Bioprocess and Biosystems Engineering

, Volume 41, Issue 7, pp 1029–1038 | Cite as

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing

  • Xiaowen Sun
  • Hefang Wu
  • Genhai Zhao
  • Zhemin Li
  • Xihua Wu
  • Hui Liu
  • Zhiming Zheng
Research Paper


The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.


Aspergillus niger Morphology Citric acid Chitin synthase RNA interference 



The present work was supported by the National High-Tech Research and Development Program of China (No.2014AA021704), Natural Science Foundation of Anhui Province (1608085QC46) and Major Projects of Science and Technology in Anhui Province (17030801036).


  1. 1.
    Meyer V (2008) Genetic engineering of filamentous fungi—progress, obstacles and future trends. Biotechnol Adv 26(2):177–185CrossRefGoogle Scholar
  2. 2.
    Wucherpfennig T, Hestler T, Krull R (2011) Morphology engineering—osmolality and its effect on Aspergillus niger morphology and productivity. Microb Cell Fact 10(1):1–15CrossRefGoogle Scholar
  3. 3.
    Buren EB et al (2014) Toolkit for visualization of the cellular structure and organelles in Aspergillus niger. ACS Synth Biol 3(12):995–998CrossRefPubMedGoogle Scholar
  4. 4.
    Lory N (2003) Analysis of gene expression in Aspergillus niger using microarray technology. Concordia University, Montreal Quebec, CanadaGoogle Scholar
  5. 5.
    Assimopoulou AN, Boskou D, Papageorgiou VP (2004) Antioxidant activities of alkannin, shikonin and Alkanna tinctoria root extracts in oil substrates. Food Chem 87(3):433–438CrossRefGoogle Scholar
  6. 6.
    Dhillon GS et al (2011) Recent advances in citric acid bio-production and recovery. Food Bioprocess Technol 4(4):505–529CrossRefGoogle Scholar
  7. 7.
    Krull R et al (2010) Morphology of filamentous fungi: linking cellular biology to process engineering using Aspergillus niger. Adv Biochem Eng Biotechnol 121:1–21PubMedGoogle Scholar
  8. 8.
    Krull R et al (2013) Characterization and control of fungal morphology for improved production performance in biotechnology. J Biotechnol 163(2):112–123CrossRefGoogle Scholar
  9. 9.
    Priegnitz BE et al (2012) The role of initial spore adhesion in pellet and biofilm formation in Aspergillus niger. Fungal Genet Biol Fg B 49(1):30–38CrossRefPubMedGoogle Scholar
  10. 10.
    Driouch H et al (2012) Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles. Biotechnol Bioeng 109(2):462–471CrossRefPubMedGoogle Scholar
  11. 11.
    Papagianni M (2004) Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv 22(3):189–259CrossRefPubMedGoogle Scholar
  12. 12.
    Paul GC, Priede MA, Thomas CR (1999) Relationship between morphology and citric acid production in submerged Aspergillus niger fermentations. Biochem Eng J 3(2):121–129CrossRefGoogle Scholar
  13. 13.
    Hille A et al (2009) Effective diffusivities and mass fluxes in fungal biopellets. Biotechnol Bioeng 103(6):1202–1213CrossRefPubMedGoogle Scholar
  14. 14.
    Posch AE, Spadiut O, Herwig C (2012) A novel method for fast and statistically verified morphological characterization of filamentous fungi. Fungal Genet Biol 49(7):499–510CrossRefPubMedGoogle Scholar
  15. 15.
    Driouch H, Sommer B, Wittmann C (2010) Morphology engineering of Aspergillus niger for improved enzyme production. Biotechnol Bioeng 105(6):1058–1068PubMedGoogle Scholar
  16. 16.
    Mcintyre M et al (2001) Metabolic engineering of the morphology of Aspergillus. Adv Biochem Eng Biotechnol 73(4):103–128PubMedGoogle Scholar
  17. 17.
    Bartnicki-Garcia S (1968) Cell wall chemistry, morphogenesis, and taxonomy of fungi. Microbiology 22(22):87–108CrossRefGoogle Scholar
  18. 18.
    Feofilova EP (2010) [The fungal cell wall: modern concepts of its composition and biological function]. Microbiology 79(6):723–733CrossRefPubMedGoogle Scholar
  19. 19.
    Li M et al (2016) Evolution and functional insights of different ancestral orthologous clades of chitin synthase genes in the fungal tree of life. Front Plant Sci 7(37):1–14Google Scholar
  20. 20.
    Muszkieta L et al (2014) Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion. Cell Microbiol 16(12):1784–1805CrossRefPubMedGoogle Scholar
  21. 21.
    Tsuizaki M et al (2009) Myosin motor-like domain of the class VI chitin synthase CsmB is essential to its functions in Aspergillus nidulans. Agric Biol Chem 73(5):1163–1167Google Scholar
  22. 22.
    Horiuchi H, Takagi M (1999) Chitin synthase genes of Aspergillus species. Contrib Microbiol 2:193–204CrossRefPubMedGoogle Scholar
  23. 23.
    Choquer M et al (2004) Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes. Eur J Biochem 271(11):2153–2164CrossRefPubMedGoogle Scholar
  24. 24.
    Borgia PT et al (1996) The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal Genet Biol 20(3):193–203CrossRefPubMedGoogle Scholar
  25. 25.
    Fukuda K et al (2009) Class III chitin synthase ChsB of Aspergillus nidulans localizes at the sites of polarized cell wall synthesis and is required for conidial development. Eukaryot Cell 8(7):945–956CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Culp DW et al (2000) The chsA gene from Aspergillus nidulans is necessary for maximal conidiation. FEMS Microbiol Lett 182(2):349–353CrossRefPubMedGoogle Scholar
  27. 27.
    Fujiwara M et al (2000) Evidence that the Aspergillus nidulans Class I and Class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development. J Biochem 127(3):359–366CrossRefPubMedGoogle Scholar
  28. 28.
    Liu H et al (2013) Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum. Appl Microbiol Biotechnol 97(8):3363–3372CrossRefPubMedGoogle Scholar
  29. 29.
    Müller C et al (2002) Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis. Appl Environ Microbiol 68(4):1827–1836CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Thomas BT, Ogunkanmi LA, Agu GC (2014) Quelling of ochratoxin a production by RNA interference. Glob J Mol Sci 9(1):7–11Google Scholar
  31. 31.
    Liu H et al (2013) Morphology engineering of Penicillium chrysogenum by RNA silencing of chitin synthase gene. Biotech Lett 35(3):423–429CrossRefGoogle Scholar
  32. 32.
    Mouyna I et al (2004) Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol Lett 237(2):317–324PubMedGoogle Scholar
  33. 33.
    Ullán RV et al (2008) RNA-silencing in Penicillium chrysogenum and Acremonium chrysogenum: validation studies using β-lactam genes expression. J Microbiol Methods 75(2):209–218CrossRefPubMedGoogle Scholar
  34. 34.
    Crawford L et al (1995) Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Biotechnolgy 13(1):58–62Google Scholar
  35. 35.
    Yelton MM, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81(5):1470–1474CrossRefPubMedGoogle Scholar
  36. 36.
    Subramanyam C, Rao SLN (1987) An enzymic method for the determination of chitin and chitosan in fungal cell walls. J Biosci 12(2):125–129CrossRefGoogle Scholar
  37. 37.
    Tavares AP et al (2014) Image analysis technique as a tool to identify morphological changes in Trametes versicolor pellets according to exopolysaccharide or laccase production. Appl Biochem Biotechnol 172(4):2132–2142CrossRefPubMedGoogle Scholar
  38. 38.
    Gupta K, Mishra PK, Srivastava P (2007) A correlative evaluation of morphology and rheology of Aspergillus terreus during lovastatin fermentation. Biotechnol Bioprocess Eng 12(2):140–146CrossRefGoogle Scholar
  39. 39.
    Ikram-ul-Haq et al (2003) The kinetic basis of the role of Ca2+ ions for higher yield of citric acid in a repeated-batch cultivation system. World J Microbiol Biotechnol 19(8):817–823CrossRefGoogle Scholar
  40. 40.
    Ikram-Ul H et al (2004) Citric acid production by selected mutants of Aspergillus niger from cane molasses. Biores Technol 93(2):125–130CrossRefGoogle Scholar
  41. 41.
    Lane JH, Eynon L (1923) Determination of reducing sugars by means of Fehling’s solution with methylene blue as internal indicator. J Soc Chem Ind 42:32–36CrossRefGoogle Scholar
  42. 42.
    Bowen AR et al (1992) Classification of fungal chitin synthases. Proc Natl Acad Sci USA 89(2):519–523CrossRefPubMedGoogle Scholar
  43. 43.
    Ichinomiya M, Horiuchi H, Ohta A (2002) Different functions of the class I and class II chitin synthase genes, chsC and chsA, are revealed by repression of chsB expression in Aspergillus nidulans. Curr Genet 42(1):51–58CrossRefPubMedGoogle Scholar
  44. 44.
    Fernandes C, Gow NAR, Gonçalves T (2015) The importance of subclasses of chitin synthase enzymes with myosin-like domains for the fitness of fungi. Fungal Biol Rev 30(1):1–14CrossRefGoogle Scholar
  45. 45.
    Roncero C, Sanchez-Diaz A, Valdivieso MH (2016) 9 Chitin synthesis and fungal cell morphogenesisGoogle Scholar
  46. 46.
    Takeshita N et al (2015) Transportation of Aspergillus nidulans Class III and V chitin synthases to the hyphal tips depends on conventional kinesin. PLoS One 10(5):e0125937CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Steel R, Martin SM, Lentz CP (1955) A standard inoculum for citric acid production in submerged culture. Can J Microbiol 1(3):150–157CrossRefGoogle Scholar
  48. 48.
    Vecht-Lifshitz SE, Magdassi S, Braun S (1990) Pellet formation and cellular aggregation in Streptomyces tendae. Biotechnol Bioeng 35(9):890–896CrossRefPubMedGoogle Scholar
  49. 49.
    Haack MB et al (2006) Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation. Appl Microbiol Biotechnol 70(4):482–487CrossRefPubMedGoogle Scholar
  50. 50.
    Spohr A et al (1998) α-Amylase production in recombinant Aspergillus oryzae during fed-batch and continuous cultivations. J Ferment Bioeng 86(1):49–56CrossRefGoogle Scholar
  51. 51.
    Müller C et al (2003) Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae. Biotechnol Bioeng 81(5):525–534CrossRefPubMedGoogle Scholar
  52. 52.
    Thomas CR, Paul GC (1996) Applications of image analysis in cell technology. Curr Opin Biotechnol 7(1):35–45CrossRefPubMedGoogle Scholar
  53. 53.
    Kelly S et al (2006) Effects of fluid dynamic induced shear stress on fungal growth and morphology. Process Biochem 41(10):2113–2117CrossRefGoogle Scholar
  54. 54.
    Posch AE, Herwig C, Spadiut O (2012) Science-based bioprocess design for filamentous fungi. Trends Biotechnol 31(1):37–44CrossRefPubMedGoogle Scholar
  55. 55.
    Berovič M et al (1991) Submerged citric acid fermentation: rheological properties of Aspergillus niger broth in a stirred tank reactor. Appl Microbiol Biotechnol 34(5):579–581CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of High Magnetic Field and Ion Beam Bioengineering, Hefei Institutes of Physical ScienceChinese Academy of SciencesHefeiPeople’s Republic of China
  2. 2.University of Science and Technology of ChinaHefeiPeople’s Republic of China

Personalised recommendations