Cell-free synthesis of polyketides

Details Cell-free synthesis of polyketides Cell-free synthesis of polyketides

1999-08-13

2001-08-14

Scheiner, Laurie (Department: 1648)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S471000, C435S252300, C435S252350, C435S320100

Reexamination Certificate

active

06274560

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to polyketides and polyketide synthases. In particular, the invention pertains to novel methods of producing polyketides and libraries of polyketides using a cell-free system.
BACKGROUND OF THE INVENTION
Polyketides are a large, structurally diverse family of natural products. Polyketides possess a broad range of biological activities including antibiotic and pharmacological properties. For example, polyketides are represented by such antibiotics as tetracyclines and erythromycin, anticancer agents including daunomycin, immunosuppressants, for example FK506 and rapamycin, and veterinary products such as monensin and avermectin.
Polyketides occur in most groups of organisms and are especially abundant in a class of mycelial bacteria, the actinomycetes, which produce various polyketides. Polyketide synthases (PKSs) are multifunctional enzymes related to fatty acid synthases (FASs). PKSs catalyze the biosynthesis of polyketides through repeated (decarboxylative) Claisen condensations between acylthioesters, usually acetyl, propionyl, malonyl or methylmalonyl. Following each condensation, they introduce structural variability into the product by catalyzing all, part, or none of a reductive cycle comprising a ketoreduction, dehydration, and enoylreduction on the &bgr;-keto group of the growing polyketide chain. PKSs incorporate enormous structural diversity into their products, in addition to varying the condensation cycle, by controlling the overall chain length, choice of primer and extender units and, particularly in the case of aromatic polyketides, regiospecific cyclizations of the nascent polyketide chain. After the carbon chain has grown to a length characteristic of each specific product, it is released from the synthase by thiolysis or acyltransfer. Thus, PKSs consist of families of enzymes which work together to produce a given polyketide. It is the controlled variation in chain length, choice of chain-building units, and the reductive cycle, genetically programmed into each PKS, that contributes to the variation seen among naturally occurring polyketides.
Two general classes of PKSs exist. These classifications are well known. See, for example, Hopwood, D. A. and Khosla, C.,
Secondary Metabolites: Their Function and Evolution
(1992) Wiley Chichester (Ciba Foundation Symposium 171) pp. 88-112.
One class, known as Type I or modular PKSs, is represented by the PKSs which catalyze the biosynthesis of complex polyketides such as erythromycin and avermectin. These “modular” PKSs include assemblies of several large multifunctional proteins carrying, between them, a set of separate active sites for each step of carbon chain assembly and modification (Cortes, J. et al.
Nature
(1990) 348:176; Donadio, S. et al.
Science
(1991) 252:675; MacNeil, D. J. et al.
Gene
(1992) 115:119). The active sites required for one cycle of condensation and reduction are clustered as “modules” (Donadio et al.
Science
(1991), supra; Donadio, S. et al.
Gene
(1992) 111:51). For example, 6-deoxyerythronolide B synthase (DEBS) consists of the three multifunctional proteins, DEBS 1, DEBS 2, and DEBS 3 (Caffrey, P. et al.
FEBS Letters
(1992) 304:225), each of which possesses two modules. (See
FIG. 1.
)
As described below, a module contains at least the minimal activities required for the condensation of an extender unit onto a growing polyketide chain; the minimal activities required are a ketosynthase (KS), an acyl transferase (AT) and an acyl carrier protein (ACP). Additional activities for further modification reactions such as a reductive cycle or cyclization may also be included in a module. Structural diversity occurs in this class of PKSs from variations in the number and type of active sites in the PKSs. This class of PKSs displays a one-to-one correlation between the number and clustering of active sites in the primary sequence of the PKS and the structure of the polyketide backbone.
The second class of PKSs, the aromatic or Type II PKSs, has a single set of iteratively used active sites (Bibb, M. J. et al.
EMBO J.
(1989) 8:2727; Sherman, D. H. et al.
EMBO J.
(1989) 8:2717; Fernandez-Moreno, M. A. et al.
J. Biol. Chem.
(1992) 267:19278). Streptomyces is an actinomycete which is an abundant producer of aromatic polyketides. In each Streptomyces aromatic PKS so far studied, carbon chain assembly requires the products of three open reading frames (ORFs). (See
FIG. 2.
) ORF1 encodes a ketosynthase (KS) and an acyltransferase (AT) active site (KS/AT); ORF2 encodes a chain length determining factor (CLF), a protein similar to the ORF1 product but lacking the KS and AT motifs; and ORF3 encodes a discrete acyl carrier protein (ACP). Some gene clusters also code for a ketoreductase (KR) and a cyclase, involved in cyclization of the nascent polyketide backbone. However, it has been found that only the KS/AT, CLF, and ACP, need be present in order to produce an identifiable polyketide.
Fungal PKSs, such as the 6-methylsalicylic acid PKS, consist of a single multidomain polypeptide which includes all the active sites required for the biosynthesis of 6-methylsalicylic acid (Beck, J. et al.
Eur. J. Biochem.
(1990) 192:487-498; Davis, R. et al.
Abstr. of the Genetics of Industrial Microorganism Meeting, Montreal, abstr.
P288 (1994)). Fungal PKSs incorporate features of both modular and aromatic PKSs.
Streptomyces coelicolor produces the blue-pigmented polyketide, actinorhodin. The actinorhodin gene cluster (act), has been cloned (Malpartida, F. and Hopwood, D. A.
Nature
(1984) 309:462; Malpartida, F. and Hopwood, D. A.
Mol. Gen. Genet.
(1986) 205:66) and completely sequenced (Fernandez-Moreno et al.
J. Biol. Chem.
(1992), supra; Hallam, S. E. et al.
Gene
(1988) 74:305; Fernandez-Moreno, M. A. et al.
Cell
(1991) 66:769; Caballero, J. et al.
Mol. Gen. Genet.
(1991) 230:401). The cluster encodes the PKS enzymes described above, a cyclase and a series of tailoring enzymes involved in subsequent modification reactions leading to actinorhodin, as well as proteins involved in export of the antibiotic and at least one protein that specifically activates transcription of the gene cluster. Other genes required for global regulation of antibiotic biosynthesis, as well as for the supply of starter (acetyl-CoA) and extender (malonyl-CoA) units for polyketide biosynthesis, are located elsewhere in the genome.
The act gene cluster from
S. coelicolor
has been used to produce actinorhodin in
S. parvulus.
Malpartida, F. and Hopwood, D. A.
Nature
(1984) 309:462.
Bartel et al.
J. Bacteriol.
(1990) 172:4816-4826, recombinantly produced aloesaponarin II using
S. galilaeus
transformed with an
S. coelicolor
act gene cluster consisting of four genetic loci, acti, actIII, actIV and actVII. Hybrid PKSs, including the basic act gene set but with ACP genes derived from granaticin, oxytetracycline, tetracenomycin and frenolicin PKSS, have also been designed which are able to express functional synthases. Khosla, C. et al.
J. Bacteriol.
(1993) 175:2197-2204. Hopwood, D. A. et al.
Nature
(1985) 314:642-644, describes the production of hybrid aromatic polyketides, using recombinant techniques. Sherman, D. H. et al.
J. Bacteriol.
(1992) 174:6184-6190, reports the transformation of various S. coelicolor mutants, lacking different components of the act PKS gene cluster, with the corresponding granaticin (gra) genes from S. violaceoruber, in trans.
Although the above described model for complex polyketide biosynthesis by modular (Type I) PKSs has been substantiated by radioisotope and stable isotope labeling experiments, heterologous expression, directed mutagenesis, and in vitro studies on partially active proteins, cell-free enzymatic synthesis of complex polyketides has proved unsuccessful despite more than 30 years of intense efforts (Caffrey et al.
FEBS Letters
(1992), supra; Aparicio, J. F. et al.
J. Biol. Chem.
(1994) 269:8524; Bevitt, D. J. et al.
Eur. J. Biochem.
(1992) 204:39; Caffrey, P. et al.
Eur. J. Biochem.
(1991) 19

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