Aspergillus niger citric acid accumulation: do we understand this well working black box?

Abstract

This Mini-Review summarizes the current knowledge on the biochemical and physiological events leading to massive citric acid accumulation by Aspergillus niger under industrially comparable conditions, thereby particularly emphasizing the roles of glycolytic flux and its control, excretion of citric acid from the mitochondria and the cytosol, and the critical fermentation variables. The potential of novel techniques for metabolic analysis and genomic approaches in understanding this fermentation is also discussed.

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Fig. 1.
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References

  1. Arisan-Atac I, Wolschek M, Kubicek CP (1996) Trehalose-6-phosphate synthase A affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux. FEMS Microbiol Lett 140:77–83

    Article  CAS  PubMed  Google Scholar 

  2. Arts E, Kubicek CP, Röhr M (1987) Regulation of phosphofructokinase from Aspergillus niger: effect of fructose-2,6-bisphosphate on the action of citrate, ammonium ions and AMP. J Gen Microbiol 133:1195–1199

    CAS  Google Scholar 

  3. Bercovitz A, Peleg Y, Battat E, Rokem JS, Goldberg I (1990) Localisation of pyruvate carboxylase in organic acid producing Aspergillus strains. Appl Environ Microbiol 56:1594–1597

    CAS  PubMed  Google Scholar 

  4. Christensen B, Nielsen J (1999) Isotopomer analysis using GC-MS. Metab Eng 1:282–290

    Article  CAS  PubMed  Google Scholar 

  5. Christensen B, Nielsen J (2000a) Metabolic network analysis of Penicillium chrysogenum using (13)C-labeled glucose. Biotechnol Bioeng 68:652–659

    Google Scholar 

  6. Christensen B, Nielsen J (2000b) Metabolic network analysis. A powerful tool in metabolic engineering. Adv Biochem Eng Biotechnol 66:209–231

    CAS  PubMed  Google Scholar 

  7. Christensen B, Nielsen J (2002) Reciprocal 13C-labeling: a method for investigating the catabolism of cosubstrates. Biotechnol Prog 18:163–166

    Article  CAS  PubMed  Google Scholar 

  8. Clark DS, Lentz CP (1961) Submerged citric acid fermentation on beet molasses: effect of pressure and recirculation of oxygen. Can J Microbiol 7:447–452

    CAS  Google Scholar 

  9. Clark DS, Ito K, Horitsu H (1966) Effect of manganese and other heavy metals on submerged citric acid fermentation of molasses. Biotechnol Bioeng 8:465–471

    CAS  Google Scholar 

  10. Cleland WW, Johnson MJ (1954) Tracer experiments on the mechanism of citric acid formation by Aspergillus niger. J Biol Chem 208:679–692

    CAS  Google Scholar 

  11. Cox PW, Thomas CR (1992) Classification and measurement of fungal pellets by automated image analysis. Biotechnol Bioeng 39:945–952

    Google Scholar 

  12. Crolla A, Kennedy KJ (2001) Optimization of citric acid production from Candida lipolytica Y-1095 using n-paraffin. J Biotechnol 89:27–40

    Article  CAS  PubMed  Google Scholar 

  13. Evans CT, Scragg AH, Ratledge C (1983) A comparative study of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts. Eur J Biochem 130:195–204

    CAS  PubMed  Google Scholar 

  14. Grayson M, Eckroth D (eds) (1979) Kirk-Othmer encyclopedia of chemical technology, vol 6, 3rd edn. Wiley, New York

  15. Grotjohann N, Huang Y, Kowallik W (2001) Tricarboxylic acid cycle enzymes of the ectomycorrhizal basidiomycete, Suillus bovinus. Z Naturforsch 56:334–342

    CAS  Google Scholar 

  16. Habison A, Kubicek CP, Röhr M (1983) Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger. Biochem J 209:669–676

    CAS  PubMed  Google Scholar 

  17. Hesse SJA, Ruijter GJG, Dijkema C, Visser J (2000) Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger. J Biotechnol 77:5–15

    Article  CAS  PubMed  Google Scholar 

  18. Hockertz S, Plönzig J, Auling G (1987) Impairment of DNA formation is an early event in Aspergillus niger under manganese starvation. Appl Microbiol Biotechnol 25:590–593

    CAS  Google Scholar 

  19. Hoischen C, Kramer R (1990) Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum. J Bacteriol 172:3409–3416

    CAS  PubMed  Google Scholar 

  20. Honecker S, Bisping B, Yang Z, Rehm H-J (1989) Influence of sucrose concentration and phosphate limitation on citric acid production by immobilized cells of Aspergillus niger. Appl Microbiol Biotechnol 31:17–24

    CAS  Google Scholar 

  21. Hossain M, Brooks JD, Maddox IS (1984) The effect of sugar source on citric acid production by Aspergillus niger. Appl Microbiol Biotechnol 19:383–391

    Google Scholar 

  22. Jaklitsch WM, Kubicek CP, Scrutton MC (1991) Intracellular organisation of citrate production in Aspergillus niger. Can J Microbiol 37:823–827

    CAS  PubMed  Google Scholar 

  23. Jernejc K, Vendramin M, Cimerman A (1989) Lipid composition of Aspergillus niger in citric acid accumulating and nonaccumulating conditions. Enzyme Microb Technol 11:452–456

    Article  CAS  Google Scholar 

  24. Jianlong W (2000) Enhancement of citric acid production by Aspergillus niger using n-dodecane as an oxygen-vector. Proc Biochem 35:1079–1083

    Article  Google Scholar 

  25. Karaffa L, Váczy K, Sándor E, Biró S, Szentirmai A, Pócsi I (2001) Cyanide-resistant alternative respiration is strictly correlated to intracellular peroxide levels in Acremonium chrysogenum. Free Radic Res 34:405–416

    CAS  PubMed  Google Scholar 

  26. Kholodenko BN, Cascante M, Westerhoff HV (1994) Control theory of metabolic channelling. Mol Cell Biochem 133:313–331

    Google Scholar 

  27. Kirimura K, Yoda M, Usami S (1999) Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L. Curr Genet 34:472–477

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  29. Kisser M, Kubicek CP, Röhr M (1980) Influence of manganese on morphology and cell-wall composition of Aspergillus niger during citric acid fermentation. Arch Microbiol 128:26–33

    CAS  PubMed  Google Scholar 

  30. Kubicek CP (1988) The role of the citric acid cycle in fungal organic acid fermentations. Biochem Soc Symp 54:113–126

    Google Scholar 

  31. Kubicek CP, Röhr M (1986) Citric acid fermentation. CRC Crit Rev Biotechnol 3:331–373

    CAS  Google Scholar 

  32. Kubicek CP, Zehentgruber O, El-Kalak H, Röhr M (1980) Regulation of citric acid production by oxygen: effects of dissolved oxygen tension on adenylate levels and respiration in Aspergillus niger. Eur J Appl Microbiol Biotechnol 9:101–116

    CAS  Google Scholar 

  33. Kubicek CP, Schreferl-Kunar G, Wohrer W, Röhr M (1988) Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger. Appl Environ Microbiol 54:633–637

    CAS  PubMed  Google Scholar 

  34. Kubicek-Pranz EM, Mozelt M, Röhr M, Kubicek CP (1990) Changes in the concentration of fructose-2,6-bisphosphate in Aspergillus niger during stimulation of acidogenesis by elevated sucrose concentrations. Biochim Biophys Acta 1033:250–255

    CAS  PubMed  Google Scholar 

  35. Ma H, Kubicek CP, Röhr M (1985) Metabolic effects of manganese deficiency in Aspergillus niger: evidence for increased protein degradation. Arch Microbiol 141:266–268

    CAS  PubMed  Google Scholar 

  36. Martin SM, Wilson PW (1951) Uptake of 14CO2 by Aspergillus niger in the formation of citric acid. Arch Biochem 27:150–157

    Google Scholar 

  37. Maxwell DP, Wang Y, McIntosh L (1999) The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells. Proc Natl Acad Sci USA 96:8271–8276

    Article  CAS  PubMed  Google Scholar 

  38. Meixner O, Mischak H, Kubicek CP, Röhr M (1985) Effects of manganese deficiency on plasma membrane lipid composition and glucose uptake in Aspergillus niger. FEMS Microbiol Lett 26:271–274

    Article  CAS  Google Scholar 

  39. Netik A, Torres NV, Riol J-M, Kubicek CP (1997) Uptake and export of citric acid by Aspergillus niger is reciprocally regulated by manganese ions. Biochim Biophys Acta 1326:287–294

    Article  CAS  PubMed  Google Scholar 

  40. Orthofer R, Kubicek CP, Röhr M (1979) Lipid levels and manganese deficiency in citric acid producing strains of Aspergillus niger. FEMS Microbiol Lett 5:403–406

    Article  CAS  Google Scholar 

  41. Palmieri F (1994) Mitochondrial carrier proteins. FEBS Lett 346:48–54

    Article  CAS  PubMed  Google Scholar 

  42. Panneman H, Ruijter GJG, Van den Broeck HC, Visser J (1998) Cloning and biochemical characterisation of Aspergillus niger hexokinase. The enzyme is strongly inhibited by physiological concentrations of trehalose-6-phosphate. Eur J Biochem 258:223–232

    CAS  PubMed  Google Scholar 

  43. Pedersen H, Hjort C, Nielsen J (2000) Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger. Mol Gen Genet 263:281–286

    Article  CAS  PubMed  Google Scholar 

  44. Peksel A (1999) Biochemical aspects of the stimulation of citric acid accumulation by the sugar concentration in Aspergillus niger. PhD thesis, TU Wien, Vienna

  45. Peksel A, Torres NV, Liu J, Juneau G, Kubicek CP (2002) 13C-NMR analysis of glucose metabolism during citric acid production by Aspergillus niger. Appl Microbiol Biotechnol 58:157–163

    CAS  PubMed  Google Scholar 

  46. Popov VN, Simonian RA, Skulachev VP, Starkov AA (1997) Inhibition of the alternative oxidase stimulates H2O2 production in plant mitochondria. FEBS Lett 415:87–90

    Article  CAS  PubMed  Google Scholar 

  47. Ratledge C (2000) Look before you clone. A comment on 'Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production' by GJG Ruijter, H Panneman, D-B Xu and J Visser, FEMS Microbiol Lett 184 (2000) 35–40. FEMS Microbiol Lett 189:317–319

    Article  CAS  PubMed  Google Scholar 

  48. Röhr M, Kubicek CP (1981) Regulatory aspects of citric acid fermentation by Aspergillus niger. Proc Biochem 16:34–37

    Google Scholar 

  49. Röhr M, Kubicek CP, Kominek J (1996) Citric acid. In: Rehm HJ, Reed G (eds) Products of primary metabolism. (Biotechnology, vol 6) Verlag Chemie, Weinheim, pp 308–345

  50. Ruijter GJG, Panneman H, Visser J (1997) Overexpression of phosphofructokinase and pyruvate kinase in citric acid producing Aspergillus niger. Biochim Biophys Acta 1334:317–326

    Article  CAS  PubMed  Google Scholar 

  51. Ruijter GJG, Vondervoort PJ van de, Visser J (1999) Oxalic acid production by Aspergillus niger: an oxalate-non-producing mutant produces citric acid at pH 5 and in the presence of manganese. Microbiology 145:2569–2576

    CAS  PubMed  Google Scholar 

  52. Ruijter GJG, Panneman H, Xu D-B, Visser J (2000) Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production. FEMS Microbiol Lett 184:35–40

    Article  CAS  PubMed  Google Scholar 

  53. Ruijter GJG, Kubicek CP, Visser J (2002) Production of organic acids by fungi. In: Osiewacz HD (ed) Industrial applications. (The Mycota, vol 10) Springer, Berlin Heidelberg New York, pp 213–230

  54. Schreferl G, Kubicek CP, Röhr M (1986) Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase. J Bacteriol 165:1019–1022

    CAS  PubMed  Google Scholar 

  55. Schuster E, Dunn-Coleman N, Frisvad JC, Van Dijck PW (2002) On the safety of Aspergillus niger. Appl Microbiol Biotechnol 59:426–435

    CAS  PubMed  Google Scholar 

  56. Schulz G, Rauch J (1975) Citronensäure. In: Ullmans (ed) Encyclopedie der technischen Chemie, vol 9, 4th edn. Verlag Chemie, Weinheim, pp 624–636

  57. Shu P, Johnson MJ (1948) Citric acid production by submerged fermentation with Aspergillus niger. Ind Eng Chem 40:1202–1205

    CAS  Google Scholar 

  58. Steinböck F, Held I, Choojun S, Röhr M, Kubicek CP (1994) Characterization and regulatory properties of a single hexokinase from the citric acid accumulating fungus Aspergillus niger. Biochim Biophys Acta 1200:215–223

    PubMed  Google Scholar 

  59. Torres N (1994a) Modelling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger. I. Model definition and stability of the steady state. Biotechnol Bioeng 44:104–111

    CAS  Google Scholar 

  60. Torres N (1994b) Modelling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger. II. Sensitivity analysis. Biotechnol Bioeng 44:112–118

    CAS  Google Scholar 

  61. Torres N, Riol-Cimas JM, Wolschek M, Kubicek CP (1996a) Glucose transport by Aspergillus niger: the low affinity carrier is only formed during growth on high glucose concentrations. Appl Microbiol Biotechnol 44:790–794

    Article  CAS  Google Scholar 

  62. Torres NV, Voit E, Gonzalez-Alcon C (1996b) Optimization of nonlinear biotechnological processes with linear programming: application to citric acid production by Aspergillus niger. Biotechnol Bioeng 49:247–258

    CAS  Google Scholar 

  63. Umbach AL, Siedow JN (2000) The cyanide-resistant alternative oxidase from the fungi Pichia stipitis and Neurospora crassa are monomeric and lack regulatory features of the plant enzyme. Arch Biochem Biophys 378:234–245

    Article  CAS  PubMed  Google Scholar 

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

    CAS  Google Scholar 

  65. Wayman FM, Mattey M (2000) Simple diffusion is the primary mechanism for glucose uptake during the production phase of the Aspergillus niger citric acid process. Biotechnol Bioeng 67:451–456

    Article  CAS  PubMed  Google Scholar 

  66. Wolschek MF, Kubicek CP (1997) The filamentous fungus Aspergillus niger contains two "differentially regulated" trehalose-6-phosphate synthase-encoding genes, tpsA and tpsB. J Biol Chem 272:2729–2735

    Article  CAS  PubMed  Google Scholar 

  67. Xu D–B, Madrid CP, Röhr M, Kubicek CP (1989) Influence of type and concentration of the carbon source on citric acid production by Aspergillus niger. Appl Microbiol Biotechnol 30:553–558

    CAS  Google Scholar 

  68. Zehentgruber O, Kubicek CP, Röhr M (1980) Alternative respiration of Aspergillus niger. FEMS Microbiol Lett 8:71–74

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are cooperating within the framework of an Austro-Hungarian Intergovernmental Science and Technology Cooperation Programme (A-26/2000). The authors are grateful to Dr. Elisabeth Sandor for drawing Fig. 2.

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Correspondence to Christian P. Kubicek.

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Karaffa, L., Kubicek, C.P. Aspergillus niger citric acid accumulation: do we understand this well working black box?. Appl Microbiol Biotechnol 61, 189–196 (2003). https://doi.org/10.1007/s00253-002-1201-7

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Keywords

  • Fermentation
  • Citric Acid
  • Gluconic Acid
  • Pyruvate Carboxylase
  • Alternative Oxidase