There has been a surge of interest in acetone-butanol-ethanol fermentations of Clostridium acetobutylicum due to its capacity to ferment many carbohydrates found in biomass. This metabolic diversity makes it a promising candidate for conversion of inexpensive, heterogeneous carbohydrate feedstocks to biofuels. Galactose is present in many such feedstocks due to its incorporation in plant cell walls. C. acetobutylicum encodes two galactose utilization pathways, the Leloir (LP) and the tagatose-6-P (T6P), and a previous study indicated genes for these pathways was differentially regulated during growth on galactose and lactose. In the current study we utilized quantitative PCR to further investigate gene expression levels and to show both pathways which were subject to carbon catabolite repression. During growth on galactose, mRNA for galactose-6-P isomerase from the T6P was induced to a greater extent than mRNA for glactokinase, the first enzyme in the LP. The galactose-6-P isomerase mRNAs were also more abundant than galactokinase mRNAs during growth on galactose. Analysis of theoretical ATP requirements to generate essential precursor metabolites indicated: 1) the LP is more efficient at generating upper glycolytic intermediates, 2) the T6P is more efficient at forming ATP, lower glycolytic intermediates and TCA cycle intermediates, 3) a combination of the two pathways is most efficient for forming precursor metabolites found in the pentose phosphate pathway. From this it can be suggested that the two pathways have different roles in the organism with the T6P generating most ATP and precursor metabolites and the LP providing upper glycolytic metabolites.
References
[1]
T. Lutke-Eversloh and H. Bahl, “Metabolic Engineering of Clostridium acetobutylicum: Recent Advances to Improve Butanol Production,” Current Opinion in Biotechnology, Vol. 22, No. 5, 2011, pp. 634-647. http://dx.doi.org/10.1016/j.copbio.2011.01.011
[2]
P. S. Nigam and A. Singh, “Production of Liquid Biofuels from Renewable Resources,” Progress in Energy and Combustion Science, Vol. 37, No. 1, 2011, pp. 52-68. http://dx.doi.org/10.1016/j.pecs.2010.01.003
[3]
J. Nolling, G. Breton, M. V. Omelchenko, K. S. Makarova, Q. D. Zeng, R. Gibson, H. M. Lee, J. Dubois, D. Y. Qiu, J. Hitti, Y. I. Wolf, R. L. Tatusov, F. Sabathe, L. Doucette-Stamm, P. Soucaille, M. J. Daly, G. N. Bennett, E. V. Koonin, D. R. Smith and G. S. C. P. Finishing, “Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum,” Journal of Bacteriology, Vol. 183, No. 16, 2001, pp. 4823-4838. http://dx.doi.org/10.1128/JB.183.16.4823-4838.2001
[4]
Y. Yu, M. Tangney, H. C. Aass and W. J. Mitchell, “Analysis of the Mechanism and Regulation of Lactose Transport and Metabolism in Clostridium acetobutylicum ATCC 824,” Applied and Environmental Microbiology, Vol. 73, No. 6, 2007, pp. 1842-1850. http://dx.doi.org/10.1128/AEM.02082-06
[5]
L. Zeng, S. Das and R. A. Burne, “Utilization of Lactose and Galactose by Streptococcus mutans: Transport, Toxicity, and Carbon Catabolite Repression,” Journal of Bacteriology, Vol. 192, No. 9, 2010, pp. 2434-2444. http://dx.doi.org/10.1128/JB.01624-09
[6]
C. E. Price, A. Zeyniyev, O. P. Kuipers and J. Kok, “From Meadows to Milk to Mucosa—Adaptation of Streptococcus and Lactococcus Species to Their Nutritional Environments,” FEMS Microbiology Reviews, Vol. 36, No. 5, 2012, pp. 949-971.
[7]
D. L. Bissett and R. L. Anderson, “Lactose and D-Ga-lactose Metabolism in Group N Streptococci: Presence of Enzymes for Both the D-Galactose 1-Phosphate and D-Tagatose 6-Phosphate Pathways,” Journal of Bacteriology, Vol. 117, No. 1, 1974, pp. 318-320.
[8]
A. R. Neves, W. A. Pool, J. Kok, O. P. Kuipers and H. Santos, “Overview on Sugar Metabolism and Its Control in Lactococcus lactis—The Input from in Vivo NMR,” FEMS Microbiology Reviews, Vol. 29, No. 3, 2005, pp. 531-554.
[9]
D. L. Bissett and R. L. Anderson, “Genetic Evidence for the Physiological Significance of the D-Tagatose 6-Phosphate Pathway of Lactose and D-Galactose Degradation in Staphylococcus aureus,” Journal of Bacteriology, Vol. 119, No. 3, 1974, pp. 698-704.
[10]
K. Bettenbrock and C. A. Alpert, “The Gal Genes for the Leloir Pathway of Lactobacillus casei 64H,” Applied and Environmental Microbiology, Vol. 64, No. 6, 1998, pp. 2013-2019.
[11]
B. M. Chassy and J. Thompson, “Regulation and Characterization of the Galactose-Phosphoenolpyruvate-Dependent Phosphotransferase System in Lactobacillus casei,” Journal of Bacteriology, Vol. 154, No. 3, 1983, pp. 1204-1214.
[12]
H. M. Holden, I. Rayment and J. B. Thoden, “Structure and Function of Enzymes of the Leloir Pathway for Ga-lactose Metabolism,” Journal of Biological Chemistry, Vol. 278, No. 45, 2003, pp. 43885-43888. http://dx.doi.org/10.1074/jbc.R300025200
[13]
N. A. Gutierrez and I. S. Maddox, “Galactose Transport in Clostridium acetobutylicum P262,” Letters in Applied Microbiology, Vol. 23, No. 2, 1996, pp. 97-100. http://dx.doi.org/10.1111/j.1472-765X.1996.tb00039.x
[14]
M. D. Servinsky, J. T. Kiel, N. F. Dupuy and C. J. Sund, “Transcriptional Analysis of Differential Carbohydrate Utilization by Clostridium acetobutylicum,” Microbiology (SGM), Vol. 156, No. 2010, pp. 3478-3491.
[15]
D. P. Wiesenborn, F. B. Rudolph and E. T. Papoutsakis, “Thiolase from Clostridium-Acetobutylicum Atcc-824 and Its Role in the Synthesis of Acids and Solvents,” Applied and Environmental Microbiology, Vol. 54, No. 11, 1988, pp. 2717-2722.
[16]
S. Rosen and H. Skaletsky, “Primer3 on the WWW for General Users and for Biologist Programmers,” Bioinformatics Methods and Protocols: Methods in Molecular Biology, Vol. 132, 2000, pp. 365-386.
[17]
M. W. Pfaffl, “A New Mathematical Model for Relative Quantification in Real-Time RT-PCR,” Nucleic Acids Research, Vol. 29, No. 9, 2001, p. e45. http://dx.doi.org/10.1093/nar/29.9.e45
[18]
J. Nolling, G. Breton, M. V. Omelchenko, K. S. Makarova, Q. Zeng, R. Gibson, H. M. Lee, J. Dubois, D. Qiu, J. Hitti, Y. I. Wolf, R. L. Tatusov, F. Sabathe, L. Doucette-Stamm, P. Soucaille, M. J. Daly, G. N. Bennett, E. V. Koonin and D. R. Smith, “Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum,” Journal of Bacteriology, Vol. 183, No. 16, 2001, pp. 4823-4838. http://dx.doi.org/10.1128/JB.183.16.4823-4838.2001
[19]
D. A. Rodionov, A. A. Mironov and M. S. Gelfand, “Transcriptional Regulation of Pentose Utilisation Systems in the Bacillus/Clostridium Group of Bacteria,” FEMS Microbiology Letters, Vol. 205, No. 2, 2001, pp. 305-314. http://dx.doi.org/10.1111/j.1574-6968.2001.tb10965.x
[20]
M. Tangney, A. Galinier, J. Deutscher and W. J. Mitchell, “Analysis of the Elements of Catabolite Repression in Clostridium acetobutylicum ATCC 824,” Journal of Molecular Microbiology and Biotechnology, Vol. 6, No. 1, 2003, pp. 6-11. http://dx.doi.org/10.1159/000073403
[21]
C. Grimmler, C. Held, W. Liebl and A. Ehrenreich, “Transcriptional Analysis of Catabolite Repression in Clostridium acetobutylicum Growing on Mixtures of D-Glucose and D-Xylose,” Journal of Biotechnology, Vol. 150, No. 3, 2010, pp. 315-323. http://dx.doi.org/10.1016/j.jbiotec.2010.09.938
[22]
C. Ren, Y. Gu, S. Hu, Y. Wu, P. Wang, Y. Yang, C. Yang, S. Yang and W. Jiang, “Identification and Inactivation of Pleiotropic Regulator CcpA to Eliminate Glucose Repression of Xylose Utilization in Clostridium acetobutylicum,” Metabolic Engineering, Vol. 12, No. 5, 2010, pp. 446-454. http://dx.doi.org/10.1016/j.ymben.2010.05.002
[23]
C. Ren, Y. Gu, Y. Wu, W. Zhang, C. Yang, S. Yang and W. Jiang, “Pleiotropic Functions of Catabolite Control Protein CcpA in Butanol-Producing Clostridium acetobutylicum,” BMC Genomics, Vol. 13, 2012, p. 349. http://dx.doi.org/10.1186/1471-2164-13-349
[24]
G. L. Lorca, Y. J. Chung, R. D. Barabote, W. Weyler, C. H. Schilling and M. H. Saier Jr., “Catabolite Repression and Activation in Bacillus subtilis: Dependency on CcpA, HPr, and HprK,” Journal of Bacteriology, Vol. 187, No. 22, 2005, pp. 7826-7839. http://dx.doi.org/10.1128/JB.187.22.7826-7839.2005
[25]
E. Noor, E. Eden, R. Milo and U. Alon, “Central Carbon Metabolism as a Minimal Biochemical Walk between Precursors for Biomass and Energy,” Molecular Cell, Vol. 39, No. 5, 2010, pp. 809-820. http://dx.doi.org/10.1016/j.molcel.2010.08.031
[26]
H. Grupe and G. Gottschalk, “Physiological Events in Clostridium acetobutylicum during the Shift from Acidogenesis to Solventogenesis in Continuous Culture and Presentation of a Model for Shift Induction,” Applied and Environmental Microbiology, Vol. 58, No. 12, 1992, pp. 3896-3902.
[27]
Q. Al-Awqati, “Protontranslocating ATPases,” Annual Review of Cell Biology, Vol. 2, 1986, pp. 179-199. http://dx.doi.org/10.1146/annurev.cb.02.110186.001143
[28]
S. C. Zeeman, J. Kossmann and A. M. Smith, “Starch: Its Metabolism, Evolution, and Biotechnological Modification in Plants,” Annual Review of Plant Biology, Vol. 61, 2010, pp. 209-234. http://dx.doi.org/10.1146/annurev-arplant-042809-112301
[29]
D. Segura, R. Mahadevan, K. Juarez and D. R. Lovley, “Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter Sulfurreducens,” Plos Computational Biology, Vol. 4, No. 2, 2008, p. e36. http://dx.doi.org/10.1371/journal.pcbi.0040036
[30]
M. Wildermuth, “Genome-Wide Analysis of Bacterial Metabolic Pathways,” Genome Biology, Vol. 1, No. 1, 2000, Reports 016.
[31]
J. Edwards and B. Palsson, “Systems Properties of the Haemophilus influenzae Rd Metabolic Genotype,” Journal of Biological Chemistry, Vol. 274, No. 25, 1999, pp. 17410-17416. http://dx.doi.org/10.1074/jbc.274.25.17410
