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Increased Hepatitis B surface antigen production by recombinant Aspergillus niger through the optimization of agitation and dissolved oxygen concentration

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Abstract

The capacity of the filamentous fungi Aspergillus niger to produce and assemble complex immunogenic viral proteins into virus-like particles (VLPs) in batch culture was enhanced by optimizing the bioprocessing parameters, agitation intensity and dissolved oxygen (dO2) concentration. Response surface methodology (RSM) and a two-factor-two-level central composite rotatable design (CCRD) were employed to evaluate the interactive response pattern between parameters and their optimum combination. The recombinant hepatitis B surface antigen (HBsAg) was used as a model VLP system to determine the effect of these parameters on biomass yield, fungal morphology, HBsAg production and bioreactor kinetics. The response surface model predicted optimum cultivation conditions at an agitation of rate of 100 rpm and a dO2 concentration of 25%, obtaining highest intracellular membrane-associated HBsAg levels of \( {\text{3}}{\text{.4}}\;{\text{mg}}\;{\text{l}}^{{{\text{ - 1}}}}_{{{\text{culture}}}} \). HBsAg production levels were increased tenfold compared to yields obtained in shake flask cultivation. Although hepatitis B VLPs mostly accumulated intracellularly, optimal bioreactor conditions resulted in significant HBsAg release in culture supernatant. These results compare favourably with other recombinant VLP systems in batch culture, and therefore, indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.

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References

  1. Andreotti PE, Ludwig GV, Peruski AH, Tuite JJ, Morse SS, Peruski LF (2003) Immunoassay of infectious agents. Biotechniques 35:850–859

  2. Bai Z, Harvey LM, White S, McNeil B (2004) Effects of oxidative stress on production of heterologous and native protein, and culture morphology in batch and chemostat cultures of Aspergillus niger (B1-D). Enzyme Microb Technol 34:10–21

  3. Box GEP, Draper NA (1959) A basis for the selection of a response surface design. J Am Stat Assoc 54:622–654

  4. Box GEP, Hunter WG, Hunter JS (1978) Statistics for experiments. Wiley, New York, pp 291–334

  5. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

  6. Braun S, Vecht-Lipshitz SE (1991) Mycelial morphology and metabolite production. Trends Biotechnol 9:63–68

  7. Cleves AE, Cooper DNW, Barondes SH, Kelly RB (1996) A new pathway for protein export in Saccharomyces cerevisiae. J Cell Biol 133:1017–1026

  8. Cregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS, Buckholz RG, Madden KR, Kellaris PA, Davis GR, Smiley BL, Cruze J, Torregrossa R, Velicelebi G, Thill GP (1987) High-level expression and efficient assembly of Hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Bio/Technology 5:479–485

  9. Cui YQ, van der Lans RG, Luyben KC (1998) Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation. Biotechnol Bioeng 57:409–419

  10. Davies RW (1994) Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli SD, Kinghorn JR (eds) Aspergillus: 50 years on. Elsevier, Amsterdam, pp 527–559

  11. de Roubin MR, Bastien L, Shen SH, Groleau D (1991) Fermentation study for the production of Hepatitis B virus Pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha. J Ind Microbiol 8:147–156

  12. Gordon CL, Khalaj V, Ram AFJ, Archer DB, Brookman JL, Trinci APJ, Jeenes DJ, Doonan JH, Wells B, Punt PJ, Van den Hondel CAMJJ, Robson GD (2000) Glucoamylase::green fluorescent protein fusions to monitor protein secretion in Aspergillus niger. Microbiology 146:415–426

  13. Gouka RJ, Punt PJ, van den Hondel CAMJJ (1997) Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1–11

  14. Gu MB, Park MH, Kim DI (1991) Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing Hepatitis B surface antigen (HBsAg). Appl Microbiol Biotechnol 35:46–50

  15. Gwynne DI, Buxton FP, Williams SA, Garven S, Davies RW (1987) Genetically engineered secretion of active human interferon and a bacterial endoglucanase from Aspergillus nidulans. Bio/Technology 5:713–719

  16. Hansson M, Nygren PA, Stahl S (2000) Design and production of recombinant subunit vaccines. Biotechnol Appl Biochem 32:95–105

  17. Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York

  18. Higashiyama K, Murakami K, Tsujimura H, Matsumoto N, Fujikawa S (1999) Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4. Biotechnol Bioeng 3:442–448

  19. Hsieh JH, Shih KY, Kung HF, Shiang M, Lee LY, Meng MH, Chang CC, Lin HM, Shih SC, Lee SY, Chow TY, Feng TY, Kuo T, Choo KB (1988) Controlled fed-batch fermentation of recombinant Saccharomyces cerevisiae to produce Hepatitis B surface antigen. Biotechnol Bioeng 32:334–340

  20. Jeenes DJ, MacKenzie DA, Roberts IN, Archer DB (1991) Heterologous protein production by filamentous fungi. Biotechnol Genet Eng Rev 9:327–367

  21. König B, Seewald C, Schügerl K (1981) Process engineering investigations of penicillin production. Eur J Appl Microbiol Biotechnol 12:205–211

  22. Li ZJ, Shukla V, Wenger KS, Fordyce AP, Pedersen AG, Gade A (2002) Effects of increased impeller power in a production scale Aspergillus oryzae fermentation. Biotechnol Prog 18:437–444

  23. Metz B, de Bruijn EW, van Suijdam JC (1981) Method for quantitative representation of the morphology of moulds. Biotechnol Bioeng 23:149–162

  24. Nielsen J, Johansen CL, Jacobsen M, Krabben P, Villadsen J (1995) Pellet formation and fragmentation in submerged cultures of Penicillium chrysogenum and its relation to penicillin production. Biotechnol Prog 11:93–98

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

  26. Papagianni M, Mattey M, Kristiansen B (1999) Hyphal vacuolation and fragmentation in batch and fed-batch culture of Aspergillus niger and its relation to citric acid production. Process Biochem 35:359–366

  27. Patel N, Thibault J (2004) Evaluation of oxygen mass transfer in Aspergillus niger fermentation using data reconciliation. Biotechnol Prog 20:239–247

  28. Paul GC, Priede MA, Thomas CR (1999) Relationship between morphology and citric acid production in submerged Aspergillus niger fermentations. Biochem Eng J 3:121–129

  29. Plüddemann A, van Zyl WH (2003) Evaluation of Aspergillus niger as host for virus-like particle production, using the Hepatitis B surface antigen as a model. Curr Genet 43:439–446

  30. Smith JJ, Lilly MD, Fox RI (1990) The effect of agitation on the morphology and penicillin production of Penicillium chrysogenum. Biotechnol Bioeng 35:1011–1023

  31. Upshall A, Kumar AA, Bailey MC, Parker MD, Favreau MA, Lewison KP, Joseph ML, Maraganore JM, McKnight GL (1987) Secretion of active human tissue plasminogen activator from the filamentous fungus Aspergillus nidulans. Bio/Technology 5:1301–1304

  32. Ujcova E, Fencl Z, Musilcova M, Seichert L (1980) Dependence of release of nucleotides from fungi on fermenter turbine speed. Biotechnol Bioeng 22:237–241

  33. Van den Hondel CAMJJ, Punt PJ (1991) Gene transfer systems and vector development for filamentous fungi. In: Peberdy JF, Caten CE, Ogden JE, Bennett JW (eds) Applied molecular genetics of fungi. Cambridge University Press, Cambridge, pp 1–28

  34. van Suijdam JC, Metz B (1981) Influence of engineering variables on the morphology of filamentous molds. Biotechnol Bioeng 23:111–148

  35. Ward PP, Piddington CS, Cunningham GA, Zhou X, Wyatt RD, Conneely OM (1995) A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic. Bio/Technology 13:498–502

  36. Wongwicharn A, McNeil B, Harvey LM (1999) Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D. Biotechnol Bioeng 65:416–424

  37. Znidarsic P, Pavko A (2001) The morphology of filamentous fungi in submerged cultivations as a bioprocess parameter. Food Technol Biotechnol 39:237–252

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Acknowledgment

E. R. James was supported by the National Research Foundation (NRF, South Africa).

Author information

Correspondence to Johann F. Görgens.

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James, E.R., van Zyl, W.H. & Görgens, J.F. Increased Hepatitis B surface antigen production by recombinant Aspergillus niger through the optimization of agitation and dissolved oxygen concentration. Appl Microbiol Biotechnol 75, 279–288 (2007). https://doi.org/10.1007/s00253-006-0812-9

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Keywords

  • VLP production
  • HBsAg
  • A. niger
  • Dissolved oxygen concentration
  • Agitation intensity