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
1999-03-17
2001-03-06
Lilling, Herbert J. (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,...
C435S123000, C435S171000, C435S256100
Reexamination Certificate
active
06197560
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the biosynthesis of cholesterol reducing agents. More specifically, the invention relates to the biosynthesis of the cholesterol lowering agent lovastatin by certain microorganisms.
BACKGROUND OF THE INVENTION
Lovastatin (mevinolin; monacolin K; &bgr;,&dgr;-dihydroxy-7-[1,2,6,7,8,8a-hexahydro-2,6-dimethyl-8-(2-methyl-butyryloxy)-naphthalen-1-yl]-heptanoic acid &dgr;-lactone) is one of the most important known cholesterol lowering agents. Lovastatin, as used herein, includes both the lactone and free hydroxy acid forms.
Its open hydroxy acid form is a potent inhibitor of the 3-hydroxy-3-methyl-glutarylcoenzyme A reductase enzyme, which catalyzes the formation of mevalonic acid, an early intermediate of cholesterol biosynthesis. Lovastatin is specifically advantageous because, as a result of its application, biosynthetic intermediates with a toxic steroid skeleton, formed at a later stage of biosynthesis fail to accumulate. Lovastatin increases the number of LDL-receptors at the surface of the cell membrane which remove the LDL cholesterol circulating in the blood, thereby inducing the lowering of blood plasma cholesterol level.
Commonly, the active ingredient is produced via fermentation. GB 2,046,737 discloses that the active ingredient can be produced by some strains belonging to the Monascus genus, e.g., by M. ruber 1005 cultivated between 7 and 40° C. As a culture medium, an aqueous solution of glucose, peptone, corn steep liquor and ammonium chloride was used. The fermentation was carried out for 10 days in aerobic conditions, and 87 mg Lovastatin was obtained from the filtrate of 5 liters of broth.
U.S. Pat. No. 4,294,926 discloses the biosynthesis of lovastatin preferably by the application of microorganisms under the deposited numbers ATCC 20541 or 20542 belonging to the
Aspergillus terreus
species on a culture medium containing carbohydrates, e.g., glucose, fructose, maltose, as carbon source; nitrogen sources, e.g., yeast, hydrolyzed yeast, hydrolyzed casein, corn steep liquor; and mineral salts, e.g., calcium carbonate, magnesium sulfate, cobalt, iron, and manganese salts at a temperature of 20-37° C. Similar procedures are described in U.S. Pat. Nos. 4,420,491, 4,342,767, 4,319,039 and 4,294,846, where the fermentations are carried out for 3-5 days on media containing 1-6% carbohydrates and 0.2-6% nitrogen sources.
German Patent No. 4,402,591 discloses biosynthesis of Lovastatin by microorganisms belonging to the Pleurotus genus, e.g.,
P. ostreatus, P. sapidus
and
P. saca
, at 25-35° C. during 7-14 days cultivation time on surface or submerge cultures.
Canadian Patent No. 2,129,416 discloses the preparation of lovastatin, or in a particular case, mevastatin, with a microorganism belonging to the Coniothyrium genus, e.g., under the deposited number
Coniothyrium fuckelii
ATCC 74227 on a culture medium containing 3-15% glucose, 0.5-4% peptone, 0.5-5% amylase, 0.2-1% ammonium sulphate, 0.01-0.1% magnesium sulphate, 0.05-0.2% antifoaming agent, 0.2-1.5% L-isoleucine, 0.2-1.5% L-aspartic acid in the pH range of 5-6. According to the examples, the active ingredient concentration of the broth was within 19-430 mg/liter.
Hungarian Patent No. HU 208,997 discloses the application of the holotype strain
Aspergillus obscurus
numbered as MV-1, deposited under the number NCAIM(P)F 001189. The fermentation is preferably carried out on a medium containing yeast extract and/or peptone and/or casein as nitrogen source(s) and glucose and/or maltose or sucrose as carbon source(s). The activity of the broth at the end of the laboratory scale cultivation is between 400-850 mg/liter.
The foregoing discussion establishes that the development work in the biosynthesis of lovastatin focused on discovery of new lovastatin-producing microorganisms rather than on the development of the fermentation procedure itself. Several references disclose that fermentations can be carried out on conventional and known media with the application of both surface and solid state cultivations. Batch-like procedures were applied where the behaviors of the procedures depended on the initial conditions. However, technical limitations, e.g., maintaining the most convenient level of ingredients, optimal dissolved oxygen supply and pH, etc., made it difficult to implement continuous corrective actions to ensure more favorable conditions. A given microorganism during the main fermentation stage, depending on its metabolism, requires different conditions/composition of media in order to obtain optimal growth and production of active ingredient. The present inventors concluded from their experiments that in the seed culture and at the beginning of the main fermentation, the quantity of active biomass is very small and variable. Thus, the yield of the fermentations are relatively low and variable. Yields reached at the end of the fermentations, which depended of course on the strain, did not exceed a lovastatin concentration of 850 mg/liter. The present inventors performed a detailed analysis of the whole fermentation procedure from the seed culture stage throughout the end of the fermentation. It was found that in the seed culture preparation stage, both in the case of the known media and execution processes, the quantity of the biomass is too low. Therefore, during the main fermentation, the metabolism of the microorganism and the morphology of the culture are not adequate.
OBJECTS OF THE INVENTION
It is, therefore, one object of the present invention is to improve the efficiency of the lovastatin-producing fermentation procedure by forcing the production ability of the microorganism via changing the conditions and the carrying out of the fermentations.
It is another object of the present invention to provide, in either or both the seed and main fermentation stage, the most convenient chemical and physiological conditions for the metabolism by the microorganism.
It is a further object of the present invention to provide, in either or both the seed and main fermentation stage, the most convenient chemical and physiological conditions for the metabolism by the microorganism by maintaining in a steady state condition, the growth rate and then, for an extended time, a maximal product formation rate.
SUMMARY OF THE INVENTION
These and other objects of the invention are achieved in one embodiment of the present invention by providing a method for producing lovastatin by a microorganism in a fermentation process having a seed culture stage and a main fermentation stage, said method comprising:
a) cultivating a microorganism biomass in said seed culture stage to produce an inoculum;
b) transferring said inoculum into a fermentation medium in said main fermentation stage; and,
c) maintaining steady stage conditions in said main fermentation stage, thereby producing a fermentation broth containing lovastatin.
In a preferred embodiment of the present invention, steady state conditions are maintained in the main fermentation stage by one or more of feeding of organic carbon sources; controlling glucose and/or total reducing sugar content; feeding of organic and/or inorganic nitrogen sources; controlling pH; controlling foam level; controlling the mass of the fermentation broth by withdrawals and feedings; and, controlling the dissolved oxygen level. Preferably, the fermentation process is conducted in a submerged culture of the microorganism and at a temperature in the range of from about 24° C. to about 30° C. In a particularly preferred embodiment of the present invention, the microorganism is an Aspergillus species. In yet other preferred embodiments of the present invention, the organic carbon source is selected from the group consisting of glucose, hydrolyzed starch and vegetable oil; the glucose content is maintained at below about 0.2% from the 60th hour of the main fermentation stage; the nitrogen sources are selected from the group consisting of corn steep liquor and ammonium hydroxide; pH is controlled to be within the range of from about 5.2 t
Balogh Gabor
Cseke Laszlo
Oláh Antal
Seress Péter
Biogal Pharmaceutical Company, Ltd.
Kenyon & Kenyon
Lilling Herbert J.
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