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

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Abstract

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.

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References

  1. Meyer V (2008) Genetic engineering of filamentous fungi—progress, obstacles and future trends. Biotechnol Adv 26(2):177–185

    Article  CAS  Google Scholar 

  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):1–15

    Article  Google Scholar 

  3. Buren EB et al (2014) Toolkit for visualization of the cellular structure and organelles in Aspergillus niger. ACS Synth Biol 3(12):995–998

    Article  CAS  PubMed  Google Scholar 

  4. Lory N (2003) Analysis of gene expression in Aspergillus niger using microarray technology. Concordia University, Montreal Quebec, Canada

  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):433–438

    Article  CAS  Google Scholar 

  6. Dhillon GS et al (2011) Recent advances in citric acid bio-production and recovery. Food Bioprocess Technol 4(4):505–529

    Article  CAS  Google Scholar 

  7. Krull R et al (2010) Morphology of filamentous fungi: linking cellular biology to process engineering using Aspergillus niger. Adv Biochem Eng Biotechnol 121:1–21

    CAS  PubMed  Google Scholar 

  8. Krull R et al (2013) Characterization and control of fungal morphology for improved production performance in biotechnology. J Biotechnol 163(2):112–123

    Article  CAS  Google Scholar 

  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):30–38

    Article  CAS  PubMed  Google Scholar 

  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):462–471

    Article  CAS  PubMed  Google Scholar 

  11. Papagianni M (2004) Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol Adv 22(3):189–259

    Article  CAS  PubMed  Google Scholar 

  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):121–129

    Article  CAS  Google Scholar 

  13. Hille A et al (2009) Effective diffusivities and mass fluxes in fungal biopellets. Biotechnol Bioeng 103(6):1202–1213

    Article  CAS  PubMed  Google Scholar 

  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):499–510

    Article  PubMed  Google Scholar 

  15. Driouch H, Sommer B, Wittmann C (2010) Morphology engineering of Aspergillus niger for improved enzyme production. Biotechnol Bioeng 105(6):1058–1068

    CAS  PubMed  Google Scholar 

  16. Mcintyre M et al (2001) Metabolic engineering of the morphology of Aspergillus. Adv Biochem Eng Biotechnol 73(4):103–128

    CAS  PubMed  Google Scholar 

  17. Bartnicki-Garcia S (1968) Cell wall chemistry, morphogenesis, and taxonomy of fungi. Microbiology 22(22):87–108

    Article  CAS  Google Scholar 

  18. Feofilova EP (2010) [The fungal cell wall: modern concepts of its composition and biological function]. Microbiology 79(6):723–733

    Article  CAS  PubMed  Google Scholar 

  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):1–14

    Google Scholar 

  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):1784–1805

    Article  CAS  PubMed  Google Scholar 

  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):1163–1167

    CAS  Google Scholar 

  22. Horiuchi H, Takagi M (1999) Chitin synthase genes of Aspergillus species. Contrib Microbiol 2:193–204

    Article  CAS  PubMed  Google Scholar 

  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):2153–2164

    Article  CAS  PubMed  Google Scholar 

  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):193–203

    Article  CAS  PubMed  Google Scholar 

  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):945–956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Culp DW et al (2000) The chsA gene from Aspergillus nidulans is necessary for maximal conidiation. FEMS Microbiol Lett 182(2):349–353

    Article  CAS  PubMed  Google Scholar 

  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):359–366

    Article  CAS  PubMed  Google Scholar 

  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):3363–3372

    Article  CAS  PubMed  Google Scholar 

  29. Müller C et al (2002) Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis. Appl Environ Microbiol 68(4):1827–1836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Thomas BT, Ogunkanmi LA, Agu GC (2014) Quelling of ochratoxin a production by RNA interference. Glob J Mol Sci 9(1):7–11

    Google Scholar 

  31. Liu H et al (2013) Morphology engineering of Penicillium chrysogenum by RNA silencing of chitin synthase gene. Biotech Lett 35(3):423–429

    Article  CAS  Google Scholar 

  32. Mouyna I et al (2004) Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol Lett 237(2):317–324

    CAS  PubMed  Google Scholar 

  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):209–218

    Article  CAS  PubMed  Google Scholar 

  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):58–62

    CAS  Google Scholar 

  35. Yelton MM, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81(5):1470–1474

    Article  CAS  PubMed  Google Scholar 

  36. Subramanyam C, Rao SLN (1987) An enzymic method for the determination of chitin and chitosan in fungal cell walls. J Biosci 12(2):125–129

    Article  CAS  Google Scholar 

  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):2132–2142

    Article  CAS  PubMed  Google Scholar 

  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):140–146

    Article  CAS  Google Scholar 

  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):817–823

    Article  CAS  Google Scholar 

  40. Ikram-Ul H et al (2004) Citric acid production by selected mutants of Aspergillus niger from cane molasses. Biores Technol 93(2):125–130

    Article  CAS  Google Scholar 

  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 42:32–36

    Article  CAS  Google Scholar 

  42. Bowen AR et al (1992) Classification of fungal chitin synthases. Proc Natl Acad Sci USA 89(2):519–523

    Article  CAS  PubMed  Google Scholar 

  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):51–58

    Article  CAS  PubMed  Google Scholar 

  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):1–14

    Article  Google Scholar 

  45. Roncero C, Sanchez-Diaz A, Valdivieso MH (2016) 9 Chitin synthesis and fungal cell morphogenesis

  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):e0125937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Steel R, Martin SM, Lentz CP (1955) A standard inoculum for citric acid production in submerged culture. Can J Microbiol 1(3):150–157

    Article  Google Scholar 

  48. Vecht-Lifshitz SE, Magdassi S, Braun S (1990) Pellet formation and cellular aggregation in Streptomyces tendae. Biotechnol Bioeng 35(9):890–896

    Article  CAS  PubMed  Google Scholar 

  49. Haack MB et al (2006) Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation. Appl Microbiol Biotechnol 70(4):482–487

    Article  CAS  PubMed  Google Scholar 

  50. Spohr A et al (1998) α-Amylase production in recombinant Aspergillus oryzae during fed-batch and continuous cultivations. J Ferment Bioeng 86(1):49–56

    Article  CAS  Google Scholar 

  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):525–534

    Article  CAS  PubMed  Google Scholar 

  52. Thomas CR, Paul GC (1996) Applications of image analysis in cell technology. Curr Opin Biotechnol 7(1):35–45

    Article  CAS  PubMed  Google Scholar 

  53. Kelly S et al (2006) Effects of fluid dynamic induced shear stress on fungal growth and morphology. Process Biochem 41(10):2113–2117

    Article  CAS  Google Scholar 

  54. Posch AE, Herwig C, Spadiut O (2012) Science-based bioprocess design for filamentous fungi. Trends Biotechnol 31(1):37–44

    Article  CAS  PubMed  Google Scholar 

  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):579–581

    Article  Google Scholar 

Download references

Acknowledgements

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).

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  1. Xiaowen Sun and Hefang Wu contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of High Magnetic Field and Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, People’s Republic of China

    Xiaowen Sun, Hefang Wu, Genhai Zhao, Zhemin Li, Xihua Wu, Hui Liu & Zhiming Zheng

  2. University of Science and Technology of China, Hefei, 230026, Anhui, People’s Republic of China

    Xiaowen Sun

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  1. Xiaowen Sun
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Correspondence to Hui Liu or Zhiming Zheng.

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Sun, X., Wu, H., Zhao, G. et al. Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing. Bioprocess Biosyst Eng 41, 1029–1038 (2018). https://doi.org/10.1007/s00449-018-1932-1

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  • DOI: https://doi.org/10.1007/s00449-018-1932-1

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