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Metabolic characteristics of citric acid synthesis by the fungus Aspergillus niger

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Abstract

The article discusses recent advances in the study of the metabolism of Aspergillus niger, a producer of citric acid, and some of its characteristics that may be responsible for the overproduction of citric acid. The paper is designed so that the entire process of citrate biosynthesis, from the absorption of glucose from a nutrient medium to the extraction of citric acid from cells, is divided into stages that are critical for the yield of the desired product. This arrangement of the material makes it possible to show the main regulatory steps in the chain of citrate synthesis. Influencing these steps enables regulation of the biosynthesis of citric acid at the level of cellular metabolism. This eventually opens promising avenues in the improvement of producing strains by means of genetic engineering. The paper also reviews the influence of some fermentation conditions (medium components, additives, and technological parameters) on metabolic processes in producing cells, which is important for an integral understanding of both biosynthesis process at the cellular level and fermentation technology.

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Abbreviations

HCF:

hexacyanoferrate

CF:

culture fluid

PPP:

pentose phosphate pathway

TCA:

tricarboxylic acid cycle

NMR:

nuclear magnetic resonance

AMP:

adenosine monophosphate

ATP:

adenosine triphosphate

CTP(citrate transport protein):

mitochondrial trasporter of citrate

NADH:

reduced nicotinamide adenosine dinucleotide

References

  1. Plassard, C. and Fransson, P., Regulation of lowmolecular weight organic acid production in fungi, Fungal Biol. Rev., 2009, vol. 23, pp. 30–39.

    Article  Google Scholar 

  2. Kirimura, K., Honda, Y., and Hattori, T., Comprehensive Biotechnology, Tokyo: Elsevier, 2011.

    Google Scholar 

  3. Dhillon, G., Brar, S.K., Verma, M., and Tyagi, R.D., Recent advances in citric acid bio-production and recovery, Food Bioproc. Technol., 2010, vol. 4, pp. 505–529.

    Article  Google Scholar 

  4. Yoshida, S. and Yokoyama, A., Identification and characterization of genes related to the production of organic acids in yeast, J. Biosci. Bioeng., 2012, vol. 113, no. 5, pp. 556–561.

    Article  CAS  PubMed  Google Scholar 

  5. Bentley, R., and Bennet, J.W., A ferment of fermentations: reflections of the production of commodity chemicals using microorganisms, Adv. Appl. Microbiol., 2008, vol. 63, pp. 1–32.

    Article  CAS  PubMed  Google Scholar 

  6. Scazzocchio, C., Encyclopedia of Microbiology, Boston: Acad. Press, 2009.

    Google Scholar 

  7. Goldman, G.H. and Osmani, S.A., The Aspergilli: Genomics, Medical Aspects, Biotechnology, and Research Methods, Florida: CRC Press, Taylor and Francis Group, 2008.

    Google Scholar 

  8. Karaffa, L. and Kubicek, C.P., Aspergillus niger citric acid accumulation: do we understand this well working black box?, Appl. Microbiol. Biotechnol., 2003, vol. 61, pp. 189–196.

    Article  CAS  PubMed  Google Scholar 

  9. Dhillon, G., Brar, S.K., Verma, M., and Tyagi, R.D., Utilization of different agro-industrial wastes for sustainable bioproduction citric acid by Aspergillus niger, Biochem. Eng. J., 2011, vol. 54, pp. 83–92.

    Article  CAS  Google Scholar 

  10. Cleland, W.W. and Johnson, M.J., Tracer experiments on the mechanism of citric acid formation by Aspergillus niger, J. Biol. Chem., 1954, vol. 208, pp. 675–698.

    Google Scholar 

  11. Shu, P., Funk, A., and Niesh, A.C., Mechanism of citric acid formation from glucose by Aspergillus niger, Can. J. Biochem. Physiol., 1954, vol. 32, pp. 68–80.

    Article  CAS  PubMed  Google Scholar 

  12. Jaklitsch, W.M., Scrutton, M.C., and Kubicek, C.P., Intracellular location of enzymes involved in citrate production by Aspergillus niger, Can. J. Microbiol., 1991, vol. 37, pp. 823–827.

    Article  CAS  PubMed  Google Scholar 

  13. Kubicek, C.P., The role of the citric acid cycle in fungal organic acid fermentaions, Biochem. Soc. Symp., 1998, vol. 54, pp. 113–126.

    Google Scholar 

  14. Peksel, A., Torres, N., Liu, J., Juneau, G., and Kubicek, C.P., 13C-NMR analysis of glucose metabolism during citric acid production by Aspergillus niger, Appl. Microbiol. Biotechnol., 2002, vol. 58, pp. 157–163.

    Article  CAS  PubMed  Google Scholar 

  15. Kubicek, C.P. and Rohr, M., Citric acid fermentation, Crit. Rev. Biotechnol., 1986, vol. 3, pp. 331–373.

    Article  CAS  Google Scholar 

  16. Torres, N., Riol-Cimas, J.M., Wolschek, M., and Kubicek, C.P., Glucose transport by Aspergillus niger: the low affinity carrier is only formed during growth on high glucose concentrations, Appl. Microbiol. Biotechnol., 1996, vol. 44, pp. 790–794.

    CAS  Google Scholar 

  17. Wayman, F. and Mattey, M., Simple diffusion in the primary mechanism for glucose uptake during the production phase of the Aspergillus niger citric acid process, Biotechnol. Bioeng., 2000, vol. 67, pp. 451–456.

    Article  CAS  PubMed  Google Scholar 

  18. Smith, J.E., Valenzuels-Perez, J., and Ng, W.S., Changes if activities of the Embden–Meyerhof–Parnas and pentose phosphate pathways during the growth cycle of Aspergillus niger, Trans. Brit. Mycol. Soc., 1971, vol. 57, pp. 57–93.

    Google Scholar 

  19. Tkacz, J.S. and Lange, L., Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine, New York: Springer US, 2004.

    Book  Google Scholar 

  20. Kubicek, C.P., Zehentgruber, O., and Rohr, M., An indirect method for studying the fine control of citric acid fermentation by Aspergillus niger, Biotech. Lett., 1979. vol. 1, p. 47.

    Article  CAS  Google Scholar 

  21. Wongchai, W. and Jefferson, W.E., Pyruvate carboxylase of Aspergillus niger: partial purification and some properties, Fed. Proc., 1974. vol. 33, p. 1378.

    Google Scholar 

  22. Kubicek, C.P. and Rohr, M., Influence of manganese on enzyme synthesis and citric acid accumulation in Aspergillus niger, Eur. J. Microbiol., 1977. vol. 4, p. 167.

    Article  CAS  Google Scholar 

  23. Agrawal, P.K., Bhatt, C.S., and Viswanathan, L., Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger, Enz. Microb. Technol., 1983. vol. 5, p. 369.

    Article  CAS  Google Scholar 

  24. Ruijter, G., Panneman, H., Xu, D.-B., and Visser, J., Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production, FEMS Microbiol. Letts., 2000, vol. 184, pp. 35–40.

    Article  CAS  Google Scholar 

  25. Ruijter, G.J., Panneman, H., and Visser, J., Overexpression of phosphokinase and pyruvate kinase in citric acid-producing Aspergillus niger, Biochim. Biophys. Acta, 1997. vol. 317, p. 1334.

    Google Scholar 

  26. Ratledge, C., Look before you clone. A comment on “Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production” by Ruijter, G.J.G., Panneman, H., Xu, D.-B., and Visser, J., FEMS Microbiol. Lett., 184 [2000] 35–40, FEMS Microbiol. Letts., 2000, vol. 189, pp. 317–319.

    CAS  PubMed  Google Scholar 

  27. Pel, H.J., de Winde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap, P.J., Turner, G., de Vries, R.P., Albang, R., Albermann, K., Andersen, M.R., Bendtsen, J.D., Benen, J.A.E., van den Berg, M., Breestraat, S., Caddick, M.X., Contreras, R., Cornell, M., Coutinho, P.M., Danchin, E.G.J., Debets, A.J.M., Dekker, P., van Dijck, P.W.M., van Dijck, A., Dijkhuizen, I., Driessen, A.J.M., d’Enfert, C., Geysens, S., Goosen, C., Groot, G.S.P., de Groot, P.W.J., Guillemette, T., Henrissat, B., Herweijer, M., ven den Hombergh, J.P.T.W., van den Hondel, C.A.M.J.J., van den Heijden, R.T.J.M., van der Kaaij, R.M., Klis, F.M., Kools, H.J., Kubicek, C.P., van Kuyk, P.A., Lauber, J., Lu, X., van der Maarel, M.J.E.C., Meulenberg, R., Menke, H., Mortimer, M.A., Nielsen, J., Oliver, S.G., Olsthoorn, M., Pal, K., van Peij, N.N.M.E., Ram, A.F.J., Rinas, U., Roubos, J.A., Sagt, C.M.J., Scmoll, M., Sun, J., Ussery, D., Varga, J., Verbecken, W., van de Vondervoort, P.J.J., Wedler, H., Wosten, H.A.B., Zeng, A.-P., van Ooyen, A.J.J., Visser, J., and Stam, H., Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88, Nat. Biotechnol., 2007, vol. 25, pp. 221–231.

    Article  PubMed  Google Scholar 

  28. Mahler, H. and Cordes, E.H., Biological Chemistry, New York: Harper and Row, 1971.

    Google Scholar 

  29. Kubicek, C.P. and Rohr, M., The role of the tricarboxylic acid cycle in citric acid accumulation by Aspergillus niger, Eur. J. Appl. Biotechnol., 1978. vol. 5, p. 263.

    Article  CAS  Google Scholar 

  30. Meixner-Monori, B., Kubicek, C.P., Habison, A., Kubicek-Pranz, E., and Rohr, M., Presence and regulation of the a-ketoglutarate dehydrogenase multien-zyme complex in the filamentous fungus Aspergillus niger, J. Bacteriol., 1985. vol. 161, p. 265.

    PubMed Central  CAS  PubMed  Google Scholar 

  31. Kubicek, C.P. and Rohr, M., Regulation of citrate synthase from the citric acid-accumulating fungus, Aspergillus niger, Biochim. Biophys. Acta, 1980, vol. 615, pp. 449–457.

    CAS  PubMed  Google Scholar 

  32. Ruijter, G.J.G., Kubicek, C.P., and Visser, J., The Mycota: a Comprehensive Treatise on Fungi As Experimental Systems for Basic and Applied Research. Industrial Applications, Berlin: Springer-Verlag, 2002.

    Google Scholar 

  33. De Jongh, W.A. and Nielsen, J., Enhanced citrate production through gene insertion in Aspergillus niger, Metabol. Eng., 2008, vol. 10, pp. 87–96.

    Article  Google Scholar 

  34. Torres, N., Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger. I. Model definition and stability ofthe steady state, Biotechnol. Bioeng., 1994, vol. 44, pp. 104–111.

    Article  CAS  PubMed  Google Scholar 

  35. Torres, N., Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger. II. Sensitivity analysis, Biotechnol. Bioeng., 1994, vol. 44, pp. 112–118.

    Article  CAS  PubMed  Google Scholar 

  36. Guebel, D.V., Torres, N., and Darias, N.V., Optimization of citric acid production by during citric acid accumulation by Aspergillus niger through a metabolic flux balance model, Electron. J. Biotechnol., 2001. vol. 4, p. 1.

    Google Scholar 

  37. Evans, C.T., Scragg, A.H., and Ratledge, C., A comparative study of citrate efflux from mitochondria of oleaginous and nonoleaginous yeasts, Eur. J. Biochem., 1983, vol. 130, pp. 195–204.

    Article  CAS  PubMed  Google Scholar 

  38. Rohr, C.J.G. and Kubicek, C.P., Regulatory aspects of citric acid fermentation by Aspergillus niger, Proc. Biochem., 1981. vol. 16, p. 34.

    Google Scholar 

  39. Sandor, A., Johnson, J.H., and Srere, P.A., Cooperation between enzyme and transporter in the inner mitochondrial membrane of yeast. Requirement for mitochondrial citrate synthase for citrate and malate transport in Saccharomyces cerevisiae, J. Biol. Chem., 1994. vol. 269, p. 29609.

    CAS  PubMed  Google Scholar 

  40. Kholodenko, B.N., Cascante, M., and Westerhoff, H.V., Control theory of metabolic channeling, Mol. Cell Biochem., 1994, vol. 133, pp. 313–331.

    Article  PubMed  Google Scholar 

  41. Jaklitsch, W.M., Kubicek, C.P., and Scrutton, M.C., Intracellular organization of citrate production in Aspergillus niger, Can. J. Microbiol., 1991, vol. 37, pp. 823–827.

    Article  CAS  PubMed  Google Scholar 

  42. Torres, N.V., Voit, E., and Gonzalez-Alcon, C., Optimization of nonlinear biotechnological process with linear programming: application to citric acid production by Aspergillus niger, Biotechnol. Bioeng., 1996. vol. 49, p. 247.

    Article  CAS  PubMed  Google Scholar 

  43. Alvarez-Vasquez, F., Alvarex-Vasquez, F., GonzalezAlcon, C., and Torres, N.V., Metabolism of citric acid production by Aspergillus niger: model definition, steady-state analysis and constrained optimization of citric acid production rate, Biotechnol. Bioeng., 2000, vol. 70, pp. 82–108.

    Article  CAS  PubMed  Google Scholar 

  44. Arisan-Atac, I., Wolschek, M.F., and Kubicek, C.P., Trehalose-6-phosphate synthase a affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux, FEMS Microbiol. Letts., 1996. vol. 140, p. 77.

    Article  CAS  Google Scholar 

  45. Habison, A., Kubicek, C.P., and Rohr, M., Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger, Biochem. J., 1983, vol. 209, pp. 669–676.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Netik, A., Torres, N.V., Riol, J.-M., and Kubicek, C.P., Uptake and export of citric acid by Aspergillus niger is reciprocally regulated by manganese ions, Biochim. Biophys. Acta, 1997, vol. 1326, pp. 287–294.

    Article  CAS  PubMed  Google Scholar 

  47. Xu, D.-B., Madrid, C.P., Rohr, M., and Kubicek, C.P., Influence of type and concentration of the carbon source on citric acid production by Aspergillus niger, Appl. Microbiol. Biotechnol., 1989, vol. 30, pp. 553–558.

    CAS  Google Scholar 

  48. Kubicek-Pranz, E.M., Mozelt, M., Rohr, M., and Kubicek, C.P., Changes in he concentration of fructose-2,6-biphosphate in Aspergillus niger during stimulation of acidogenesis by elevated sucrose concentrations, Biochim. Biophys. Acta, 1990, vol. 1033, pp. 250–255.

    Article  CAS  PubMed  Google Scholar 

  49. Peksel, A., Biochemical aspects of the stimulation of citric acid accumulation by the sugar concentration in Aspergillus niger, PhD Thesis, TUWein: Vienna, 1999.

    Google Scholar 

  50. Ma, H., Kubicek, C.P., and Rohr, M., Metabolic effects of manganese deficiency in Aspergillus niger: evidence for increased protein, Arch. Microbiol., 1985, vol. 141, pp. 266–268.

    Article  CAS  PubMed  Google Scholar 

  51. Hockertz, S., Plonzig, J., and Auling, G., Impairment of DNA formation is an early event in Aspergillus niger under manganese starvation, Appl. Microbiol. Biotechnol., 1987, vol. 25, pp. 590–593.

    CAS  Google Scholar 

  52. Jerneijc, K., Vendramin, M., and Cimerman, A., Lipid composition of Aspergillus niger in citric acid accumulating and nonaccumulating conditions, Enz. Microb. Technol., 1989, vol. 11, pp. 452–456.

    Article  Google Scholar 

  53. Kisser, M., Kubicek, C.P., and Rohr, M., Influence of manganese on morphology and cell-wall composition of Aspergillus niger during citric acid fermentation, Arch. Microbiol., 1980, vol. 128, pp. 26–33.

    Article  CAS  PubMed  Google Scholar 

  54. Cox, P.W. and Thomas, C.R., Classification and measurement of fungal pellets by automated image analysis, Biotechnol. Bioeng., 1992, vol. 39, pp. 945–952.

    Article  CAS  PubMed  Google Scholar 

  55. Bruchmann, E.E., Enzymochemische Untersuchungen uber Schimmelpilzgarungen. II Einige Hemmstoffe der Aconitase und ihre Bedeutung fur die Citronensaureanhaufung durch Aspergillus niger in Submerskultur, Biochim. Z., 1961. vol. 335, p. 806.

    Google Scholar 

  56. Bruchmann, E.E., Action of hydrogen peroxide of accumulation of citric acid by Aspergillus niger, Naturwissenschafie, 1966, vol. 53, pp. 226–227.

    Article  CAS  Google Scholar 

  57. Jianlong, W., Enhancement of citric acid production by Aspergillus niger using n-dodecane as an oxygen vector, Proc. Biochem., 2000, vol. 35, pp. 1079–1083.

    Article  Google Scholar 

  58. Kirimura, K., Yoda, M., Shimizu, H., Sugano, S., Mizuno, M., Kino, K., and Usami, S., Contribution of cyanide-insensitive respiratory pathway catalyzed by the alternative oxidase to citric acid production in Aspergillus niger, Biosci. Biotechnol. Biochem., 2000, vol. 64, pp. 2034–2039.

    Article  CAS  PubMed  Google Scholar 

  59. Wallrath, J., Schmidt, J., and Weiss, H., Concomitant loss of respiratory chain NADH:ubiquinone reductase [complex I] and citric acid accumulation in Aspergillus niger, Appl. Microbiol. Biotechnol., 1991, vol. 36, pp. 76–81.

    Article  CAS  Google Scholar 

  60. Kirimura, K., Yoda, M., and Usami, S., Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223l, Curr. Genet., 1999, vol. 6, pp. 474–477.

    Google Scholar 

  61. Maxwell, D.P., Wang, Y., and McInosh, L., The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells, Proc. Natl. Acad. Sci. USA, 1999, vol. 6, pp. 827–8276.

    Google Scholar 

  62. Karaffa, L., Vaczy, K., Sandor, E., Biro, S., Szentirmai, A., and Posci, I., Cyanide-resistant alternative respiration is strictly correlated to intracellular peroxide levels in Acremonium chrysogenum, Free Radic. Res., 2001, vol. 34, pp. 405–416.

    Article  CAS  PubMed  Google Scholar 

  63. Sluse, F.E. and Jarmuszkiewicz, W., Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role, Brazil. J. Med. Biol. Res, 1998, vol. 31, pp. 733–747.

    Article  CAS  Google Scholar 

  64. Hesse, S.J.A., Ruijter, G.J.C., Dijkema, C., and Visser, J., Measurement of intracellular [compartmental] pH by 31P NMR in Aspergillus niger, J. Biotechnol., 2000, vol. 77, pp. 5–15.

    Article  CAS  PubMed  Google Scholar 

  65. Papagianni, M., Advances in citric acid fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling, Biotechnol. Adv., 2007, vol. 25, pp. 244–263.

    Article  CAS  PubMed  Google Scholar 

  66. Kristiansen, B. and Sinclair, C.G., Production of citric acid in batch culture, Biotechnol. Bioeng., 1978, vol. 20, pp. 1711–1722.

    Article  CAS  Google Scholar 

  67. Mitsushima, K., Shinmyo, A., and Enatsu, T., Control of citrate and 2-oxoglutarate formation in Candida lipolityca mitochondria by adenine nucleotides, Biochim. Biophys. Acta, 1979. vol. 582, p. 246.

    Article  Google Scholar 

  68. Millis, N.F., Trumpy, B.H., and Palmer, B.M., The effect of lipids on citric acid production by Aspergillus niger, J. Gen. Microbiol., 1963. vol. 30, p. 365.

    Article  CAS  Google Scholar 

  69. Haq, I.-U., Sikander, A., Qadeer, M.A., and Iqbal, J., Stimulatory effect of alcohols [methanol and ethanol] on citric acid productivity by a 2-deoxy-d-glucose resistant culture of Aspergillus niger GCB-47, Biores. Technol., 2003, vol. 886, pp. 227–233.

    Article  Google Scholar 

  70. Barrington, S. and Kim, J.-W., Response surface optimization of medium components for citric acid production by Aspergillus niger NRRL 567 grown in peat moss, Biores. Technol., 2008, vol. 99, pp. 368–377.

    Article  CAS  Google Scholar 

  71. Jianlong, W., Production of citric acid by immobilized Aspergillus niger using a rotating biological contactor [RBC], Biores. Technol., 2000, vol. 75, pp. 245–247.

    Article  CAS  Google Scholar 

  72. Pazouki, M., Sinha, J., and Panda, T., Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose, Bioproc. Eng., 2000, vol. 22, pp. 353–361.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. St. Petersburg State Chemical and Pharmaceutical Academy, St. Petersburg, 197376, Russia

    K. V. Alekseev & V. P. Komov

  2. St. Petersburg Academic Univeristy, St. Petersburg, 194021, Russia

    M. V. Dubina

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  1. K. V. Alekseev
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Original Russian Text © K.V. Alekseev, M.V. Dubina, V.P. Komov, 2014, published in Biotekhnologiya, 2014, No. 5, pp. 8–18.

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Alekseev, K.V., Dubina, M.V. & Komov, V.P. Metabolic characteristics of citric acid synthesis by the fungus Aspergillus niger . Appl Biochem Microbiol 51, 857–865 (2015). https://doi.org/10.1134/S0003683815090021

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