Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold
OwnerChaitan Khosla, Camilla M. Kaoinventors
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Abstract
Combinatorial libraries of polyketides can be obtained by suitable manipulation of a host modular polyketide synthase gene cluster such as that which encodes the PKS for erythromycin. The combinatorial library is useful as a source of pharmaceutically active compounds. In addition, novel polyketides and antibiotics are prepared using this method.
Claims28
A method to prepare a nucleic acid with a nucleotide sequence encoding a modified PKS from a nucleotide sequence encoding a naturally occurring modular PKS wherein said naturally occurring modular PKS contains first regions which encode enzymatic activities and second regions which encode scaffolding amino acid sequences, which method comprises modifying at least one said first region.
A multiplicity of cell colonies comprising a library of colonies wherein each colony of the library contains an expression vector comprising a nucleotide sequence encoding a modular PKS derived from a naturally occurring PKS gene cluster wherein at least one enzymatic activity has been deleted and/or replaced by a different version of said activity or is mutated so as to result in a polyketide other than that produced by said naturally occurring PKS and
- wherein the nucleotide sequence contained in each colony in the library encodes a different PKS.
A multiplicity of cell colonies comprising a library of colonies wherein each colony of the library contains a modular PKS derived from a naturally occurring PKS wherein at least one enzymatic activity has been deleted or replaced by a different version of said activity or is produced from a mutated form of said gene so as to result in a polyketide other than that produced by said naturally occurring PKS, and
- each colony in the library contains a different PKS.
A multiplicity of polyketides which comprises a combinatorial library of polyketides which results from culturing colonies containing polyketide synthases derived from a naturally occurring PKS wherein at least one enzymatic activity has been deleted and/or replaced by a different version of said activity or is mutated so as to result in a polyketide other than that produced by said naturally occurring PKS, wherein each PKS in said library produces a different polyketide.
A compound of the formula:
- 9
- including the glycosylated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
- each of R1 and R2 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- X1 is H2, HOH or ═O;
- with the provisos that:
- at least one of R1 and R2 must be alkyl (1-4C);
- and
- the compound is other than compounds 1, 2, 3, 5 and 6 of FIG. 6A;
- or of the formula:
- 10
- including the glycosylated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-8C;
- each of R1, R2 and R3 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- each of X1 and X2 is independently H2, HOH or ═O;
- with the proviso that:
- at least two of R1, R2 and R3 are alkyl (1-4C);
- or of the formula:
- 11
- including the glycosylated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
- each of R1, R2 and R3 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- each of X1, X2 and X3 is independently H2, HOH or ═O;
- with the provisos that:
- at least one of R1 and R2 must be alkyl (1-4C);
- and
- the compound is other than compound 8 of FIG. 6A;
- or of the formula:
- 12
- including the glycosylated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-8C;
- each of R1, R2 and R3 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- each of X* and X2 is independently H2, HOH or ═O;
- with the proviso that:
- at least one of R2 and R3 is alkyl (1-4C);
- and
- the compound is other than compound 9 of FIG. 6A;
- or of the formula:
- 13
- including the glycosylated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
- each of R1-R5 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- R6 is alkyl (1-5C);
- each of X1 and X3 and X6 is independently H2, HOH or ═O;
- with the proviso that:
- at least two of R1-R4 are alkyl (1-4C);
- or of the formula:
- 14
- including the glycoslated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
- each of R1-R5 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- R6 is alkyl (1-5C);
- X2 is OH or H;
- each X1, X3, X4 and X5 is independently H2, HOH or ═O;
- or X4 is H and
- the compound of formula (8) has a π-bond between positions 9-10, with the proviso that:
- if X2 is H, at least one of X3 and X4 is HOH or ═O.
A compound of the formula:
- 15
- including the glycosylated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
- each of R1, R2, R3, R4 and R5 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- each of X1, X2, X3 and X4 is independently H2, HOH or ═O;
- or
- X1 or X2 or X3 or X4 is H and the compound of formula (5) contains a π-bond at positions 8-9 or 6-7 or 4-5 or 2-3;
- with the proviso that:
- at least two of R1-R5 are alkyl (1-4C);
- and
- the compound is other than compound 13 or 14 of FIG. 6A or compound 205, 210-213 of FIG. 11.
A compound of the formula:
- 16
- including the glycosylated and isolated stereoisomeric forms thereof;
- wherein R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
- each of R1-R6 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
- each of X1-X5 is independently H2, HOH or ═O;
- or
- each of X1-X4 is independently H and the compound of formula (5) contains a π-bond in the ring adjacent to the position of said X at 2-3,4-5, 6-7,8-9 and/or 10-11;
- with the proviso that:
- at least two of R1-R6 are alkyl (1-4C);
- and
- the compound is other than compounds 17, 24 or 28 of FIG. 6B, compound 301-311 of FIG. 12(A) or compound 312-322 of FIG. 12(B).
Dependencies are inferred from the claim text.