Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1997-12-23
2001-03-13
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S069100, C435S069700, C435S252300, C435S252350, C435S320100, C536S023200, C536S023400
Reexamination Certificate
active
06200813
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel polynucleotide sequences, proteins encoded therefrom which are involved in the biosynthesis of polyketides, methods for directing the biosynthesis of novel polyketides using those polynucleotide sequences and novel derivatives produced therefrom. In particular, the invention relates to the production of novel polyketide derivatives through manipulation of the genes encoding polyketide synthases.
BACKGROUND OF THE INVENTION
Polyketides are a large class of natural products that includes many important antibiotic, antifungal, anticancer, antihelminthic, and immunosuppressant compounds such as erythromycins, tetracyclines, amphotericins, daunorubicins, avermectins, and rapamycins. Their synthesis proceeds by an ordered condensation of acyl esters to generate carbon chains of varying length and substitution pattern that are later converted to mature polyketides. This process has long been recognized as resembling fatty acid biosynthesis, but with important differences. Unlike a fatty acid synthase, a typical polyketide synthase is programmed to make many choices during carbon chain assembly: for example, the choice of “starter” and “extender” units, which are often selected from acetate, propionate or butyrate residues in a defined sequence by the polyketide synthase. The choice of using a full cycle of reduction-dehydration-reduction after some condensation steps, omitting it completely, or using one of two incomplete cycles (reduction alone or reduction followed by dehydration) is additionally programmed, and determines the pattern of keto or hydroxyl groups and the degree of saturation at different points in the chain. Finally, the stereochemistry for the substituents at many of the carbon atoms is programmed by the polyketide synthase.
Streptomyces and the closely related Saccharopolyspora genera are producers of a prodigious diversity of polyketide metabolites. Because of the commercial significance of these compounds, a great amount of effort has been expended in the study of Streptomyces and Saccharopolyspora genetics. Consequently, much is known about these organisms and several cloning vectors and techniques exist for their transfornation.
Although many polyketides have been identified, there remains the need to obtain novel polyketide structures with enhanced properties. Current methods of obtaining such molecules include screening of natural isolates and chemical modification of existing polyketides, both of which are costly and time consuming. Current screening methods are based on gross properties of the molecules, i.e. antibacterial, antifungal activity, etc., and both a priori knowledge of the structure of the molecules obtained or predetermination of enhanced properties are virtually impossible. Chemical modification of preexisting structures has been successfully employed to obtain novel polyketides, but still suffers from practical limitations to the type of compounds obtainable, largely connected to the poor yield of multistep synthesis and available chemistry to effect modifications. Modifications which are particularly difficult to achieve are those involving additions or deletions of carbon side chains. Accordingly, there exists a considerable need to obtain molecules wherein such changes can be specified and performed in a cost effective manner and with high yield.
The present invention solves these problems by providing reagents (specifically, polynucleotides, vectors comprising the polynucleotides and host cells comprising the vectors) and methods to generate novel polyketides by (de novo biosynthesis rather than by chemical modification.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compounds of the formula:
wherein R
1
, R
2
, R
3
, R
4
, R
5
, and R
6
are independently selected from Q wherein Q is selected from the group consisting of (a) —H, (b) -Me, (c) -Et, and (d) —OH; L
1
and L
2
are independently —H or —OH; L
3
is D-desosamine or —OH; and L
4
is L-mycarose, L-cladinose or —OH with the proviso that when R
1
-R
5
are -Me, R
6
is other than —H or —Me. Preferred compounds of the invention are those in Q is selected from the group consisting of (a), (b) and (c) above or (a), (b) and (d) above or (a), (c) and (d) above or (b), (c) and (d) above or (a) and (b) above or (a) and (c) above or (a) and (d) above or (b) and (c) above or (c) and (d) above and L
1
, L
2
, L
3
and L
4
are as defined above. Other preferred compounds include those in which R
1
, R
2
, R
3
, R
4
, R
5
and R
6
are all —H or -Et or —OH and L
1
, L
2
, L
3
and L
4
are as defined above. Still other preferred compounds include didesmethyl, tridesmethyl, tetradesmethyl, pentadesmethyl and hexadesmethyl derivatives of the compounds of formula X and particularly, di- tri-, tetra-, penta- and hexadesmethyl derivatives of erythromycins A and B. Other especially preferred compounds of formula X include 6,10-didesmethyl-6-ethylerythromycin A, 10,12-didesmethyl-12-deoxy-12-ethylerythromycin A, 10,12-didesmethyl- 12-deoxy- 10-hydroxyerythromycin A, 6,10,12-tridesmethyl-6,12-diethylerythromycin A, 6,10,12-tridesmethyl-6-deoxy-6,12-diethylerythromycin A, 10-desmethylerythronolide B, 10-desmethyl-6-deoxyerythronolide B, 12-desmethylerythronolide B, 12-desmethyl-6-deoxyerythronolide B, 12-desmethyl-12-ethylerythronolide B, 6-desmethyl-6-deoxy-6-ethylerythronolide B, 10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A, 10-desmethyl-6,12-dideoxyerythromycin A, 12-desmethylerythromycin A, 12-desmethyl-12-deoxyerythromycin A, 12-desmethyl-6,12-dideoxyerythromycin A, 6-desmethyl-6-ethylerythromycin A, 12-desmethyl-12-ethylerythromycin A, 12-desmethyl- 12-deoxy-12-ethylerythromycin A, 10-desmethyl-10-hydroxyerythromycin A, 12-desmethyl-12-epihydroxyerythromycin A, 10,12-didesmnethylerythromycin A, 10,12-didesmethyl-12-deoxyerythromycin A, 10,12-didesmethyl-6,12-dideoxyerythromycin A, 10-desmethylerythronolide B, 10-desmethyl-6-deoxyerythronolide B, 12-desmethylerythronolide B, 12-desmethyl-6-deoxyerythronolide B, 10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A, 10-desmethyl-6,12-dideoxyerythromycin A, 12-desmethylerythromycin A, 12-desmethyl-12-deoxyerythromycin A, 12-desmethyl-6,12-dideoxyerythromycin A, 10,12-didesmiiethylerythromycini A, 10,12-didesmethyl-12-deoxyerythromycin A, and 10,12-didesmethyl-6,12-dideoxyerythromycin A. Most preferred compounds include 10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A, and 12-desmethyl-12-deoxyerythromycin A.
In another aspect, the present invention provides an isolated polynucleotide sequence or fragment thereof which encodes an enzymatically active acyltransferase domain from a PKS selected from
Streptomyces hygroscopicus, Streptomyces venezuelae
, and
Streptonmyces caelestis
. Preferably, the polynucleotide sequence is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:29 or SEQ ID NO:30. In another preferred embodiment, the polynucleotide sequence encodes an acyltransferase domain selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.
The present invention also provides a vector comprising a polynucleotide sequence or fragment thereof which encodes which encodes an enzymatically active acyltransferase domain from Streptomyces. Preferably, the polynucleotide sequence is selected from those described above and the Streptomyces is
Streptomyces hygroscopicus, Streptomyces venezuelae
, or
Streptomyces caelestis
. A particularly preferred vector is pCS5. Other vectors of the invention include pUC18/LigAT2, pEryAT1/LigAT2, pEryAT2/LigAT2, pUC18/venAT, pEryAT1/venAT, pUC19/rapAT14, pEryAT1/rapAT14, pEryAT2/rapAT14, pUC/5′-flank/ethAT, pUC/ethAT/C-6, pEAT4, pUC18/NidAT6, and pEryAT2/NidAT6.
In another aspect, the invention provides host cells transformed with a vector as described above. The host cell may be a bacterial cell and preferably is selected from the group consisting of
E. coli
and Bacillus species. Alternatively, the host cell is a polyketide-producing microorganism. A preferred pol
Kakavas Stephan J.
Katz Leonard
Pereda-Lopez Ana
Ruan Xiaoan
Stassi Diane L.
Abbott Laboratories
Achutamurthy Ponnathapu
Casuto Dianne
Moore William W.
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