US5672491A
Expired / Lapsed
US
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Recombinant production of novel polyketides
Assignee: John Innes Centre
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Filing: 1994-05-06
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Granted: 1997-09-30
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Expires: 2014-05-06
Abstract
Abstract Novel polyketides and novel methods of efficiently producing both new and known polyketides, using recombinant technology, are disclosed. In particular, a novel host-vector system is described which is used to produce polyketide synthases which in turn catalyze the production of a variety of polyketides.
Inventors
- 👤 Chaitan Khosla
- 👤 David A. Hopwood
- 👤 Suzanne Ebert-Khosla
- 👤 Robert McDaniel
- 👤 Hong Fu
- 👤 Camilla Kao
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 16:21
CPC Classifications
C; C07; C07D; C07D407/00; C07D407/02; C07D407/06; C07D309/00; C07D309/34; C07D309/36; C07D311/00; C07D311/02; C07D311/78; C07D311/92; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/52; C12N9/00; C12N9/93; C12P; C12P17/00; C12P17/02; C12P17/06; C12P17/16; C12P17/162; C12P19/00; C12P19/44; C12P19/60; C12P19/62; C12P7/00; C12P7/24; C12P7/26
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
262 words
A method for producing a complete recombinant modular polyketide synthase i (PKS) gene cluster comprising: a. inserting a first portion of a modular PKS gene cluster into a donor plasmid and inserting a second portion of a modular PKS gene cluster into a recipient plasmid, wherein the first and second portions collectively encode a complete modular PKS gene cluster, and further wherein: i. the donor plasmid expresses a gene which encodes a first selection marker and is capable of replication at a first, permissive temperature and incapable of replication at a second, non-permissive temperature; ii. the recipient plasmid expresses a gene which encodes a second selection marker; and iii. the donor plasmid comprises regions of DNA complementary to regions of DNA in the recipient plasmid, such that homologous recombination can occur between the first portion of the modular PKS gene cluster and the second portion of the modular PKS gene cluster, whereby a complete modular PKS gene cluster can be generated; b. transforming the donor plasmid and the recipient plasmid into a host cell and culturing the transformed host cell at the first, permissive temperature and under conditions which allow the growth of host cells which express the first and the second selection markers, to generate a first population of cells; and c. culturing the first population of cells at the second, non-permissive temperature and under conditions which allow the growth of cells which express the first selection marker, to generate a second population of cells which includes host cells which contain a recombinant plasmid comprising a complete modular PKS gene cluster.
Claim 2
Independent Claim
262 words
A method for producing a complete recombinant modular polyketide synthase i (PKS) gene cluster comprising: a. inserting a first portion of a modular PKS gene cluster into a donor plasmid and inserting a second portion of a modular PKS gene cluster into a recipient plasmid, wherein the first and second portions collectively encode a complete modular PKS gene cluster, and further wherein: i. the donor plasmid expresses a gene which encodes a first selection marker and is capable of replication at a first, permissive temperature and incapable of replication at a second, non-permissive temperature; ii. the recipient plasmid expresses a gene which encodes a second selection marker; and iii. the donor plasmid comprises regions of DNA complementary to regions of DNA in the recipient plasmid, such that homologous recombination can occur between the first portion of the modular PKS gene cluster and the second portion of the modular PKS gene cluster, whereby a complete modular PKS gene cluster can be generated; b. transforming the donor plasmid and the recipient plasmid into a host cell and culturing the transformed host cell at the first, permissive temperature and under conditions which allow the growth of host cells which express the first and the second selection markers, to generate a first population of cells; and c. culturing the first population of cells at the second, non-permissive temperature and under conditions which allow the growth of cells which express the first selection marker, to generate a second population of cells which includes host cells which contain a recombinant plasmid comprising a complete modular PKS gene cluster.
Claim 3
Dependent Claim
39 words
The method of claim 1, wherein the method further comprises after step (c) culturing the second population of cells at the first, permissive temperature and under conditions which allow the growth of cells which express the first selection marker.
Claim 4
Dependent Claim
25 words
The method of claim 1, wherein the first and second portions of the PKS gene cluster are derived from the 6-deoxyerythronolide B synthase gene cluster.
Claim 5
Dependent Claim
50 words
A method for producing a recombinant polyketide synthase which method comprises transferring the recombinant plasmid from step c of claim 1 into host cells to generate transformed cells containing said plasmid; and culturing the transformed cells under conditions wherein the complete PKS gene cluster present in the cells is expressed.
Claim 6
Dependent Claim
121 words
A host cell containing nucleic acid molecules, wherein said molecules comprise at least one module that encodes a modular polyketide synthase (PKS) functional in catalyzing the synthesis of a polyketide, said module comprising at least one nucleotide sequence which encodes a PKS acyl transferase (AT) activity; at least one nucleotide sequence which encodes a PKS ketoacyl carrier protein synthase (KS) activity; and at least one nucleotide sequence which encodes a PKS acyl carrier protein (ACP) activity; said module operatively linked to a control sequence, whereby a functional modular PKS is produced in said cell, with the proviso that said module or said control sequence is heterologous to the host cell, and wherein said host completely lacks its own PKS gene cluster.
Claim 7
Dependent Claim
121 words
A host cell containing nucleic acid molecules, wherein said molecules comprise at least one module that encodes a modular polyketide synthase (PKS) functional in catalyzing the synthesis of a polyketide, said module comprising at least one nucleotide sequence which encodes a PKS acyl transferase (AT) activity; at least one nucleotide sequence which encodes a PKS ketoacyl carrier protein synthase (KS) activity; and at least one nucleotide sequence which encodes a PKS acyl carrier protein (ACP) activity; said module operatively linked to a control sequence, whereby a functional modular PKS is produced in said cell, with the proviso that said module or said control sequence is heterologous to the host cell, and wherein said host completely lacks its own PKS gene cluster.
Claim 8
Dependent Claim
18 words
The host cell of claim 5 wherein said nucleic acid molecules comprise a complete modular PKS gene cluster.
Claim 9
Dependent Claim
30 words
The host cell of claim 5 wherein said host cell has been modified so as completely to lack a PKS gene cluster normally present in the unmodified said host cell.
Claim 10
Dependent Claim
13 words
The host cell of claim 7 wherein said host cell is S. coelicolor.
Claim 11
Dependent Claim
88 words
The host cell of claim 5 wherein said module comprises: at least one nucleotide sequence encoding PKS ketoreductase (KR) activity; or at least one nucleotide sequence encoding PKS ketoreductase (KR) activity and at least one nucleotide sequence encoding PKS dehydrase (DH) activity; or at least one nucleotide sequence encoding PKS ketoreductase (KR) activity and at least one nucleotide sequence encoding PKS dehydrase (DH) activity, and at least one nucleotide sequence encoding PKS enoyl reductase (ER) activity; and optionally, at least one nucleotide sequence encoding PKS thioesterase (TE) activity.
Claim 12
Dependent Claim
43 words
The host cell of claim 9 wherein said module comprises: at least one nucleotide sequence encoding PKS ketoreductase (KR) activity and at least one nucleotide sequence encoding PKS dehydrase (DH) activity; and at least one nucleotide sequence encoding PKS enoyl reductase (ER) activity.
Claim 13
Dependent Claim
15 words
The host cell of claim 5 wherein said module is a 6-deoxyerythronolide B synthase module.
Claim 14
Dependent Claim
19 words
The host cell of claim 11 wherein said nucleic acid molecule comprises the complete 6-deoxyerythronolide B synthase gene cluster.
Claim 15
Independent Claim
26 words
A method to produce a functional polyketide synthase which method comprises culturing the cells of any of claims 5-12 under conditions wherein said module is expressed.
Claim 16
Independent Claim
35 words
A method to produce a polyketide which method comprises culturing the cells of any of claims 5-12 under conditions wherein said module is expressed to produce functional PKS protein so as to produce said polyketide.
Claim 17
Dependent Claim
90 words
A recombinant vector comprising one or more modules of a modular replacement PKS gene cluster wherein a module comprises at least one nucleotide sequence which encodes a PKS acyl transferuse (AT) activity; at least one nucleotide sequence Which encodes a PKS ketoacyl carrier protein synthase (KS) activity; and at least one nucleotide sequence which encodes a PKS acyl carrier protein (ACP) activity; said module operably linked to control sequences for expression of each said module wherein at least one control sequence is heterologous to at least one of said modules.
Claim 18
Dependent Claim
90 words
A recombinant vector comprising one or more modules of a modular replacement PKS gene cluster wherein a module comprises at least one nucleotide sequence which encodes a PKS acyl transferuse (AT) activity; at least one nucleotide sequence Which encodes a PKS ketoacyl carrier protein synthase (KS) activity; and at least one nucleotide sequence which encodes a PKS acyl carrier protein (ACP) activity; said module operably linked to control sequences for expression of each said module wherein at least one control sequence is heterologous to at least one of said modules.
Claim 19
Dependent Claim
21 words
The recombinant vector of claim 15 wherein at least one module is a module of the 6-deoxyerythronolide B synthase gens cluster.
Claim 20
Dependent Claim
14 words
The recombinant vector of claim 15 which comprises a complete modular PKS gens duster.
Claim 21
Dependent Claim
21 words
The recombinant vector of claim 17 wherein the complete modular PKS gene cluster is the complete 6-deoxyerythronolide B synthase gens cluster.
Claim 22
Dependent Claim
9 words
The recombinant vector of claim 18 which is pCK7.
Claim 23
Dependent Claim
17 words
A host cell that has been modified to contain the recombinant vector of any of claims 15-19.
Claim 24
Dependent Claim
29 words
The host cell of claim 20 wherein said host has been modified so as completely to lack a PKS gene cluster normally present in the unmodified said host cell.
Claim 25
Dependent Claim
24 words
A method to produce a modular PKS which method comprises culturing the cells of claim 20 under conditions wherein each said module is expressed.
Claim 26
Dependent Claim
21 words
The method of claim 22 wherein said at least one module is a module of the 6-deoxyerythronolide B synthase gens cluster.
Claim 27
Dependent Claim
16 words
The method of claim 23 wherein said modules comprise the complete 6-deoxyerythronolide B synthase gene cluster.
References Cited (Backward Citations)
US4935340A
Cited
WO1993013663A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-09-29 | 2001-03-31 | 2001-09-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-09-29 | 2005-03-31 | 2005-09-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-09-29 | 2009-03-31 | 2009-09-30 | $7700.00 | Window Closed / Lapsed |