Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
1999-12-21
2001-07-17
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S041000, C435S171000, C435S176000, C435S177000, C435S178000, C435S179000, C435S180000, C435S256300
Reexamination Certificate
active
06261811
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for accelerating the synthesis of several or many natural products from fungi and in particular, to the synthesis of natural products using spores which have been immobilized onto/into loofah sponge. It has been unexpectedly discovered that the use of the loofah sponge as an immobilizing support can accomodate the growth of natural product producing fungi from spores at a rate and in an amount which has not been possible before the present invention. The method of the present invention preferably takes place in a standard bioreactor and unepectedly produces large quantities of the desired natural product. In the present invention, the method of using immobilized spores into/onto loofah to synthesize natural products occur for a period of at least about 4-10 days, more preferably about 25 days and even more preferably about 50 days or even longer. In a preferred embodiment of the present invention, spores of
Penicillium cyclopium
which have been immobilized onto/into loofah sponge are used to produce compactin, which is a hypocholesteremic agent.
BACKGROUND OF THE INVENTION
Fungi produce secondary metabolites during growth in response to environmental stress, and these compounds are synthesized from precurors derived from primary metabolites. Many of these products have significant economic value as growth regulators, as antimicrobial agents and as hypocholesterolemic agents. Compactin, a lovastatin analogue, which may be isolated from several fungi, including
Penicillium cyclopium
, functions as a specific competitive inhibitor of the enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase. Compactin and other compounds, such as simvastatin and pravastatin, have been shown to appreciably lower serum cholesterol levels.
Compactin is an antihypercholesterolemic agent which may affect DNA replication and also may enhance adhesion of tumor cells. Cyclopenol and cyclopenin are also produced by
P. Cyclopium.
A number of natural products including antibiotics, growth regulators and related pharmaceutical products are produced by isolating natural product (preferably, secondary) metabolites from fungi. The art is therefore always searching for ways to improve productivity and lower costs in providing these products.
It is an object of the invention to provide a method which results in enhanced or accelerated production of natural products from fungal spores.
It is an object of the present invention to provide an immobilization vehicle or platform to produce natural products from spores in submerged or bioreactor systems.
It is an object of the invention to provide a method which results in an enhanced or accelerated production or synthesis of compactin from
Penicillium cyclopium.
It is a further object of the invention to provide an immobilization vehicle or platform for
Penicillium cyclopium
for use to make compactin in submerged and/or bioreactor systems.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to a method for accelerating the synthesis or production of natural products (preferably, secondary metabolites) from fungi using spores immobilized into/onto loofah sponge or a related support. The method of the present invention comprises immobilizing a natural product producing fungal spore into/onto loofah sponge or a related support and then exposing the sponge to nutrient media in a submerged or bioreactor system in order to promote/accelerate the growth of fungi from the spores thereby producing natural product metabolites which may be readily isolated from fermented media in which the fungi has been grown and/or from the fungi. In a preferred embodiment of the pesent invention, a method of making compactin comprises immobilizing spores of
Penillium cyclopium
into/onto loofah sponge or a related support; exposing the immobilized spores to nutrient media containing glucose in a static system or alternatively, at a constant flow rate preferably for a period of at least about 4-10 days, more preferably about 25 days up to 50 days or more; and then isolating compactin from the fungi which grow from the spores and/or the fermented media in which the fungi grow. The method of the present invention may be used in a submerged system or in a standard bioreactor, which has preferably been adapted to accommodate air flow (oxygen) and to enhance the concentration of dissolved oxygen in the nutrient feed stock. Preferably, the method is used in a vertical bioreactor which is adapted to accommodate enhanced air flow and enhanced flow of nutrients in the nutrient media through the system. The combination of increased air (oxygen) flow and nutrient feed stock flow results in greater production of natural product (preferably, compactin) according to the present invention.
In a preferred embodiment of the present invention, the biosynthesis of compactin is conducted by preferably immobilizing the spores into/onto the outer and/or inner layers or small pieces of the outer layer of loofah sponge, which immobilized spores are then suspended in a column to form a bioreactor. The immobilized spores are then exposed to a constant flow of nutrient media and air through the suspended supports within the bioreactor. After a period of time, for example at 6, 8, 12, 16, 24, 48, 72, etc. hour intervals or longer, the media in which the spores are grown is then removed from the bioreactor and replenished with fresh media. The media which is removed from the reactor (otherwise known as “wort” media) contains substantial quantities of the desired natural product. The desired natural product is then isolated from the wort media using standard isolation techniques, including extraction, thin layer chromatography, thick layer chromatography, column chromatography, liquid chromatography, countercurrent distribution and the like and is subsequently and preferably, crystallized to produce the desired product in high purity. In an alternative embodiments used to produce natural products which may accumulate in the fungi, after a period of growth, the fungi which grow from the spores are then harvested, and the metabolites are isolated from the fungi and crystallized to produce the desire natural product.
In the present invention, it is preferred that a bioreactor system be used. The bioreactor may be a continuous-type system where the immobilized spores are exposed to a constant flow of nutrient media and air. Effluent from the bioreactor contains a substantial amount of natural product. This effluent is the wort from which the natural product is isolated. The bioreactor may alternatively be a batch-type (static) system with fungal spores being immobilized into or onto loofah sponge, immersed in nutrient media and exposed to a flow of air. The wort is isolated and natural product separated from the wort at varying intervals in this system. Isolation is similar to that used in the continuous reactor.
REFERENCES:
patent: 4929452 (1990-05-01), Hamdy
Bazaraa et al. “Bioreactor for Continuous Synthesis of Compactin byPenicillium Cyclopium”, Journal of Industrial Microbiology&Biotechnology,21: 192-202, 1998.
Satoh et al. “Regulation of N-Acetylkanamycin Amidohydrolase in the Idiophase in Kanamycin Fermentation”,Agr. Biol. Chem.,40 (1): p. 191, 1976.
Robbers et al. “Physiological Studies on Erogt: Further Studies on the Induction of Alkaloid Synthesis by Tryptophan and Its Inhibition by Phosphate”,Journal of Bacteriology,112 (2): p. 791, 1972.
Vliet et al. “Vastatins Have a Distinct Effect on Sterol Synthesis and Progesterone Secretion in Human Granulosa Cells in Vitro”,Biochimica et Biophysica Acta,1301: 237-241, 1996.
Bartman et al. “Mycophenolic Acid Production byPenicillium brevicompactumon Solid Media”,Applied and Environmental Microbiology,41 (3): p. 729, 1981.
Bird et al. “Disposition of Mycophenolic Acid, Brevianamide A, Asperphenamate, and Ergosterol in Solid Cultures ofPenicillium brevicompactum”, Applied and Environmental Microbiology,43 (2): p. 345, 1982.
Gallo et al. “Regulation of Secondary Metabolite Biosynthesis: Ca
Coleman Henry D.
Naff David M.
Sapone William J.
Sudol R. Neil
University of Georgia Research Foundation Inc.
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