Yeast synthetic biology platform for identifying shikimate pathway enzyme inhibitors
Provided are compositions and methods for compound discovery. Modified yeast that have their endogenous yeast shikimate pathway disrupted or deleted, and replaced with homologous pathway genes from one or more distinct organisms, are provided and used in assays of test agents. The homologous pathway genes are designed to supplement the disrupted or deleted shikimate pathway genes. The assays are designed to identify whether or not the test agents can interfere with the function of enzymes in the shikimate pathway from organisms that are distinct from the yeast avatar hosts. In embodiments, the disruption/deletion of the yeast endogenous shikimate pathway results in the yeast being incapable of producing chorismic acid.
1. A method for identification of a test agent that can inhibit one or more enzymes that are part of a shikimate pathway, the method comprising introducing the test agent into modified Saccharomyces yeast, the modified Saccharomyces yeast comprising:
i) a disruption of an endogenous Saccharomyces yeast gene that encodes the shikimate pathway enzyme, and
ii) a supplemental gene encoding a heterologous enzyme that is homologous to the enzyme encoded by the endogenous Saccharomyces yeast gene;
wherein an inhibition of growth of the modified Saccharomyces yeast relative to a control is indicative that the test agent inhibits the shikimate pathway enzyme, and wherein the control comprises growth of the modified Saccharomyces yeast into which the test agent is introduced, and wherein the growth of the modified Saccharomyces yeast is performed on a medium that is supplemented with one or more aromatic amino acids such that growth of the modified yeast is independent of the function of the supplemental gene.
2. The method of claim 1 , wherein a plurality of distinct test agents are introduced into a plurality of distinct Saccharomyces yeast cultures.
3. The method of claim 2 , wherein the plurality of test agents are distinct test agents.
4. The method of claim 1 , wherein the endogenous Saccharomyces yeast gene that is disrupted or deleted is ARO1.
5. The method of claim 1 , wherein the supplemental gene is homologous to the endogenous Saccharomyces yeast gene that is disrupted or deleted and is from a species that is infectious to mammals, insects, birds, fish or plants.
6. The method of claim 1 , wherein the supplemental gene is from a prokaryotic pathogen.
7. The method of claim 1 , wherein the supplemental gene is from a eukaryotic pathogen.