Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-05
2001-10-16
Achutamurthy, Ponnathapu (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S320100
Reexamination Certificate
active
06303767
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polyketides and the polyketide synthase (“PKS”) enzymes that produce them. The invention also relates generally to genes encoding PKS enzymes and to recombinant host cells containing such genes and in which expression of such genes leads to the production of polyketides. Thus, the invention relates to the fields of chemistry, molecular biology, and agricultural, medical, and veterinary technology.
BACKGROUND OF THE INVENTION
Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Avermectin, candicidin, epothilone, erythromycin, FK-506, FK-520, narbomycin, oleandomycin, picromycin, rapamycin, spincoyn, tetracycline, and tylosin are examples of such compounds. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low expression of polyketides in wild-type cells that produce them naturally, there has been considerable interest in finding improved or alternate means to produce polyketide compounds.
This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. For example, the following publications relate generally to the cloning of all or parts of the genes coding for the expression of PKS enzymes or other enzymes that act on polyketides of significant commercial interest or potential.
Avermectin
U.S. Pat. No. 5,252,474 to Merck.
MacNeil et al., 1993
, Industrial Microorganisms: Basic and Applied Molecular Genetics
, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.
MacNeil et al., 1992
, Gene
115: 119-125, Complex Organization of the
Streptomyces avermitilis
genes encoding the avermectin polyketide synthase.
Candicidin (FRO008)
Hu et al., 1994
, Mol. Microbiol
. 14: 163-172.
Epothilone
U.S. patent application Ser. No. 60/130,560, filed Apr. 22, 1999, and Ser. No. 60/122,620, filed Mar. 3, 1999.
Erythromycin
PCT Pub. No. 93/13663 to Abbott.
U.S. Pat. No. 5,824,513 to Abbott.
Donadio et al., 1991
, Science
252:675-9.
Cortes et al., Nov. 8, 1990
, Nature
348:176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of
Saccharopolyspora erythraea.
Glycosylation Enzymes
PCT Pat. App. Pub. No. 97/23630 to Abbott.
FK-506
Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506
, Eur. J. biochem
. 256: 528-534.
Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506
, Eur. J. Biochem
. 244: 74-80.
Methyltransferase
U.S. Pat. No. 5,264,355, issued Nov. 23, 1993, Methylating enzyme from Streptomyces MA6858. 31-O-desmethyl-FK506 methyltransferase.
Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase 40 genes involved in the biosynthesis of the immunosuppressants FK506 and FK520
, J. Bacteriol
. 178: 5243-5248.
FK-520
U.S. patent application Ser. No. 60/139,650, filed Jun. 17, 1999, and Ser. No. 60/123,810, filed Mar. 11, 1999. See also Nielsen et al., 1991
, Biochem
. 30:5789-96 (enzymology of pipecolate incorporation).
Lovastatin
U.S. Pat. No. 5,744,350 to Merck.
Nemadectin
MacNeil et al., 1993, supra.
Niddamycin
Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from
Streptomyces caelestis, J. Bacteriol
. 179: 7515-7522.
Oleandomycin
Swan et al., 1994, Characterisation of a
Streptomyces antibioticus
gene encoding a type I polyketide synthase which has an unusual coding sequence,
Mol. Gen. Genet
. 242: 358-362.
U.S. patent application Ser. No. 60/120,254, filed Feb. 16, 1999.
Olano et al., 1998, Analysis of a
Streptomyces antibioticus
chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring,
Mol. Gen. Genet
. 259(3): 299-308.
Platenolide
EP Pat. App. Pub. No. 791,656 to Lilly.
Rapamycin
Schwecke et al., August 1995, The biosynthetic gene cluster for the polyketide rapamycin,
Proc. Natl. Acad. Sci. USA
92:7839-7843.
Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in
Streptomyces hygroscopicus
: analysis of the enzymatic domains in the modular polyketide synthase,
Gene
169: 9-16.
Rifamycin
August et al., Feb. 13, 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of
Amycolatopsis mediterranei
S669
, Chemistry
&
Biology
, 5(2): 69-79.
Soraphen
U.S. Pat. No. 5,716,849 to Novartis.
Schupp et al., 1995
, J. Bacteriology
177: 3673-3679. A
Sorangium cellulosum
(Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polyketide Synthase Genes from Actinomycetes.
Spiramycin
U.S. Pat. No. 5,098,837 to Lilly.
Activator Gene
U.S. Pat. No. 5,514,544 to Lilly.
Tylosin
EP Pub. No. 791,655 to Lilly.
Kuhstoss et al., 1996
, Gene
183:231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.
U.S. Pat. No. 5,876,991 to Lilly.
Tailoring enzymes
Merson-Davies and Cundliffe, 1994
, Mol. Microbiol
. 13: 349-355. Analysis of five tylosin biosynthetic genes from the tylBA region of the
Streptomyces fradiae
genome.
Each of the above-referenced patent applications, patents, and publications is incorporated by reference herein.
The cloning of PKS genes has been accompanied by advances in technology allowing one to manipulate a known PKS gene(s) either to produce the polyketide synthesized by the corresponding PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketide produced from a known PKS. See, e.g., PCT publication Nos. WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 5,672,491; and 5,712,146; and Fu et al., 1994
, Biochemistry
33: 9321-9326; McDaniel et al., 1993
, Science
262: 1546-1550; and Cane et al., Oct. 2, 1998, Harnessing the Biosynthetic Code: Combinations, Permutations, and Mutations,
Science
282: 63-68, each of which is incorporated herein by reference.
PKS enyzmes are similar to, but distinct from, the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. Two major types of PKS enzymes are found in nature: these types are commonly referred to as Type I or “modular” and Type II “aromatic” PKS enzymes. A third type sometimes referred to in the scientific literature is a “fungal PKS”; however, for purposes of the present invention, this type is to be considered a Type I PKS. These types differ in their composition and mode of synthesis of the polyketide synthesized. Type I PKSs are typically found in nature as complexes of multiple very large proteins. In this type, a set of separate catalytic active sites (each active site is termed a “domain”, and a set thereof is termed a “module”) exists for each cycle of carbon chain elongation and modification in the polyketide synthesis pathway.
The active sites and modules of a typical Type I PKS enzyme are shown in
FIG. 9
of PCT patent publication No. WO 95/08548, which depicts a model of 6-deoxyerythronolide B synthase (“DEBS”), which is involved in the synthesis of erythromycin. Six separate modules, each catalyzing a round of condensation and modification of a 2-carbon unit, are present in DEBS. The number and type of catalytic domains that are present in each module varies, and the total of 6 extender modules and a loading module is provided on 3 separate proteins (designated DEBS-1, DEBS-2, and DEBS-3, with 2 modules
Betlach Melanie C.
McDaniel Robert
Achutamurthy Ponnathapu
Favorito Carolyn
Kaster Kevin
Kerr Kathleen
Kosan Biosciences, Inc.
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