Title:
RECOMBINANT METHODS AND MATERIALS FOR PRODUCING EPOTHILONE AND EPOTHILONE DERIVATIVES
Document Type and Number:
WIPO Patent Application WO/2000/031247
Kind Code:
A2
Abstract:
Recombinant nucleic acids that encode all or a portion of the epothilone polyketide synthase (PKS) of sorangium cellulosum are used to express recombinant PKS genes in host cells for the production of epithilones, epothilone derivatives, and polyketides that are useful as cancer chemotherapeutics, fungicides, and immunosuppressants.
Inventors:
JULIEN BRYAN
KATZ LEONARD
KHOSLA CHAITAN
TANG LI
ZIERMANN RAINER
Application Number:
PCT/US1999/027438
Publication Date:
June 02, 2000
Filing Date:
November 19, 1999
Assignee:
KOSAN BIOSCIENCES INC (US)
International Classes:
A61K31/365; A61K31/427; C12N15/09; A61K31/529; A61K31/537; A61P35/00; A61P43/00; C07D277/22; C07D277/28; C07D313/00; C07D417/06; C07D493/04; C07D493/08; C07D498/08; C07D513/08; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12N9/00; C12N15/52; C12P17/16; C12P17/18; C12R1/38; (IPC1-7): C12N15/00
Domestic Patent References:
| WO1999054330A1 | 1999-10-28 | | | |
| WO1999054319A1 | 1999-10-28 | | | |
| WO1999054318A1 | 1999-10-28 | | | |
| WO1999043653A1 | 1999-09-02 | | | |
| WO1999043320A1 | 1999-09-02 | | | |
| WO1999042602A2 | 1999-08-26 | | | |
| WO1999040047A1 | 1999-08-12 | | | |
| WO1999027890A2 | 1999-06-10 | | | |
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Foreign References:
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| US41055199A | 1999-10-01 | | | |
| USPP10709398P | 1998-11-05 | | | |
| US5712146A | 1998-01-27 | | | |
Other References:
K. GERTH ET AL., J. ANTIBIOTICS, vol. 49, 1996, pages 560 - 563
D. BOLLAG ET AL., CANCER RES., vol. 55, 1995, pages 2325 - 2333
SU ET AL.: "Structure-activity relationships of the epothilones and the first in vivo comparison with paclitaxel", ANGEW. CHEM. INT. ED. ENGL, 1997
MACNEIL ET AL.: "Industrial Microorganisms: Basic and Applied Molecular Genetics", 1993, ASM, article "A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin", pages: 245 - 256
MACNEIL ET AL.: "Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase", GENE, vol. 115, 1992, pages 119 - 125
IKEDA; OMURA: "Avermectin biosynthesis. Candicidin (FR008)", CHEM. RES., vol. 97, 1997, pages 2599 - 2609
HU ET AL., MOL. MICROBIOL., vol. 14, 1994, pages 163 - 172
DONADIO ET AL., SCIENCE, vol. 252, 1991, pages 675 - 9
CORTES ET AL.: "An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea", NATURE, vol. 348, 8 November 1990 (1990-11-08), pages 176 - 8
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MOTAMEDI ET AL.: "Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK-506", EUR. J. BIOCHEM., vol. 244, 1997, pages 74 - 80
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CASPERS ET AL., CELLULAR AND MOLECULAR BIOLOGY, vol. 40, no. 5, 1994, pages 635 - 644
Attorney, Agent or Firm:
Murashige, Kate H. (N.W. Washington, DC, US)
Claims:
Claims
| 1. |
An isolated recombinant nucleic acid compound that comprises a nucleotide sequence encoding at least a domain of an epothilone polyketide synthase (PKS) protein and/or encoding a functional region of an epothilone modification enzyme. |
| 2. |
The nucleic acid of claim 1, wherein said domain is selected from the group consisting of a loading domain, a thioesterase domain, an NRPS, an AT domain, a KS domain, an ACP domain, a KR domain, a DH domain, and an ER domain, a methyl transferase domain and a functional oxidase domain. |
| 3. |
The nucleic acid of claim 1 or 2 that comprises the coding sequence of an epoA gene, and/or the coding sequence of an epoB gene, and/or the coding sequence of an epoC gene, and/or the coding sequence of an epoD gene, and/or the coding sequence of an epoE gene, and/or the coding sequence of an epoF gene, and/or the coding sequence of an epoK gene, and/or the coding sequence of an epoL gene. |
| 4. |
The nucleic acid of any of claims 13 that further comprises a promoter positioned to transcribe said encoding nucleotide sequence in host cells in which said promoter is operable. |
| 5. |
The nucleic acid of claim 4, wherein said promoter is a promoter from a Sorangium gene, or from a Myxococcus gene, or from a Streptomyces gene, or from an epothilone PKS gene, or from a pilA gene, or from an actinorhodin PKS gene. |
| 6. |
The nucleic acid of any of claims 15 that is a recombinant DNA expression vector. |
| 7. |
Host cells which contain the nucleic acid of any of claims 46. |
| 8. |
The cells of claim 7 which are Sorangium cells, or Myxococcus cells, or Pseudomonas cells, or Streptomyces cells. |
| 9. |
A method to produce a polyketide which method comprises culturing the cells of claim 7 or 8 under conditions wherein the encoding nucleotide sequence is expressed to obtain a functional PKS. |
| 10. |
A recombinant Sorangium cellulosum host cell that contains a mutated gene for an epothilone PKS protein or epothilone modification enzyme, wherein said mutated gene was inserted in whole or in part into genomic DNA of said cell by homologous recombination with a recombinant vector comprising all or a part of an epothilone PKS gene or epothilone modification gene. |
| 11. |
The recombinant host cell of claim 10 that makes epothilone C or D but not A or B due to a mutation inactivating or deleting an epoK gene, or makes epothilone A or C but not B or D due to a mutation in epoD altering module 4 AT domain specificity, or makes epothilone B or D but not A or C due to a mutation in epoD altering module 4 AT domain specificity, or makes epothilone C but not epothilone A, B or D due to a mutation in epoD altering module 4 AT domain specificity and a mutation in epoK, or makes epothilone D but not epothilone A, B or C due to a mutation in epoD altering module 4 AT domain specificity and a mutation in epoK. |
| 12. |
Recombinant Streptomyces or Myxococcus host cells that express an epothilone PKS gene or an epothilone modification enzyme gene, optionally comprising one or more of said epothilone PKS or modification enzyme genes integrated into their chromosomal DNA and/or one or more of said epothilone PKS or modification enzyme genes on an extrachromosomal expression vector. |
| 13. |
The host cells of claim 12 or 13 that are S. coelicolor CH999. |
| 14. |
A method to produce an epothilone or epothilone derivative which comprises culturing the cells of claims 12 or 13. |
| 15. |
A modified functional epothilone PKS wherein said modification comprises at least one of : replacement of at least one AT domain with an AT domain of different specificity; inactivation of the NRPSlike module 1 or of the KS2 catalytic domain; inactivation of at least one activity in at least one pcarbonyl modification domain; addition of at least one of KR, DH and ER activity in at least one ßcarbonyl modification domain; and replacement of the NRPS module 1 with an NRPS of different specificity. |
| 16. |
The modified PKS of claim 15 contained in a cell or contained in a cellfree system, wherein said cell or system contains additional enzymes for modification of the product of said epothilone PKS. |
| 17. |
The modified PKS of claim 16 wherein said modifying enzymes comprise at least one of a methyltransferase, an oxidase or a glycosylation enzyme. |
| 18. |
A method to prepare an epothilone derivative which method comprises providing substrates including extender units to the modified PKS of any of claims 1517. |
| 19. |
A modified functional epothilone PKS wherein said modification comprises inactivation of the NRPS of module 1 or the KS2 of module 2 thereof. |
| 20. |
A method to make an epothilone derivative which method comprises contacting the modified PKS of claim 19 with a module 2 substrate or a module 3 substrate and extender units. |
| 21. |
Recombinant host cells which comprise the modified PKS of any of claims 1517 or 19. |
| 22. |
The cells of claim 21 that produce an epothilone derivative selected from the group consisting of 16desmethyl epothilones, 14methyl epothilones, 11hydroxyl epothilones, 10methyl epothilones, 8,9anhydro epothilones, 9hydroxyl epothilones, 9 keto epothilones, 8desmethyl epothilones, and 6desmethyl epothilones. |
| 23. |
A compound selected from the group consisting of 16desmethyl epothilones, 14methyl epothilones, 11hydroxyl epothilones, 10methyl epothilones, 8,9 anhydro epothilones, 9hydroxyl epothilones, 9keto epothilones, 8desmethyl epothilones, and 6desmethyl epothilones. |
| 24. |
A recombinant PKS enzyme that comprises one or more domains, modules, or proteins of a nonepothilone PKS and one or more domains, modules, or proteins of an epothilone PKS, and/or contains a loading domain that comprises a KSQ domain. |
| 25. |
The PKS enzyme of claim 24, wherein said PKS comprises a DEBS loading domain and 5 modules of DEBS and an NRPS of the epothilone PKS, wherein said PKS comprises all of a nonepothilone PKS with an MT domain of the epothilone PKS. |
| 26. |
A compound of the formula: including the glycosylated forms thereof and stereoisomeric forms where the stereochemistry is not shown, wherein A is a substituted or unsubstituted straight, branched chain or cyclic alkyl, alkenyl or alkynyl residue optionally containing 13 heteroatoms selected from O, S and N; or wherein A comprises a substituted or unsubstituted aromatic residue; R2 represents H, H, or H, lower alkyl, or lower alkyl, lower alkyl; X5 represents =O or a derivative thereof, or H, OH or H, NR2 wherein R is H, alkyl or acyl, or H, OCOR2, H, OCONR2 wherein R is H or alkyl, or is H, H; R6 represents H or lower alkyl, and the remaining substituent on the corresponding carbon is H; X7 represents OR, or NR2, wherein R is H, alkyl or acyl or is OCOR, or OCONR2 wherein R is H or alkyl or X7 taken together with X9 forms a carbonate or carbamate cycle, and wherein the remaining substituent on the corresponding carbon is H; R8 represents H or lower alkyl and the remaining substituent on the carbon is H; X9 represents =O or a derivative thereof, or H, OR or H, NR2 wherein R is H, alkyl or acyl, or is H, OCOR or H, OCONR2, wherein R is H or alkyl, or represents H, H or wherein X9 together with X7 or with X''can form a cyclic carbonate or carbamate; Rlo is H, H or H, lower alkyl, or lower alkyl, lower alkyl; X''is =0 or a derivative thereof, or H, OR, or H, NR2 wherein R is H, alkyl or acyl or H, OCOR or H, OCONR2 wherein R is H or alkyl, or is H, H or wherein X11 in combination with X9 may form a cyclic carbonate or carbamate; R12 is H, H, or H, lower alkyl, or lower alkyl, lower alkyl; X13 is =O or a derivative thereof, or H, OR or H, NR2 wherein R is H, alkyl or acyl or is H, OCOR or H, OCONR2 wherein R is H or alkyl; R14 is H, H, or H, lower alkyl, or lower alkyl, lower alkyl; R16 is H or lower alkyl; and wherein optionally H or another substituent may be removed from positions 12 and 13 and/or 8 and 9 to form a double bond, wherein said double bond may optionally be converted to an epoxide. |
| 27. |
A compound of the formula wherein both Z are O or one Z is N and the other Z is O and the remaining substituents are defined as in claim 26. |
| 28. |
A recombinant vector selected from the group consisting of pKOS35 70.8A3, pKOS3570.1A2, pKOS3570.4, pKOS3579.85, pKOS039124R, and pKOS039126R. |
Description:
INTERNATIONAL SEARCH REPORT----------------- ; Interr nnal Application No PCT/US 99/27438 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. T TANG, L. ET AL.:"Cloning and 1-22,24, heterologous expression of the epithilone 28 gene cluster." SCIENCE, vol. 287,28 January 2000 (2000-01-28), pages 640-42, XP00213541 the whole document A WO 98 22461 A (BIOTECHNOLOG FORSCHUNG GMBH 1-22,24, ; GERTH KLAUS (DE); HOEFLE GERHARD (DE)) 28 28 May 1998 (1998-05-28) the whole document A CHOU T. C. ET AL. :"DESOXYEPITHILONE B: 1-22,24, AN EFFICACIOUS MICROTUBULE-TARGETED 28 ANTITUMOR AGENT WITH A PROMISING IN VIVO PROFILE RELATIVE TO EPOTHILONE B." PROC. NATL. ACAD. SCI. USA, vol. 95, no. 16, 4 August 1998 (1998-08-04), pages 9642--7, XP000910107 the whole document Int itional application No. INTERNATIONAL SEARCH REPORT PCT/US 99/27438 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This International Search Report has not been established in respect of certain claims under Article 17 (2) (a) for the following reasons: 1.Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: 2. je Claims Nos.: because they relate to parts of the International Application that do not comply with the prescribed requirements to such an extent that no meaningful Intemational Search can be carried out, specifically: 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4 (a). Box 11 Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: See additional sheet 1. As all required additional search fees were timely paid by the applicant, this International Search Report covers all searchable claims. 2. As ai) searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this International Search Report covers only those claims for which fees were paid, specifically claims Nos.: 4. No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is restncted to the invention first mentioned in the claims; it is covered by claims Nos.: 1-22,24,28 Remark on Protest The additional search fees were accompanied by the applicant's protest. L-J C No protest accompanied the payment of additional search fees. LJ FURTHER INFORMATION CONTINUED FROM PCT/ISA/210 1. Claims: 1-22,24,28 nucleic acid encoding at least a domain of an epothilone polyketide synthase and/or modification enzyme.
2. Claims: 23,25-27 epothilone compounds (16 inventions). INTERNATIONAL SEARCH REPORT !..,,------- Inten.-al Applicafion No ,rmation on patent famiiy memioers Patent document Publication Patent family Publication cited in search report date member (s) date WO 9966028 A 23-12-1999 AU 4611699 A 05-01-2000 WO 0022139 A 20-04-2000 DE 19846493 A 13-04-2000 AU 6512699 A 01-05-2000 WO 9822461 A 28-05-1998 AU 5483798 A 10-06-1998 BR 9713363 A 25-01-2000 CN 1237970 A 08-12-1999 CZ 9901750 A 15-09-1999 EP 0941227 A 15-09-1999 NO 992338 A 14-05-1999 PL 333435 A 06-12-1999 ZA 9710384 A 18-05-1999
