Recombinant production of novel polyketides

Details Recombinant production of novel polyketides Recombinant production of novel polyketides

1999-03-05

2002-06-04

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

C435S471000, C435S486000, C435S183000, C435S189000, C435S232000, C435S252300, 53, 53

Reexamination Certificate

active

06399382

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to polyketides and polyketide synthases. In particular, the invention pertains to the recombinant production of polyketides using a novel host-vector 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. 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. One class, known as Type I PKSs, is represented by the PKSs for macrolides such as erythromycin. These “complex” or “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). Structural diversity occurs in this class 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, called Type II PKSs, is represented by the synthases for aromatic compounds. Type II PKSs have 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). ORF1 encodes a ketosynthase (KS) and an acyltransferase (AT) active site; ORF2 encodes a protein similar to the ORF1 product but lacking the KS and AT motifs; and ORF3 encodes a discrete acyl carrier protein (ACP).
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, M. A. et al.
J. Biol. Chem
. (1992) 267:19278; 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 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.
However, no one to date has described the recombinant production of polyketides using genetically engineered host cells which substantially lack their entire native PKS gene clusters.
SUMMARY OF THE INVENTION
The present invention provides for novel polyketides and novel methods of efficiently producing both new and known polyketides, using recombinant technology. In particular, a novel host-vector system is used to produce PKSs which in turn catalyze the production of a variety of polyketides. Such polyketides are useful as antibiotics, antitumor agents, immunosuppressants and for a wide variety of other pharmacological purposes.
Accordingly, in one embodiment, the invention is directed to a genetically engineered cell which expresses a polyketide synthase (PKS) gene cluster in its native, nontransformed state, the genetically engineered cell substantially lacking the entire native PKS gene cluster.
In another embodiment, the invention is directed to the genetically engineered cell as described above, wherein the cell comprises:
(a) a replacement PKS gene cluster which encodes a PKS capable of catalyzing the synthesis of a polyketide; and
(b) one or more control sequences operatively linked to the PKS gene cluster, whereby the genes in the gene cluster can be transcribed and translated in the genetically engineered cell,
with the proviso that when the replacement PKS gene cluster comprises an entire PKS gene set, at least one of the PKS genes or control elements is heterologous to the cell.
In particularly preferred embodiments, the genetically engineered cell is
Streptomyces coelicolor
, the cell substantially lacks the entire native actinorhodin PKS gene cluster and the replacement PKS gene cluster comprises a first gene encoding a PKS ketosynthase and a PKS acyltransferase active site (KS/AT), a second gene encoding a PKS chain length determining factor (CLF), and a third gene encoding a PKS acyl carrier protein (ACP).
In another embodiment, the invention is directed to a method for producing a recombinant polyketide comprising:
(a) providing a population of cells as described above; and
(b) culturing the population of cells under conditions whereby the replacement PKS gene cluster present in the cells, is expressed.
In still another embodiment, the invention is directed to a method for producing a recombinant polyketide comprising:
a. inserting a first portion of a replacement PKS gene cluster into a donor plasmid and inserting a second portion of a replacement PKS gene cluster into a recipient plasmid, wherein the first and second portions collectively encode a complete replacement PKS gene cluster, and further wherein:
i. the donor p

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