Journal of Microbiology and Biotechnology

  • 제16권6호
  • /
  • Pages.870-879
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  • 2006
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Formation of Succinic Acid by Klebsiella pneumoniae MCM B-325 Under Aerobic and Anaerobic Conditions

  • Thakker Chandresh (Microbial Science Division, Agharkar Research Institute) ;
  • Bhosale Suresh (Microbial Science Division, Agharkar Research Institute) ;
  • Ranade Dilip (Microbial Science Division, Agharkar Research Institute)
  • 발행 : 2006.06.01
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초록

The present study describes the formation of succinic acid by a nonvirulent, highly osmotolerant Klebsiella pneumoniae strain SAP (succinic acid producer), its profile of metabolites, and enzymes of the succinate production pathway. The strain produced succinate along with other metabolites such as lactate, acetate, and ethanol under aerobic as well as anaerobic growth conditions. The yield of succinate was higher in the presence of $MgCO_3$ under $N_2$ atmosphere as compared with that under $CO_2$ atmosphere. Analysis of intracellular metabolites showed the presence of a smaller PEP pool than that of pyruvate. Oxaloacetate, citrate, and $\alpha$-ketoglutarate pools were considerably larger than those of isocitrate and fumarate. In order to understand the synthesis of succinate, the enzymes involved in end-product formation were studied. Levels of phosphoenolpyruvate carboxykinase, fumarate reductase, pyruvate kinase, and acetate kinase were higher under anaerobic growth conditions. Based on the profiles of the metabolites and enzymes, it was concluded that the synthesis of succinate took place via oxaloacetate, malate, and fumarate in the strain under anaerobic growth conditions. The strain SAP showed potential for the bioconversion of fumarate to succinate under $N_2$ atmosphere in the presence of $MgCO_3$. At an initial fumarate concentration of 10 g/l, 7.1 g/l fumarate was converted to 7 g/l succinate with a molar conversion efficiency of 97.3%. The conversion efficiency and succinate yield were increased in the presence of glucose. Cells grown on fumarate contained an 18-fold higher fumarate reductase activity as compared with the activity obtained when grown on glucose.

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참고문헌

  1. Ashworth, J. M. and H. L. Kornberg. 1966. The anaplerotic fixation of carbon dioxide by Escherichia coli. Proc. R. Soc. Ser. B. 165: 179-188
  2. Banul, J., D. Clifton, M. Kretschmer, and D. G. Fraenkel. 1993. Glucose metabolism in Escherichia coli and the effect of increased amount of aldolase. Biochemistry 32: 4685-4692 https://doi.org/10.1021/bi00068a029
  3. Brown, T. D. K., M. C. Jones-Mortimer, and H. L. Kornberg. 1977. The enzymic interconversion of acetate and acetylcoenzyme A in Escherichia coli. J. Gen. Microbiol. 102: 327-336 https://doi.org/10.1099/00221287-102-2-327
  4. Chao, Y. P., R. Patnaik, W. D. Roof, R. F. Young, and J. C. Liao. 1993. Control of gluconeogenesis by pps and pck in Escherichia coli. J. Bacteriol. 175: 6939-6944 https://doi.org/10.1128/jb.175.21.6939-6944.1993
  5. Clark, D. P. 1989. The fermentation pathways of Escherichia coli. FEMS Microbiol. Rev. 63: 223-234 https://doi.org/10.1016/0168-6445(89)90033-8
  6. Clark, J. M. and R. L. Switzer. 1977. Experimental Biochemistry, pp. 67-85. 2nd Ed. W. H. Freeman and Company, San Francisco, CA
  7. Gokarn, R. R., M. A. Eiteman, and E. Altman. 2000. Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase. Appl. Environ. Microbiol. 66: 1844-1850 https://doi.org/10.1128/AEM.66.5.1844-1850.2000
  8. Gokarn, R. R., M. A. Eiteman, and J. Sridhar. 1997. Succinic acid production by anaerobic microorganisms. Am. Chem. Soc. Symp. Ser. 666: 237-253
  9. Goldberg, I., K. Lonberg-Holm, E. A. Bagley, and B. Stieglitz. 1983. Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase. Appl. Environ. Microbiol. 45: 1838-1847
  10. Gottschalk, G. 1985. Bacterial Metabolism, 2nd Ed. Springer-Verlag, New York, U.S.A
  11. Gray, C. T., J. W. T. Wimpenny, and M. R. Mossman. 1966. Regulation of metabolism in facultative bacteria. II. Effects of aerobiosis, anaerobiosis and nutrition on the formation of Krebs cycle enzymes in Escherichia coli. Biochim. Biophys. Acta 117: 33-41 https://doi.org/10.1016/0304-4165(66)90149-8
  12. Hong, S. H., J. S. Kim, S. Y. Lee, Y. H. In, S. S. Choi, J. K. Rih, C. H. Kim, H. Jeong, C. G. Hur, and J. J. Kim. 2004. The genome sequence of the capnophilic rumen bacterium Mannheimia succiniciproducens. Nat. Biotechnol. 22: 1275-1281 https://doi.org/10.1038/nbt1010
  13. Hungate, R. E. 1970. A roll tube method for cultivation of strict anaerobes, pp. 117-132. In J. R. Norris and D. W. Ribbons (eds.), Methods in Microbiology, vol. 3B. Academic Press, New York, U.S.A
  14. Kim, P., M. Laivenieks, C. Vieille, and J. G. Zeikus. 2004. Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli. Appl. Environ. Microbiol. 70: 1238-1241 https://doi.org/10.1128/AEM.70.2.1238-1241.2004
  15. Lee, P. C., S. Y. Lee, S. H. Hong, and H. N. Chang. 2002. Isolation and characterization of a new succinic acid producing bacterium, Mannheimia succiniciproducens MBEL55E from bovine rumen. Appl. Microbiol. Biotechnol. 58: 663-668 https://doi.org/10.1007/s00253-002-0935-6
  16. Lee, P. C., W. G. Lee, S. Y. Lee, and H. N. Chang. 1999. Effects of medium components on the growth of Anaerobiospirillum succinogenes and succinic acid production. Proc. Biochem. 35: 49-55
  17. Lorowitz, W. and D. Clark. 1982. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase. J. Bacteriol. 152: 935-938
  18. Macy, J. M., L. G. Ljungdahl, and G. Gottschalk. 1978. Pathway of succinate and propionate formation in Bacteroides fragilis. J. Bacteriol. 134: 84-91
  19. Park, D. H. and J. G. Zeikus. 1999. Utilization of electrically reduced neutral red by Actinobacillus succinogenes: Physiological function of neutral red in membrane-driven fumarate reduction and energy conservation. J. Bacteriol. 181: 2403-2410
  20. Park, D. H., M. Laivenieks, M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1999. Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production. Appl. Environ. Microbiol. 65: 2912-2917
  21. Podkovyrov, S. M. and J. G. Zeikus. 1993. Purification and characterization of phosphoenolpyruvate carboxykinase, a catabolic $CO_2$-fixing enzyme, from Anaerobiospirillum succiniciproducens. J. Gen. Microbiol. 139: 223-228 https://doi.org/10.1099/00221287-139-2-223
  22. Ryu, H. W., J. S. Yun, and K. H. Kang. 1998. Isolation and characterization of the Enterococcus sp. RKY1 for biosynthesis of succinic acid. Kor. J. Appl. Microbiol. Biotechnol. 26: 545-550
  23. Ryu, H. W., K. H. Kang, and J. S. Yun. 1999. Bioconversion of fumarate to succinate using glycerol as a carbon source. Appl. Biochem. Biotechnol. 77-79: 511-520
  24. Samuelov, N. S., R. Lamed, S. Lowe, and J. G. Zeikus. 1991. Influence of $CO_2-HCO_{3}^{-}$ levels and pH on growth, succinate production, and enzyme activities of Anaerobiospirillum succiniciproducens. Appl. Environ. Microbiol. 57: 3013-3019
  25. Sridhar, J., M. A. Eiteman, and J. W. Wiegel. 2000. Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin. Appl. Environ. Microbiol. 66: 246-251 https://doi.org/10.1128/AEM.66.1.246-251.2000
  26. Van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167: 332-342 https://doi.org/10.1007/s002030050452
  27. Winstrom, L. O. 1978. Succinic acid and succinic anhydride, pp. 848-864. In H. F. Mark, D. F. Othmer, C. G. Overberger, and G. T. Seaborg (eds.), Kirk-Othmer Encyclopedia of Chemical Technology, vol. 21. Wiley, New York, U.S.A
  28. Wood, W. A. 1961. Fermentation of carbohydrates and related compounds, pp. 59-149. In I. C. Gunsalus and R. Y. Stanier (eds.), The Bacteria, vol. 2. Academic Press, New York
  29. Yang, C., Q. Hua, T. Baba, H. Mori, and K. Shimizu. 2003. Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockout. Biotechnol. Bioeng. 84: 129-144 https://doi.org/10.1002/bit.10692
  30. Zeikus, J. G. 1980. Chemical and fuel production by anaerobic bacteria. Annu. Rev. Microbiol. 34: 423-464 https://doi.org/10.1146/annurev.mi.34.100180.002231
  31. Zeikus, J. G., P. Elankovan, and A. Grethlein. 1995. Utilizing fermentation as a processing alternative - succinic acid from renewable resources. Chem. Proc. 58: 71-73