Recombinant megalomicin biosynthetic genes and uses thereof

Details Recombinant megalomicin biosynthetic genes and uses thereof Recombinant megalomicin biosynthetic genes and uses thereof

2000-10-04

2003-02-25

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252350, C435S254110, C435S325000, C435S419000, C435S320100, C536S023100, C536S023200, C536S023700

Reexamination Certificate

active

06524841

ABSTRACT:

FIELD OF THE INVENTION
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 OF THE INVENTION
Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994,
Biochemistry
33: 9321-9326; McDaniel et al., 1993,
Science
262: 1546-1550; and Rohr, 1995,
Angew. Chem. Int. Ed. Engl.
34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes.
Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomicin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or “domains.” These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying &bgr;-carbon processing activities (see O'Hagan, D.
The polyketide metabolites;
E. Horwood: New York, 1991, incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based
Streptomyces coelicolor
expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994,
Science,
265: 509-512, McDaniel et al., 1993,
Science
262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism,
Saccharopolyspora erythraea,
allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a “clean” host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of &bgr;-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998,
Curr. Opin. Microbiol.
1: 319-329; Carreras and Santi, 1998,
Curr. Opin. Biotech.
9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that 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 polyketides produced from known PKS gene clusters.
Megalomicin is a macrolide antibiotic produced by
Micromonospora megalomicea,
a member of the Actinomycetales family of soil bacteria that produces many types of biologically active compounds. Megalomicin is a glycoside of erythromycin A, a widely used antibacterial drug with little or no antimalarial activity. Megalomicin has antibacterial properties similar to those of erythromycin, and in 1998, it was discovered also to have potent antiparasitic activity and low toxicity. The antiparasitic activity may be related to the effect megalomicin has on protein trafficking in eukaryotes, where it appears to inhibit vesicular transport between the medial and trans-Golgi, resulting in under-sialylation of proteins. Hence, megalomicin offers an exciting opportunity to develop a new class of antiparasitic drugs with a different mechanism of action than the drugs currently in use and, therefore, possibly active against drug-resistant forms of
Plasmodium falciparum.
The number and diversity of megalomicin derivatives have been limited due to the inability to manipulate the PKS genes, which have not previously been available in recombinant form. Genetic systems that allow rapid engineering of the megalomicin biosynthetic genes would be valuable for creating novel compounds for pharmaceutical. agricultural, and veterinary applications. The production of such compounds could be more readily accomplished if the heterologous expression of the megalomicin biosynthetic genes in
Streptomyces coelicolor
and
S. lividans
and other host cells were possible. The present invention meets these and other needs.
SUMMARY OF THE INVENTION
The present invention provides recombinant methods and materials for expressing PKS enzymes and polyketide modification enzymes derived in whole and in part from the megalomicin biosynthetic genes in recombinant host cells. The invention also provides the polyketides produced by such PKS enzymes. The invention provides in recombinant form all of the genes for the proteins that constitute the complete PKS that ultimately results, in
Micromonospora megalomicea,
in the production of megalomicin. Thus, in one embodiment, the invention is directed to recombinant materials comprising nucleic acids with nucleotide sequences encoding at least one domain, module, or protein encoded by a megalomicin PKS gene. In one preferred embodiment of the invention, the DNA compounds of the invention comprise a coding sequence for at least one and preferably two or more of the domains of the loading module and extender modules 1 through 6, inclusive, of the megalomicin PKS.
In one embodiment, the invention provides a recombinant expression vector that comprises a heterologous promoter positioned to drive expression of one or more of the megalomicin biosynthetic genes. In a preferred embodiment, the promoter is derived from another PKS gene. In a related embodiment, the invention provides recombinant host cells comprising one or more expression vectors that produce(s) megalomicin or a megalomicin derivative or precursor. In a preferred embodiment, the host cell is
Streptomyces lividans
or
S. coelicolor.
In another embodiment, the invention provides a recombina

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