Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-18
2002-05-21
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S125000, C435S006120, C530S350000, C536S023100
Reexamination Certificate
active
06391583
ABSTRACT:
CROSS-REFERENCES TO RELATED APPLICATION
Not applicable.
BACKGROUND OF THE INVENTION
Cholesterol and other lipids are transported in body fluids by low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Substances that effectuate mechanisms for lowering LDL-cholesterol may serve as effective antihypercholesterolemic agents because LDL levels are positively correlated with the risk of coronary artery disease.
MEVACOR (lovastatin; mevinolin) and ZOCOR (simvastatin) are members of a group of active antihypercholesterolemic agents that function by inhibiting the rate-limiting step in cellular cholesterol biosynthesis, namely the conversion of hydroxymethylglutarylcoenzyme A (HMG-CoA) into mevalonate by HMG-CoA reductase.
The general biosynthetic pathway of a naturally occurring HMG-CoA reductase inhibitor has been outlined by Moore, et al., who showed that the biosynthesis of mevinolin (lovastatin) by Aspergillus terreus ATCC 20542 begins with acetate and proceeds via a polyketide pathway (R. N. Moore, et al.,
J. Amer. Chem. Soc.
107:3694-3701, 1985). Endo, et al. described similar biosynthetic pathways in
Pencillium citrinum
NRRL 8082 and
Monascus ruber
M-4681 (A. Y. Endo, et al.,
J. Antibiot.
38:444-448, 1985).
The recent commercial introduction of microbial HMG-CoA reductase inhibitors has fostered a need for high yielding production processes. Methods of improving process yield have included scaling up the process, improving the culture medium and simplifying the isolation.
Previous attempts to increase the biosynthesis of HMG-CoA reductase inhibitors at the level of gene expression have focused on increasing the concentration triol polyketide synthase (TPKS), a multifunctional protein with at least six activities as evidenced by the product of the enzymatic activity (Moore, supra, 1985). TPKS is believed to be the rate-limiting enzymatic activity(ies) in the biosynthesis of the HMG-CoA reductase inhibitor compounds.
U.S. Pat. No. 5,744,350 identifies a DNA encoding triol polyketide synthase (TPKS) from
Aspergillus terreus.
“NPKS” is now preferred to TPKS as the acronym for “nonaketide polyketide synthase.”
SUMMARY OF THE INVENTION
In one embodiment, the present invention is a method of increasing the production of lovastatin in a lovastatin-producing organism. The method comprises the steps of transforming the organism with a nucleic acid sequence comprising the D4B segment, preferably comprising nucleotides 579-33,000 of SEQ ID NO:18 and 1-5,349 of SEQ ID NO:19. The nucleic acid sequence is transcribed and translated and an increase in lovastatin production occurs. Preferably, this increase is at least 2-fold.
In a preferred form of the present invention, the lovastatin-producing organism is selected from the group consisting
A. terreus
ATCC 20542 and ATCC 20541.
In another embodiment, the method comprises the step of transforming the organism with the corresponding D4B segment isolated from a non-
A. terreus
lovastatin-producing organism.
In another embodiment, the present invention is a method of increasing the production of lovastatin in a lovastatin-producing organism, comprising the step of transforming the organism with the LovE gene, wherein the nucleic acid sequence is transcribed and translated and wherein an increase in lovastatin production occurs.
In another embodiment of the present invention, one may increase the production of monacolin J in a non-lovastatin-producing organism comprising the steps of transforming the organism with a nucleic acid sequence comprising the D4B segment. As a further step, one may additionally transform the organism with an entire LovF gene. If the entire LovF gene is added to the D4B segment, the organism will produce lovastatin.
In another embodiment, the present invention is the lovastatin production gene cluster, preferably SEQ ID NOs:18 and 19, and the individual genes comprising that cluster.
It is an object of the present invention to provide a method for increasing lovastatin and monacolin J production in both lovastatin-producing and non-lovastatin producing organisms.
Other objects, features and advantages of the present invention will become apparent after review of the specification, claims and drawings.
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J.T. Kealey, et al., “Production of a Polyketide Natural product in Nonpolyketide-producing Prokaryotic and Eukaryotic Hosts,”Proc. Natl. Acad. Sci. USA95:505-509, 1998.
J. Kennedy, et al., “Modulation of Polyketide Synthase Activity by Accessory Proteins During Lovastatin Biosynthesis,”Science284:1368-1372, 1999.
M. Manzoni, et al., “Production and Purification of Statins fromAspergillus terreusStrains,”Biotechnol. Tech. 12(7):529-532, 1998.
Vinci et al., Journal of Industrial Microbiology, vol. 8, No. 2, pp. 113-119, 1991.*
Alignment: Vinci et al., Acession No. Q92323, 1995.*
Murphy, et al., “Hypothetical 59.3 KDA Protein C17C9.16C in Chromosome I fromSchizosaccharomyces pombe” 1996 (Database).
Oliver, et al., “Putative Tricarboxylate Transport Protein C19G12.05 from Fission Yeast,” 1998 (Database).
Van Peij, et al., “Beta-xylosidase, x1nD Gene fromAspergillus nidulans,” 1997 (Database).
Hutchinson Charles R.
Kennedy Jonathan
Park Cheonseok
Carlson Karen Cochrane
Quarles & Brady LLP
Robinson Hope A.
Wisconsin Alumni Research Foundation
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