# Patent

## Title
Biosynthetic gene cluster for leptomycins

## Abstract

Polypeptides and domains of leptomycin polyketide synthase and the nucleic acids encoding them are provided. Methods to prepare leptomycin, leptomycin analogs, and leptomycin derivatives are described, as are methods to prepare other polyketides using the nucleic acids encoding leptomycin polyketide synthase domains or modifying enzymes.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of allowed application Ser. No. 10/937,730, filed 8 Sep. 2004 now U.S. Pat. No. 7,288,396, which claims benefit under 35 U.S.C. §119 to U.S. provisional applications No. 60/502,423 (filed 11 Sep. 2003) and No. 60/553,384 (filed 15 Mar. 2004), the entire contents of each prior application being incorporated herein by reference.


STATEMENT OF GOVERNMENT INTEREST
This invention was made with Government support under Grant No. 1 R43 CA108372-01 awarded by the National Institutes of Health. The Government has certain rights in this invention.


FIELD OF THE INVENTION
This invention relates to materials and methods for biosynthesis of leptomycins, leptomycin derivatives and analogs, and other useful polyketides. The invention finds application in the fields of molecular biology, recombinant DNA technology, chemistry, human and veterinary medicine, and agriculture.
BACKGROUND OF THE INVENTION
Polyketides are complex natural products that are produced by microorganisms such as fungi and mycelial bacteria. There are about 10,000 known polyketides, from which numerous pharmaceutical products in many therapeutic areas have been derived, including: adriamycin, epothilone, erythromycin, mevacor, rapamycin, tacrolimus, tetracycline, rapamycin, and many others. However, polyketides are made in very small amounts in microorganisms and are difficult to make or modify chemically. For this and other reasons, biosynthetic methods are preferred for production of therapeutically active polyketides. 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; 5,712,146 and 6,410,301; Fu et al., 1994, Biochemistry 33: 9321-26; McDaniel et al., 1993, Science 262: 1546-1550; Kao et al., 1994, Science, 265:509-12, and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34: 881-88, each of which is incorporated herein by reference.
The biosynthesis of polyketides may be accomplished by heterologous expression of Type I or modular polyketide synthase enzymes (PKSs). Type I PKSs are large multifunctional protein complexes, the protein components of which are encoded by multiple open reading frames (ORF) of PKS gene clusters. Each ORF of a Type I PKS gene cluster can encode one, two, or more modules of ketosynthase activity. Each module activates and incorporates a two-carbon (ketide) unit into the polyketide backbone. Each module also contains multiple ketide-modifying enzymatic activities, or domains. The number and order of modules, and the types of ketide-modifying domains within each module, determine the structure of the resulting product. Polyketide synthesis may also involve the activity of nonribosomal peptide synthetases (NRPSs) to catalyze incorporation of an amino acid-derived building block into the polyketide, as well as post-synthesis modification, or tailoring enzymes. The modification enzymes modify the polyketide by oxidation or reduction, addition of carbohydrate groups or methyl groups, or other modifications.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker regions. These regions collectively can be considered to define boundaries of the various domains. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKSs by various available methodologies. Using this method, new polyketide synthases (which produce novel polyketides) can be produced. It will be recognized from the foregoing that genetic manipulation of PKS genes and heterologous expression of PKSs can be used for the efficient production of known polyketides, and for production of novel polyketides structurally related to, but distinct from, known polyketides (see references above, and Hutchinson, 1998, Curr. Opin. Microbiol. 1:319-29; Carreras and Santi, 1998, Curr. Opin. Biotech. 9:403-11; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference).
One valuable class of polyketides includes the leptomycins and their analogs (FIG. 1). These compounds are selective inhibitors of protein export from the cell nucleus and thus affect the cellular location of proteins. The function of many key proteins and transcription factors involved in cell growth can be regulated by their cellular location. For instance, the tumor suppressor p53 normally resides in the cell nucleus where its activation promotes cell-cycle arrest and apoptotic cell death. Mislocation of p53 into the cytoplasm, especially its dominant negative mutant forms, is associated with development of many types of cancer. Nuclear factor κB (NFκB) is a transcriptional activator that targets genes involved in cell proliferation and apoptosis. It is constitutively activated in certain cancer cells, aiding tumor resistance to radiation and cancer chemotherapy drugs. NFκB resides in the cytoplasm in an inactive form complexed with the inhibitor of nuclear factor IκB; upon stimulation by factors such as TNF-α or CD-40 ligand, events are set in place that remove IκB and allow importation of NFκB into the cell nucleus.
Leptomycin B (LMB; also known as CI-940 or elactocin) and the ratjadones (FIG. 2) are the only known low molecular weight inhibitor of nuclear transport. Because of the structural similarities, the kazusamycins, leptofuranins and callystatins are also implicated. Callystatins come from a marine sponge whereas all the other compounds are bacterial metabolites. All of these molecules are exceptionally potent, typically displaying IC50 values in the 100 picomolar to 10 nanomolar range.
Protein export from the cell nucleus requires a nuclear export signal (NES) as a domain in the exported protein, CRMI (exportin-1) to recognize the NES and Ran, a Ras-like GTPase. In the nucleus CRMI forms a complex with the NES-protein and Ran/GTP, then the complex is translocated through the nuclear pore complex into the cytoplasm. There, the Ran GTPase activating protein (RanGAP), found only in the cytoplasm, promotes hydrolysis of Ran/GTP to Ran/GDP, causing release of the NES-protein.
The high potency and novel mechanism of action prompted an investigation of the antitumor activity of LMB in mouse murine and xenograph cancer models. Activity was observed at low doses against adriamycin, amsacrine and mitoxantrone resistant P388 leukemia, other leukemias, B16 melanoma, Ridgway osteogenic and M5076 sarcomas and mammary adenocarinoma. Acute toxicity appeared to be gastrointestinal and was exacerbated upon more frequent or oral administration of the drug. The maximum tolerated dose (MTD) in mice ranged from 0.12 to 1 mg/kg, as a function of dosing schedule.
LMB has also attracted considerable interest as a biochemical tool to study the role and regulation of nucleo-cytoplasmic shuttling proteins and for its potential therapeutic use in combination with other drugs. Vigneri and Wang, “Induction of apoptosis in chronic myelogenous leukemia cells through nuclear entrapment of BCR-ABL tyrosine kinase,” Nature Medicine (2001) 7:228-234, describes combined treatment of cultured CML cells with STI-571 and LMB. STI-571 effectively masks the ability of Bcr-AbI to be retained preferentially in the cytoplasm; upon nuclear importation of the drug-inactivated protein, LMB inhibits nuclear export of Bcr-AbI and withdrawal of STI-571 releases the ability of the constitutively activated AbI component to induce apoptosis. While the effect of either drug alone is fully reversible (STI-571 does not permanently inhibit Bcr-AbI and nuclear export is restored by synthesis of fresh CRM1), their combined use caused irreversible and complete killing of the Bcr-Abl transformed cells. Such treatment also preferentially eliminated mouse bone marrow cells that express Bcr-Abl. This strategy can overcome the main limitation of acute CML treatment with STI-571, which is acquired drug resistance due to mutation or overexpression of Bcr-AbI.
LMB has other types of potential therapeutic uses. Because it can promote nuclear retention of the p53 tumor suppressor protein, treatment with LMB can lead to p53 activation in the nucleus, which results in cell-cycle arrest and apoptosis. Combined use LMB and actinomycin D can reactivate p53 and prevent its degradation by HPV E6 protein in cervical carcinoma cells infected with human papillomavirus. LMB can also potentiate the effect of rapamycin, an emerging cancer drug, by blocking nuclear export of mTOR, the protein kinase target of rapamycin that controls the activity of two transcription factors. The antiviral activity of LMB has been elucidated as resulting from inhibition of the nuclear export of the HIV-1 Rev protein and Rev-dependent unspliced and partially spliced mRNA, which is an early step in viral replication. LMB interferes with cyclinB1/Cdc2, cyclinD1/CDK4 and TGF-beta dependent signaling also, suggesting possible uses against cancers with aberrant signaling involving these actors. A synthetic HIV-1 Rev inhibitor, PKF050-638 (FIG. 2), has been developed that mimics the activity of LMB.
Two limitations have to be overcome to increase the potential for development of LMB into an effective anticancer or antiviral drug. One, a reliable source of pure drug must be developed, because “The use of LMB . . . has been hampered by the variability of the quality of LMB production lots” (D. Daelemans et al. 2002, “A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export” Proc. Natl. Acad Sci. USA 99: 14440-5). This is not surprising given the close structural similarity of leptomycin-like compounds isolated from their natural sources (FIG. 1). In fact, at least 5 different forms of leptomycins have been detected in the culture extracts of the ATCC 39366 strain and 6 forms in another LMB producer. Two, a less toxic form of LMB would be more appealing for drug development studies. Even though the drug's effects have been reported to be fully reversible, toxicity is likely to be mechanism-related and exhibited in different bodily tissues given the widespread role of CRM1-mediated protein export. The available SAR data (FIG. 2) are insufficient for designing a less toxic analog. Analog production and evaluation will require both chemical and microbiological approaches, because little effort towards the total synthesis of LMB has been reported.
The following data suggest that analogs with an acceptable therapeutic index could be found. LMB displayed an approx. 250-fold difference in activity between a Rev-dependent assay and cytotoxicity to the same cells in vitro and PKF050-638 had a 75-fold difference in the same two assays (FIG. 2). These data show that LMB itself can have a good therapeutic window in certain instances. It is thus likely that less toxic LMB analogs can be discovered as a consequence of differential binding to CRMI or pharmacokinetic behavior that modulates their distribution, half-life or metabolism.
Given the promise of leptomycin B in the treatment of conditions and diseases characterized by undesired cellular hyperproliferation, there thus exists an unmet need for a production system that can provide large quantities of leptomycin B in a form substantially free of minor congeners and other impurities. The present invention meets this need by providing the biosynthetic genes responsible for the production of leptomycins and providing for their expression in heterologous hosts. Further, there is an unmet need for analogs of leptomycins potentially useful in the treatment of viral diseases. The present invention meets this need by providing the means for biological generation of leptomycin analogs through genetic engineering of the biosynthetic genes.
SUMMARY OF THE INVENTION
The present invention provides recombinant nucleic acids encoding polyketide synthases and polyketide modification enzymes. The recombinant nucleic acids of the invention are useful in the production of polyketides, including but not limited to leptomycin and leptomycin analogs and derivatives in recombinant host cells.
In one aspect, the invention provides the nucleic acids involved in leptomycin biosynthesis in isolated, purified, recombinant, or synthetic form, including but not limited to sequences incorporated into a vector or into the chromosome of a host cell. The biosynthesis of leptomycin is performed by a modular PKS and polyketide modification enzymes. The leptomycin polyketide synthase (herein also “leptomycin PKS” or “leptomycin synthase”) is made up of several proteins, each having one or more modules. The modules have domains with specific synthetic functions.
In another aspect, the present invention provides domains and modules of the leptomycin PKS and corresponding nucleic acid sequences encoding them and/or parts thereof. Such compounds are useful in the production of hybrid PKS enzymes and the recombinant genes that encode them.
In another aspect, the present invention provides modifying genes of leptomycin biosynthetic gene cluster in recombinant form, including but not limited to isolated form and incorporated into a vector or the chromosomal DNA of a host cell. Such compounds are useful in the production of leptomycins, leptomycin analogs, and leptomycin derivatives according to the methods of the invention.
In another aspect the invention provides a recombinant PKS wherein at least 10, 15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of leptomycin polyketide synthase. Preferably at least an entire domain of a module of leptomycin synthase is included. Representative leptomycin PKS domains useful in this aspect of the invention include, for example, KR, DH, ER, AT, ACP and KS domains. In one embodiment of the invention, the PKS is assembled from polypeptides encoded by DNA molecules that comprise coding sequences for PKS domains, wherein at least one encoded domain corresponds to a domain of leptomycin PKS. In such DNA molecules, the coding sequences are operably linked to control sequences so that expression therefrom in host cells is effective. In this manner, leptomycin PKS coding sequences or modules and/or domains can be made to encode PKS to biosynthesize compounds having antibiotic or other useful bioactivity other than leptomycin.
In one embodiment, the invention provides a recombinant DNA molecule that comprises a sequence encoding a chimeric polyketide synthase composed of at least a portion of the leptomycin PKS and at least a portion of a second PKS for a polyketide other than leptomycin. Such chimeric genes are useful in the production of leptomycin analogs, leptomycin derivatives, and other polyketides.
In another aspect, the present invention provides recombinant host cells that contain the nucleic acids of the invention. In one embodiment, the host cell provided by the invention is a Streptomyces host cell that produces a leptomycin modification enzyme and/or a domain, module, or protein of the leptomycin PKS. Methods for the genetic manipulation of Streptomyces are described in Kieser et al, “Practical Streptomyces Genetics,” The John Innes Foundation, Norwich (2000), which is incorporated herein by reference in its entirety. In other embodiments, the host cells provided by the invention are eubacterial cells such as Escherichia coli, yeast cells such as Saccharomyces cerevisiae, or myxobacterial cells such as Myxococcus xanthus. 
In another embodiment, the invention provides a recombinant Streptomyces host cell that produces leptomycin in its native state, wherein at least one domain-encoding region of the endogenous leptomycin PKS gene is deleted, inactivated, or replaced. Also provided is a recombinant Streptomyces host cell that produces leptomycin in its native state, wherein at least one polypeptide-encoding open reading frame of the leptomycin PKS gene cluster is deleted or otherwise inactivated.
In another aspect, the invention also provides methods for producing leptomycins, leptomycin analogs and derivatives, and other polyketides using the nucleic acids, proteins, vectors, and host cells of the invention.
These and other aspects of the present invention are described in more detail in the Detailed Description of the Invention, below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows various members of the leptomycin family of natural polyketides.
FIG. 2 shows biological activity results for several members of the leptomycin family, and the structure of ratjadone.
FIG. 3 shows the expected organization of the leptomycin PKS and a possible pathway for biosynthesis. Biosynthetic relationships of members of the leptomycin family are also indicated.
FIG. 4 shows the organization of the leptomycin biosynthetic gene cluster as deduced from SEQ ID NO:1. FIG. 4A shows the organization of the portion of the gene cluster comprising LepA. FIG. 4B shows the organization of the portion of the gene cluster comprising LepB. FIG. 4C shows the organization of the portion of the gene cluster comprising LepC, LepD, LepE, and LepF.
FIG. 5 shows the DNA sequence of the leptomycin biosynthetic gene cluster.
FIG. 6 shows SEQ ID NO: 4, the polypeptide product of lepA, a gene in the leptomycin PKS cluster.
FIG. 7 shows SEQ ID NO: 5, the polypeptide product of lepB, a gene in the leptomycin PKS cluster.
FIG. 8 shows SEQ ID NO: 6, the polypeptide product of lepC, a gene in the leptomycin PKS cluster.
FIG. 9 shows SEQ ID NO: 7, the polypeptide product of lepD, a gene in the leptomycin PKS cluster.
FIG. 10 shows SEQ ID NO: 8, the polypeptide product of lepE, a gene encoding a cytochrome P450-type oxidase.
FIG. 11 shows SEQ ID NO: 9, the polypeptide product of lepF, a gene encoding a tetR-like transcriptional regulator.



The following references provide background on the leptomycins and are hereby incorporated by reference:

    1) Wolff B, Sanglier J J, Wang Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem Biol. (1997) 4:139-147.
    2) Lain S, Midgley C, Sparks A, Lane E B, Lane D P. An inhibitor of nuclear export activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODs. Exp Cell Res. (1999) 248:457-72
    3) Hietanen S, Lain S, Krausz E, Blattner C, Lane D P. Activation of p53 in cervical carcinoma cells by small molecules. Proc Natl Acad Sci USA. (2000) 97:8501-8506.
    4) Kim J E, Chen J. Cytoplasmic-nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin-sensitive signaling and translation initiation. Proc Natl Acad Sci USA. (2000) 97:14340-14345.
    5) Park I H, Bachmann R, Shirazi H, Chen J. Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin. J Biol Chem. (2002) 277:31423-31429.
    6) Daelemans D, Afonina E, Nilsson J, Werner G, Kjems J, De Clercq E, Pavlakis G N, Vandamme A M. A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export. Proc Natl Acad Sci USA. (2002) 99:14440-14445.
    7) Wang, Y, Ponelle M et al., Novel leptomycins for a Streptomyces strain A92-308902. Inhibitors of the nucelo-cytoplasmic translocation of the HIV-1 regulatory protein Rev. Helv Chim Acta (1997) 80:2157-2167.
    8) Kalesse M, M. Christmann. The chemistry and biology of the leptomycin family. Synthesis (2002) 8:981-1003.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides recombinant materials for the production of polyketides. In one aspect, the invention provides recombinant nucleic acids encoding at least one domain of a polyketide synthase required for leptomycin biosynthesis. Methods and host cells for using these genes to produce a polyketide in recombinant host cells are also provided.
The nucleotide sequences encoding leptomycin PKS domains, modules and polypeptides of the present invention were isolated from Streptomyces sp. ATCC 39366 as described in Example 1. Alternatively, the DNA sequences provided herein may be obtained through gene synthesis as described in U.S. Patent Application 20040166567, which is incorporated herein by reference. Given the valuable properties of leptomycin and its derivatives and analogs, means to produce useful quantities of these molecules in a highly pure form is of great potential value. The compounds produced may be used as antitumor agents or for other therapeutic uses, and/or a intermediates for further enzymatic or chemical modification, and/or as agents for in vitro inhibition of protein phosphatase. The nucleotide sequences of the leptomycin biosynthetic gene cluster encoding domains, modules and polypeptides of leptomycin synthase, and modifying enzymes, and other polypeptides can be used, for example, to make both known and novel polyketides.
In one aspect of the invention, purified and isolated DNA molecules are provided that comprise one or more coding sequences for one or more domains or modules of leptomycin synthase. Examples of such encoded domains include leptomycin synthase KR, DH, ER, AT, ACP, and KS domains. In one aspect, the invention provides DNA molecules in which sequences encoding one or more polypeptides of leptomycin synthase are operably linked to expression control sequences that are effective in suitable host cells to produce leptomycin, its analogs or derivatives, or novel polyketides.
The sequence of the leptomycin gene cluster was assembled from sequences deduced from the cosmids pKOS279-128.PF27, pKOS279-128.2L78, and pKOS279-130.PFA42. The gene cluster is found to comprise six open reading frames (ORFs), named lepA, lepB, lepC, lepD, lepE, and lepF. The polyketide synthase is encoded by lepABCD, and is comprised of eleven modules terminating in a thioesterase domain. The lepA gene encodes modules 0-4, where module 0 is the loading module; the lepB gene encodes modules 5-8; lepC encodes modules 9-10; and lepD encodes module 11 and the terminating thioesterase domain. The lepE gene encodes a cytochrome P450-type oxidase, presumably responsible for oxidation of the C24 methyl group. The lepF gene appears to be a regulatory gene.
Tables 1 and 2 provide a description of genes in the leptomycin PKS gene cluster including sequences encoding encoding modules, domains and ORFs, as deduced from two contigs assembled from sequences of pKOS279-125.2L78. The nucleotide sequences of the two contigs are provided in the attached Sequence Listing, and have been assigned SEQ ID NOS: 1 and 2, respectively.
As indicated in Table 1, the nucleic acid having SEQ ID NO:1 was found to encode portions of two ORFs. ORF1, SEQ ID NO:6, comprises nucleotides <1 to 17260 of the SEQ ID NO:1. The start of ORF1 (LepA) lies upstream of the beginning of SEQ ID NO:1. The nucleic acid encodes a polypeptide comprising a portion of module 1 (a portion of KR1 and all of ACP1), and the complete modules 2, 3, and 4 of leptomycin synthase. The sequence of ORF2 comprises nucleotides 17546 to >29467 of the SEQ ID NO:1. The end of ORF2 (LepB) lies downstream of the end of SEQ ID NO:1. The nucleic acid sequence encodes a polypeptide comprising the complete modules 5 and 6 and a portion of module 7 (the beginning of KS7) of leptomycin synthase. The modules encoded by the nucleic acid of SEQ ID NO:1 are indicated in Table 1.
Table 2 provides the ORF, module, and domain descriptions for the second contig, the nucleic acid of SEQ ID NO:2. One partial ORF has been identified, encoding a polypeptide comprising a portion of module 7 (part of AT7, and all of DH7, ER7, KR7, and ACP7) and all of module 8 of leptomycin synthase. The modules encoded by the nucleic acid of SEQ ID NO:2 and domains within each module are indicated in Table 2.
Subsequent sequencing provided the complete sequence of the leptomycin biosynthetic gene cluster, given below as SEQ ID NO:3. The PKS modules encoded by the nucleic acid of SEQ ID NO:3 and domains within each module are indicated in Table 3. The ORFs encoding the PKS have been designated LepA, LepB, LepC, and LepD. LepA comprises a loading module, referred to as “module 0,” which comprises a ketosynthase domain wherein there is a glutamine in place of the expected active-site cysteine (“KSq”), and thus likely funcations as a decarboxylase. LepD comprises module 11 together with a thioesterase (TE) domain.
The LepE gene, corresponding to nucleotides 64703-65881 of SEQ ID NO:3 encodes a cytochrome-P450 type oxidase. The LepF gene, corresponding to nucleotides 66124-66564 of SEQ ID NO:3 encodes a putative tetR-family transcriptional regulator.
In another aspect of the invention, the polypeptides encoded by the above-described leptomycin PKS genes are provided as LepA (FIG. 6; SEQ ID NO:4), LepB (FIG. 7; SEQ ID NO:5), LepC (FIG. 8; SEQ ID NO:6), LepD (FIG. 9; SEQ ID NO:7), LepE (FIG. 10; SEQ ID NO:8), and LepF (FIG. 11; SEQ ID NO:9). These polypeptides may be in isolated, purified, or recombinant form, either singly or present in any combination comprising each other or other polyketide synthase polypeptides.







TABLE 1







ORFs, modules, and domains of the leptomycin PKS determined


from the nucleotide sequence determined from the T3-side of


the insert from cosmid pKOS279-125.2L78 (SEQ ID NO: 1).











Nucleotide



feature
sequence location

















Contig 1

  1-29467



ORF 1


  <1-17260




module 1

 <1-661





KR1
 <1-358





ACP1
404-661




module 2

 722-6868





KS2
 722-1999





AT2
2306-3352





DH2
3386-3991





ER2
4910-5770





KR2
5753-6571





ACP2
6611-6868




module 3

 6929-12172





KS3
6929-8206





AT3
8537-9595





DH3
 9629-10204





KR3
11057-11881





ACP3
11915-12172




module 4

12236-17260





KS4
12236-13513





AT4
13823-14869





DH4
14903-15493





KR4
16298-16807





ACP4
17003-17260



ORF2 (start)


 17546->29467




module 5

17546-22879





KS5
17648-18925





AT5
19328-20299





DH5
20333-20932





KR5
21758-22603





ACP5
22622-22879




module 6

22961-28144





KS6
22961-24241





AT6
24551-25603





DH6
25652-26206





KR6
27014-27868





ACP6
27887-28144




module 7

 28199->29467





KS7
 28199->29467

















TABLE 2







ORFs, modules, and domains of the leptomycin PKS determined


from the nucleotide sequence determined from the T7-side of


the insert from cosmid pKOS279-125.2L78 (SEQ ID NO: 2).










feature
sequence location

















contig 2

  1-9724



ORF 2 (end)


  <1->9724




module 7

 <1-4501





AT7
 <1-967





DH7
1001-1585





ER7
2528-3382





KR7
3380-4225





ACP7
4244-4501




module 8

4559-9703





KS8
4559-5836





AT8
6152-7213





DH8
7250-7822





KR8
8639-9409





ACP8
9446-9703

















TABLE 3







Complete list of ORFs, modules, and domains of the


leptomycin PKS determined from SEQ ID NO: 3.











Nucleotide



feature
sequence location
















LepA


 370-25686




module 0
KSq(0)
 439-1725





AT(0)
2080-3147





ACP(0)
3220-3481




module 1

3535-8844





KS(1)
3535-4812





AT(1)
5143-6204





DH(1)
6241-6831





KR(1)
7759-8547





ACP(1)
8587-8844




module 2

 8905-15048





KS2
 8905-10182





AT2
10489-11535





DH2
11569-12147





ER2
13093-13953





KR2
13936-14751





ACP2
14791-15048




module 3

15109-20361





KS3
15109-16386





AT3
16717-17775





DH3
17809-16384





KR3
19237-20070





ACP3
20104-20361




module 4

20425-25449





KS4
20425-21702





AT4
22012-23058





DH4
23092-23682





KR4
24487-24996





ACP4
25192-25449



LepB


25735-48024




module 5

25837-31068





KS5
25837-27114





AT5
27427-28488





DH5
28522-29121





KR5
29947-30792





ACP5
30811-31068




module 6

31150-36333





KS6
31150-32430





AT6
32740-33792





DH6
33841-34395





KR6
35203-36057





ACP6
36076-36333




module 7

36388-42570





KS7
36388-37665





AT7
37981-39036





DH7
39070-39654





ER7
40597-41451





KR7
41449-42294





ACP7
42313-42570




module 8

42628-47772





KS8
42628-43905





AT8
44221-45282





DH8
45319-45891





KR8
46708-47478





ACP8
47515-47772



LepC


48110-58357




module 9

48209-53437





KS9
48209-49417





AT9
49775-50824





DH9
50864-51454





KR9
52325-53140





ACP9
53180-53437




module 10

53501-58111





KS10
53501-54781





AT10
55115-56173





KR10
56975-57637





ACP10
57854-58111



LepD


58243-64173




module 11

58543-59847





KS11
58543-59847





AT11
60250-61257





KR11
62143-62931





ACP11
62995-63252




TE

63253-64170










In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one domain, alternatively at least one module, alternatively at least one polypeptide, involved in the biosynthesis of a leptomycin.
In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to at least one of SEQ ID NOS: 1, 2, and 3 or their complement. [Hereinafter, each reference to a nucleic acid sequence is also intended to refer to and include the complementary sequence, unless otherwise stated or apparent from context.] In an embodiment the subsequence comprises a sequence encoding a complete leptomycin PKS domain, module or polypeptide.
In one aspect, the present invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes an open reading frame, module or domain having an amino acid sequence identical or substantially similar to an ORF, module or domain encoded by SEQ ID NOS: 1, 2 or 3. Generally, a polypeptide, module or domain having a sequence substantially similar to a reference sequence has substantially the same activity as the reference protein, module or domain (e.g., when integrated into an appropriate PKS framework using methods known in the art). In certain embodiments, one or more activities of a substantially similar polypeptide, module or domain are modified or inactivated as described below.
In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ID NOs:1, 2 or 3, e.g., a polypeptide, module or domain involved in the biosynthesis of a leptomycin, wherein said nucleotide sequence comprises at least 10, 20, 25, 30, 35, 40, 45, or 50 contiguous base pairs identical to a sequence of SEQ ID NOS: 1, 2 or 3. In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ID NOS: 1, 2 or 3, e.g., a polypeptide, module or domain involved in the biosynthesis of a leptomycin, wherein said polypeptide, module or domain comprises at least 10, 15, 20, 30, or 40 contiguous residues of a corresponding polypeptide, module or domain.
It will be understood that SEQ ID NOS: 1, 2 and 3 were determined using the insert of various cosmids. Accordingly, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to a ORF encoding sequence of the insert of one or more of these cosmids.
Those of skill will recognize that, due to the degeneracy of the genetic code, a large number of DNA sequences encode the amino acid sequences of the domains, modules, and proteins of the leptomycin PKS, the enzymes involved in leptomycin modification and other polypeptides encoded by the genes of the leptomycin biosynthetic gene cluster. The present invention contemplates all such DNAs. For example, it may be advantageous to optimize sequence to account for the codon preference of a host organism. The invention also contemplates naturally occurring genes encoding the leptomycin PKS that are polymorphic or other variants.
As used herein, the terms “substantial identity,” “substantial sequence identity,” or “substantial similarity” in the context of nucleic acids, refers to a measure of sequence similarity between two polynucleotides. Substantial sequence identity can be determined by hybridization under stringent conditions, by direct comparison, or other means. For example, two polynucleotides can be identified as having substantial sequence identity if they are capable of specifically hybridizing to each other under stringent hybridization conditions. Other degrees of sequence identity (e.g., less than “substantial”) can be characterized by hybridization under different conditions of stringency. “Stringent hybridization conditions” refers to conditions in a range from about 5° C. to about 20° C. or 25° C. below the melting temperature (Tm) of the target sequence and a probe with exact or nearly exact complementarity to the target. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., Berger and Kimmel, 1987, Methods In Enzymology, Vol. 152: Guide To Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory). Typically, stringent hybridization conditions for probes greater than 50 nucleotides are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 50° C., preferably at least about 60° C. As noted, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed. Exemplary conditions include hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 pH 7.0, 1 mM EDTA at 65° C.; wash with 2×SSC, 1% SDS, at 50° C.
Alternatively, substantial sequence identity can be described as a percentage identity between two nucleotide or amino acid sequences. Two nucleic acid sequences are considered substantially identical when they are at least about 70% identical, or at least about 80% identical, or at least about 90% identical, or at least about 95% or 98% identical. Two amino acid sequences are considered substantially identical when they are at least about 60%, sequence identical, more often at least about 70%, at least about 80%, or at least about 90% sequence identity to the reference sequence. Percentage sequence (nucleotide or amino acid) identity is typically calculated using art known means to determine the optimal alignment between two sequences and comparing the two sequences. Optimal alignment of sequences may be conducted using the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by the BLAST algorithm of Altschul (1990) d. Mol. Biol. 215: 403-410; and Shpaer (1996) Genomics 38:179-191, or by the Needleham et al. (1970) J. Mol. Biol. 48: 443-453; and Sankoff et al., 1983, Time Warps, String Edits, and Macromolecules, The Theory and Practice of Sequence Comparison, Chapter One, Addison-Wesley, Reading, Mass.; generally by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.; BLAST from the National Center for Biotechnology Information at the World Wide Web (ncbi.nlm.nih.gov). In each case default parameters are used (for example the BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands).
The invention methods may be directed to the preparation of an individual polyketide. The polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it. The resulting polyketides may be further modified to convert them to other useful compounds. Examples of chemical structures of that can be made using the materials and methods of the present invention include known analogs, such as those described in Kalesse & Christmann, 2002, “The Chemistry and Biology of the Leptomycin Family” Synthesis (8):981-1003 (incorporated herein by reference) and the references cited therein, and novel molecules produced by modified or chimeric PKSs comprising a portion of the leptomycin PKS sequence, molecules produced by the action of polyketide modifying enzymes from the leptomycin PKS cluster on products of other PKSs, molecules produced by the action on products of the leptomycin PKS of polyketide modifying enzymes from other PKSs, and the like.
As noted, in one aspect the invention provides recombinant PKS wherein at least 10, 15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of leptomycin polyketide synthase. A polyketide synthase “derived from” a naturally occurring PKS contains the scaffolding encoded by all the portion employed of the naturally occurring synthase gene, contains at least two modules that are functional, and contains mutations, deletions, or replacements of one or more of the activities of these functional modules so that the nature of the resulting polyketide is altered. This definition applies both at the protein and genetic levels. Particular embodiments include those wherein a KS, AT, KR, DH, or ER has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS, and derivatives where at least one noncondensation cycle enzymatic activity (KR, DH, or ER) has been deleted or wherein any of these activities has been added or mutated so as to change the ultimate polyketide synthesized. There are at least five degrees of freedom for constructing a polyketide synthase in terms of the polyketide that will be produced. See, U.S. Pat. No. 6,509,455 for a discussion.
As can be appreciated by those skilled in the art, polyketide biosynthesis can be manipulated to make a product other than the product of a naturally occurring PKS biosynthetic cluster. For example, AT domains can be altered or replaced to change specificity. The variable domains within a module can be deleted and or inactivated or replaced with other variable domains found in other modules of the same PKS or from another PKS. See e.g., Katz & McDaniel, Med Res Rev 19: 543-558 (1999) and WO 98/49315. Similarly, entire modules can be deleted and/or replaced with other modules from the same PKS or another PKS. See e.g., Gokhale et al., Science 284: 482 (1999) and WO 00/47724 each of which are incorporated herein by reference. Protein subunits of different PKSs also can be mixed and matched to make compounds having the desired backbone and modifications. For example, subunits of 1 and 2 (encoding modules 1-4) of the pikromycin PKS were combined with the DEBS3 subunit to make a hybrid PKS product (see Tang et al., Science, 287: 640 (2001), WO 00/26349 and WO 99/6159).
Mutations can be introduced into PKS genes such that polypeptides with altered activity are encoded. Polypeptides with “altered activity” include those in which one or more domains are inactivated or deleted, or in which a mutation changes the substrate specificity of a domain, as well as other alterations in activity. Mutations can be made to the native sequences using conventional techniques. The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. (See, e.g., Kunkel, T. A. Proc Natl Acad Sci USA (1985) 82 448; Geisselsoder et al. BioTechniques (1987) 5:786.) Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. (See Zoller and Smith, Methods in Enzymology (1983) 100: 468). Primer extension is effected using DNA polymerase. The product of the extension reaction is cloned, and those clones containing the mutated DNA are selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. (See, e.g., Dalbie-McFarland et al. Proc Natl Acad Sci USA (1982) 79:6409). PCR mutagenesis can also be used for effecting the desired mutations. Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid,
In addition to providing mutated forms of regions encoding enzymatic activity, regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS synthase can be recovered, for example, using PCR techniques with appropriate primers. By “corresponding” activity encoding regions is meant those regions encoding the same general type of activity—e.g., a ketoreductase activity in one location of a gene cluster would “correspond” to a ketoreductase-encoding activity in another location in the gene cluster or in a different gene cluster; similarly, a complete reductase cycle could be considered corresponding—e.g., KR/DH/ER could correspond to KR alone.
If replacement of a particular target region in a host polyketide synthase is to be made, this replacement can be conducted in vitro using suitable restriction enzymes or can be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene. One such system involving plasmids of differing temperature sensitivities is described in PCT application WO 96/40968. Another useful method for modifying a PKS gene (e.g., making domain substitutions or “swaps”) is a RED/ET cloning procedure developed for constructing domain swaps or modifications in an expression plasmid without first introducing restriction sites. The method is related to ET cloning methods (see, Datansko & Wanner, 2000, Proc. Natl. Acad. Sci. U.S.A. 97, 6640-45; Muyrers et al, 2000, Genetic Engineering 22:77-98). The RED/ET cloning procedure is used to introduce a unique restriction site in the recipient plasmid at the location of the targeted domain. This restriction site is used to subsequently linearize the recipient plasmid in a subsequent ET cloning step to introduce the modification. This linearization step is necessary in the absence of a selectable marker, which cannot be used for domain substitutions. An advantage of using this method for PKS engineering is that restriction sites do not have to be introduced in the recipient plasmid in order to construct the swap, which makes it faster and more powerful because boundary junctions can be altered more easily.
In a further aspect, the invention provides methods for expressing chimeric or hybrid PKSs and products of such PKSs. For example, the invention provides (1) encoding DNA for a chimeric PKS that is substantially patterned on a non-leptomycin producing enzyme, but which includes one or more functional domains, modules or polypeptides of leptomycin PKS; and (2) encoding DNA for a chimeric PKS that is substantially patterned on the leptomycin PKS, but which includes one or more functional domains, modules, or polypeptides of another PKS or NRPS.
With respect to item (1) above, in one embodiment, the invention provides chimeric PKS enzymes in which the genes for a non-leptomycin PKS function as accepting genes, and one or more of the above-identified coding sequences for leptomycin domains or modules are inserted as replacements for one or more domains or modules of comparable function. Construction of chimeric molecules is most effectively achieved by construction of appropriate encoding polynucleotides. In making a chimeric molecule, it is not necessary to replace an entire domain or module accepting of the PKS with an entire domain or module of leptomycin PKS: subsequences of a PKS domain or module that correspond to a peptide subsequence in an accepting domain or module, or which otherwise provide useful function, may be used as replacements. Accordingly, appropriate encoding DNAs for construction of such chimeric PKS include those that encode at least 10, 15, 20 or more amino acids of a selected leptomycin domain or module.
Recombinant methods for manipulating modular PKS genes to make chimeric PKS enzymes are described in U.S. Pat. Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos. 98/49315 and 97/02358. A number of genetic engineering strategies have been used with DEBS to demonstrate that the structures of polyketides can be manipulated to produce novel natural products, primarily analogs of the erythromycins (see the patent publications referenced supra and Hutchinson, 1998, Curr Opin Microbiol. 1:319-329, and Baltz, 1998, Trends Microbiol. 6:76-83). In one embodiment, the components of the chimeric PKS are arranged onto polypeptides having interpolypeptide linkers that direct the assembly of the polypeptides into the functional PKS protein, such that it is not required that the PKS have the same arrangement of modules in the polypeptides as observed in natural PKSs. Suitable interpolypeptide linkers to join polypeptides and intrapolypeptide linkers to join modules within a polypeptide are described in PCT publication WO 00/47724.
A partial list of sources of PKS sequences for use in making chimeric molecules, for illustration and not limitation, includes Avermectin (U.S. Pat. No. 5,252,474; MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256; MacNeil et al., 1992, Gene 115: 119-25); Candicidin (FRO008) (Hu et al., 1994, Mol. Microbiol. 14: 163-72); Epothilone (U.S. Pat. No. 6,303,342); Erythromycin (WO 93/13663; U.S. Pat. No. 5,824,513; Donadio et al., 1991, Science 252: 675-79; Cortes et al., 1990, Nature 348: 176-8); FK-506 (Motamedi et al., 1998, Eur. J. Biochem. 256: 528-34; Motamedi et al., 1997, Eur. J. Biochem. 244:74-80); FK-520 (U.S. Pat. No. 6,503,737; see also Nielsen et al., 1991, Biochem. 30:5789-96); Lovastatin (U.S. Pat. No. 5,744,350); Nemadectin (MacNeil et al., 1993, supra); Niddamycin (Kakavas et al., 1997, J. Bacteriol. 179:7515-22); Oleandomycin (Swan et al., 1994, Mol. Gen. Genet. 242:358-62; U.S. Pat. No. 6,388,099; Olano et al., 1998, Mol. Gen. Genet. 259:299-308); Platenolide (EP Pat. App. 791,656); Rapamycin (Schwecke et al., 1995, Proc. Natl. Acad. Sci. USA 92:7839-43); Aparicio et al., 1996, Gene 169:9-16); Rifamycin (August et al., 1998, Chemistry & Biology, 5:69-79); Soraphen (U.S. Pat. No. 5,716,849; Schupp et al., 1995, J. Bacteriology 177: 3673-79); Spiramycin (U.S. Pat. No. 5,098,837); Tylosin (EP 0 791,655; Kuhstoss et al., 1996, Gene 183:231-36; U.S. Pat. No. 5,876,991). Additional suitable PKS coding sequences remain to be discovered and characterized, but will be available to those of skill (e.g., by reference to GenBank).
The leptomycin PKS-encoding polynucleotides of the invention may also be used in the production of libraries of PKSs (i.e., modified and chimeric PKSs comprising at least a portion of the leptomycin PKS sequence. The invention provides libraries of polyketides by generating modifications in, or using a portion of, the leptomycin PKS so that the protein complexes produced by the cluster have altered activities in one or more respects, and thus produce polyketides other than the natural leptomycin product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method. By providing a large number of different genes or gene clusters derived from a naturally occurring PKS gene cluster, each of which has been modified in a different way from the native PKS cluster, an effectively combinatorial library of polyketides can be produced as a result of the multiple variations in these activities. Expression vectors containing nucleotide sequences encoding a variety of PKS systems for the production of different polyketides can be transformed into the appropriate host cells to construct a polyketide library. In one approach, a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected for successful transformants. Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide. A variety of strategies can be devised to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide. The number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products. The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large. The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity. See, for example,
Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of candidate polyketides. The polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like. The supernatants per se can be used for screening, or partial or complete purification of the polyketides can first be effected. Typically, such screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members of the library can be tested against a desired target. In this event, screens wherein the biological response of the target is measured can be included.
As noted above, the DNA compounds of the invention can be expressed in host cells for production of proteins and of known and novel compounds. Preferred hosts include fungal systems such as yeast and procaryotic hosts, but single cell cultures of, for example, mammalian cells could also be used. A variety of methods for heterologous expression of PKS genes and host cells suitable for expression of these genes and production of polyketides are described, for example, in U.S. Pat. Nos. 5,843,718 and 5,830,750; WO 01/31035, WO 01/27306, and WO 02/068613; and U.S. patent application Ser. Nos. 10/087,451 (published as US2002000087451); 60/355,211; and 60/396,513 (corresponding to published application 20020045220).
Appropriate host cells for the expression of the hybrid PKS genes include those organisms capable of producing the needed precursors, such as malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-ACP, and having phosphopantotheinylation systems capable of activating the ACP domains of modular PKSs. See, for example, U.S. Pat. No. 6,579,695. However, as disclosed in U.S. Pat. No. 6,033,883, a wide variety of hosts can be used, even though some hosts natively do not contain the appropriate post-translational mechanisms to activate the acyl carrier proteins of the synthases. Also see WO 97/13845 and WO 98/27203. The host cell may natively produce none, some, or all of the required polyketide precursors, and may be genetically engineered so as to produce the required polyketide precursors. Such hosts can be modified with the appropriate recombinant enzymes to effect these modifications. Suitable host cells include Streptomyces, E. coli, yeast, and other procaryotic hosts that use control sequences compatible with Streptomyces spp. Examples of suitable hosts that either natively produce modular polyketides or have been engineered so as to produce modular polyketides include but are not limited to actinomyctes such as Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces fradiae, Streptomyces ambofaciens, and Saccharopolyspora erythraea, eubacteria such as Escherichia coli, myxobacteria such as Myxococcus xanthus, and yeasts such as Saccharomyces cerevisiae. 
In one embodiment, any native modular PKS genes in the host cell have been deleted to produce a “clean host,” as described in U.S. Pat. No. 5,672,491, incorporated herein by reference. In a variant of this embodiment, a host cell that produces leptomycin, a leptomycin analog, or a leptomycin derivative in its native state (e.g., Streptomyces sp. ATCC 39366) is engineered so as to delete or inactivate at least one domain in the leptomycin PKS gene cluster so as to produce a host cell that no longer produces leptomycin, a leptomycin analog, or a leptomycin derivative. Such a host cell can subsequently be transformed with a gene comprising an active variant of the deleted or inactivated domain, thus restoring polyketide production by complementation. When the active variant of the deleted or inactivated domain is derived from a second PKS gene cluster that produces a polyketide other than leptomycin, such complementation results in the production of a leptomycin analog or derivative. In one embodiment, one or more complete genes (ORFs) of the native leptomycin synthase are deleted from or inactivated in the host cell, which is subsequently complemented by transformation with engineered forms of the deleted or inactivated genes (ORFs). Methods for performing such complementation experiments are known in the art, for example as described in U.S. Pat. No. 6,505,737 which is incorporated herein by reference.
In some embodiments, the host cell expresses, or is engineered to express, a polyketide “tailoring” or “modifying” enzyme. Once a PKS product is released, it is subject to post-PKS tailoring reactions. These reactions are important for biological activity and for the diversity seen among polyketides. Tailoring enzymes normally associated with polyketide biosynthesis include oxygenases, glycosyl- and methyl-transferases, acyltransferases, halogenases, cyclases, aminotransferases, and hydroxylases. In addition to biosynthetic accessory activities, secondary metabolite clusters often code for activities such as transport. In the case of leptomycin biosynthesis (FIG. 3), tailoring enzymes are expected to include at least one P450 hydroxylase for oxidation of the C24 methyl group to a carboxylic acid. Tailoring enzymes may also be involved in the introduction of the cis-alkene at C8-C9.
Tailoring enzymes for modification of a product of the leptomycin PKS, a non-leptomycin PKS, or a chimeric PKS, can be those normally associated with leptomycin biosynthesis or “heterologous” tailoring enzymes. Tailoring enzymes can be expressed in the organism in which they are naturally produced, or as recombinant proteins in heterologous hosts. In some cases, the structure produced by the heterologous or hybrid PKS may be modified with different efficiencies by post-PKS tailoring enzymes from different sources. In such cases, post-PKS tailoring enzymes can be recruited from other pathways to obtain the desired compound. For example, the tailoring enzymes of the leptomycin PKS gene cluster can be expressed heterologously to modify polyketides produced by non-leptomycin synthases or can be inactivated in the Leptomycin producer.
Alternatively, the unmodified polyketide compounds can be produced in the recombinant host cell, and the desired modification (e.g., oxidation) steps carried out in vitro (e.g., using purified enzymes, isolated from native sources or recombinantly produced) or in vivo in a converting cell different from the host cell (e.g., by supplying the converting cell with the unmodified polyketide).
It will be apparent to the reader that a variety of recombinant vectors can be utilized in the practice of aspects of the invention. As used herein, “vector” refers to polynucleotide elements that are used to introduce recombinant nucleic acid into cells for either expression or replication. Selection and use of such vehicles is routine in the art. An “expression vector” includes vectors capable of expressing DNAs that are operatively linked with regulatory sequences, such as promoter regions. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
The vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes may be chosen to contain control sequences operably linked to the resulting coding sequences in a manner that expression of the coding sequences may be effected in an appropriate host. Suitable control sequences include those that function in eucaryotic and procaryotic host cells. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This can be done individually, or using a pool of isolated encoding nucleotide sequences, which can be inserted into host vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies.
Suitable control sequences for single cell cultures of various types of organisms are well known in the art. Control systems for expression in yeast are widely available and are routinely used. Control elements include promoters, optionally containing operator sequences, and other elements depending on the nature of the host, such as ribosome binding sites. Particularly useful promoters for procaryotic hosts include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including those from Type I or aromatic (Type II) PKS gene clusters. Examples are act promoters, tcm promoters, spiramycin promoters, and the like. However, other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, are also useful. Additional examples include promoters derived from biosynthetic enzymes such as for tryptophan (trp), the β-lactamase (bla), bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can be used.
As noted, particularly useful control sequences are those which themselves, or with suitable regulatory systems, activate expression during transition from growth to stationary phase in the vegetative mycelium. The system contained in the plasmid identified as pCK7, i.e., the actI/actIII promoter pair and the actII-ORF4 (an activator gene), is particularly preferred. Particularly preferred hosts are those which lack their own means for producing polyketides so that a cleaner result is obtained. Illustrative control sequences, vectors, and host cells of these types include the modified S. coelicolor CH999 and vectors described in PCT publication WO 96/40968 and similar strains of S. lividans. See U.S. Pat. Nos. 5,672,491; 5,830,750, 5,843,718; and 6,177,262, each of which is incorporated herein by reference.
Other regulatory sequences may also be desirable which allow for regulation of expression of the PKS sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
Selectable markers can also be included in the recombinant expression vectors. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes which confer antibiotic resistance or sensitivity to the plasmid. Alternatively, several polyketides are naturally colored, and this characteristic provides a built-in marker for screening cells successfully transformed by the present constructs.
The various PKS nucleotide sequences, or a mixture of such sequences, can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements or under the control of a single promoter. The PKS subunits or components can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunits so that hybrid or chimeric PKSs can be generated. The design of such restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR. Methods for introducing the recombinant vectors of the present invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl2 or other agents, such as divalent cations, lipofection, DMSO, protoplast transformation, conjugation, and electroporation.
Thus, the present invention provides recombinant DNA molecules and vectors comprising those recombinant DNA molecules that encode at least a portion of the leptomycin PKS and that, when transformed into a host cell and the host cell is cultured under conditions that lead to the expression of said leptomycin PKS enzymes, results in the production of polyketides including but not limited to leptomycin and/or analogs or derivatives thereof in useful quantities. The present invention also provides recombinant host cells comprising those recombinant vectors.
Suitable culture conditions for production of polyketides using the cells of the invention will vary according to the host cell and the nature of the polyketide being produced, but will be know to those of skill in the art. See, for example, the examples below and WO 98/27203 “Production of Polyketides in Bacteria and Yeast” and WO 01/83803 “Overproduction Hosts For Biosynthesis of Polyketides.”
The polyketide product produced by host cells of the invention can be recovered (i.e., separated from the producing cells and at least partially purified) using routine techniques (e.g., extraction from broth followed by chromatography).
The compositions, cells and methods of the invention may be directed to the preparation of an individual polyketide or a number of polyketides. The polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it. It will be understood that the resulting polyketides may be further modified to convert them to other useful compounds. For example, an ester linkage may be added to produce a “pharmaceutically acceptable ester” (i.e., an ester that hydrolyzes under physiologically relevant conditions to produce a compound or a salt thereof). Illustrative examples of suitable ester groups include but are not limited to formates, acetates, propionates, butyrates, succinates, and ethylsuccinates.
The polyketide product can be modified by addition of a protecting group, for example to produce prodrug forms. A variety of protecting groups are disclosed, for example, in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, New York (1999). Prodrugs are in general functional derivatives of the compounds that are readily convertible in vivo into the required compound. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs,” H. Bundgaard ed., Elsevier, 1985.
Similarly, improvements in water solubility of a polyketide compound can be achieved by addition of groups containing solubilizing functionalities to the compound or by removal of hydrophobic groups from the compound, so as to decrease the lipophilicity of the compound. Typical groups containing solubilizing functionalities include, but are not limited to: 2-(dimethylaminoethyl)amino, piperidinyl, N-alkylpiperidinyl, hexahydropyranyl, furfuryl, tetrahydrofurfuryl, pyrrolidinyl, N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl, morpholinyl, N-alkylaziridinylmethyl, (1-azabicyclo[1.3.0]hex-1-yl)ethyl, 2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl, 2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl, 2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl.
In addition to post synthesis chemical or biosynthetic modifications, various polyketide forms or compositions can be produced, including but not limited to mixtures of polyketides, enantiomers, diastereomers, geometrical isomers, polymorphic crystalline forms and solvates, and combinations and mixtures thereof can be produced
Many other modifications of polyketides produced according to the invention will be apparent to those of skill, and can be accomplished using techniques of pharmaceutical chemistry.
Prior to use the PKS product (whether modified or not) can be formulated for storage, stability or administration. For example, the polyketide products can be formulated as a “pharmaceutically acceptable salt.” Suitable pharmaceutically acceptable salts of compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoic acid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonic acid, or the like. Where the compounds carry one or more acidic moieties, pharmaceutically acceptable salts may be formed by treatment of a solution of the compound with a solution of a pharmaceutically acceptable base, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, ammonia, alkylamines, or the like.
Prior to administration to a mammal the PKS product will be formulated as a pharmaceutical composition according to methods well known in the art, e.g., combination with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a medium that is used to prepare a desired dosage form of a compound. A pharmaceutically acceptable carrier can include one or more solvents, diluents, or other liquid vehicles; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents; preservatives; solid binders; lubricants; and the like. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) and Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe ed. (American Pharmaceutical Assoc. 2000), disclose various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
The composition may be administered in any suitable form such as solid, semisolid, or liquid form. See Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th edition, Lippicott Williams & Wilkins (1991). In an embodiment, for illustration and not limitation, the polyketide is combined in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use. The carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.
EXAMPLES
The following Examples are intended to illustrate, but not limit, the scope of the invention.
Example 1
Gene Library Construction
Growth of Organism and Extraction of Genomic DNA
For genomic DNA extraction, a spore stock of Streptomyces sp ATCC 39366 (obtained from the American Type Culture Collection, Manassas, Va.) was inoculated into 35 ml of liquid R5 medium three days and grown in 30° C. A 10 ml portion of the cell suspension was centrifuged 5,000×g. The pellet was suspended into 3.5 ml of buffer I (Tris, 50 mM, pH7.5; 20 mM EDTA, 150 μg/ml RNase (Sigma-Aldrich) and 1 mg/ml of lysozyme (Sigma)). After incubation of the mixture at 37° C. for about 30 min, the salt concentration was adjusted by adding 850 μl of 5 M NaCl solution, then the mixture was extracted two times with phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol) with gentle agitation followed by centrifugation for 10 min at 12,000×g. The genomic DNA in the supernatant was precipitated with 1 vol of isopropanol and redissolved in 800 μl of water.
Genomic Library Preparation
Approximately 10 μg of genomic DNA was partially digested with Sau3A1 (a series digestions with different dilutions of the enzyme) and the digested DNAs were run on an agarose gel with DNA standards. One of the conditions used was found to have generated fragments of size 30-45 kb. The DNA from this digestion was ligated with pSuperCos-1 (Stratagene), pre-linearized with BamHI and XbaI and the ligation mixture was packaged using a Gigapack XIII (Stragene) in vitro packaging Kit and the mixture was subsequently used for infection of Escherichia coli DH5α employing protocols supplied by the manufacturer.
Identification of Leptomycin Biosynthetic Gene Cluster
To find the gene cluster for leptomycin biosynthesis, cosmids from 475 E. coli transductants resulted from the above ligation mixture were sequenced using convergent primersT7cos (5′-CATAATACGACTCACTATAGGG) (SEQ ID NO:10) and T3cos-1 (5′-TTCCCCGAAAAGTGCCAC) (SEQ ID NO:11). After BLAST analysis, the sequences revealed that 4 cosmids carried DNA inserts with both ends encoding type I PKS (polyketide synthetase) genes. Restriction analysis of these four cosmids with BamHI showed 3 cosmids having overlapping inserts; the fourth cosmid (pKOS279-125.2L78) was distinct. Cosmid pKOS279-125.2L78 and pKOS279-125.3L71 from the 3 overlapping cosmids were sequenced. The incomplete sequences of pKOS279-125.2L78 revealed 6 complete modules and three incomplete modules.
From the 475 cosmids sequenced, also it was found that 16 cosmids carry inserts with PKS genes at one of their ends. While the above cosmids were under sequenced, DNA fragments encoding PKS genes from these 16 cosmids were pulled out by PCR and labeled with DIG (digoxigenin, Roche). The DIG-labeled PCR products were used to screen about 2000 E. coli transductants resulting from the ligation mixture of SuperCos-1 and partially-digested genomic DNA from the leptomycin producer. The in situ hybridization revealed up 89 positive transductants, and the cosmids in these clones were verified to contain PKS inserts by sequencing using T7cos and T3cos-1 primers (SEQ ID NO:57 and 58, respectively).
After DNA sequences of pKOS279-125.2L78 were available, these end sequences were analyzed using BLAST. DNA Blast revealed 3 interesting cosmids (pKOS279-128.PF26, pKOS279-128.PF27 and pKOS279-128.PF48. These 3 cosmids all have inserts extending to cover upstream of KR1ACP1 and reaching non-PKS genes (see FIG. 4).
All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.
Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention. Citation of publications and patent documents is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description are for purposes of illustration and not limitation of the following claims.

## Claims

1. An isolated DNA molecule comprising a polynucleotide having a nucleotide sequence that is at least 95.0% identical to 58243 to 64170 of SEQ ID NO:3, wherein said polynucleotide encodes a polypeptide having thioesterase activity.

2. An isolated DNA molecule comprising a polynucleotide sequence encoding SEQ ID NO:7.

3. The isolated DNA molecule of claim 2, wherein said polynucleotide comprises nucleotides 58243 to 64170 of SEQ ID NO:3.

4. A recombinant polyketide synthase (PKS) comprising the isolated nucleic acid molecule of claim 2.

5. An isolated recombinant host cell comprising the PKS of claim 4.

6. A recombinant vector comprising the isolated nucleic acid molecule of claim 2.

7. An isolated recombinant host cell comprising the vector of claim 6.

8. A method of making an isolated polypeptide comprising:
(a) culturing the isolated recombinant host cell of claim 5 or claim 7 under conditions such that said polypeptide is expressed; and
(b) recovering said polypeptide.

9. The isolated DNA molecule of claim 2, wherein said nucleic acid sequence further comprises at least a portion of a PKS sequence.

10. The isolated DNA molecule of claim 9, wherein said nucleic acid sequence further comprises at least a portion of a second PKS sequence.

11. An isolated DNA molecule comprising the complete complementary sequence of the polynucleotide provided in claim 2.