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Overproduction hosts for biosynthesis of polyketidesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Compound Containing Saccharide Radical, Preparing O-glycoside (e.g., Glucosides, Etc.), Oxygen Of The Saccharide Radical Is Directly Bonded To A Nonsaccharide Heterocyclic Ring Or A Fused- Or Bridged-ring System Which Contains A Nonsaccharide Heterocyclic Ring (e.g., Coumermycin, Novobiocin, Etc.), The Hetero Ring Has Eight Or More Ring Members And Only Oxygen As Ring Hetero Atoms (e.g., Erythromycin, Spiramycin, Nystatin, Etc.)Overproduction hosts for biosynthesis of polyketides description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050287645, Overproduction hosts for biosynthesis of polyketides. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. Ser. No. 09/847,526 filed May 1, 2001, now allowed, which claims the benefit of U.S. provisional patent application 60/201,287, filed May 2, 2000. The entire contents of these documents are incorporated herein by reference. TECHNICAL FIELD [0003] The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology. BACKGROUND ART [0004] Polyketides are an important class of natural products responsible for the development of many human therapeutic, veterinary, and agricultural products (e.g. FK506, lovastatin, and avermectin). The enzymes which synthesize these compounds, polyketide synthases (PKSs), have been the target of various molecular engineering methods aimed at producing either improved analogs of existing pharmaceuticals or combinatorial libraries of novel polyketides. Modular PKSs--such as the 6-deoxyerythronolide B synthase (DEBS) shown in FIG. 1--have been altered by such techniques to produce new polyketide structures derived by genetic manipulation of one or more of the enzymatic domains contained in such PKS enzymes (see U.S. Pat. No. 5,962,290 and PCT Pub. No. 98/49315, supra). [0005] These first-generation successes have since led to a rapid proliferation of genetically engineered PKSs that produce novel polyketides or `unnatural` natural products (see PCT Pub. Nos. 99/61599, 00/024907, and 00/026349, each of which is incorporated herein by reference). Recent work has culminated in the generation of libraries of .about.100 macrocyclic compounds, illustrating the potential to create libraries with significant complexity and diversity (see PCT Pub. Nos. 00/063361 and 00/024907, each of which is incorporated herein by reference). [0006] The ability to manipulate predictably the catalytic activities of these multifunctional enzymes represents significant technological achievements in protein engineering. However, one of the current challenges to the construction of very large compound libraries (>1000 compounds) is the decline in production levels associated with many genetically modified PKSs, particularly those in which multiple domains have been modified (PCT Pub. Nos. 00/063361 and 00/024907, supra). While it is desirable to use PKS structure-activity knowledge to help guide more optimal engineering of PKSs, due to the complexity and size of these enzymes, the current understanding is relatively limited, and progress has been slow. It is therefore important to develop complementary approaches that do not rely on a detailed understanding of the enzymatic architecture to improve combinatorial biosynthesis technologies. [0007] One possibility leverages the significant resources that are generally devoted over the course of many years towards establishing commercial processes to produce natural product pharmaceuticals. Almost inevitably, extensive strain improvement and process development programs are undertaken to increase the yield of compound from the natural producing organism, often achieving greater than 100-fold increases in titers. A number of microorganisms have been optimized through random mutagenesis for bulk production of highly valuable compounds, including penicillins, macrolide antibiotics, and lovastatins. Although this conventional approach to strain improvement could be applied to strains carrying engineered PKSs, it is a labor-intensive process, especially given the potentially large numbers of mutant strains that could be generated by combinatorial biosynthesis. However, if overproduction capabilities of existing industrial strains could be applied to increase titers of polyketides derived from engineered PKSs, it would present an attractive and economical solution. Not only would overproduction increase the accessibility of compounds produced at levels currently too low for screening, but also the size of libraries created could be enlarged, because more modifications could be introduced into a PKS before productions levels became too low. The broad applicability of such strains, however, depends on what factors are responsible for overproduction, and whether production from a recombinant PKS would be increased in an overproduction background. [0008] The present invention meets the need for a generic host cell capable of producing a polyketide at levels significantly higher than in other host cells. SUMMARY OF THE INVENTION [0009] In a first embodiment, the present invention provides a method for producing a first polyketide, said method comprising expressing polyketide synthase (PKS) genes encoding a PKS that produces the first polyketide in a cell that has been optimized for the production of a second polyketide. [0010] In one aspect, this method involves the production of a first polyketide that is a derivative of the second polyketide and altering the PKS genes in the overproducing cell such that those genes express a PKS that produces the first polyketide. [0011] In another aspect, this method involves the introduction of genes that express a PKS that produces the first polyketide into the overproducing cell. In a preferred mode, the genes that express the PKS that produces the second polyketide are deleted or otherwise rendered inactive before or after introduction of the genes that encode the first PKS. [0012] In a preferred embodiment of this method, the overproducing cell produces the second polyketide at a level greater than 1 g/L, preferably greater than 2.5 g/L, more preferably greater than 5 g/L, and most preferably greater than 10 g/L. In a preferred embodiment of this method, the overproducing cell produces the first polyketide at a level greater than 10 mg/L, preferably greater than 25 mg/L, more preferably greater than 50 mg/L, and most preferably greater than 100 mg/L. [0013] In another embodiment, the present invention provides a generic overproducing host cell from which the genes encoding the second polyketide have been deleted and in which the genes encoding the PKS that produces the first polyketide can be readily introduced and expressed. [0014] In one aspect, this generic overproducing host cell is derived from a Saccharopolyspora erythraea host cell that produces erythromycins at a level exceeding 2.5 g/L and is modified either by deletion of all or substantially all of the eryA genes or by mutational inactivation of the ketosynthase (KS) domain of module 1 of the DEBS PKS. [0015] In another embodiment, the present invention provides a generic overproducing host cell that has been modified to express the PKS that produces the first polyketide. In a preferred embodiment, the host cell contains an attachment site for the integrating phage phiC31 that facilitates transformation of the strain. In one embodiment the host cell has been modified by genetic alteration to include such an attachment site. [0016] In one aspect, this generic overproducing host cell is derived from a Saccharopolyspora erythraea host cell that produces erythromycins at a level exceeding 2.5 g/L and is modified either by alteration of the eryA genes to encode a PKS that produces 10,11-anhydro-6-deoxyerythronolide B or by mutational inactivation of the ketosynthase (KS) domain of module 1 of the DEBS-PKS. [0017] These and other embodiments, modes, and aspects of the invention are described in more detail in the following description, the examples, and claims set forth below. BRIEF DESCRIPTION OF THE DRAWINGS [0018] FIG. 1 shows key steps in erythromycin biosynthesis, including the modular organization of the three proteins (DEBS1, DEBS2, and DEBS3) that comprise DEBS, the intermediates formed at each step in the synthesis of 6-deoxyerythronolide B (6-dEB), and the structures of the erythromycins resulting from modification of 6-dEB (erythromycin C is not shown). [0019] FIG. 2 shows the replacement in DEBS of the KR domain of module 2 with the KR and DH domains of module 4 of the rapamycin PKS and the products formed by the resulting recombinant PKS and action of polyketide modification enzymes in Saccharopolyspora erythraea. [0020] FIG. 3 illustrates production of erythromycin derivatives by precursor-directed biosynthesis, showing two diketides (compounds 4 and 5) and the products formed (compounds 6-11) upon providing them to a Saccharopolyspora erythraea host cell of the invention containing a KS1.degree. mutation. Continue reading about Overproduction hosts for biosynthesis of polyketides... 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