Methods of making polyketides using hybrid polyketide synthases
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsRajesh S. Gokhale, Stuart Tsuji, Chaitan Khosla
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Abstract
Linking sequences which modulate cross-talk between modules of Type I polyketide synthases have been identified. Thus, arbitrarily chosen modules can be mixed and matched by supplying the appropriate linkers to obtain desired polyketide synthases and new polyketides. The modules are provided suitable linkers so that the polyketide chain is passed from one module to the other in the correct sequence. Synthetic peptides which mimic linkers can be used to inhibit the synthesis of polyketides. Kinetic channeling, both intrapolypeptide and interpolypeptide, of diketide intermediates in a Type I polyketide synthase can occur.
Claims12
A method to prepare a desired polyketide which method comprises incubating required substrates with a hybrid modular polyketide synthase (PKS) comprising at least a first extender module and a second extender module of a different PKS from said first module,
- wherein said extender modules are defined as consisting of the amino acid sequence from the N-terminus of the ketosynthase (KS) domain through the C-terminus of the acyl transferase protein (ACP) domain;
- wherein the C-terminus of said first module is covalently linked to the N-terminus of a intra-molecular linker (RAL) and the N-terminus of the second module is covalently linked to the C-terminus of said RAL, and
- wherein said RAL is defined as the amino acid sequence between the C-terminus of an upstream ACP domain and the N-terminus of an adjacent downstream KS domain;
- said ACP and KS domains occupying adjacent modules in the same reading frame;
- wherein either said first module or second module is not covalently linked to said RAL in a naturally occurring polyketide synthase;
- whereby the RAL effects the transfer of a nascent polyketide chain from said first module to said second module.
A method to prepare a desired polyketide which method comprises incubating required substrates with a hybrid modular polyketide synthase (PKS) comprising at least a first extender module and a second extender module of a different PKS from said first module,
- wherein said extender modules are defined as consisting of the amino acid sequence from the N-terminus of the ketosynthase (KS) domain through the C-terminus of the acyl transferase protein (ACP) domain;
- wherein the C-terminus of said first module is covalently linked to the N-terminus of a inter-molecular linker (ERL) and the N-terminus of the second module is covalently linked to the C-terminus of said ERL, and
- wherein said ERL is defined as a contiguous polypeptide comprising, in order, (1) the amino acid sequence beginning at the C-terminus of the ACP domain of the most downstream module of a first open reading frame and (2) the amino acid sequence upstream of the N-terminus of the most upstream KS domain of a second open reading frame, which second open reading frame is immediately adjacent to and downstream of said first open reading frame;
- and
- wherein either said first module or second module is not covalently linked to said ERL in a naturally occurring polyketide synthase;
- whereby the ERL effects the transfer of a nascent polyketide chain from said first module to said second module.
Dependencies are inferred from the claim text.