Heterofermentative lactic acid bacterium Lactobacillus brevis may be considered as a promising host for heterologous butanol synthesis because of tolerance to butanol and ability to ferment pentose and hexose sugars from wood hydrolysates that are cheap and renewable carbohydrate source. Carbon and electron flow was evaluated in two L. brevis strains in order to assess metabolic potential of these bacteria for heterologous butanol synthesis. Conditions required for generation of acetyl-CoA and NADH which are necessary for butanol biosynthesis have been determined. Key enzymes controlling direction of metabolic fluxes in L. brevis in various redox conditions were defined. In anaerobic glucose fermentation, the carbon flow through acetyl-CoA is regulated by aldehyde dehydrogenase ALDH possessing low affinity to NADH and activity (KmNADH= 200 μM, Vmax= 0.03 U/mg of total cell protein). Aerobically, the NADH-oxidase NOX (KmNADH= 25 μM, Vmax = 1.7 U/mg) efficiently competes with ALDH for NADH that results in formation of acetate instead of acetyl-CoA. In general, external electron acceptors (oxygen, fructose) and pentoses decrease NADH availability for native ethanol and recombinant butanol enzymes and therefore reduce carbon flux through acetyl-CoA. Pyruvate metabolism was studied in order to reveal redirection possibilities of competitive carbon fluxes towards butanol synthesis. The study provides a basis for the rational development of L. brevis strains producing butanol from wood hydrolysate.
 C. Jin, M. Yao, H. Liu, F. Chia-fon, C. F. Lee and J. Ji, “Progress in the Production and Application of n-Butanol as a Biofuel,” Renewable and Sustainable Energy Reviews, Vol. 15, No. 8, 2011, pp. 4080-4106. doi:10.1016/j.rser.2011.06.001
 E. McCoy, E. B. Fred, W. H. Peterson and E. G. Hastings, “A Cultural Study of the Acetone Butyl Alcohol Organism,” The Journal of Infectious Diseases, Vol. 39, No. 6, 1926, pp. 457-483. doi:10.1093/infdis/39.6.457
 S. Atsumi, A. F. Cann, M. R. Connor, C. R. Shen, K. M. Smith, M. P. Brynildsen, K. J. Chou, T. Hanai and J. C. Liao, “Metabolic Engineering of Escherichia coli for 1-Butanol Production,” Metabolic Engineering, Vol. 10, No. 6, 2008, pp. 305-311. doi:10.1016/j.ymben.2007.08.003
 M. Inui, M. Suda, S. Kimura, K. Yasuda, H. Suzuki, H. Toda, S. Yamamoto, S. Okino, N. Suzuki and H. Yukawa, “Expression of Clostridium acetobutylicum Butanol Synthetic Genes in Escherichia coli,” Applied Microbiology and Biotechnology, Vol. 77, No. 6, 2008, pp. 1305-1316. doi:10.1007/s00253-007-1257-5
 E. J. Steen, R. Chan, N. Prasad, S. Myers, C. J. Petzold, A. Redding, M. Ouellet and J. D. Keasling, “Metabolic Engineering of Saccharomyces cerevisiae for the Production of n-Butanol,” Microbial Cell Factories, Vol. 3, 2008, pp. 7-36.
 D. R. Nielsen, E. Leonard, S.-H. Yoon, H.-C. Tseng, C. Yuan and K. L. J. Prather, “Engineering Alternative Butanol Production Platforms in Heterologous Bacteria,” Metabolic Engineering, Vol. 11, No. 4-5, 2009, pp. 262-273. doi:10.1016/j.ymben.2009.05.003
 O. V. Berezina, N. V. Zakharova, A. Brandt, S. V. Yarotsky, W. H. Schwarz and V. V. Zverlov, “Reconstructing the Clostridial n-Butanol Metabolic Pathway in Lactobacillus brevis,” Applied Microbiology and Biotechnology, Vol. 87, No. 2, 2010, pp. 635-646. doi:10.1007/s00253-010-2480-z
 B. B. Bond-Watts, R. J. Bellerose and M. C. Y Chang, “Enzyme Mechanism as a Kinetic Control Element for Designing Synthetic Biofuel Pathways,” Nature Chemical Biology, Vol. 7, No. 4, 2011, pp. 222-227. doi:10.1038/nchembio.537
 C. R. Shen, E. I. Lan, Y. Dekishima, A. Baez, K. M. Cho and J. C. Liao, “Driving Forces Enable High-titer Anaerobic 1-Butanol Synthesis in Escherichia coli,” Applied and Environmental Microbiology, Vol. 77, No. 14, 2011, pp. 2905-2915. doi:10.1128/AEM.03034-10
 M. Rakkolainen, M. Iakovlev, A.-L. Terasvuori, E. Sklavounos, G. Jurgens, T. B. Granstrom and A. van Heiningen, “SO2-Ethanol-Water Fractinations of Forest Biomass and Implications for Biofuel Production by ABE Fermentation,” Cellulose Chemistry and Technology, Vol. 44, No. 4-6, 2010, pp. 139-145.
 E. Sklavounos, M. Iakovlev, M. Rakkolainen, A. L. Terasvuori, G. Jurgens, T. Granstrom and A. van Heiningen, “Conditioning of SO2-Ethanol-Water Spent Liquor from Spruce for the Production of Chemicals by ABE Fermentation,” Holzforschung, Vol. 65, No. 4, 2011, pp. 551-558. doi:10.1515/hf.2011.103
 S. A. Survase, E. Sklavounos, G. Jurgens, A. van Heiningen and T. Granstrom, “Continuous Acetone-Butanol-Ethanol Fermentation Using SO2-Ethanol-Water Spent Liquor from Spruce,” Bioresource Technology, Vol. 102, No. 23, 2011, pp. 10996-11002. doi:10.1016/j.biortech.2011.09.034
 E. N. Miller, L. R. Jarboe, P. C. Turner, P. Pharkya, L. P. Yomano, S. W. York, D. Nunn, K. T. Shanmugam and L. O. Ingram, “Furfural Inhibits Growth by Limiting Sulfur Assimilation in Ethanologenic Escherichia coli Strain LY180,” Applied and Environmental Microbiology, Vol. 75, No. 19, 2009, pp. 6132-6141. doi:10.1128/AEM.01187-09
 E. Palmqvist and B. Hahn-Hagerdal, “Fermentation of Lignocellulosic Hydrolysates II: Inhibitors and Mechanisms of Inhibition,” Bioresource Technology, Vol. 74, No. 1, 2000, pp. 25-33. doi:10.1016/S0960-8524(99)00161-3
 O. Kandler and N. Weiss, “Regular, Nonsporing Gram-Positive Rods: Lactobacillus,” In: P. H. A. Sneath, N. Mair, M. E. Sharpe and J. G. Holt, Eds., Bergey’s Manual of Systematic Bacteriology, Williams & Wilkins, Baltimore, Vol. 2, 1986, pp. 1209-1234.
 O. V. Berezina, S. P. Sineoky, G. A. Velikodvorskaya, W. H. Schwarz and V. V. Zverlov, “Extracellular Glycosyl Hydrolase Activity of the Clostridium Strains Producing Acetone, Butanol, and Ethanol,” Applied Biochemistry and Microbiology, Vol. 44, 2008, pp. 42-47. doi:10.1134/S0003683808010079
 P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson and D. C. Klenk, “Measurement of Protein Using Bicinchoninic Acid,” Analytical Biochemistry, Vol. 150, No. 1, 1985, pp. 76-85. doi:10.1016/0003-2697(85)90442-7
 M. M. Bradford, “A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,” Analytical Biochemistry, Vol. 72, 1976, pp. 248-254. doi:10.1016/0003-2697(76)90527-3
 D. Kessler, I. Leibrecht and J. Knappe, “Pyruvate-Formate-Lyase-Deactivase and Acetyl-CoA Reductase Activities of Escherichia coli Reside on a Polymeric Protein Particle Encoded by adhE,” FEBS Letters, Vol. 281, No. 1-2, 1991, pp. 59-63. doi:10.1016/0014-5793(91)80358-A
 M. G. Hartmanis and S. Gatenbeck, “Intermediary Metabolism in Clostridium acetobutylicum: Levels of Enzymes Involved in the Formation of Acetate and Butyrate,” Applied and Environmental Microbiology, Vol. 47, No. 6, 1984, pp. 1277-1283.
 A. Molina-Gutierrez, V. Stippl, A. Delgado, M. G. Ganzle and R. F. Vogel, “In Situ Determination of the Intracellular pH of Lactococcus lactis and Lactobacillus plantarum During Pressure Treatment,” Applied and Environmental Microbiology, Vol. 68, No. 9, 2002, pp. 4399-4406. doi:10.1128/AEM.68.9.4399-4406.2002
 H. Yahui, L. Yan, H. Hongwu and Y. Junlin, “A New Optimized Spectrophotometric Assay for the Measurement of Pyruvate Dehydrogenase’s Activity,” The 1st International Conference on Bioinformatics and Biomedical Engineering, 2007.
 N. Asanuma, M. lwamoto and T. Hino, “Structure and Transcriptional Regulation of the Gene Encoding Pyruvate Formate-Lyase of a Ruminal Bacterium, Streptococcus bovis,” Microbiology, Vol. 145, No. 1, 1999, pp. 151-157. doi:10.1099/13500872-145-1-151
 H. Taniai, K. Iida, M. Seki, M. Saito, S. Shiota, H. Nakayama and S. Yoshida, “Concerted Action of Lactate Oxidase and Pyruvate Oxidase in Aerobic Growth of Streptococcus pneumoniae: Role of Lactate as an Energy Source,” Journal of Bacteriology, Vol. 190, No. 10, 2008, pp. 3572-3579. doi:10.1128/JB.01882-07
 T. C. Hoppner and H. W. Doelle, “Purification and Kinetic Characteristics of Pyruvate Decarboxylase and Ethanol Dehydrogenase from Zymomonas mobilis in Relation to Ethanol Production,”European Journal of Applied Microbiology and Biotechnology,” Vol. 17, No. 3, 1983, pp. 152-157. doi:10.1007/BF00505880
 W. de Koning and K. van Dam, “A Method for the Determination of Changes of Glycolytic Metabolites in Yeast on a Subsecond Time Scale Using Extraction at Neutral pH,” Analytical Biochemistry, Vol. 204, No. 1, 1992, pp. 118-123. doi:10.1016/0003-2697(92)90149-2
 L. Axelsson, “Lactic Acid Bacteria: Classification and Physiology,” In: S. Salminen and A. Wright, Eds., Lactic Acid Bacteria. Microbiology and Functional Aspects, 2nd Edition, Marcel Dekker Inc., New York, 1998, pp. 1-72.
 K. Makarova, A. Slesarev, Y. Wolf, A. Sorokin, et al., “Comparative Genomics of the Lactic Acid Bacteria,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 42, 2006, pp. 15611-15616. doi:10.1073/pnas.0607117103
 Y. Wang, K. J. Addess, J. Chen, L. Y. Geer, J. He, S. He, S. Lu, T. Madej, A. Marchler-Bauer, P. A. Thiessen, N. Zhang and S. H. Bryant, “MMDB: Annotating Protein Sequences with Entrez’s 3D-structure Database,” Nucleic Acids Research, Vol. 35, 2007, pp. 205-210. doi:10.1093/nar/gkl952
 A. Jansch, S. Freiding, J. Behr and R. F. Vogel, “Contribution of the NADH-Oxidase (Nox) to the Aerobic Life of Lactobacillus sanfranciscensis DSM20451T,” Food Microbiology, Vol. 28, No. 1, 2011, pp. 29-37. doi:10.1016/j.fm.2010.08.001
 H. Richter, I. Hamann and G. Unden, “Use of the Mannitol Pathway in Fructose Fermentation of Oenococcus oeni due to Limiting Redox Regeneration Capacity of the Ethanol Pathway,” Archives of Microbiology, Vol. 179, No. 4, 2003, pp. 227-233.
 I. L. Benito de Cardenas, O. V. Ledesma, A. A. Pesce de Ruiz Holgado and G. Oliver, “Effect of Lactate on the Growth and Production of Diacetyl and Acetoin by Lactobacilli,” Journal of Dairy Science, Vol. 68, No. 8, 1985, pp. 1897-1901. doi:10.3168/jds.S0022-0302(85)81047-X
 M. G. Murphy, L. O’Connor, D. Walsh and S. Condon, “Oxygen Dependent Lactate Utilization by Lactobacillus plantarum,” Archives of Microbiology, Vol. 141, No. 1, 1985, pp. 75-79. doi:10.1007/BF00446743
 P. Goffin, F. Lorquet, M. Kleerebezem and P. Hols, “Major Role of NAD-dependent Lactate Dehydrogenases in Aerobic Lactate Utilization in Lactobacillus plantarum during Early Stationary Phase,” Journal of Bacteriology, Vol. 186, No. 19, 2004, pp. 6661-6666. doi:10.1128/JB.186.19.6661-6666.2004
 B. Sedewitz, K. H. Schleifer and F. Gotz, “Physiological Role of Pyruvate Oxidase in the Aerobic Metabolism of Lactobacillus plantarum,” Journal of Bacteriology, Vol. 160, No. 1, 1984, pp. 462-465.
 F. Lorquet, P. Goffin, L. Muscariello, J. B. Baudry, V. Ladero, M. Sacco, M. Kleerebezem and P. Hols, “Characterization and Functional Analysis of the poxB gene, which Encodes Pyruvate Oxidase in Lactobacillus plantarum,” Journal of Bacteriology, Vol. 186, No. 12, pp. 6661-6666.
 P. Goffin, L. Muscariello, F. Lorquet, A. Stukkens, D. Prozzi, M. Sacco, M. Kleerebezem and P. Hols, “Involvement of Pyruvate Oxidase Activity and Acetate Production in the Survival of Lactobacillus plantarum during the Stationary Phase of Aerobic Growth,” Applied and Environmental Microbiology, Vol. 72, No. 12, 2006, pp. 7933-7940. doi:10.1128/AEM.00659-06
 T. D. Thomas and K. W. Turner, “Carbohydrate Fermentation by Streptococcus cremoris and Streptococcus lactis Growing in Agar Gels,” Applied and Environmental Microbiology, Vol. 41, No. 6, 1981, pp. 1289-1294.
 S. Takahashi, K. Abbe and T. Yamada, “Purification of Pyruvate Formate-Lyase from Streptococcus mutans and its Regulatory Properties,” Journal of Bacteriology, Vol. 149, No. 3, 1982, pp. 1034-1040.
 F. Li, J. Hinderberger, H. Seedorf, J. Zhang, W. Buckel and R. K. Thauer, “Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri,” Journal of Bacteriology, Vol. 190, No. 3, 2008, pp. 843-850. doi:10.1128/JB.01417-07