Abstract
Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising cellulose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H2 and CO2. Very little is known about the factors that determine metabolic fluxes influencing H2 synthesis in anaerobic, cellulolytic bacteria like C. thermocellum. We have begun to investigate the relationships between genome content, gene expression, and end-product synthesis in C. thermocellum cultured under different conditions. Using bioinformatics tools and the complete C. thermocellum 27405 genome sequence, we identified genes encoding key enzymes in pyruvate catabolism and H2-synthesis pathways, and have confirmed transcription of these genes throughout growth on α-cellulose by reverse transcriptase polymerase chain reaction. Bioinformatic analyses revealed two putative lactate dehydrogenases, one pyruvate formate lyase, four pyruvate:formate lyase activating enzymes, and at least three putative pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as “Energy-converting Hydrogenases”, or via NAD(P)Hdependent Fe-only hydrogenases which would permit H2 production from NADH generated during the glyceraldehyde-3-phosphate dehydrogenase reaction. Furthermore, our findings show the presence of a gene cluster putatively encoding a membrane integral NADH:Fd oxidoreductase, suggesting a possible mechanism in which electrons could be transferred between NADH and ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H2 synthesis is the first step in developing strategies to increase hydrogen yields from biomass. Our studies have outlined the likely pathways leading to hydrogen synthesis in C. thermocellum strain 27405, but the actual functional roles of these gene products during pyruvate catabolism and in H 2 synthesis remain to be elucidated, and will need to be confirmed using both expression analysis and protein characterization.
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References
Lamed R and Zeikus G (1980) Ethanol production by thermophilic bacteria: Relationship between fermentation product yields of and catabolic enzyme activities in Clostridium thermocellum and Thermoanerobium brockii. J Bacteriol 144:569–578
Lynd LR and Grethlein HG (1987) Hydrolysis of dilute acid pretreated hardwood and purified microcyrstalline cellulose by cell-free broth from Clostridium thermocellum. Biotechnol Bioeng 29:92–100
Ng TK, Weimer PJ and Zeikus JG (1977) Cellulolytic and physiological properties of Clostridium thermocellum. Arch Microbiol 114:1–7
Patni NJ and Alexander JK (1971a) Catabolism of fructose and mannitol by Clostridium thermocellum: Presence of phosphoenolpyruvate:fructose phosphotransferase, fructose-1-phosphate kinase, phosphoenol-pyruvate:mannitol phosphotransferase, and mannitol-1-phosphate dehydrogenase in cell extracts. J Bacteriol 105:226–231
Patni NJ and Alexander JK (1971b) Utilization of glucose by Clostridium thermocellum: Presence of glucokinase and other glycolytic enzymes in cell extracts. J Bacteriol 105:220–225
Thauer RK, Jungermann KA and Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180
Sparling R, Islam R, Cicek N, Carere C, Chow H and Levin DB (2006) Formate synthesis by Clostridium thermocellum during anaerobic fermentation. Can J Microbiol 52:681–688
Demain AL, Newcomb M and Wu JHD (2005) Cellulase, Clostridia, and ethanol. Microbiol Mol Biol Rev 69:124–154
Lynd LR, Weimer PJ, van Zyl WH and Pretorius IS (2002) Microbial cellulose utilization: Fundamentals and biotechnology. Micro Mol Biol Rev 66:506–577
Lynd LR, Grethlein HG and Wolkin RH (1989) Fermentation of cellulose substrates in batch and continuous culture by Clostridium thermocellum. App Environ Microbiol 55:3131–3139
Islam R, Cicek N, Sparling R and Levin DB (2006) Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405. Appl Microbiol Biotechnol 72(3):576–583
Levin DB, Sparling R, Islam R and Cicek N (2006) Hydrogen production by Clostridium thermocellum 27405 from cellulosic biomass substrates. Int J Hydrogen Energy 31(11):1496–1503
Charon MH, Volbeda A, Chabriére E, Pieulle L and Fontecilla-Camps JC (1999) Structure and electron transfer mechanism of pyruvate:ferredodin oxidoreductase. Curr Opin Struct Biol 9:663–669
Hallenbeck PC and Benemann JR (2002) Biological hydrogen production; fundamentals and limiting processes. Int J Hydrogen Energy 27:1185–1193
Hallenbeck PC (2005) Fundamentals of the fermentative production of hydrogen. Water Sci Technol 52:21–29
Sauter M and Sawers G (1990) Transcriptional analysis of the gene encoding Pyruvate formate lyase activating enzyme of Escherichia coli. Mol Microbiol 4:355–363
Bradford MM (1976) A rapid and sensitive method for the estimation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Sirko A, Zehelein E, Freundlich M and Sawers G (1993) Integration host factor is required for anaerobic pyruvate induction of pfl operon expression in Escherichia coli. J Bacteriol 175 :5769–5
Özkan M, Ylmaz E, Lynd LR and Özcengiz G (2004) Cloning and Expression of the Clostridium thermocellum L-lactate Dehydrogenase in Escherichia coli and Enzyme Characterization. Can J Microbiol 50:845–851
Weidner G and Sawers G (1996) Molecular characterization of the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum. J Bacteriol 178:2440–2444
Meinecke B, Bertram J and Gottschalk G (1989) Purification and characterization of the pyruvate-ferredoxin oxidoreductase of Clostridium acetobutylicum. Arch Microbiol 152:244–250
Desai SG, Steven DM, Prince HL, Guerinot ML, Lynd LH (1999) Clostridium thermocellum hydrogenase 1. GenBank accession # Q9XC55. Direct Submission
Soboh B, Linder D and Hedderich R (2004) A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis. Microbiology 150:2451–2463
Vanoni MA, Verzotti E, Zanetti G and Curti B (1996) Properties of the recombinant b subunit of glutamate synthase. European J Biochem 236:937–946
Forzi L, Koch J, Guss AM, Radosevich CG, Metcalf W and Hedderich R (2005) Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants. FEBS Journal 272:4741–4753
Bruggemann H, Baumer S, Fricke WF, Wiezer A, Liesegang H, Decker I, Herzberg C, Martinez-Arias R, Merkl R, Henne A and Gottschalk G (2003) The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci USA 100:1316–1321
Guedon E, Payot S, Desvaux M and Petitdemanger H (1999) Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium. J Bacteriol 181:3262–3269
Dabrock B, Bahl H and Gottschalk G (1992) Parameters affecting solvent production in Clostridium pasteurianum. Appl Environ Microbiol 58:1233–1239
Viles F and Silverman L (1949) Determination of starch and cellulose. Anal Chem 21:950–953
Thauer RK, Kirchniawy FH and Jungermann KA (1972) Properties and function of the pyruvate-formate-lyase reaction in clostridiae. Eur J Biochem 23:282–290
Vasconcelos I, Girbal L and Soucaille P (1994) Regulation of carbon and electron flow in Clostridium acetobutyliticum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol. J Bacteriol 176(5):1443–1450
Kletzin A and Adams MWW (1996) Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosis and pyruvate ferredoxin oxidoreductase from Thermotoga maritime. J Bacteriol 178:248–257
Kunow J, Linder D and Thauer RK (1995) Pyruvate:ferredoxin oxidoreductase from sulfate reducing Archaeoglubus fulgidis: molecular composition, catalytic properties and sequence alignments. Arch Microbiol 63:21–28
Hughes NJ, Chalk PA, Clayton CL and Kelly DJ (1995) Identification of carboxylation enzymes and characterization of a novel four-subunit Pyruvate:Flavodoxin Oxidoreductase from Helicobacter pylori. J Bacteriol 177(14):3953–3959
Peters JW, Lanzilotta WN, Lemon BJ and Seefeldt LC (1998) X-ray crystal structure of the Fe-Only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 Angstrom resolution. Science, 282:1853–1858
Vignais PM, Billoud B and Meyer J (2001) Classification and phylogeny of hydrogenases. FEMS Microbiol Reviews 25:455–501
Jungermann K, Thauer RK, Leimenstoll G and Decker K (1973) Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia. Biochimica et Biophysica Acta — Bioenergetics, 305:268–280
Chen YP and Yoch DC (1989) Isolation, characterization and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b. J Bacteriol 171:5012–5016
Nakamura Y, Kaneko T, Sato S, Ikeuchi M, Katoh H, Sasamoto S, Watanabe A, Iriguchi M, Kawashima K, Kimura T, Kishida Y, Kiyokawa C, Kohara M, Matsumoto M, Matsuno A, Nakazaki N, Shimpo S, Sugimoto M, Takeuchi C, Yamada M and Tabata S (2002) Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. DNA Res 9(4):123–130
Desai SG, Guerinot ML and Lynd LR (2004) Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485. Appl Microbiol Biotechnol 65(5):600–605
Nolling J, Breton G, Omelchenko MV, Markarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, Wolf YI, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV and Smith DR (2001) Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183(16):4823–4838
Myers GS, Rasko DA, Cheung JK, Ravel J, Seshadri R, De-Boy RT, Ren Q, Varga J, Awad MM, Brinkac LM, Daugherty SC, Haft DH, Dodson RJ, Madupu R, Nelson WC, Rosovitz MJ, Sullivan SA, Khouri H, Dimitrov GI, Watkins KL, Mulligan S, Benton J, Radune D, Fisher DJ, Atkins HS, Hiscox T, Jost BH, Billington SJ, Songer JG, McClane BA, Titball RW, Rood JI, Melville SB and Paulsen IT (2006) Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16(8):1031–1040
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Carere, C.R., Kalia, V., Sparling, R. et al. Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405. Indian J Microbiol 48, 252–266 (2008). https://doi.org/10.1007/s12088-008-0036-z
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DOI: https://doi.org/10.1007/s12088-008-0036-z