Abstract
Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bartus RT, Dean RL, Beer B, Lippa AS (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 217: 408–417
Behal V, Neuzil J, Hostalek Z (1983) Effect of tetracycline derivatives and some cations on the activity of anhydrotetracycline. Biotechnol Lett 5:537–542
Beutler HO (1985) L-Glutamate, colorimetric method with glutamate dehydrogenase and diaphorase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 8, 3rd edn. Verlag Chemie, Weinheim Deerfield Beach Basel, pp 369–376
Box GP, Hunter WG, Hunter JS (1978) Response surface methods. In: Statisticsfor experimentors. Hafner Publishing Company, New York, pp 510–539
Bu'Lock JD (1961) Intermediary metabolism and antibiotic synthesis. Adv Appl Microbiol 3:293–342
Cazin J Jr, Suter M, Butler JE (1988) Production of streptavidin in a synthetic medium. J Immunol Methods 113:75–81
Chemnitius F (1927) Zur Darstellung des Physostigmins. J Prakt Chem 116:59–64
Cimburkova E, Zima J, Noval J, Vanek Z (1988) Nitrogen regulation of avermectins biosynthesis in Streptomyces avermitilis in a chemically defined medium. J Basic Microbiol 28:491–499
Corkin S (1981) Acetylcholine, aging and Alzheimer's disease implications for treatment. Trends Neurosci 4:287–290
Davis KL, Mohs RC, Tinklenberg JR, Pfefferbaum A, Hollister LE, Koppel BS (1978) Physostigmine: improvement of long-term memory processes in normal humans. Science 201:272–274
Daoust DR (1973) Fermentation process for producing physostigmine. US patent 3734832
Demain AL (1968) Regulatory mechanisms and the industrial production of microbial metabolites. Lloydia 31:395–418
Elsayed NM, Ryabik JRG, Ferraris S, Wheeler CR, Korte DW Jr (1989) Determination of physostigmine in plasma by high performance liquid chromatography and fluorescence detection. Anal Biochem 177:207–211
Engelheart E, Loewi O (1930) Fermentative Azetylcholinspaltung im Blut und ihre Hemmung durch Physpstimin. Arch Exp Pathol Pharmakol 150:1–13
Garcia-Ochoa F, Santos VE, Fritsch AP (1992) Nutritional study of Xanthomonas campestris in xanthan gum production by factorial design of experiments. Enzyme Microb Technol 14:991–996
Greasham R, Inamine E (1986) Nutritional improvement of processes. In: Demain AL, Solomon NA (eds) Manual of industrial microbiology and biotechnology. American Society of Microbiology, Washington, DC, pp 41–48
Hambleton LG (1977) Semiautomated method for simultaneous determination of phosphorus, calcium, and crude protein in animal feeds. J Assoc Assoc Chem 60:845–852
Hara M, Asano K, Kawamoto I, Takiguchi T, Katsumata S, Takashi K, Nakano H (1989) Leinamycin, a new antitumor antibiotic from Streptomyces: producing organism: fermentation and isolation. J Antibiot (Tokyo) 42:1768–1774
Iwasa T, Harada S, Sato Y (1979) Method of production of physostigmine. Japan patent S54-62390
Lebrihi A, Lamsaif D, Lefebvre G, Germain P (1992) Effects of ammonium ions on spiramycin biosynthesis in Streptomyces ambofaciens. Appl Microbiol Biotechnol 37:382–387
Lhomme B, Roux JC (1991) Utilization of experimental designs for optimization of Rhizopus arrhizus culture. Bioresource Technol 35:301–312
Liras P, Villanueva JR, Martin JF (1977) Sequential expression of macromolecule biosynthesis and candicidin formation in Streptomyces griseus. J Gen Microbiol 102:269–277
Matthes K (1930) The action of blood on acetylcholine. J Physiol (Lond) 70:338–348
Miller TL, Churchill BW (1986) Substrates for large-scale fermentations. In: Demain AL, Solomon NA (eds) Manual of industrial microbiology and biotechnology. American Society of Microbiology, Washington, DC, pp 122–136
Omura S, Tanaka Y, Mamada H, Masuma R (1984) Effect of ammonium ion, inorganic phosphate and amino acids on the biosynthesis of protylonolide, a precursor of tylosin aglycone. J Antibiot (Tokyo) 37:494–502
Roseiro JC, Esgalhado ME, Amaral Collaco MT, Emery AN (1992) Medium development for xanthan production. Process Biochem 27:167–175
Shirato S, Motoyama H (1966) Fermentation studies with Streptomyces griseus: synthetic medium for the production of streptomycin. Appl Microbiol 14:706–710
Thal L, Fuld PA, Maseur MS, Sharpless NS (1983) Oral physostigmine and lecithin improve memory in Alzheimer's disease. Ann Neurol 13:491–496
Thal L, Masur D, Blau A (1989) Chronic oral physostigmine without lecithin improves memory in Alzheimer's disease. J Am Geriat Soc 37:42–48
Van Staden JF (1983) Simultaneous determination of protein (nitrogen), phosporus, and calcium in animal feed stuffs by multichannel flow-injection analysis. J AOAC 66:718–726
Vu-Trong K, Bhuwapathanapun S, Gray PP (1980) Metabolic regulation in tylosin-producing Streptomyces fradiae: regulatory role of adenylate nucleotide pool and enzymes involved in biosynthesis of tylonolide precursors. Antimicrob Agents Chemother 17:519–525
Walker MS, Walker JB (1971) Streptomycin biosynthesis: separation and substrate specificities of phosphatases acting on guanidinodeoxyscyllo-inositol phosphate and streptomycin-(streptidino) phosphate. J Biol Chem 246:7034–7040
Wallace KK, Payne GF, Speedie MK (1990) Ammonium effects on streptonigrin biosynthesis by Streptomyces flocculus.J Ind Microbiol 6:43–48
Weinberg ED (1970) Biosynthesis of secondary metabolites: role of trace metals. Adv Microb Physiol 4:1–44
Weinberg ED (1971) Secondary metabolism: raison d'etre. Perspect Biol Med 14:565–577
Wijnberg J, Speckamp WN (1978) New total synthesis of dl-physostigmine (dl-eserine) via regioselective NaBH4-reduction of imides. Tetrahedron 34:2399–2404
Williams WK, Katz E (1977) Development of a chemically defined medium for the synthesis of actinomycin D by Streptomyces parvulus. Antimicrobiol Agents Chemother 11:281–290
Young MD, Kemp LL, Bader FG (1985) Effects of phosphate, glucose, and ammonium on cell growth and lincomycin production by Streptomyces lincolnensis in chemically defined media. Biotechnol Bioeng 27:327–333
Zhang J, Wolfe S, Demain AL (1989a) Phosphate regulation of ACV synthesis and cephalosporin biosynthesis in Streptomyces clavuligerus. FEMS Microbiol Lett 57:145–150
Zhang J, Wolfe S, Demain AL (1989b) Ammonium ions repress δ-(L-α-aminoadipyl)-l-cysteinyl-d-valine synthetase in Streptomyces clavuligerus NRRL 3585. Can J Microbiol 35:399–402
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Zhang, J., Martin, C., Shifflet, M.A. et al. Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus . Appl Microbiol Biotechnol 44, 568–575 (1996). https://doi.org/10.1007/BF00172487
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00172487