Patent Application
20190264184
Polyketide natural products are produced biosynthetically by polyketide synthases (PKSs), e.g., type I polyketide synthases, in conjunction with other tailoring enzymes. Polyketide synthases (PKSs) are a family of large, multi-domain proteins whose catalytic functions are organized into modules to produce polyketides. The basic functional unit of polyketide synthase clusters is the module, which encodes a 2-carbon extender unit, e.g., derived from malonyl-CoA. The modules generally present in a polyketide synthase include i) a loading module; ii) extending modules; and iii) releasing modules. Within the module, the minimal domain architecture required for polyketide chain extension and elongation includes the ketosynthase (KS), acyl-transferase (AT) and the ACP (acyl-carrier protein) domains, and the specific chemistry of each module is encoded by the AT domain and by the presence of the β-ketone processing domains: ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains. Polyketide synthase biosynthesis proceeds by two key mechanisms: polyketide chain elongation with a polyketide synthase extending module and translocation of the polyketide intermediate between modules. Productive chain elongation depends on the concerted function of the numerous catalytic domains both within and between modules.
Combinatorial biosynthesis is a general strategy that has been employed to engineer polyketide synthase (PKS) gene clusters to produce novel drug candidates (Weissman and Leadlay, Nature Reviews Microbiology, 2005). To date, these strategies have relied on engineering PKS domain deletions and/or domain swaps within a module or by swapping an entire module from another cluster to produce a chimeric cluster. The problem with this approach is that protein engineering of the polyketide megasynthases via wholesale domain and/or module replacement, insertion, or deletion can perturb the “assembly line” architecture of the PKS, thus drastically reducing the amount of polyketide synthesized.
The present disclosure provides compositions and methods for use in combinatorial biosynthesis of polyketides without a significant loss of compound production by module swapping between polyketide synthase genes. Bioinformatics approaches may be used to predict module interface compatibility and therefore, the likelihood that a heterologous module may be swapped into a PKS gene. The resulting compatibility information may be used to engineer a polyketide synthase with an increased likelihood of functioning in assembly-line polyketide biosynthesis.
Accordingly, in one aspect, the disclosure provides an engineered polyketide synthase that includes one or more heterologous modules with altered enzymatic activity relative to a reference polyketide, wherein the engineered polyketide synthase is capable of producing a polyketide when expressed under conditions suitable to allow expression of a compound by the engineered polyketide synthase and wherein the one or more heterologous modules do not substantially inhibit polyketide translocation during polyketide biosynthesis.
In another aspect, the disclosure provides an engineered polyketide synthase including one or more heterologous modules with altered enzymatic activity relative to a reference polyketide, wherein the engineered polyketide synthase is capable of producing a polyketide when expressed under conditions suitable to allow expression of a compound by the engineered polyketide synthase and wherein the one or more heterologous modules include linking sequences which are compatible to the linking sequences of the modules adjacent thereto.
In another aspect, the disclosure provides an engineered polyketide synthase including one or more heterologous modules with altered enzymatic activity relative to a reference polyketide, wherein the engineered polyketide synthase is capable of producing a polyketide when expressed under conditions suitable to allow expression of a compound by the engineered polyketide synthase and wherein the polyketide expression level of the engineered polyketide synthase is at least 1% (e.g., at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%) of the polyketide expression level of the reference polyketide synthase.
In some embodiments, the polyketide expression level of the engineered polyketide synthase is at least 1-10% (e.g. at least 1-10%, at least 11-20%, at least 21-30%, at least 31-40%, at least 41-50%, at least 51-60%, at least 61-70%, at least 71-80%, at least 81-90%, at least 91-100%, at least 101-110%, at least 1111-120%, at least 121-130%, at least 131-140%, at least 141-150%). In some embodiments, the engineered polyketide synthase includes one or more heterologous modules with native linking sequences.
In some embodiments, the engineered polyketide synthase may include one, two, three, or more heterologous modules. In some embodiments in which the engineered polyketide synthase contains multiple heterologous modules, the heterologous modules may be adjacent in the engineered polyketide synthase. In some embodiments in which the polyketide synthase contains multiple heterologous modules, any of the modules may be separated by one or more native modules in the engineered polyketide synthase.
In some embodiments of any of the above described aspects, at least one of the one or more heterologous modules is an elongation module which modifies a β-carbonyl unit in the variable region of the polyketide.
In some embodiments of any of the above described aspects, at least one of the one or more heterologous modules includes a portion having at least 90% identity to any one of SEQ ID NO: 1-174.
In some embodiments of any of the above described aspects, at least one of the one or more heterologous modules includes a portion having the sequence of any one of SEQ ID NO: 1-174.
In another aspect, the disclosure provides a chimeric polyketide synthase, wherein at least one module of the chimeric polyketide synthase has been modified as compared to a polyketide synthase having the sequence of SEQ ID NO: 175-176.
In some embodiments, the disclosure provides a chimeric polyketide synthase where at least one module includes a portion having at least 90% identity to any one of SEQ ID NO: 1-174.
In another aspect, the disclosure provides a nucleic acid encoding any one of the above described polyketide synthases.
In some embodiments of any of the above described aspects, the nucleic acid encoding any one of the above described polyketide synthases further encodes an LAL in which the sequence encoding the LAL is operatively linked to the sequence encoding the polyketide synthase.
In some embodiments, the LAL may be a heterologous LAL.
In some embodiments, the LAL may include a portion having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to SEQ ID NO: 177. In some embodiments, the LAL may include a portion having the sequence of SEQ ID NO: 177. In some embodiments, the disclosure provides a nucleic in which the LAL has the sequence of SEQ ID NO: 177. In some embodiments, the LAL lacks a TTA inhibitory codon in an open reading frame.
In some embodiments of any of the foregoing nucleic acids, the nucleic acid includes an LAL binding site, in which the sequence encoding the LAL binding site is operatively linked to the sequence encoding the polyketide synthase.
In some embodiments, the LAL binding site includes a portion having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to the sequence of SEQ ID NO: 178 (CTAGGGGGTTGC). In some embodiments, the LAL binding site includes a portion having the sequence of SEQ ID NO: 178 (CTAGGGGGTTGC). In some embodiments, the LAL binding site has of the sequence of SEQ ID NO: 178 (CTAGGGGGTTGC). In some embodiments of the above described aspect, the LAL binding site has the sequence of SEQ ID NO: 179 (GGGGGT).
In some embodiments of any of the foregoing nucleic acids, the binding of an LAL to the LAL binding site promotes expression of the polyketide synthase.
In some embodiments of any of the foregoing nucleic acids, the nucleic acid encoding any one of the above described polyketide synthases, further encodes a nonribosomal peptide synthase.
In some embodiments of any of the foregoing nucleic acids, the nucleic acid encoding any one of the above described polyketide synthases further encodes a P450 enzyme.
In some embodiments of any of the foregoing nucleic acids, the nucleic acid encoding any one of the above described polyketides and a first P450 enzyme, further encodes a second P450 enzyme.
In another aspect, the disclosure provides an expression vector including any of the foregoing nucleic acids. In some embodiments, the expression vector may be an artificial chromosome, e.g., a bacterial artificial chromosome.
In another aspect, the disclosure provides a host cell including any of the above described expression vectors.
In another aspect, the disclosure provides a host cell including any of the foregoing polyketide synthases, in which the polyketide synthase is heterologous to the host cell.
In some embodiments of any of the foregoing host cells, the host cell naturally lacks an LAL and/or an LAL binding site.
In some embodiments of any of the foregoing host cells, the host cell includes an LAL capable of binding to an LAL binding site and regulating expression of a polyketide synthase. In some embodiments, the LAL and/or LAL binding site may be heterologous to the cell. In some embodiments, the host cell includes an LAL with a portion having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to the sequence of SEQ ID NO: 177.
In some embodiments of any of the foregoing host cells, t he host cell is a bacterium, e.g., an actinobacterium, such as an actinobacterium selected from the group consisting of Streptomyces ambofaciens, Streptomyces hygroscopicus, or Streptomyces malayensis. In some embodiments in which the host cells is an actinobacterium, the actinobacterium is S1391, S1496, or S2441.
In some embodiments of any of the foregoing host cells, the host cell has been modified to enhance expression of a polyketide synthase. For example, the host cell has been modified to enhance expression of a compound-producing protein by (i) deletion of an endogenous gene cluster which expresses a compound-producing protein; (ii) insertion of a heterologous gene cluster which expresses a compound-producing protein; (iii) exposure of the host cell to an antibiotic challenge; and/or (iv) introduction of a heterologous promoter that results in an at least 2-fold increase in expression of a compound compared to the homologous promoter.
In another aspect, the disclosure provides a method of producing a polyketide by culturing any of the foregoing host cells under suitable conditions.
In another aspect, the disclosure provides a method of producing a polyketide by culturing a host cell engineered to express any of the foregoing polyketide synthases under conditions suitable for the polyketide synthase to produce a polyketide.
In another aspect, the disclosure provides a method of producing a compound, the method including: (a) providing a parent polyketide synthase sequence capable of producing a compound; (b) determining the compatibility of at least one module of a second polyketide synthase with at least two modules of the parent polyketide synthase; (c) producing a nucleic acid encoding a modified polyketide synthase, wherein the modified polyketide synthase includes at least one module of a second polyketide synthase which has been determined to be compatible with the at least two modules of the parent polyketide synthase.
In another aspect, the disclosure provides a method of producing a compound, the method including: (a) providing a parent nucleic acid encoding a parent polyketide synthase; (b) modifying the parent nucleic acid to create a modified nucleic acid encoding a modified polyketide synthase capable of producing a compound, wherein the modification produces a modified polyketide synthase including at least one heterologous module.
In another aspect, the disclosure provides a method of producing a compound, the method including: (a) providing a parent polynucleotide sequence capable of producing a compound; (b) identifying one or more heterologous modules suitable for replacement of one or more modules in the parent polynucleotide sequence; (c) producing a nucleic acid encoding a modified polyketide synthase, wherein the modified polyketide synthase includes at least one heterologous module identified in step (b).
In another aspect, the disclosure provides a method of producing a plurality of engineered polyketide synthases, wherein each of the plurality of polynucleotides corresponds to an engineered polyketide synthase, and wherein each of the plurality of polynucleotides includes one or more heterologous modules with altered enzymatic activity relative to a reference polyketide. The method includes the steps of: (a) providing a parent polynucleotide sequence encoding a polyketide synthase; (b) identifying one or more modules for replacement in the parent polynucleotide sequence; (c) identifying two or more heterologous modules suitable for replacement for each of the modules identified in step (b); (d) generating a plurality of polynucleotides, wherein each of the plurality of polynucleotides corresponds to an engineered polyketide synthase, and wherein each of the plurality of polynucleotides includes a heterologous module selected from the two or more heterologous modules identified in step (c) in replacement of each of the one or more modules to be replaced identified in step (b).
A “polyketide synthase” refers to an enzyme belonging to the family of multi-domain enzymes capable of producing a polyketide. A polyketide synthase may be expressed naturally in bacteria, fungi, plants, or animals.
As used herein, the term “engineered polyketide synthase” is used to describe a non-natural polyketide synthase whose design and/or production involves action of the hand of man. For example, in some embodiments, an “engineered” polyketide synthase is prepared by production of a non-natural polynucleotide which encodes the polyketide synthase.
A cell that is “engineered to contain” and/or “engineered to express” refers to a cell that has been modified to contain and/or express a protein that does not naturally occur in the cell. A cell may be engineered to contain a protein, e.g., by introducing a nucleic acid encoding the protein by introduction of a vector including the nucleic acid.
The term “gene cluster that produces a small molecule” or “gene cluster that produces a compound,” as used herein, refers to a cluster of genes which encodes one or more compound-producing proteins.
The term “heterologous,” as used herein, refers to a relationship between two or more proteins, nucleic acids, compounds, and/or cell that is not present in nature. For example, the LAL having the sequence of SEQ ID NO: 177 is naturally occurring in the S18 Streptomyces strain and is thus homologous to that strain and would thus be heterologous to the S12 Streptomyces strain.
The terms “homologous” or “native,” as used interchangeably herein, refer to a relationship between two or more proteins, nucleic acids, compounds, and/or cells that is present naturally. For example, the LAL having the sequence of SEQ ID NO: 177 is naturally occurring in the S18 Streptomyces strain and is thus homologous to that strain.
The term “recombinant,” as used herein, refers to a protein that is produced using synthetic methods.
As used herein, the term “reference polyketide synthase” refers to a polyketide synthase that has a sequence having at least 80% identity (e.g., at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) to the sequence of an engineered polyketide synthase except to the sequence of the one or more modules which are modified.
As used here, the term “compatibility” refers to a measure of the likelihood of two adjacent modules to form a competent module-module junction, in which polyketide translocation is not substantially inhibited. A heterologous module may be considered compatible if it meets at least one of the following criteria: 1) the module is present in the same module clade as one or more adjacent modules of the reference PKS, as determined by the module-level phylogeny classification described in the detailed description of the invention; 2) the module is assigned a score of greater than or equal to 0.90 in the inter-module covariation analysis algorithm described in the detailed description of the invention; or 3) the module belongs to the same functional clade or sub-clade as one or more adjacent modules of the reference PKS, as determined by the evolutionary trace methodology outlined in the detailed description of the invention.
As used here, the term “linking sequence” refers to a sequence directly upstream or downstream of an inter-modular junction. For example, in a single module swap, the ACP for the upstream homologous module, the ACP and KS-AT didomain of the inserted heterologous module, and the KS of the downstream homologous module may all be considered linking sequences.
As used herein, the term “module” refers to a region of a polyketide synthase that includes multiple domains. Modules present in a polyketide synthase may include i) a loading module; ii) extending modules; and iii) releasing and/or cyclization modules, depending on whether the final polyketide is linear or cyclic. The domains which may be included in a given module include, but are not limited to, acyltransferase (AT), acyl carrier protein (ACP), keto-synthase (KS), ketoreductase (KR), dehydratase (DH), enoylreductase (ER), methyltransferase (MT), sulfhydrolase (SH), and thioesterase (TE).
As used here, the term “acceptor module” refers to a homologous module within a PKS cluster subject to engineering by module swapping. In the resulting engineered PKS cluster, the acceptor module is absent.
As used here, the term “donor module” refers to a heterologous module that is introduced into an engineered PKS cluster.
As used here, the term “module swapping” refers to the exchange of one or more heterologous donor modules for one or more homologous acceptor modules.
As used here, the term “does not substantially inhibit polyketide translocation” refers to the ability of a heterologous PKS module to function in a biosynthetic assembly line. For example, a heterologous loading module does not substantially inhibit polyketide translocation if the loading module is able to load a starter unit onto its ACP domain and pass the starter unit to the KS domain of the adjacent (n+1) extender module. A heterologous extender module does not substantially inhibit polyketide translocation if the extender module is able to receive a starter unit or polyketide chain from the previous (n−1) module, catalyze the addition of an extender unit, and pass the elongated polyketide chain to the adjacent (n+1) module. In some embodiments, a heterologous module does not substantially inhibit polyketide translocation if the engineered PKS that includes the heterologous module produces a compound in levels that are detectable by a highly sensitive detection method, e.g., LC-TOF mass spectrometry.
An extender unit, e.g., a malonyl-CoA, is loaded onto the acyl carrier protein domain of the current module catalyzed by another acyltransferase domain. The polyketide chain is then elongated by subsequent extender modules after being passed from the acyl carrier protein domain of module n to the ketosynthase domain of the n+1 module. The acyl carrier protein bound extender unit reacts with the polyketide chain bound to the ketosynthase domain with expulsion of CO2 to produce an extended polyketide chain bound to the acyl carrier protein. Each added extender unit may then be modified by β-ketoprocessing domains, i.e., ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons).
The present invention describes compositions and methods for the production of polyketide compounds by an engineered polyketide synthase that includes one or more heterologous modules. The present invention also describes methods for predicting the compatibility of linking sequences of heterologous module-module junctions to produce an engineered polyketide synthase that does not substantially inhibit translocation during polyketide biosynthesis.
Compounds that may be produced with the methods of the invention include, but are not limited to, polyketides and polyketide macrolide antibiotics such as erythromycin; hybrid polyketides/non-ribosomal peptides such as rapamycin and FK506; carbohydrates including aminoglycoside antibiotics such as gentamicin, kanamycin, neomycin, tobramycin; benzofuranoids; benzopyranoids; flavonoids; glycopeptides including vancomycin; lipopeptides including daptomycin; tannins; lignans; polycyclic aromatic natural products, terpenoids, steroids, sterols, oxazolidinones including linezolid; amino acids, peptides and peptide antibiotics including polymyxins, non-ribosomal peptides, β-lactams antibiotics including carbapenems, cephalosporins, and penicillin; purines, pteridines, polypyrroles, tetracyclines, quinolones and fluoroquinolones; and sulfonamides.
Polyketide synthases (PKSs) are a family of multi-domain enzymes that produce polyketides. Type I polyketide synthases are large, modular proteins which include several domains organized into modules. The modules generally present in a polyketide synthase include i) a loading module; ii) extending modules; and iii) releasing and/or cyclization modules depending on whether the final polyketide is linear or cyclic. The domains which generally are found in the modules are acyltransferase (AT), acyl carrier protein (ACP), keto-synthase (KS), ketoreductase (KR), dehydratase (DH), enoylreductase (ER), methyltransferase (MT), sulfhydrolase (SH), and thioesterase (TE).
A polyketide chain and the starter groups are generally bound to the thiol groups of the active site cysteines in the ketosynthase domain (the polyketide chain) and acyltransferase domain (the loading group and malonyl extender units) through a thioester linkage. Binding to acyl carrier protein (ACP) is mediated by the thiol of the phosphopantetheinyl group, which is bound to a serine hydroxyl of ACP, to form a thioester linkage to the growing polyketide chain. The growing polyketide chain is handed over from one thiol group to another by trans-acylations and is released after synthesis by hydrolysis or cyclization.
The synthesis of a polyketide begins by a starter unit, being loaded onto the acyl carrier protein domain of the PKS catalyzed by the acyltransferase in the loading module. An extender unit, e.g., a malonyl-CoA, is loaded onto the acyl carrier protein domain of the current module catalyzed by another acyltransferase domain. The polyketide chain is then elongated by subsequent extender modules after being passed from the acyl carrier protein domain of module n to the ketosynthase domain of the n+1 module. The acyl carrier protein bound extender unit reacts with the polyketide chain bound to the ketosynthase domain with expulsion of CO2 to produce an extended polyketide chain bound to the acyl carrier protein. Each added extender unit may then be modified by β-ketoprocessing domains, i.e., ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons). Once the synthesis of the polyketide is complete, a thioesterase domain in the releasing modules hydrolyzes the completed polyketide chain from the acyl carrier protein of the last extending module. The compound released from the PKS may then be further modified by other proteins, e.g., nonribosomal peptide synthase. In some cases, the biosynthetic cluster harbors polyketide megasynthases and a non-ribosomal peptide synthase (NRPS). This hybrid architecture is referred to as hybrid PKS/NRPS.
PKS biosynthesis proceeds by two key mechanisms: polyketide chain elongation within a module and translocation between modules (
β-ketone processing domains are the domains in a PKS which result in modification of the elongation groups added during the synthesis of a polyketide. Each β-ketone processing domain is capable of changing the oxidation state of an elongation group. The β-ketone processing domains include ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons).
The present disclosure provides methods and compositions related to engineered polyketide synthases produced by swapping modules between related PKS clusters. Polyketide translocation is controlled by protein-protein interactions at the inter-modular junctions. In some embodiments, module swapping is guided by bioinformatic predictions to determine which modules have the highest probability of functioning in assembly-line polyketide biosynthesis. Multiple bioinformatics methods are used to determine the structural information in PKS sequence alignments to predict protein-protein interactions that mediate polyketide translocation at the inter-modular junction. The present disclosure includes a DNA assembly strategy to swap one or more heterologous donor modules for one or more acceptor modules to generate hybrid PKS clusters.
In some embodiments, module swapping is achieved by single, di- or tri-, or multi-module capture. In some embodiments, module swapping may be performed by exchange of the loading module. In some embodiments, module swapping may be performed by exchange of one or more extender modules. In some embodiments, module swapping may be performed by exchange of one or more releasing or cyclization modules. In some embodiments, two or more heterologous donor modules may replace a single acceptor module which may result in the production of a ring-expanded compound. In some embodiments, a single heterologous donor module may replace two or more acceptor modules which may result in a contracted ring compound. In some embodiments, the engineered polyketide synthases may produce novel compounds.
In some embodiments, the pooled capture and transfer of single, di- or tri-, or multi-module units enables the production of combinatorial libraries of engineered polyketide synthases. A dimodule unit, for example, consists of two heterologous modules, each of which may be independently selected from a pool of heterologous modules. A trimodule unit, example, consists of three heterologous modules, each of which may be independently selected from a pool of heterologous modules. One or more modules of a polyketide synthase may be replaced with a single, di-, tri-, or multi-module unit, where the single, di-, tri- or multi-module unit is selected from a pool of single- di-, tri- or multi-module units produced by combinatorial synthesis. Exemplary methods for the production of combinatorial libraries of engineered polyketide synthases (e.g., dimodule and trimodule combinatorial libraries) are provided in Examples 2 and 4.
In some embodiments of the invention, single-molecule long-read sequencing technology (e.g., Nanopore sequencing or SMRT sequencing) may be used to characterize libraries of engineered polyketide synthases which are produced by any of the methods described herein. In particular, single-molecule long-read sequencing (e.g., Nanopore sequencing or SMRT sequencing) may be used to characterize (e.g., deconvolute) combinatorial libraries of engineered polyketide synthases (e.g., combinatorial libraries of engineered polyketides synthases which are produced by pooled capture and transfer of single, di- or tri-, or multi-module units). Single-molecule long-read sequencing enables the identification of the module or modules which are incorporated into the combinatorial library. This further enables the prediction of the chemistry of the resulting plurality of engineered polyketide synthases. The predicted enzymatic chemistry can therefore be connected to the compounds produced by the engineered polyketide synthases. The resulting compounds may be identified by chemical methods of analysis known to one of skill in the art (e.g., mass spectrometry or high performance liquid chromatography). Furthermore, the predicted enzymatic chemistry can be connected to the function of the resulting compounds (e.g., binding to a target protein or inducing a phenotype, such as a cell based phenotype). Accordingly, long-read sequencing of a genetically encoded molecule may allow for genotypic-phenotypic linkage.
Single-molecule long-read sequencing technologies may be considered to include any sequencing technology which enables the sequencing of a single molecule of a biopolymer (e.g., a polynucleotide such as DNA or RNA), and which enables read lengths of greater than 2 kilobases (e.g., greater than 5 kilobases, greater than 10 kilobases, greater than 20 kilobases, greater than greater than 50 kilobases, or greater 100 kilobases). Single-molecule long-read sequencing technologies may enable the sequencing of multiple single molecules of DNA or RNA in parallel. Single-molecule long-read sequencing technologies may include sequencing technologies that rely on individual compartmentalization of each molecule of DNA or RNA being sequenced.
Nanopore sequencing is an exemplary single-molecule long-read sequencing technology that may be used to characterize libraries of engineered polyketide synthases that are prepared by any of the methods described herein. Nanopore sequencing enables the long-read sequencing of single molecules of of biopolymers (e.g., polynucleotides such as DNA or RNA). Nanopore sequencing relies on protein nanopores set in an electrically resistant polymer membrane. An ionic current is passed through the nanopores by setting a voltage across this membrane. If an analyte (e.g., a biopolymer such as DNA or RNA) passes through the pore or near its aperture, this event creates a characteristic disruption in current. The magnitude of the electric current density across a nanopore surface depends on the composition of DNA or RNA (e.g., the specific base) that is occupying the nanopore. Therefore, measurement of the current makes it possible to identify the sequence of the molecule in question. Exemplary methods for the use of Nanopore sequencing to characterize combinatorial libraries of engineered polyketide synthases are provided in Example 3.
Single molecule real-time (SMRT) sequencing (PacBio) is an exemplary single-molecule long-read sequencing technology that may be used to characterize libraries of engineered polyketide synthases that are prepared by any of the methods described herein. SMRT is a parallelized single molecule DNA sequencing method. SMRT utilizes a zero-mode waveguide (ZMW). A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
The present disclosure provides complementary bioinformatic approaches for the prediction of functional protein-protein interactions at the module-module junction (
Sequence divergence between polyketide modules and inter-module linkers suggests importance in module-module compatibility. In some embodiments, a module-level phylogenic map may be constructed by multiple sequence alignment of PKS modules. For example, a module-level phylogenic map was generated by multiple sequence alignments of complete FK-family modules (
Inter-module residue covariation across the intermodular junction was computed to generate an algorithm to rank order intermodule compatibility (
To generate a score for a potential single module substitution, the following alignments are retrieved from the original multiple alignment: the ACP for the upstream domain, the ACP and KS-AT didomain for the inserted module, and the KS for the downstream module. These are used to synthesize two rows compatible with the original multiple alignment: one with the ACP of the upstream module and KS-AT of the inserted module and a second with the ACP of the inserted module and KS-AT of the downstream module. For each I,J pair in the saved coupling matrix, the amino acids at position I and J in the synthesized alignment are retrieved (aaI, aaJ). The mutual information for this amino acid pair within the alignment is multiplied by the coupling score to generate a raw score. The raw scores are computed for each I,J pair in the saved coupling matrix and for each of the two synthesized alignments. The sum of the raw scores for the heterologous donor domain is divided by the sum of the raw scores for the homologous native domain to generate a normalized percentage score. Candidate swaps with the same chemistry are ranked by this score. In the case of multiple module swaps, the process is expanded, e.g., if N donor domains are to be swapped in, then one synthetic alignment is generated for the preceding module's ACP domain and the first donor module's KS-AT didomain, another for the first donor modules' ACP domain and the second donor module's KS-AT didomain and so forth, concluding with the final donor domain's ACP and the first module of the recipient synthase downstream of the breakpoint. Scores are computed and normalized in the same manner: the scores for the swapped modules are normalized for the score computed for the native modules. In some embodiments, a heterologous module is compatible if the module is assigned a score of greater than or equal to 0.90 in the inter-module covariation analysis algorithm described herein.
Evolutionary Trace Analysis to Identify Modules within Functional Clades or Sub-Clades
As an additional test of module compatibility, evolutionary trace analysis may be used to identify modules that belong to the same functional clade or sub-clade (
The Large ATP-binding regulators of the LuxR family of transcriptional activators (LALs) are known transcriptional regulators of polyketides such as FK506 or rapamycin. The LAL family has been found to have an active role in the induction of expression of some types of natural product gene clusters, for example PikD for pikromycin production and RapH for rapamycin production. Binding of the LAL or multiple LALs in a complex to specific sites in the promoters of genes within a gene cluster that produces a small molecule (e.g., a polyketide synthase gene cluster) potentiates expression of the gene cluster and hence promotes production of the compound (e.g., a polyketide). In some embodiments, LALs may be used for the regulation of the expression of engineered PKS clusters.
LALs include three domains, a nucleotide-binding domain, an inducer-binding domain, and a DNA-binding domain. A defining characteristic of the structural class of regulatory proteins that include the LALs is the presence of the AAA+ ATPase domain. Nucleotide hydrolysis is coupled to large conformational changes in the proteins and/or multimerization, and nucleotide binding and hydrolysis represents a “molecular timer” that controls the activity of the LAL (e.g., the duration of the activity of the LAL). The LAL is activated by binding of a small-molecule ligand to the inducer binding site. In most cases the allosteric inducer of the LAL is unknown. In the case of the related protein MalT, the allosteric inducer is maltotriose. Possible inducers for LAL proteins include small molecules found in the environment that trigger compound (e.g., polyketide) biosynthesis. The regulation of the LAL controls production of compound-producing proteins (e.g., polyketide synthases) resulting in activation of compound (e.g., polyketide) production in the presence of external environmental stimuli. Therefore, there are gene clusters that produce small molecules (e.g., PKS gene clusters) which, while present in a strain, do not produce compound either because (i) the LAL has not been activated, (ii) the strain has LAL binding sites that differ from consensus, (iii) the strain lacks an LAL regulator, or (iv) the LAL regulator may be poorly expressed or not expressed under laboratory conditions. Since the DNA binding region of the LALs of the known PKS LALs are highly conserved, the known LALs may be used interchangeably to activate PKS gene clusters other than those which they naturally regulate. In some embodiments, the LAL is a fusion protein.
In some embodiments, an LAL may be modified to include a non-LAL DNA-binding domain, thereby forming a fusion protein including an LAL nucleotide-binding domain and a non-LAL DNA-binding domain. In certain embodiments, the non-LAL DNA-binding domain is capable of binding to a promoter including a protein-binding site positioned such that binding of the DNA-binding domain to the protein-binding site of the promoter promotes expression of a gene of interest (e.g., a gene encoding a compound-producing protein, as described herein). The non-LAL DNA binding domain may include any DNA binding domain known in the art. In some instances, the non-LAL DNA binding domain is a transcription factor DNA binding domain. Examples of non-LAL DNA binding domains include, without limitation, a basic helix-loop-helix (bHLH) domain, leucine zipper domain (e.g., a basic leucine zipper domain), GCC box domain, helix-turn-helix domain, homeodomain, srf-like domain, paired box domain, winged helix domain, zinc finger domain, HMG-box domain, Wor3 domain, OB-fold domain, immunoglobulin domain, B3 domain, TAL effector domain, Cas9 DNA binding domain, GAL4 DNA binding domain, and any other DNA binding domain known in the art. In some instances, the promoter is positioned upstream to the gene of interest, such that the fusion protein may bind to the promoter and induce or inhibit expression of the gene of interest. In certain instances, the promoter is a heterologous promoter introduced to the nucleic acid (e.g., a chromosome, plasmid, fosmid, or any other nucleic acid construct known in the art) containing the gene of interest. In other instances, the promoter is a pre-existing promoter positioned upstream to the gene of interest. The protein-binding site within the promoter may, for example, be a non-LAL protein-binding site. In certain embodiments, the protein-binding site binds to the non-LAL DNA binding domain, thereby forming a cognate DNA binding domain/protein-binding site pair.
In some embodiments, the LAL is encoded by a nucleic acid having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 180-212 or has a sequences with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to any one of SEQ ID Nos: 180-212.
In some embodiments, a gene cluster (e.g., a PKS gene cluster) includes one or more promoters that include one or more LAL binding sites. The LAL binding sites may include a polynucleotide consensus LAL binding site sequence (e.g., as described herein). In some instances, the LAL binding site includes a core AGGGGG (SEQ ID NO: 213) motif. In certain instances, the LAL binding site includes a sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) homology to SEQ ID NO: 213. The LAL binding site may include mutation sites that have been restored to match the sequence of a consensus or optimized LAL binding site. In some embodiments, the LAL binding site is a synthetic LAL binding site. In some embodiments, synthetic LAL binding sites may be identified by (a) providing a plurality of synthetic nucleic acids including at least eight nucleotides; (b) contacting one or more of the plurality of nucleotides including at least eight nucleotides with one or more LALs; (c) determining the binding affinity between a nucleic acid of step (a) and an LAL of step (b), wherein a synthetic nucleic acid is identified as a synthetic LAL binding site if the affinity between the synthetic nucleic acid and an LAL is greater than X. The identified synthetic LAL binding sites may then be introduced into a host cell in a compound-producing cluster (e.g., a PKS cluster).
In some embodiments, a pair of LAL binding site and a heterologous LAL or a heterologous LAL binding site and an LAL that have increased expression compared to a natural pair may be identified by (a) providing one or more LAL binding sites; (b) contacting one or more of the LAL binding sites with one or more LALs; (c) determining the binding affinity between a LAL binding site and an LAL, wherein a pair having increased expression is identified if the affinity between the LAL binding site and the LAL is greater than the affinity between the LAL binding site and its homologous LAL and/or the LAL at its homologous LAL binding site. In some embodiments, the binding affinity between the LAL binding site and the LAL is determined by determining the expression of a protein or compound by a cell which includes both the LAL and the LAL binding site.
In some embodiments, the recombinant LAL is a constitutively active LAL. For example, the amino acid sequence of the LAL has been modified in such a way that it does not require the presence of an inducer compound for the altered LAL to engage its cognate binding site and activate transcription of a compound producing protein (e.g., polyketide synthase). Introduction of a constitutively active LAL to a host cell would likely result in increased expression of the compound-producing protein (e.g., polyketide synthase) and, in turn, increased production of the corresponding compound (e.g., polyketide).
FK gene clusters are arranged with a multicistronic architecture driven by multiple bidirectional promoter-operators that harbor conserved (in single or multiple, and inverted to each other and/or directly repeating) GGGGGT (SEQ ID NO: 179) motifs presumed to be LAL binding sites. Bidirectional LAL promoters may be converted to unidirectional ones (UniLALs) by strategically deleting one of the opposing promoters, but maintaining the tandem LAL binding sites (in case binding of LALs in the native promoter is cooperative, as was demonstrated for MalT). Functionally this is achieved by removal of all sequences 3′ of the conserved GGGGGT (SEQ ID NO: 179) motif present on the antisense strand (likely containing the −35 and −10 promoter sequences), but leaving intact the entire sequence on the sense strand. As a consequence of this deletion, transcription would be activated in one direction only. The advantages of this feed-forward circuit architecture would be to tune and/or maximize LAL expression during the complex life cycle of Streptomyces vegetative and fermentation growth conditions
In some embodiments, the host cell is a bacteria such as an Actiobacterium. For example, in some embodiments, the host cell is a Streptomyces strain. In some embodiments, the host cell is Streptomyces anulatus, Streptomyces antibioticus, Streptomyces coelicolor, Streptomyces peucetius, Streptomyces sp. ATCC 700974, Streptomyces canus, Streptomyces nodosus, Streptomyces (multiple sp.), Streptoalloteicus hindustanus, Streptomyces hygroscopicus, Streptomyces avermitilis, Streptomyces viridochromogenes, Streptomyces verticillus, Streptomyces chartruensis, Streptomyces (multiple sp.), Saccharothrix mutabilis, Streptomyces halstedii, Streptomyces clavuligerus, Streptomyces venezuelae, Strteptomyces roseochromogenes, Amycolatopsis orientalis, Streptomyces clavuligerus, Streptomyces rishiriensis, Streptomyces lavendulae, Streptomyces roseosporus, Nonomuraea sp., Streptomyces peucetius, Saccharopolyspora erythraea, Streptomyces filipinensis, Streptomyces hygroscopicus, Micromonospora purpurea, Streptomyces hygroscopicus, Streptomyces narbonensis, Streptomyces kanamyceticus, Streptomyces coffinus, Streptomyces lasaliensis, Streptomyces lincolnensis, Dactosporangium aurantiacum, Streptomyces toxitricini, Streptomyces hygroscopicus, Streptomyces plicatus, Streptomyces lavendulae, Streptomyces ghanaensis, Streptomyces cinnamonensis, Streptomyces aureofaciens, Streptomyces natalensis, Streptomyces chattanoogensis L10, Streptomyces lydicus A02, Streptomyces fradiae, Streptomyces ambofaciens, Streptomyces tendae, Streptomyces noursei, Streptomyces avermitilis, Streptomyces rimosus, Streptomyces wedmorensis, Streptomyces cacaoi, Streptomyces pristinaespiralis, Streptomyces pristinaespiralis, Actinoplanes sp. ATCC 33076, Streptomyces hygroscopicus, Lechevalieria aerocolonegenes, Amycolatopsis mediterranei, Amycolatopsis lurida, Streptomyces albus, Streptomyces griseolus, Streptomyces spectabilis, Saccharopolyspora spinosa, Streptomyces ambofaciens, Streptomyces staurosporeus, Streptomyces griseus, Streptomyces (multiple species), Streptomyces acromogenes, Streptomyces tsukubaensis, Actinoplanes teichomyceticus, Streptomyces glaucescens, Streptomyces rimosus, Streptomyces cattleya, Streptomyces azureus, Streptoalloteicus hindustanus, Streptomyces chartreusis, Streptomyces fradiae, Streptomyces coelicolor, Streptomyces hygroscopicus, Streptomyces sp. 11861, Streptomyces virginiae, Amycolatopsis japonicum, Amycolatopsis balhimycini, Streptomyces albus J1074, Streptomyces coelicolor M1146, Streptomyces lividans, Streptomyces incarnates, Streptomyces violaceoruber, or Streptomyces griseofuscus. In some embodiments, the host cell is an Escherichia strain such as Escherichia coli. In some embodiments, the host cell is a Bacillus strain such as Bacillus subtilis. In some embodiments, the host cell is a Pseudomonas strain such as Pseudomonas putitda. In some embodiments, the host cell is a Myxococcus strain such as Myxococcus xanthus.
Inter-module residue covariation analysis and evolutionary trace analysis were used to predict 10 heterologous donor modules that would successfully replace module 3 of the PKS that produces Compound 1 (
A single module was swapped to produce an engineered PKS by replacing module 3 of the PKS that produces Compound 1 with module 3 of Streptomyces strain S317. The donor S317 module 3 was PCR amplified and Gibson cloned into position 3 of the PKS that produces Compound 1 (
To replace module 4 in the PKS that produces Compound 1, module swapping prediction algorithms based on inter-module covariation were used to generate a list of 16 modules encoding 4 chemistries. Gibson-based subcloning into module 4 was not as efficient as module 3. Gibson cloning, which involves a ssDNA intermediate, is difficult in high GC-rich regions, and direct ligation of donor modules to restriction sites with 4 bp overhangs may not be sensitive to local GC content. Therefore AM and SpeI sites were introduced at new positions in the inter-module flanking region to generate a direct ligation acceptor BAC. This direct ligation acceptor BAC was linearized by digestion with AflII and SpeI, and 12 donor modules were gel-purified, digested with AflII and XbaI and subcloned by ligation.
Single module swaps of either module 3 or module 4 in the PKS that produces Compound 1 generated novel Compounds 2-5 (
Pooled transfer of dimodule libraries was used to simultaneously replace modules 3 and 4 in the PKS that produces Compound 1 and generate a plurality of engineered PKS clusters (
Replicate BACs encoding single module and dimodule swaps were conjugated to optimized Streptomyces producer strain S2441 and solid-phase extracted samples were subjected to LC-TOF mass spectrometry with the expected protein binding partner, purified FKBP12 protein. Further analysis confirmed that dimodule library generation is capable of engineering PKS clusters that express novel compounds in high yield (
A 650-member combinatorial library of engineered derivatives of the PKS that produces Compound 1 was produced by dimodule swapping. A total of 31 modules were amplified for transfer the module 3 position and 25 modules for the module 4 position of the PKS that produces Compound 1 (
A subset of the library corresponding to 15 different donor modules at the module 3 position and 15 different donor modules at the module 4 position produced a potential combinatorial library of 225 novel PKS clusters and resulting novel compounds (the 15×15 dimodule library). Because the dimodule library was assembled as a pool, rarefaction analysis was performed to determine how many clones needed to be conjugated, fermented, and extracted to effectively sample >90% of the diversity of the library. Rarefaction analysis indicated that 650 clones corresponded to a statistical sampling >90% of the dimodule library (
A library corresponding to 15 different donor modules at the module 3 position and 15 different donor modules at the module 4 position (the 15×15 dimodule library), produced according to the methods of Example 2, was characterized by Nanopore sequencing (
The combinatorial module swap protocols were modified to generate trimodule assemblies in the PKS that produces Compound 7 (
Trimodule assembly leverages the technical advances of the dimodule protocol with an additional “proof-reading” Gibson cloning step to insert the captured trimodule assembly into the PKS that produces Compound 7 (
A heterologous dimodule donor assembly encoding mDEK chemistry and K chemistry was swapped into module 3, a single module acceptor, of the PKS that produces Compound 1 by the methods described above (
Rapamycin is a natural product synthesized by a mixed polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system. Rapamycin shares a common structural motif with related natural product FK506 which is responsible for binding to FK506-binding proteins (FKBPs). During biogenesis of Rapamycin, loading modules bind and load a 4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid starter unit via a CaiC domain, which functions as a carboxylic acid ligase (CL) like domain (
The X23 PKS cluster produces Compound 9 and Compound 10 (
It is to be understood that while the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and alterations are within the scope of the following claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any polynucleotide or protein encoded thereby; any method of production; any method of use) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/58800 | 10/27/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62414410 | Oct 2016 | US |
