulovorans lives under anaerobic conditions because photosynthesis is not operated under the natural growth condition of C. cellulovorans (wood chip). It has been reported that CO2 fixation is useful to maintain redox balance in microorganisms that have the Calvin cycle or TCA cycle (McKinlay and Harwood 2010); therefore CO2 fixation could be a common mechanism to regulate redox balance.
Notably, lactate production in cultivation supernatants after 4 days was 474 mg/l (= 31.6 mol per 100 mol glucose), which is about twice of that reported in the previous study (Sleat et al. 1984), where cellobiose was used as a carbon source.
Here, we demonstrated that, accompanied with CO2 fixation, C. cellulovorans produced several kinds of organic acids and that the TCA cycle was partly operated in a reductive manner at the metabolite level. These presented results would provide important information for the application of C. cellulovorans as industrial cellulosome-producing bacteria.
This research was supported by CREST and the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN).
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