Bacteria-induced static batch fungal fermentation of the diterpenoid cyathin A₃, a small-molecule inducer of nerve growth factor

• Published in: Journal of industrial microbiology & biotechnology 2011-05, Vol.38 (5), p.607-615
• Main Authors: Dixon, Emma, Schweibenz, Tatiana, Hight, Alison, Kang, Brian, Dailey, Allyson, Kim, Sarah, Chen, Meng-Yang, Kim, Yura, Neale, Sarah, Groth, Ashley, Ike, Trish, Khan, Sarah, Schweibenz, Brandon, Lieu, David, Stone, David, Orellana, Tania, Couch, Robin D
• Format: Article
• Language: English
• Subjects: absorption; agitation; antimicrobial properties; Bacillus; bacteria; batch fermentation; Biochemistry; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; biosynthesis; Biotechnology; carbon; Cyathus; cytotoxicity; diterpenoids; enzyme-linked immunosorbent assay; Escherichia coli; Escherichia coli K12; Fundamental and applied biological sciences. Psychology; fungi; Genetic Engineering; high performance liquid chromatography; humans; Inorganic Chemistry; lead; Life Sciences; mass spectrometry; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; Microbiology; nerve growth factor; neuroglia; Original Paper; therapeutics
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-010-0805-7

Cyathin A₃, produced by the fungus Cyathus helenae, is a member of the cyathane family of diterpene natural products. While many of the cyathanes display antibacterial/antimicrobial activity or have cytotoxic activity against human cancer cell lines, their most exciting therapeutic potential is derived from their ability to induce nerve growth factor (NGF) release from glial cells, making the cyathanes attractive lead molecules for the development of neuroprotective therapeutics to prevent/treat Alzheimer's disease. To investigate if cyathin A₃ has NGF-inducing activity, we set out to obtain it using published C. helenae bench-scale fungal fermentations. However, to overcome nonproducing fermentations, we developed an alternative, bacteria-induced static batch fermentation approach to the production of cyathin A₃, as described in this report. HPLC, UV absorption spectra, and mass spectrometry identify cyathin A₃ in fungal fermentations induced by the timely addition of Escherichia coli K12 or Bacillus megabacterium. Pre-filtration of the bacterial culture abolishes cyathin A₃ induction, suggesting that bacteria-associated media changes or physical interaction between the fungus and bacteria underlie the induction mechanism. Through alteration of incubation conditions, including agitation, the timing of induction, and media composition, we optimized the fermentation to yield nearly 1 mg cyathin A₃/ml media, a sixfold increase over previously described yields. Additionally, by comparison of fermentation profiles, we reveal that cyathin A₃ biosynthesis is regulated by carbon catabolite repression. We have used an enzyme-linked immunosorbent assay to illustrate that cyathin A₃ induces NGF release from cultured glial cells, and therefore cyathin A₃ warrants further examination in the development of neuroprotective therapeutics.