YEAST SYNTHETIC BIOLOGY PLATFORM FOR IDENTIFYING SHIKIMATE PATHWAY ENZYME INHIBITORS
1. A modified yeast comprising genomic modification(s) comprising disruption and/or deletion of all or a segment of least one endogenous gene that encodes a shikimate pathway enzyme, the modified yeast further comprising at least one supplemental gene encoding a heterologous enzyme that is homologous to the disrupted and/or deleted endogenous yeast gene, wherein the supplemental gene is from a distinct organism, and whereby the supplemental gene restores biosynthesis of chorismic acid to the modified yeast.
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 modified yeast comprising genomic modification(s) comprising disruption and/or deletion of all or a segment of least one endogenous gene that encodes a shikimate pathway enzyme, the modified yeast further comprising at least one supplemental gene encoding a heterologous enzyme that is homologous to the disrupted and/or deleted endogenous yeast gene, wherein the supplemental gene is from a distinct organism, and whereby the supplemental gene restores biosynthesis of chorismic acid to the modified yeast.
This application claims priority to U.S. provisional patent application No. 62/653,600, filed Apr. 6, 2018, the disclosure of which is incorporated herein by reference.
The present invention relates generally to modified fungi such as yeast that are suitable for screening compounds to determine if the compounds affect the multi-enzyme shikimate pathway, and for identifying components of the multi-enzyme pathway.
The rise of drug resistant microorganisms and the emergence of new pathogens together pose an enormous threat to human health and security. There is a desperate need to identify new antimicrobial compounds and their molecular targets. While high-throughput screens to identify antimicrobials are possible for many pathogens, on the whole screens are cumbersome because of biosafety requirements and the need to establish organism-specific assays amenable to high-throughput automation, a challenge for slow growing organisms like Mycobacterium tuberculosis or for difficult-to-culture pathogens such as apicomplexans. Thus, there is an ongoing and unmet need for alternatives to existing approaches to identify compounds that are candidates for use as antimicrobial agents, and for compounds that can target other unwanted organisms. The present disclosure is pertinent to this need.
The present disclosure provides compositions and methods that relate to cell-based platforms for compound discovery. The disclosure provides in certain aspects modified yeast that have the endogenous yeast shikimate pathway disrupted or deleted, and replaced with homologous pathway genes from one or more distinct organisms. In embodiments, the disruption/deletion of the yeast endogenous shikimate pathway results in the yeast being incapable of producing chorismic acid. The modified yeast are accordingly considered to be avatars that are useful for, among other purposes, screening test agents to assess the effects of the agents on proteins encoded by the homologous genes. Thus, embodiments of the disclosure facilitate, for example, pathogen DNA sequence to yeast-based pathway screens. In embodiments, the disclosure provides approaches to identify useful compounds, such as broad-spectrum anti-infective agents. Among other advantages, the disclosure provides for high-throughput screening of any of a wide variety of test agents, removing the need to work directly with pathogenic organisms, and the capability to analyze pathways from pathogens or other organisms that pose difficult challenges to lab-based culturing.
In view of the foregoing, it will be apparent to those skilled in the art that the present disclosure provides in one aspect a modified yeast comprising genomic modification(s) which comprise a disruption and/or deletion of all or a segment of least one endogenous gene that encodes a shikimate pathway enzyme. In embodiments, the disruption and/or deletion of all or a segment of least one endogenous gene that encodes a shikimate pathway enzyme is directed to at least one gene that encodes at least one of the following enzymes: DAHPS, 3-deoxyarabinoheptulosonate-7-phosphate synthase; DHQS, dehydroquinate synthase; DHQD, dehydroquinate dehydratase; SDH, shikimate dehydrogenase; SHK, shikimate kinase; EPSPS, 5-enolpyruvyl shikimate-3-phosphate synthase; or chorismate synthase. In embodiments, the endogenous yeast gene that is disrupted or deleted is selected from the group consisting of ARO1, ARO2, ARO3 and ARO4, and combinations thereof.
The yeast are engineered such that they contain at least one supplemental gene which encodes a heterologous enzyme that is homologous to the disrupted and/or deleted endogenous yeast gene. One or more supplemental genes can be used, and can originate in one, or more than one, distinct organisms. In certain embodiments, the supplemental gene is homologous to the endogenous yeast gene that is disrupted or deleted and is from a species that is infectious to mammals, insects, birds, fish or plants, or is from a prokaryotic pathogen, or is from a eukaryotic pathogen.
The supplemental gene(s) restore biosynthesis of chorismic acid to the modified yeast. In certain embodiments, the supplemental gene is present on an episomal element. The episomal element may contain a selectable marker, and/or may contain an essential gene of the yeast that is distinct from the gene encoding the endogenous yeast shikimate pathway enzyme. In the latter configuration, the essential yeast gene can be relocated from a chromosome of the yeast to the episomal element.
In another aspect, the disclosure provides a method for identification of a test agent that can inhibit one or more enzymes that are part of the shikimate pathway. The method comprises introducing the test agent into modified yeast as described above, wherein an inhibition of growth of the modified yeast relative to a control is indicative that the test agent inhibits one or more of the shikimate pathway enzymes. This approach is suitable for multiplex and high throughput approaches, and for testing a single agent, small sets of agents, and large numbers of distinct agent.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
The disclosure includes all proteins and contiguous segments of them described herein, all polynucleotides, and complementary and reverse-complementary sequences thereof.
The present disclosure provides novel, cell-based platforms for compound discovery. The approach is focused on the shikimate pathway that exhibits the following properties: (i) inhibition of the pathway causes microorganism (or other organism) growth inhibition or death; (ii) conservation in structure across species to provide useful antimicrobial spectrum; and (iii) structural differences or even absence from human cells to avoid mechanism-based toxicity.
In embodiments, the present disclosure provides modified yeast, as further described below. In embodiments, the yeast is a Saccharomyces, such as Saccharomyces cerevisiae. In various implementations the disclosure provides bespoke yeast “avatars” for screening against proteins expressed from heterologous gene sequences that are endogenous to pathogens, or other unwanted organisms, or any organism of interest. It is expected that these engineered cells will permit standardized, systematic sequence-to-screen approaches for anti-infective discovery, and analysis of test compounds for other purposes as described herein, going directly from pathogen DNA sequence to yeast-based pathway screens. Additional validation can be performed if desired using the actual organism from which shikimate pathway genes are adapted. The biological framework described herein can include a built in counter-screen against general yeast toxins and a mechanism to distinguish broad- and narrow-spectrum antibiotics.
For use in the assays of this disclosure that are described further below, a modified yeast is used. In certain embodiments, the modified yeast comprises genomic modification(s) comprising disruption and/or deletion of all or a segment of least one endogenous shikimate pathway gene. In embodiments, such disruptions/deletions result in rendering the yeast incapable of producing chorismic acid. In embodiments, the heterologous genes encoding shikimate pathway enzymes functionally complement the disrupted/deleted shikimate pathway genes, meaning they enable biosynthesis of chorismic acid. In embodiments, the heterologous genes code for enzymes that are homologous to the proteins encoded by the endogenous genes, but are from a distinct organism. The modified yeast thus comprise heterologous shikimate pathway genes, which are also referred to herein as supplemental genes. “Endogenous” means the shikimate genes are present on an unmodified chromosome of a yeast. “Heterologous” means one or more shikimate pathway genes that are not present in chromosomes of unmodified yeast.
As noted above, the heterologous genes code for proteins that are homologous to the proteins encoded by the endogenous yeast genes. “Homologous” means the shikimate pathway gene(s) encode enzyme(s) that perform the same or similar function as the proteins encoded by the endogenous yeast shikimate pathway genes. Those skilled in the art can readily determine whether or not any particular gene encodes a protein that is homologous to a protein encoded by another gene. Further, functional complementation in the modified yeast strain for the biosynthesis of chorismic acid indicates the gene(s) encoding the protein is homologous. In embodiments, the homologous gene that is expressed in the modified yeast codes for a protein that is from 50-100% identical to the protein expressed in the original, distinct organism. Homology may be, for example, across the entire length of the protein, or may be limited to one or more functional domains of the protein. Yeast modified according to this disclosure accordingly comprise supplemental shikimate pathway genes from organisms that are not the same species as the modified yeast.
In embodiments, the endogenous shikimate pathway proteins encoded by the gene(s) that is/are disrupted or deleted in the modified yeast, any one or combination of which may be replaced with a homologous gene from a different organism as described herein, are selected from the group of genes consisting of genes encoding the following enzymatic functions in S. cerevisiae: DAHPS, 3-deoxyarabinoheptulosonate-7-phosphate synthase (EC 188.8.131.52); DHQS, dehydroquinate synthase (EC 184.108.40.206); DHQD, dehydroquinate dehydratase (EC 220.127.116.11); SDH, shikimate dehydrogenase (EC 18.104.22.168); SHK, shikimate kinase (EC 22.214.171.124); EPSPS, 5-enolpyruvyl shikimate-3-phosphate synthase (EC 126.96.36.199); chorismate synthase (EC 188.8.131.52), and combinations thereof.
In embodiments, the disclosure uses modified yeast that comprise a disruption or deletion of any one or a combination of gene is selected from the group consisting of ARO1, ARO2, ARO3 and ARO4, and combinations thereof. Each of these yeast genes is known in the art, and their nucleotide sequences can be readily identified. Further, each of these genes is identified herein by its Kyoto Encyclopedia of Genes and Genomes (KEGG) reference number. KEGG is a publicly accessible database, available at www.genome.jp/kegg/. Each KEGG reference number can be used to access the nucleotide sequences of the yeast genes, as well as homologous genes from many different organisms. All polynucleotide sequences and all protein sequences associated with the KEGG “EC” numbers of this disclosure are incorporated by reference herein, as they exist on the filing date of this application or patent. Those skilled in the art will also recognize that additional entries for homologous genes may be entered into the KEGG database from time to time, and can be analyzed for use in embodiments of this disclosure. In embodiments, the homologous genes from distinct organisms that are used to modify yeast as described herein can be codon-optimized before being introduced into the yeast. In embodiments, the homologous gene(s) are introduced into the yeast, or are otherwise configured, such that the homologous gene is present on an episomal element. In embodiments, the episomal element optionally comprises a selectable marker, and/or an essential gene of the yeast that is distinct from the genes encoding the endogenous yeast shikimate pathway enzymes, and that has been relocated from a chromosome of the yeast to the episomal element. Compositions and methods for introducing supplemental genes, such as onto episomal elements, for disrupting endogenous genes, and for relocating genes from a native chromosome to an episomal element, are known in the art and can be adapted to implement embodiments described herein by those skilled in the art when provided the benefit of this disclosure.
In more detail, a yeast-based drug screening system of this disclosure is believed to be differentiated from existing yeast drug-screening platforms by expressing unique enzymatic activities of the shikimate metabolic pathway that together provide an essential function in yeast. Each enzymatic activity represents a unique druggable target. By screening at the pathway level, this effectively multiplexes the screen compared with a biochemical assay developed for an individual target. Pathway-level multiplexing targeting the shikimate pathway increases the chances of identifying hits in a drug screen by up to seven-fold. Thus, there is strong potential to identify useful compounds, which can include but are not necessarily limited to broad-spectrum anti-infective agents. Further, the disclosure allows for testing species-specificity of an identified compound using a series of yeast avatars, each expressing an enzymatic pathway transplanted from a different pathogen or other organism of interest. A yeast strain lacking its native, homologous pathway, by disrupting or deleting one or more genes that are required for pathway function, provides a counterscreen against off-target yeast cytotoxicity. Other advantages of the present disclosure include but are not necessarily limited to enabling high-throughput screening, avoiding the handling of pathogenic organisms, the ability to screen using pathways from pathogens or other organisms that are difficult to impossible to culture in the lab (e.g. parasites), the test candidate compound screen hits will be specific to the engineered shikimate pathway, and resistance mechanisms can be readily identified via drug selection and sequencing of metabolic pathways encoded by genetic elements from survivors.
The shikimate pathway is known in the art. It is composed of seven enzymatic steps (
While the lack of growth when the shikimate pathway is targeted is due in part to the inability to synthesize aromatic amino acids, the shikimate pathway is also linked to other metabolic networks and thus the efficacy of compounds targeting this pathway may be even greater than expected for simple starvation for lack of amino acids. Some evidence suggests that compounds targeting the shikimate pathway may be expected to induce stasis rather than cell death (i.e. bacteriostatic vs. bacteriocidal). Specifically, shikimate pathway mutants have been used to construct attenuated mutants of S. typhimurium, Salmonella typhi, Shigella flexneri, Pasteurella multocida, and A. salmonicida that can be exploited as live vaccines (18-24). In the absence of chorismate (and downstream molecules) the organism may scavenge aromatic amino acids from host cells to prevent immediate death, or via a starvation response may simply stop growing but not die. In the case of an infectious organism, this could allow the immune system of a host to mount an antibody-mediated response, resulting in clearance of the infection and also preventing future reinfection. Further, this outcome would avoid the absolute selective pressure associated with outright death, reducing emergence of early arising drug resistant strains. Further, the invention is not necessarily limited to testing candidates for use as antibiotics because it can be adapted, given the benefit of this disclosure, to identify agents that can function as herbicides and insecticides, or for use against any organism for which the shikimate pathway is essential to viability. Thus, it is considered that the disclosure can be used to identify, for example, test agents/compounds that affect shikimate pathways for a wide variety of pathogenic and/or destructive or otherwise unwanted organisms, such as pathogenic prokaryotes, and/or invasive species of any type that rely on the shikimate pathway, and/or parasites that rely on the shikimate pathway that can infect humans and/or non-human mammals. Non-human mammals include but are not limited to domesticated companion mammals, such as felines and canines, and agriculturally important animals, such as cattle, pigs and horses, and also for avian animals, such as agriculturally important fowl, and also for aquatic animals, including but not necessarily limited to agriculturally and/or environmentally important fish and shellfish. The disclosure can also be used to test compounds for activity against shikimate pathway enzymes in photosynthetic organisms, such as algae, and multicellular plants, including but not necessarily limited to invasive and/or otherwise unwanted plants such as weeds, and any organism that is parasitic or otherwise deleterious to multicellular plants.
In embodiments, the disclosure provides for determining whether or not the homologous genes and/or the proteins encoded by the genes are functional in the yeast avatars, such as illustrated by Examples 2 and 3, and
In embodiments, the disclosure provides for determining which particular genes/proteins are the target(s) of test agents. This is demonstrated, for example, in Example 4 and
In embodiments, this disclosure provides for determining which particular genes/proteins are functional or non-functional in yeast. This is demonstrated, for example, in Example 2 and
The disclosure is readily scalable for high-throughput approaches, which magnify the multiplex target screening capacity, and can be automated. The disclosure thus provides for concurrent assessment of the effects of many test agents against a plurality of distinct shikimate pathways, thereby facilitating simultaneous evaluation of thousands of interactions between test agents and shikimate pathways taken from a diversity of organisms. Accordingly, the disclosure is suitable for simultaneously testing and identifying agents that have antimicrobial properties, anti-parasite properties (where the parasite may be prokaryotic or eukaryotic), herbicidal compounds, as well as compounds that could have effects against, for example, organisms that have a commensal relationship with the host. For instance, a compound that can selectively target a shikimate pathway that is present in a pathogenic bacteria, but is not present or is less effected by the compound in a non-pathogenic and/or commensal bacteria, could be of particular usefulness.
In embodiments a method of this disclosure comprises adding at least one test agent to a culture of modified yeast cells, wherein the cells express a heterologous shikimate pathway. If desired, the culture of cells can be incubated for a period of time to assess one or more phenotypes, such as a growth phenotype, before adding the test agent. Generally, the cell culture comprising the modified fungus, such as yeast, and the test agent may be incubated together for a period of time. The incubations can be performed for any desirable amount of time, such as from at least one minute, to at least 1-16 hours, including all time values there between to the minute, and all ranges there between, or over a period of at least one to several days. The incubation can be performed at any desirable temperature, with any other controllable conditions, such as controlled humidity, air flow, oxygen content, and the like. The cell culture can be a liquid or solid medium or semi-solid medium, such as a liquid cell culture, or semi-solid culture medium of the type used in a petri or other culture dish. In embodiments, the cell culture comprises a liquid culture or semi-solid medium which is separated into a plurality of reaction chambers, such as in a high-throughput configuration. In an embodiment, the plurality of reaction chambers comprises up to or at least 384 reaction chambers. Into each reaction chamber a distinct test agent may be added, and a change in the cell culture due to the presence of the test agent can be observed. In alternative embodiments, a plurality of culture plates can be used, and individual yeast colonies can be assayed with distinct test agents, the effects of which can be assessed using any suitable technique, such as by human or automated inspection for visually detectable differences in, for example, colony size and/or cell morphology. In an embodiment, a change in the growth of the modified yeast identifies the test agent as a modulator of the heterologous shikimate pathway. In an embodiment, inhibition of growth and/or lethality of the modified yeast indicates the test compound inhibits the function of at least one enzyme that is part of the heterologous shikimate pathway. Any measurement of the effect of a test compound on modified yeast as described herein can be compared to any suitable control. In an embodiment, the control comprises a modified yeast wherein the endogenous shikimate pathway is disrupted/deleted, but wherein endogenous pathway genes are not supplemented with a homologous shikimate pathway counterparts from a distinct organism. In embodiments the control comprises a culture to which a test agent has not been added. In embodiments, the control comprises a culture to which a compound with a known effect has been added. In embodiments, the control comprises a culture to which has been added (or has not been added) one or more compounds that are essential for growth of a modified yeast, such as amino acids.
The following specific examples are provided to illustrate the invention, but are not intended to be limiting in any way.
This example provides a demonstration of a plurality of modified S. cerevisiae strains, and use of the strains for analyzing the effects of a representative test compound.
In this representative modified yeast, most of the endogenous shikimate pathway, a single gene encoding the penta-functional protein ARO1, is deleted (
Glyphosate is a known inhibitor of EPSPS, the sixth enzyme in the shikimate pathway (7). A panel of yeast avatars was tested for ability to grow on glyphosate (
This example illustrates a strategy to individually analyze enzymatic activity of heterologous shikimate pathways that do not complement aro1Δ yeast cells plated on medium lacking aromatic amino acids.
As discussed above, five steps of the endogenous shikimate pathway are provided by a single gene, ARO1, which encodes a penta-functional protein (
This example provides evidence of complementation of heterologous step 1 enzymatic activity in yeast.
A yeast strain lacking ARO3 and ARO4 (aro3Δ aro4Δ) was built by deleting these two genes using standard homologous recombination-dependent methods with selectable markers. The aro3Δ aro4Δ can no longer grow on medium lacking aromatic amino acids since the first step of the shikimate pathway is no longer present (
This example provides a demonstration that small molecule-mediated inhibition of heterologous pathways can be linked to a specific enzymatic activity or activities within the pathway.
Glyphosate is a well-known inhibitor of the shikimate pathway (
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While the invention has been described through specific embodiments, routine modifications will be apparent to those skilled in the art and such modifications are intended to be within the scope of the present invention.