Recombinant production of novel polyketides
AssigneesLeland Stanford Junior University, The John Innes Institute
InventorsChaitan Khosla, David A. Hopwood, Suzanne Ebert-Khosla, Robert McDaniel, Hong Fu, Camilla Kao
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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.
Claims24
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;
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- 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.
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.
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.
Dependencies are inferred from the claim text.