Effects of oxidative stress on production of heterologous and native protein, and culture morphology in batch and chemostat cultures of Aspergillus niger (B1-D)

Abstract The effects of addition of menadione (MD) to induce elevated intracellular superoxide levels (superoxide stress) and of elevated oxygen availability (oxygen stress) was investigated in batch and chemostat cultures of a recombinant Aspergillus niger (B1-D). Culture responses to the two stressors were clearly distinct in terms of the changes in recombinant protein production, overall protein synthesis, and morphology. MD addition resulted in a significant increase in intracellular O 2 − levels, but gassing with O 2 -enriched air (∼25%) led to decreased intracellular O 2 − levels. Under elevated O 2 availability, the activity of the alternative respiratory pathway was enhanced, reducing endogenous O 2 − generation, but the consequent decreased energy availability in such cultures is proposed to be the main reason leading to the decreased intracellular protein content and foreign protein secretion in batch cultures and chemostat cultures at low dilution rates ( D −1 ). The negative effects of O 2 enrichment on protein synthesis were less pronounced at high D due to the relative dominance of the main respiratory pathway at high growth rates. Under stressed cultivation conditions, the mean number of tips per clump correlated well with the secretion of recombinant protein. Morphological change with O 2 enrichment was not directly caused by the increased O 2 − generation as proposed in some previous studies, but possibly by the significantly altered culture metabolism.

[1]  T. Imanaka,et al.  Recombinant Microbes for Industrial and Agricultural Applications , 1993 .

[2]  Michael Schmidt,et al.  Concomitant loss of respiratory chain NADH: ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger , 1991, Applied Microbiology and Biotechnology.

[3]  N. Matsumoto,et al.  Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4. , 1999, Biotechnology and bioengineering.

[4]  D. Mackenzie,et al.  Physiological optimization of secreted protein production by Aspergillus niger. , 1994, Enzyme and microbial technology.

[5]  G. C. Paul,et al.  Characterisation of mycelial morphology using image analysis. , 1998, Advances in biochemical engineering/biotechnology.

[6]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[7]  G. Robson,et al.  Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture. , 1998, Biotechnology and bioengineering.

[8]  L. Harvey,et al.  Morphological and enzymatic responses of a recombinant Aspergillus niger to oxidative stressors in chemostat cultures. , 2003, Journal of biotechnology.

[9]  D. Mackenzie,et al.  Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture , 1990, Applied Microbiology and Biotechnology.

[10]  L. Harvey,et al.  Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D. , 1999, Biotechnology and bioengineering.

[11]  A. Friboulet,et al.  Editorial: Whither catalytic antibodies? , 1998 .

[12]  L. Harvey,et al.  Use of the chemiluminescent probe lucigenin to monitor the production of the superoxide anion radical in a recombinant Aspergillus niger (B1-D). , 2001, Biotechnology and bioengineering.

[13]  Archer Db.  Enzyme production by recombinant Aspergillus. , 1994 .

[14]  G. Robson,et al.  Growth‐rate‐independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325 , 2000, Biotechnology and bioengineering.

[15]  G. Stephanopoulos,et al.  Metabolic Engineering: Principles And Methodologies , 1998 .

[16]  A. H. Stouthamer,et al.  Growth and product formation in chemostat and recycling cultures by Aspergillus niger N402 and a glucoamylase overproducing transformant, provided with multiple copies of the glaA gene. , 1993, Journal of general microbiology.

[17]  D. Mackenzie,et al.  Strategies for improving heterologous protein production from filamentous fungi , 2004, Antonie van Leeuwenhoek.

[18]  S. Kerscher.  Diversity and origin of alternative NADH:ubiquinone oxidoreductases. , 2000, Biochimica et biophysica acta.

[19]  Amanullah,et al.  Effects of agitation intensity on mycelial morphology and protein production in chemostat cultures of recombinant Aspergillus oryzae. , 1999, Biotechnology and bioengineering.

[20]  D. Hollomon,et al.  Fungal respiration: a fusion of standard and alternative components. , 2001, Biochimica et biophysica acta.

[21]  K. Luyben,et al.  Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation. , 1998, Biotechnology and bioengineering.

[22]  J. Siedow,et al.  The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity. , 2000, Biochimica et biophysica acta.

[23]  Brian McNeil,et al.  Physiological responses of chemostat cultures of Aspergillus niger (B1-D) to simulated and actual oxidative stress. , 2003, Biotechnology and bioengineering.

[24]  K. Kirimura,et al.  Enhancement and repression of cyanide-insensitive respiration in Aspergillus niger. , 1996, FEMS microbiology letters.

[25]  A. H. Stouthamer,et al.  Bioenergetic consequences of glucoamylase production in carbon-limited chemostat cultures ofAspergillus niger , 2004, Antonie van Leeuwenhoek.

[26]  J. Kruszewska.  Heterologous expression of genes in filamentous fungi. , 1999, Acta biochimica Polonica.

[27]  J. Turrens.  Superoxide Production by the Mitochondrial Respiratory Chain , 1997, Bioscience reports.

[28]  M. Angelova,et al.  Effect of Dissolved Oxygen Concentration on Superoxide Dismutase Production by Humicola lutea Cells , 1999 .

[29]  T. Nyström.  To be or not to be: the ultimate decision of the growth‐arrested bacterial cell , 1998 .