COMPOSITIONS AND METHODS FOR THE PRODUCTION OF COMPOUNDS

Abstract

The present disclosure provides proteins, nucleic acids, vectors, and host molecules useful for the production of compounds of interest, and methods for their use.

Claims

1. An engineered polyketide synthase comprising 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.

2. An engineered polyketide synthase comprising 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 comprise linking sequences which are compatible to the linking sequences of the modules adjacent thereto.

3. An engineered polyketide synthase comprising 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% of the polyketide expression level of the reference polyketide synthase.

4. The engineered polyketide synthase of any one of claims 1 to 3, wherein the one or more heterologous modules comprise native linking sequences.

5. The engineered polyketide synthase of any one of claims 1 to 4, wherein the engineered polyketide synthase comprises two or more heterologous modules.

6. The engineered polyketide synthase of claim 5, wherein the two or more heterologous modules are adjacent.

7. The engineered polyketide synthase of any one of claims 1 to 6, wherein the engineered polyketide synthase comprises three or more heterologous modules.

8. The engineered polyketide synthase of claim 7, wherein the three or more heterologous modules are adjacent.

9. The engineered polyketide synthase of any one of claims 1 to 8, wherein the heterologous module is an elongation module which modifies a -carbonyl unit in the variable region of the polyketide.

10. The engineered polyketide synthase of any one of claims 1 to 9, wherein at least one of the one or more heterologous modules comprises a portion having at least 90% identity to any one of SEQ ID NO: 1-174.

11. The engineered polyketide synthase of any one of claims 1 to 10, wherein at least one of the one or more heterologous modules comprises a portion having the sequence of any one of SEQ ID NO: 1-174.

12. 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.

13. The chimeric polyketide synthase of claim 12, wherein the at least one module comprises a portion having at least 90% identity to any one of SEQ ID NO: 1-174.

14. A nucleic acid encoding a polyketide synthase of any one of claims 1 to 13.

15. The nucleic acid of claim 15, wherein the nucleic acid further encodes an LAL, wherein the sequence encoding the LAL is operatively linked to the sequence encoding the polyketide synthase.

16. The nucleic acid of claim 15, wherein the LAL is a heterologous LAL.

17. The nucleic acid of claim 15 or 16, wherein LAL comprises a portion having at least 80% identity to SEQ ID NO: 177.

18. The nucleic acid of claim 17, wherein the LAL comprises a portion having the sequence of SEQ ID NO: 177.

19. The nucleic acid of claim 18, wherein the LAL has the sequence of SEQ ID NO: 177.

20. The nucleic acid of any one of claims 14 to 19, wherein the nucleic acid encoding the LAL lacks a TTA inhibitory codon in an open reading frame.

21. The nucleic acid of any one of claims 14 to 20, wherein the nucleic acid further comprises an LAL binding site, wherein the sequence encoding the LAL binding site is operatively linked to the sequence encoding the polyketide synthase.

22. The nucleic acid of claim 21, wherein the LAL binding site comprises a portion having at least 80% sequence identity to the sequence of SEQ ID NO: 178.

23. The nucleic acid of claim 22, wherein the LAL binding site comprises a portion having the sequence of SEQ ID NO: 178.

24. The nucleic acid of claim 23, wherein the LAL binding site has of the sequence of SEQ ID NO: 178.

25. The nucleic acid of claim 21, wherein the LAL binding site has the sequence GGGGGT (SEQ ID NO: 179).

26. The nucleic acid of any one of claims 21 to 25, wherein the binding of an LAL to the LAL binding site promotes expression of the polyketide synthase.

27. The nucleic acid of any one of claims 14 to 26, wherein the nucleic acid further encodes a nonribosomal peptide synthase.

28. The nucleic acid of any one of claims 14 to 27, wherein the nucleic acid further encodes a first P450 enzyme.

29. The nucleic acid of claim 28, wherein the nucleic acid further encodes a second P450 enzyme.

30. An expression vector comprising a nucleic acid of any one of claims 14 to 29.

31. The expression vector of claim 30, wherein the expression vector is an artificial chromosome.

32. The expression vector of claim 31, wherein the artificial chromosome is a bacterial artificial chromosome.

33. A host cell comprising an expression vector of any one of claims 30 to 32.

34. A host cell comprising a polyketide synthase of any one of claims 1 to 13, wherein the polyketide is heterologous to the host cell.

35. The host cell of claim 33 or 34, wherein the host cell naturally lacks an LAL.

36. The host cell of any one of claims 33 to 35, wherein the host cell naturally lacks an LAL binding site.

37. The host cell of any one of claims 33 to 36, wherein the host cell comprises an LAL capable of binding to an LAL binding site and regulating expression of a polyketide synthase.

38. The host cell of claim 37, wherein the LAL is heterologous.

39. The host cell of claim 37 or 38, wherein the LAL comprises a portion having at least 80% identity to the sequence of SEQ ID NO: 177.

40. The host cell of any one of claims 33 to 39, wherein the host cell is a bacterium.

41. The host cell of claim 40, wherein the bacterium is an actinobacterium.

42. The host cell of claim 41, wherein the actinobacterium is Streptomyces ambofaciens, Streptomyces hygroscopicus, or Streptomyces malayensis.

43. The host cell of claim 42, wherein the actinobaceterium is S1391, S1496, or S2441.

44. The host cell of any one of claims 33 to 43, wherein the host cell has been modified to enhance expression of a polyketide synthase.

45. The host cell of claim 44, wherein 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.

46. A method of producing a polyketide, the method comprising culturing a host cell of any one of claims 33 to 45 under suitable conditions.

47. A method of producing a polyketide, the method comprising culturing a host cell engineered to express a polyketide synthase of any one of claims 1 to 13 under conditions suitable for polyketide synthase to produce a polyketide.

48. A method of producing a compound, the method comprising: 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 comprises 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.

49. A method of producing a compound, the method comprising: (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 comprising at least one heterologous module.

50. A method of producing a compound, the method comprising: (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 comprises at least one heterologous module identified in step (b).

51. A method of producing 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 comprises one or more heterologous modules with altered enzymatic activity relative to a reference polyketide, wherein the method comprises: (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 comprises 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).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0053] FIGS. 1A and 1B are schematics illustrating the mechanisms by which PKS biosynthesis proceed. FIG. 1A depicts polyketide chain elongation and -carbonyl processing within a module. FIG. 1B depicts translation between modules.

[0054] FIG. 2A is a diagram depicting complementary bioinformatics approaches to the prediction of functional protein-protein interactions at the module-module junction.

[0055] FIG. 2B is a phylogenetic tree resulting from multiple sequence alignments of complete FK-family modules.

[0056] FIGS. 2C-2E depict how inter-module residue covariation is used to generate an algorithm that ranks module-module junction compatibility. FIG. 2C is a diagram that illustrates the upstream and downstream module-module junctions used to determine the compatibility of a given heterologous module. FIG. 2D is a correlation map that depicts the alignment of the ACP domain of a given module and the KS-AT didomain of a second module. FIG. 2E depicts the compatibility score resulting from inter-domain residue covariation analysis for a series of heterologous modules. Scores are normalized to the homologous module for the polyketide synthase in question, which is given a score of 1.00.

[0057] FIGS. 2F and 2G depict how evolutionary trace analysis is used to predict module-module junction compatibility. FIG. 2F is a phylogenetic tree generated by multiple sequence alignments of FK-family KS and ACP domains, in which group-specific residues have been concatenated into functional clades or sub-clades. The distance between modules can be used to predict module-module junction compatibility. FIG. 2G is a schematic depicting the compatibility relationships predicted by evolutionary trace analysis between KS and ACP domains for the FK-family.

[0058] FIG. 3A is a schematic depicting a single module swap in which a donor module replaces either module 3 or module 4 of the PKS gene cluster that produces Compound 1.

[0059] FIG. 3B is an image of the engineered PKS that includes the heterologous module 3 from the S17 Streptomyces strain in place of the homologous module 3 in the PKS that produces Compound 1. The engineered PKS module 3 now includes an ER domain, and thus, the resulting compound produced by the engineered PKS, Compound 2, is reduced relative to Compound 1.

[0060] FIG. 3C is an image depicting compounds, e.g., Compound 2, Compound 3, Compound 4, and Compound 5, produced by single module swaps of either module 3 or module 4 in the PKS that produces Compound 1 with compatible heterologous modules.

[0061] FIG. 4A is a schematic depicting combinatorial swapping of a dimodule unit.

[0062] FIG. 4B is a schematic depicting the synthesis of dimodule units from exogenous donor modules by a first round of Gibson assembly. The dimodule product is shown as analyzed by DNA gel electrophoresis.

[0063] FIG. 4C is a schematic depicting dimodule capture, amplification, and enrichment in a shuttle vector. Dimodule units resulting from a first round of Gibson assembly are captured in a shuttle vector by a second round of Gibson assembly. This allows for the dimodule assembly to be amplified, enriched, and ligated into the intended PKS.

[0064] FIG. 4D is a schematic depicting the construction of dimodule libraries by combinatorial synthesis.

[0065] FIG. 4E is an image depicting the possible resulting compounds that may be generated by an exemplary dimodule library swapped into module 3 and module 4 of the PKS that produces Compound 1.

[0066] FIG. 4F depicts oversampling required for sufficient coverage of a large combinatorial dimodule library. FIG. 4F is a graphical representation of the oversampling required to achieve 90% or greater coverage of a 225 member dimodule combinatorial library. 18% of the 650 sampled clones were found to have produced polyketide compounds resulting from the engineered PKS cluster, as determined by LC-TOF mass spectrometry analysis.

[0067] FIG. 4G is a schematic depicting a method of preparing combinatorial dimodule libraries and characterizing the resulting libraries using NanoPore sequencing.

[0068] FIG. 4H is a schematic depicting the core informatics workflow for deconvoluting the sequences of combinatorial dimodule libraries by NanoPore sequencing.

[0069] FIGS. 5A and 5B depict the construction of trimodule libraries by combinatorial synthesis. FIG. 5A is a schematic illustrating a trimodule swap of modules 4, 5, and 6 of the PKS cluster that produces Compound 7, to produce a theoretical library size of 2,197 engineered polyketide synthases. FIG. 5b is an image of high efficiency trimodule assembly by Gibson assembly as analyzed by DNA gel electrophoresis.

[0070] FIG. 6A is a schematic illustrating a module swap that results in ring expansion by exchanging a single module acceptor for a dimodule donor. The resulting expanded ring compound produced by the engineered PKS, Compound 8, is also depicted.

[0071] FIG. 6B is a spectrogram that shows the production of an expanded ring compound, Compound 8, as analyzed by LC-TOF mass spectrometry.

[0072] FIG. 7A is schematic depicting the enzymatic domains of five PKS loading modules, including Rapamycin and novel PKS cluster, X23. Also shown is the starter unit associated with each loading module.

[0073] FIG. 7B depicts the compounds produced by engineered PKS clusters resulting from single module swaps in the X23 PKS cluster. The products include Compound 11 and 12, which are produced by an engineered PKS that contains a heterologous loading module.

DETAILED DESCRIPTION OF THE INVENTION

[0074] 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

[0075] 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, -lactams antibiotics including carbapenems, cephalosporins, and penicillin; purines, pteridines, polypyrroles, tetracyclines, quinolones and fluoroquinolones; and sulfonamides.

Proteins

Polyketide Synthases

[0076] 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).

[0077] 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.

[0078] 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 CO.sub.2 to produce an extended polyketide chain bound to the acyl carrier protein. Each added extender unit may then be modified by -ketoprocessing domains, i.e., ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H.sub.2O 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.

Polyketide Synthase Extender Modules

[0079] PKS biosynthesis proceeds by two key mechanisms: polyketide chain elongation within a module and translocation between modules (FIGS. 1A and 1B). The basic functional unit of polyketide synthase clusters is the extender module, which encodes a 2-carbon extender unit derived from malonyl-CoA. Within the extender module, the minimal domain architecture required for polyketide chain 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 beta-carbonyl processing domains: ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains. Productive chain elongation depends on the concerted function of numerous domains

-Ketone Processing Domains

[0080] -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 -ketone processing domain is capable of changing the oxidation state of an elongation group. The -ketone processing domains include ketoreductase (which reduces the carbonyl of the elongation group to a hydroxy), dehydratase (which expels H.sub.2O to produce an alkene), and enoylreductase (which reduces alkenes to produce saturated hydrocarbons).

Module Swapping to Produce Engineered Polyketide Synthases

[0081] 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.

[0082] 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.

Combinatorial Libraries of Engineered Polyketide Synthases

[0083] 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.

Characterization of Engineered PKS Libraries by Single-Molecule Long-Read Sequencing

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

Computational Approaches for the Prediction of Functional Inter-Modular Junctions

[0088] The present disclosure provides complementary bioinformatic approaches for the prediction of functional protein-protein interactions at the module-module junction (FIG. 2A). In some embodiments, these bioinformatic approaches serve as the predictive basis for the design of chimeric PKS proteins by module swapping.

Module-Level Phylogeny

[0089] 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 (FIG. 2B). This enabled the identification of 10 module clades including 8 elongation, 1 loading, and 1 off-loading. In some embodiments, a heterologous module is compatible if it is present in the same module clade as the adjacent modules.

Inter-Module Residue Covariation

[0090] Inter-module residue covariation across the intermodular junction was computed to generate an algorithm to rank order intermodule compatibility (FIGS. 2C-2E). Type I polyketide synthase protein sequences were extracted from Genbank and an internal database using Hidden Markov Models trained on the ketosynthase (KS) and acyl carrier protein (ACP) domains. Shorter peptide sequences, starting with the ACP of a module and extending through the KS and acyl transferase (AT) of the following module, were extracted to generate a multiple alignment. Positions not aligning to an amino acid from PDB entry 2JU1 (for the ACP) or 2HG4 (for KS and AT and associated linkers) were removed to compress the multiple alignment. Evolutionary couplings were then calculated using the package FreeContact. These couplings take the form of a score matrix with two indices: the first amino acid position in the multiple alignment (I) and the second amino acid position in the multiple alignment (J, which is always greater than I) and the amino acid at position J. I,J pairs with a score above a specified cutoff and in which I is within the ACP and J within the KS-AT didomain are saved.

[0091] 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

[0092] 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 (FIGS. 2F-2G). For example, phylogenetic trees with uniform branch lengths were constructed based on multiple sequence alignments of FK-family KSs and ACPs. For every non-terminal node in a tree, a vertical cutoff was applied by which terminal nodes were partitioned into groups based on shared parental nodes at the cutoff. Residues globally conserved across all groups and residues locally conserved within groups, but specific to a given group, were identified as functional residues. Globally conserved residues suggest rules that likely must be observed for all members of the FK-family. Group-specific residues suggest guidelines that may provide predictive power for engineering within the FK class. For each tree, the earliest cutoff at which the number of group-specific residues exceeded the number of globally conserved residues was selected for further analysis. Group-specific residues were concatenated into functional clades and unrooted phylogenetic trees of the clades were constructed. Distances between terminal nodes in the phylogenetic tree were used to create an evolutionary distance score (EDS). The KS and ACP EDSs between a homologous acceptor module and a proposed heterologous donor module were calculated and used to predict engineering compatibility. KS and ACP clade classifications were then used to create network maps of neighboring KSs and ACPs weighted by the frequency a given KS-ACP or ACP-KS pair was observed in FK-family polyketides. Superimposing a proposed module swap onto the network map was used to predict engineering compatibility with upstream ACPs and downstream KSs. In some embodiments, a heterologous module is compatible if the module belongs to the same functional evolutionary clade or sub-clade as one or more adjacent modules in the reference PKS.

Regulation of Polyketide Synthase Expression

[0093] 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

[0094] 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.

[0095] 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.

[0096] 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.

TABLE-US-00004 SEQ ID NO: 180 ATGCCTGCCGTGGAGTGCTATGAACTGGACGCCCGCGATGACGAGCTCAG AAAACTGGAGGAGGTTGTGACCGGGCGGGCCAACGGCCGGGGTGTGGTGG TCACCATCACCGGACCGATCGCCTGCGGCAAGACCGAACTGCTCGACGCA GCCGCCGCGAAGGCCGACGCCATCACGTTACGAGCGGTCTGCTCCGCGGA GGAACAGGCACTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGG CGCTCGCCTCCCACGCGCTGGAGCCGGCCTGCCCGACCCTCCCGGGCGAG CACCTGTCGCCGGAGGCCGAGAACCGGCTGCGCAGCGACCTCACCCGTAC CCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCGGCATCGACGAGT CACACGCGAACGCTTTGTGTCTGCTCCACCTGGCCCGAAGGGTCGGCTCG GCCCGGATCGCCATGGTCCTCACCGAGTTGCGCCGGCTCACCCCGGCCCA CTCACAGTTCCAGGCCGAGCTGCTCAGCCTGGGGCACCACCGCGAGATCG CGCTGCGCCCGCTCAGCCCGAAGCACACCGCCGAGCTGGTCCGCGCCGGT CTCGGTCCCGACGTCGACGAGGACGTGCTCACGGGGTTGTACCGGGCGAC CGGCGGCAACCTGAACCTCACCCGCGGACTGATCAACGATGTGCGGGAGG CCTGGGAGACGGGAGGGACGGGCATCAGCGCGGGCCGCGCGTACCGGCTG GCATACCTCGGTTCCCTCTACCGCTGCGGCCCGGTCCCGTTGCGGGTCGC ACGGGTGGCCGCCGTGCTGGGCCAGAGCGCCAACACCACCCTGGTGCGCT GGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCAACCGAGATCCTC ACCGAAGGCGGCCTGCTGCACGACCTGCGGTTCCCGCACCCGGCGGCCCG TTCGGTGGTACTCAACGACATGTCCGCCCAGGAACGACGCCGCCTGCACC GGTCCGCTCTGGAAGTGCTGGACGACGTGCCCGTGGAAGTGGTCGCGCAC CACCAGGTCGGCGCCGGTCTCCTGCACGGCCCGAAGGCCGCCGAGATATT CGCCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGTTGGACACCGCGT CCGACTATCTGCAACTGGCCCACCAGGCCTCCGACGACGCCGTCACCGGG ATGCGGGCCGAGGCCGTGGCGATCGAGCGCCGCCGCAACCCGCTGGCCTC GAGCCGGCACCTCGACGAGCTGACCGTCGTCGCCCGTGCCGGGCTGCTCT TCCCCGAGCACACGGCGCTGATGATCCGCTGGCTGGGCGTCGGCGGGCGG TCCGGCGAGGCAGCCGGGCTGCTGGCCTCGCAGCGCCCCCGTGCGGTCAC CGACCAGGACAGGGCCCATATGCGGGCCGCCGAGGTATCGCTCGCGCTGG TCAGCCCCGGCACGTCCGGCCCGGACCGGCGGCCGCGTCCGCTCACGCCG GATGAGCTCGCGAACCTGCCGAAGGCGGCCCGGCTCTGCGCGATCGCCGA CAATGCCGTCATGTCGGCCCTGCGCGGTCGTCCCGAGCTCGCCGCGGCCG AGGCGGAGAACGTCCTGCAGCACGCCGACTCGGCGGCGGCCGGCACCACC GCCCTCGCCGCGCTGACCGCCTTGCTGTACGCGGAGAACACCGACACCGC TCAGCTCTGGGCCGACAAGCTGGTCTCCGAGACCGGGGCGTCGAACGAGG AGGAGGCGGGCTACGCGGGGCCGCGCGCCGAAGCCGCGTTGCGTCGCGGC GACCTGGCCGCGGCGGTCGAGGCAGGCAGCACCGTTCTGGACCACCGGCG GCTCTCGACGCTCGGCATCACCGCCGCGCTACCGCTGAGCAGCGCGGTGG CCGCCGCCATCCGGCTGGGCGAGACCGAGCGGGCGGAGAAGTGGCTCGCC CAGCCGCTGCCGCAGGCCATCCAGGACGGCCTGTTCGGCCTGCACCTGCT CTCGGCGCGCGGCCAGTACAGCCTCGCCACGGGCCAGCACGAGTCGGCGT ACACGGCGTTTCGCACCTGCGGGGAACGTATGCGGAACTGGGGCGTTGAC GTGCCGGGTCTGTCCCTGTGGCGCGTCGACGCCGCCGAGGCGCTGCTGCA CGGCCGCGACCGGGACGAGGGCCGACGGCTCGTCGACGAGCAACTCACCC GTGCGATGGGACCCCGTTCCCGCGCCTTGACGCTGCGGGTGCAGGCGGCG TACAGCCCGCCGGCGAAGCGGGTCGACCTGCTCGATGAAGCGGCCGACCT GCTGCTCTCCTGCAACGACCAGTACGAGCGGGCACGGGTGCTCGCCGACC TGAGCGAGACGTTCAGCGCGCTCCGGCACCACAGCCGGGCGCGGGGACTG CTTCGGCAGGCCCGGCACCTGGCCGCCCAGCGCGGCGCGATACCGCTGCT GCGCCGACTCGGGGCCAAGCCCGGAGGCCCCGGCTGGCTGGAGGAATCCG GCCTGCCGCAGCGGATCAAGTCGCTGACCGACGCGGAGCGGCGGGTGGCG TCGCTGGCCGCCGGCGGACAGACCAACCGCGTGATCGCCGACCAGCTCTT CGTCACGGCCAGCACGGTGGAGCAGCACCTCACGGACGTCTCCACTGGGT CAAGGCCGCCAGCACCTGCCGCCGAACTCGTCTAG SEQ ID NO: 181 ATGCCTGCCGTGGAGTGCTATGAACTGGACGCCCGCGATGACGAGCTCAG AAAACTGGAGGAGGTTGTGACCGGGCGGGCCAACGGCCGGGGTGTGGTGG TCACCATCACCGGACCGATCGCCTGCGGCAAGACCGAACTGCTCGACGCA GCCGCCGCGAAGGCCGACGCCATCACGCTGCGAGCGGTCTGCTCCGCGGA GGAACAGGCACTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGG CGCTCGCCTCCCACGCGCTGGAGCCGGCCTGCCCGACCCTCCCGGGCGAG CACCTGTCGCCGGAGGCCGAGAACCGGCTGCGCAGCGACCTCACCCGTAC CCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCGGCATCGACGAGT CACACGCGAACGCTTTGTGTCTGCTCCACCTGGCCCGAAGGGTCGGCTCG GCCCGGATCGCCATGGTCCTCACCGAGTTGCGCCGGCTCACCCCGGCCCA CTCACAGTTCCAGGCCGAGCTGCTCAGCCTGGGGCACCACCGCGAGATCG CGCTGCGCCCGCTCAGCCCGAAGCACACCGCCGAGCTGGTCCGCGCCGGT CTCGGTCCCGACGTCGACGAGGACGTGCTCACGGGGTTGTACCGGGCGAC CGGCGGCAACCTGAACCTCACCCGCGGACTGATCAACGATGTGCGGGAGG CCTGGGAGACGGGAGGGACGGGCATCAGCGCGGGCCGCGCGTACCGGCTG GCATACCTCGGTTCCCTCTACCGCTGCGGCCCGGTCCCGTTGCGGGTCGC ACGGGTGGCCGCCGTGCTGGGCCAGAGCGCCAACACCACCCTGGTGCGCT GGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCAACCGAGATCCTC ACCGAAGGCGGCCTGCTGCACGACCTGCGGTTCCCGCACCCGGCGGCCCG TTCGGTGGTACTCAACGACATGTCCGCCCAGGAACGACGCCGCCTGCACC GGTCCGCTCTGGAAGTGCTGGACGACGTGCCCGTGGAAGTGGTCGCGCAC CACCAGGTCGGCGCCGGTCTCCTGCACGGCCCGAAGGCCGCCGAGATATT CGCCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGTTGGACACCGCGT CCGACTATCTGCAACTGGCCCACCAGGCCTCCGACGACGCCGTCACCGGG ATGCGGGCCGAGGCCGTGGCGATCGAGCGCCGCCGCAACCCGCTGGCCTC GAGCCGGCACCTCGACGAGCTGACCGTCGTCGCCCGTGCCGGGCTGCTCT TCCCCGAGCACACGGCGCTGATGATCCGCTGGCTGGGCGTCGGCGGGCGG TCCGGCGAGGCAGCCGGGCTGCTGGCCTCGCAGCGCCCCCGTGCGGTCAC CGACCAGGACAGGGCCCATATGCGGGCCGCCGAGGTATCGCTCGCGCTGG TCAGCCCCGGCACGTCCGGCCCGGACCGGCGGCCGCGTCCGCTCACGCCG GATGAGCTCGCGAACCTGCCGAAGGCGGCCCGGCTCTGCGCGATCGCCGA CAATGCCGTCATGTCGGCCCTGCGCGGTCGTCCCGAGCTCGCCGCGGCCG AGGCGGAGAACGTCCTGCAGCACGCCGACTCGGCGGCGGCCGGCACCACC GCCCTCGCCGCGCTGACCGCCTTGCTGTACGCGGAGAACACCGACACCGC TCAGCTCTGGGCCGACAAGCTGGTCTCCGAGACCGGGGCGTCGAACGAGG AGGAGGCGGGCTACGCGGGGCCGCGCGCCGAAGCCGCGTTGCGTCGCGGC GACCTGGCCGCGGCGGTCGAGGCAGGCAGCACCGTTCTGGACCACCGGCG GCTCTCGACGCTCGGCATCACCGCCGCGCTACCGCTGAGCAGCGCGGTGG CCGCCGCCATCCGGCTGGGCGAGACCGAGCGGGCGGAGAAGTGGCTCGCC CAGCCGCTGCCGCAGGCCATCCAGGACGGCCTGTTCGGCCTGCACCTGCT CTCGGCGCGCGGCCAGTACAGCCTCGCCACGGGCCAGCACGAGTCGGCGT ACACGGCGTTTCGCACCTGCGGGGAACGTATGCGGAACTGGGGCGTTGAC GTGCCGGGTCTGTCCCTGTGGCGCGTCGACGCCGCCGAGGCGCTGCTGCA CGGCCGCGACCGGGACGAGGGCCGACGGCTCGTCGACGAGCAACTCACCC GTGCGATGGGACCCCGTTCCCGCGCCTTGACGCTGCGGGTGCAGGCGGCG TACAGCCCGCCGGCGAAGCGGGTCGACCTGCTCGATGAAGCGGCCGACCT GCTGCTCTCCTGCAACGACCAGTACGAGCGGGCACGGGTGCTCGCCGACC TGAGCGAGACGTTCAGCGCGCTCCGGCACCACAGCCGGGCGCGGGGACTG CTTCGGCAGGCCCGGCACCTGGCCGCCCAGCGCGGCGCGATACCGCTGCT GCGCCGACTCGGGGCCAAGCCCGGAGGCCCCGGCTGGCTGGAGGAATCCG GCCTGCCGCAGCGGATCAAGTCGCTGACCGACGCGGAGCGGCGGGTGGCG TCGCTGGCCGCCGGCGGACAGACCAACCGCGTGATCGCCGACCAGCTCTT CGTCACGGCCAGCACGGTGGAGCAGCACCTCACGGACGTCTCCACTGGGT CAAGGCCGCCAGCACCTGCCGCCGAACTCGTCTAG SEQ ID NO: 182 GTGGTTCCTGAAGTGCGAGCAGCCCCCGACGAACTGATCGCCCGCGATGA CGAGCTGAGCCGCCTCCAACGGGCACTCACCAGGGCGGGGAGCGGAAGGG GCGGCGTCGTCGCCATCACCGGGCCCATCGCCAGCGGAAAGACGGCGCTG CTCGACGCCGGAGCGGCCAAGTCCGGCTTCGTCGCACTCCGTGCGGTGTG CTCCTGGGAAGAGCGCACTCTGCCGTACGGGATGCTGGGCCAGCTCTTCG ACCATCCCGAACTGGCCGCCCAGGCGCCGGACCTTGCCCACTTCACGGCT TCGTGCGAGAGCCCTCAGGCCGGTACCGACAACCGCCTGCGGGCCGAGTT CACCCGCACCCTGCTGGCGCTCGCCGCGGACTGGCCCGTCCTGATCGGCA TCGACGACGTGCACCACGCCGACGCGGAATCACTGCGCTGTCTGCTCCAC CTCGCCCGCCGCATCGGCCCGGCCCGCATCGCGGTCGTACTGACCGAGCT GCGCAGACCGACGCCCGCCGACTCCCGCTTCCAGGCGGAACTGCTGAGCC TGCGCTCCTACCAGGAGATCGCGCTCAGACCGCTCACCGAGGCGCAGACC GGCGAACTCGTACGTCGGCACCTCGGCGCGGAGACCCACGAGGACGTCTC CGCCGATACGTTCCGGGCGACCGGCGGGAACCTGCTCCTCGGGCACGGTT TGATCAATGACATCCGGGAGGCGCGGACAGCGGGACGGCCGGGGGTCGTC GCGGGGCGGGCGTACCGGCTCGCGTACCTCAGCTCGCTCTACCGCTGCGG CCCGAGCGCGCTGCGTGTCGCCCGGGCGTCCGCCGTGCTCGGCGCGAGCG CCGAAGCCGTGCTCGTCCAGCGGATGACCGGACTGAACAAGGACGCGGTC GAACAGGTCTATGAGCAGCTGAACGAGGGACGGCTGCTGCAGGGCGAGCG GTTTCCGCACCCGGCGGCCCGCTCCATCGTCCTTGACGACCTGTCGGCCC TGGAACGCAGAAACCTGCACGAGTCGGCGCTGGAGCTGCTGCGGGACCAC GGCGTGGCCGGCAACGTGCTCGCCCGCCACCAGATCGGCGCCGGCCGGGT GCACGGCGAGGAGGCCGTCGAGCTGTTCACCGGGGCCGCACGGGAGCACC ACCTGCGCGGTGAACTGGACGACGCGGCCGGATACCTGGAACTCGCCCAC CGTGCCTCCGACGACCCCGTCACGCGCGCCGCACTACGCGTCGGCGCCGC CGCGATCGAGCGCCTCTGCAATCCGGTACGGGCAGGCCGGCATCTGCCCG AGCTGCTCACCGCGTCGCGCGCGGGACTGCTCTCCAGCGAGCACGCCGTG TCGCTCGCCGACTGGCTGGCGATGGGCGGGCGCCCGGGCGAGGCGGCCGA GGTCCTCGCGACGCAGCGTCCCGCGGCCGACAGCGAGCAGCACCGCGCAC TCCTGCGCAGCGGCGAGTTGTCCCTCGCGCTGGTCCACCCCGGCGCGTGG GATCCGTTGCGCCGGACCGATCGGTTCGCCGCGGGCGGGCTCGGCTCGCT TCCCGGACCCGCCCGGCACCGCGCGGTCGCCGACCAAGCCGTCATCGCGG CGCTGCGTGGACGTCTCGACCGGGCGGACGCCAACGCGGAGAGCGTTCTC CAGCACACCGACGCCACGGCGGACCGGACCACGGCCATCATGGCGTTGCT GGCCCTGCTCTACGCGGAGAACACCGATGCTGTCCAGTTCTGGGTCGACA AACTGGCCGGTGACGAGGGCACCAGGACACCGGCCGACGAGGCGGTCCAC GCGGGGTTCAACGCCGAGATCGCGCTGCGCCGCGGCGACTTGATGAGAGC CGTCGAGTACGGCGAGGCAGCGCTCGGCCACCGGCACCTGCCCACCTGGG GAATGGCCGCCGCTCTGCCGCTGAGCAGCACCGTGGTTGCCGCGATCCGG CTCGGCGACCTCGACAGGGCCGAGCGGTGGCTCGCCGAGCCGCTGCCGCA GCAGACGCCGGAGAGCCTCTTCGGGCTGCACCTGCTCTGGGCCCGCGGGC AGCACCACCTCGCGACCGGGCGGCACGGGGCGGCGTACACGGCGTTCAGG GAATGCGGCGAGCGGATGCGGCGGTGGGCCGTCGACGTGCCGGGCCTGGC CCTGTGGCGGGTCGACGCCGCCGAATCGCTGCTGCTGCTCGGCCGTGACC GTGCCGAAGGACTGCGGCTCGTCTCCGAGCAGCTGTCCCGGCCGATGCGC CCTCGCGCGCGCGTGCAGACGTTACGGGTACAGGCGGCCTACAGTCCGCC GCCCCAACGGATCGACCTGCTCGAAGAGGCCGCCGACCTGCTGGTCACCT GCAACGACCAGTACGAACTGGCAAACGTACTCAGCGACTTGGCAGAGGCC TCCAGCATGGTCCGGCAGCACAGCAGGGCGCGGGGTCTGCTCCGCCGGGC ACGGCACCTCGCCACCCAGTGCGGCGCCGTGCCGCTCCTGCGGCGGCTCG GCGCGGAACCCTCGGACATCGGCGGAGCCTGGGACGCGACGCTGGGACAG CGGATCGCGTCACTGACGGAGTCGGAGCGGCGGGTGGCCGCGCTCGCCGC GGTCGGGCGTACGAACAGGGAGATCGCCGAGCAGCTGTTCGTCACGGCCA GCACGGTGGAACAGCACCTCACGAACGTGTTCCGCAAACTGGCGGTGAAG GGCCGCCAGCAGCTTCCGAAGGAACTGGCCGACGTCGGCGAGCCGGCGGA CCGCGACCGCCGGTGCGGGTAG SEQ ID NO: 183 ATGGTTCCTGAAGTGCGAGCAGCCCCCGACGAACTGATCGCCCGCGATGA CGAGCTGAGCCGCCTCCAACGGGCACTCACCAGGGCGGGGAGCGGAAGGG GCGGCGTCGTCGCCATCACCGGGCCCATCGCCAGCGGAAAGACGGCGCTG CTCGACGCCGGAGCGGCCAAGTCCGGCTTCGTCGCACTCCGTGCGGTGTG CTCCTGGGAAGAGCGCACTCTGCCGTACGGGATGCTGGGCCAGCTCTTCG ACCATCCCGAACTGGCCGCCCAGGCGCCGGACCTTGCCCACTTCACGGCT TCGTGCGAGAGCCCTCAGGCCGGTACCGACAACCGCCTGCGGGCCGAGTT CACCCGCACCCTGCTGGCGCTCGCCGCGGACTGGCCCGTCCTGATCGGCA TCGACGACGTGCACCACGCCGACGCGGAATCACTGCGCTGTCTGCTCCAC CTCGCCCGCCGCATCGGCCCGGCCCGCATCGCGGTCGTACTGACCGAGCT GCGCAGACCGACGCCCGCCGACTCCCGCTTCCAGGCGGAACTGCTGAGCC TGCGCTCCTACCAGGAGATCGCGCTCAGACCGCTCACCGAGGCGCAGACC GGCGAACTCGTACGTCGGCACCTCGGCGCGGAGACCCACGAGGACGTCTC CGCCGATACGTTCCGGGCGACCGGCGGGAACCTGCTCCTCGGGCACGGTT TGATCAATGACATCCGGGAGGCGCGGACAGCGGGACGGCCGGGGGTCGTC GCGGGGCGGGCGTACCGGCTCGCGTACCTCAGCTCGCTCTACCGCTGCGG CCCGAGCGCGCTGCGTGTCGCCCGGGCGTCCGCCGTGCTCGGCGCGAGCG CCGAAGCCGTGCTCGTCCAGCGGATGACCGGACTGAACAAGGACGCGGTC GAACAGGTCTATGAGCAGCTGAACGAGGGACGGCTGCTGCAGGGCGAGCG GTTTCCGCACCCGGCGGCCCGCTCCATCGTCCTTGACGACCTGTCGGCCC TGGAACGCAGAAACCTGCACGAGTCGGCGCTGGAGCTGCTGCGGGACCAC GGCGTGGCCGGCAACGTGCTCGCCCGCCACCAGATCGGCGCCGGCCGGGT GCACGGCGAGGAGGCCGTCGAGCTGTTCACCGGGGCCGCACGGGAGCACC ACCTGCGCGGTGAACTGGACGACGCGGCCGGATACCTGGAACTCGCCCAC CGTGCCTCCGACGACCCCGTCACGCGCGCCGCACTACGCGTCGGCGCCGC CGCGATCGAGCGCCTCTGCAATCCGGTACGGGCAGGCCGGCATCTGCCCG AGCTGCTCACCGCGTCGCGCGCGGGACTGCTCTCCAGCGAGCACGCCGTG TCGCTCGCCGACTGGCTGGCGATGGGCGGGCGCCCGGGCGAGGCGGCCGA GGTCCTCGCGACGCAGCGTCCCGCGGCCGACAGCGAGCAGCACCGCGCAC TCCTGCGCAGCGGCGAGTTGTCCCTCGCGCTGGTCCACCCCGGCGCGTGG GATCCGTTGCGCCGGACCGATCGGTTCGCCGCGGGCGGGCTCGGCTCGCT TCCCGGACCCGCCCGGCACCGCGCGGTCGCCGACCAAGCCGTCATCGCGG CGCTGCGTGGACGTCTCGACCGGGCGGACGCCAACGCGGAGAGCGTTCTC CAGCACACCGACGCCACGGCGGACCGGACCACGGCCATCATGGCGTTGCT GGCCCTGCTCTACGCGGAGAACACCGATGCTGTCCAGTTCTGGGTCGACA AACTGGCCGGTGACGAGGGCACCAGGACACCGGCCGACGAGGCGGTCCAC GCGGGGTTCAACGCCGAGATCGCGCTGCGCCGCGGCGACTTGATGAGAGC CGTCGAGTACGGCGAGGCAGCGCTCGGCCACCGGCACCTGCCCACCTGGG GAATGGCCGCCGCTCTGCCGCTGAGCAGCACCGTGGTTGCCGCGATCCGG CTCGGCGACCTCGACAGGGCCGAGCGGTGGCTCGCCGAGCCGCTGCCGCA GCAGACGCCGGAGAGCCTCTTCGGGCTGCACCTGCTCTGGGCCCGCGGGC AGCACCACCTCGCGACCGGGCGGCACGGGGCGGCGTACACGGCGTTCAGG GAATGCGGCGAGCGGATGCGGCGGTGGGCCGTCGACGTGCCGGGCCTGGC CCTGTGGCGGGTCGACGCCGCCGAATCGCTGCTGCTGCTCGGCCGTGACC GTGCCGAAGGACTGCGGCTCGTCTCCGAGCAGCTGTCCCGGCCGATGCGC CCTCGCGCGCGCGTGCAGACGCTGCGGGTACAGGCGGCCTACAGTCCGCC GCCCCAACGGATCGACCTGCTCGAAGAGGCCGCCGACCTGCTGGTCACCT GCAACGACCAGTACGAACTGGCAAACGTACTCAGCGACTTGGCAGAGGCC TCCAGCATGGTCCGGCAGCACAGCAGGGCGCGGGGTCTGCTCCGCCGGGC ACGGCACCTCGCCACCCAGTGCGGCGCCGTGCCGCTCCTGCGGCGGCTCG GCGCGGAACCCTCGGACATCGGCGGAGCCTGGGACGCGACGCTGGGACAG CGGATCGCGTCACTGACGGAGTCGGAGCGGCGGGTGGCCGCGCTCGCCGC GGTCGGGCGTACGAACAGGGAGATCGCCGAGCAGCTGTTCGTCACGGCCA GCACGGTGGAACAGCACCTCACGAACGTGTTCCGCAAACTGGCGGTGAAG GGCCGCCAGCAGCTTCCGAAGGAACTGGCCGACGTCGGCGAGCCGGCGGA CCGCGACCGCCGGTGCGGGTAG SEQ ID NO: 184 GTGATAGCGCGCTTATCTCCCCCAGACCTGATCGCCCGCGATGACGAGTT CGGTTCCCTCCACCGGGCGCTCACCCGAGCGGGGGGCGGGCGGGGCGTCG TCGCCGCCGTCACCGGGCCGATCGCCTGCGGCAAGACCGAACTCCTCGAC GCCGCCGCGGCCAAGGCCGGCTTCGTCACCCTTCGCGCGGTGTGCTCCAT GGAGGAGCGGGCCCTGCCGTACGGCATGCTCGGCCAGCTCCTCGACCAGC CCGAGCTGGCCGCCCGGACACCGGAGCTGGTCCGGCTGACGGCATCGTGC GAAAACCTGCCGGCCGACGTCGACAACCGCCTGGGGACCGAACTCACCCG CACGGTGCTGACGCTCGCCGCGGAGCGGCCCGTACTGATCGGCATCGACG ACGTGCACCACGCCGACGCGCCGTCGCTGCGCTGCCTGCTCCACCTCGCG CGCCGCATCAGCCGGGCCCGTGTCGCCATCGTGCTGACCGAGCTGCTCCG GCCGACGCCCGCCCACTCCCAATTCCGGGCGGCACTGCTGAGTCTGCGCC ACTACCAGGAGATCGCGCTGCGCCCGCTCACCGAGGCGCAGACCACCGAA CTCGTGCGCCGGCACCTCGGCCAGGACGCGCACGACGACGTGGTGGCCCA GGCGTTCCGGGCGACCGGCGGCAACCTGCTCCTCGGCCACGGCCTGATCG ACGACATCCGGGAGGCACGGACACGGACCTCAGGGTGCCTGGAAGTGGTC GCGGGGCGGGCGTACCGGCTCGCCTACCTCGGGTCGCTCTATCGTTGCGG CCCGGCCGCGCTGAGCGTCGCCCGAGCTTCCGCCGTGCTCGGCGAGAGTG TCGAACTCACCCTCGTCCAGCGGATGACCGGCCTCGACACCGAGGCGGTC GAGCAGGCCCACGAACAGCTGGTCGAGGGGCGGCTGCTGCGGGAAGGGCG GTTCCCGCACCCCGCGGCCCGCTCCGTCGTACTCGACGACCTCTCCGCCG CCGAGCGGCGTGGCCTGCACGAGCTGGCGCTGGAACTGCTGCGGGACCGC GGCGTGGCCAGCAAGGTGCTCGCCCGCCACCAGATGGGTACCGGCCGGGT GCACGGCGCCGAGGTCGCCGGGCTGTTCACCGACGCCGCGCGCGAGCACC ACCTGCGCGGCGAGCTCGACGAGGCCGTCACCTACCTGGAGTTCGCCTAC CGGGCCTCCGACGACCCCGCCGTCCACGCCGCACTGCGCGTCGACACCGC CGCCATCGAGCGGCTCTGCGATCCCGCCAGATCCGGCCGGCATGTGCCCG AGCTGCTCACCGCGTCGCGGGAACGGCTCCTCTCCAGCGAGCACGCCGTG TCGCTCGCCTGCTGGCTGGCGATGGACGGGCGGCCGGGCGAGGCCGCCGA GGTCCTGGCGGCCCAGCGCTCCGCCGCCCCGAGCGAGCAGGGCCGGGCGC ACCTGCGCGTCGCGGACCTGTCCCTCGCGCTGATCTATCCCGGCGCGGCC GATCCGCCGCGTCCGGCCGATCCGCCGGCCGAGGACGAGGTCGCCTCGTT TTCCGGAGCCGTCCGGCACCGCGCCGTCGCCGACAAGGCCCTGAGCAACG CGCTGCGCGGCTGGTCCGAACAGGCCGAGGCCAAAGCCGAGTACGTGCTC CAGCACTCCCGGGTCACGACGGACCGGACCACGACCATGATGGCGTTGCT GGCCCTGCTCTACGCCGAGGACACCGATGCCGTCCAGTCCTGGGTCGACA AGCTGGCCGGTGACGACAACATGCGGACCCCGGCCGACGAGGCGGTCCAC GCGGGGTTCCGCGCCGAGGCCGCGCTGCGCCGCGGCGACCTGACCGCCGC CGTCGAATGCGGCGAGGCCGCGCTCGCCCCCCGGGTCGTGCCCTCCTGGG GGATGGCCGCCGCATTGCCGCTGAGCAGCACCGTGGCCGCCGCGATCCGA CTGGGCGACCTGGACCGGGCGGAGCGGTGGCTCGCCGAGCCGTTGCCGGA GGAGACCTCCGACAGCCTCTTCGGACTGCACATGGTCTGGGCCCGTGGGC AACACCATCTCGCGGCCGGGCGGTACCGGGCGGCGTACAACGCGTTCCGG GACTGCGGGGAGCGGATGCGACGCTGGTCCGTCGACGTGCCGGGCCTGGC CCTGTGGCGGGTCGACGCCGCCGAAGCGCTTCTGCTGCTCGGCCGCGGCC GTGACGAGGGGCTGAGGCTCATCTCCGAGCAGCTGTCCCGGCCGATGGGG TCCCGGGCGCGGGTGATGACGCTGCGGGTGCAGGCGGCCTACAGTCCGCC GGCCAAGCGGATCGAACTGCTCGACGAGGCCGCCGATCTGCTCATCATGT GCCGCGACCAGTACGAGCTGGCCCGCGTCCTCGCCGACATGGGCGAAGCG TGCGGCATGCTCCGGCGGCACAGCCGTGCGCGGGGACTGTTCCGCCGCGC ACGGCACCTCGCGACCCAGTGCGGAGCCGTGCCGCTCCTCCGGCGGCTCG GTGGGGAGTCCTCGGACGCGGACGGCACCCAGGACGTGACGCCGGCGCAG CGGATCACATCGCTGACCGAGGCGGAGCGGCGGGTGGCGTCGCACGCCGC GGTCGGGCGCACCAACAAGGAGATCGCCAGCCAGCTGTTCGTCACCTCCA GCACGGTGGAACAGCACCTCACCAACGTGTTCCGCAAGCTGGGGGTGAAG GGCCGTCAGCAACTGCCCAAGGAACTGTCCGACGCCGGCTGA SEQ ID NO: 185 ATGATAGCGCGCCTGTCTCCCCCAGACCTGATCGCCCGCGATGACGAGTT CGGTTCCCTCCACCGGGCGCTCACCCGAGCGGGGGGCGGGCGGGGCGTCG TCGCCGCCGTCACCGGGCCGATCGCCTGCGGCAAGACCGAACTCCTCGAC GCCGCCGCGGCCAAGGCCGGCTTCGTCACCCTTCGCGCGGTGTGCTCCAT GGAGGAGCGGGCCCTGCCGTACGGCATGCTCGGCCAGCTCCTCGACCAGC CCGAGCTGGCCGCCCGGACACCGGAGCTGGTCCGGCTGACGGCATCGTGC GAAAACCTGCCGGCCGACGTCGACAACCGCCTGGGGACCGAACTCACCCG CACGGTGCTGACGCTCGCCGCGGAGCGGCCCGTACTGATCGGCATCGACG ACGTGCACCACGCCGACGCGCCGTCGCTGCGCTGCCTGCTCCACCTCGCG CGCCGCATCAGCCGGGCCCGTGTCGCCATCGTGCTGACCGAGCTGCTCCG GCCGACGCCCGCCCACTCCCAATTCCGGGCGGCACTGCTGAGTCTGCGCC ACTACCAGGAGATCGCGCTGCGCCCGCTCACCGAGGCGCAGACCACCGAA CTCGTGCGCCGGCACCTCGGCCAGGACGCGCACGACGACGTGGTGGCCCA GGCGTTCCGGGCGACCGGCGGCAACCTGCTCCTCGGCCACGGCCTGATCG ACGACATCCGGGAGGCACGGACACGGACCTCAGGGTGCCTGGAAGTGGTC GCGGGGCGGGCGTACCGGCTCGCCTACCTCGGGTCGCTCTATCGTTGCGG CCCGGCCGCGCTGAGCGTCGCCCGAGCTTCCGCCGTGCTCGGCGAGAGTG TCGAACTCACCCTCGTCCAGCGGATGACCGGCCTCGACACCGAGGCGGTC GAGCAGGCCCACGAACAGCTGGTCGAGGGGCGGCTGCTGCGGGAAGGGCG GTTCCCGCACCCCGCGGCCCGCTCCGTCGTACTCGACGACCTCTCCGCCG CCGAGCGGCGTGGCCTGCACGAGCTGGCGCTGGAACTGCTGCGGGACCGC GGCGTGGCCAGCAAGGTGCTCGCCCGCCACCAGATGGGTACCGGCCGGGT GCACGGCGCCGAGGTCGCCGGGCTGTTCACCGACGCCGCGCGCGAGCACC ACCTGCGCGGCGAGCTCGACGAGGCCGTCACCTACCTGGAGTTCGCCTAC CGGGCCTCCGACGACCCCGCCGTCCACGCCGCACTGCGCGTCGACACCGC CGCCATCGAGCGGCTCTGCGATCCCGCCAGATCCGGCCGGCATGTGCCCG AGCTGCTCACCGCGTCGCGGGAACGGCTCCTCTCCAGCGAGCACGCCGTG TCGCTCGCCTGCTGGCTGGCGATGGACGGGCGGCCGGGCGAGGCCGCCGA GGTCCTGGCGGCCCAGCGCTCCGCCGCCCCGAGCGAGCAGGGCCGGGCGC ACCTGCGCGTCGCGGACCTGTCCCTCGCGCTGATCTATCCCGGCGCGGCC GATCCGCCGCGTCCGGCCGATCCGCCGGCCGAGGACGAGGTCGCCTCGTT TTCCGGAGCCGTCCGGCACCGCGCCGTCGCCGACAAGGCCCTGAGCAACG CGCTGCGCGGCTGGTCCGAACAGGCCGAGGCCAAAGCCGAGTACGTGCTC CAGCACTCCCGGGTCACGACGGACCGGACCACGACCATGATGGCGTTGCT GGCCCTGCTCTACGCCGAGGACACCGATGCCGTCCAGTCCTGGGTCGACA AGCTGGCCGGTGACGACAACATGCGGACCCCGGCCGACGAGGCGGTCCAC GCGGGGTTCCGCGCCGAGGCCGCGCTGCGCCGCGGCGACCTGACCGCCGC CGTCGAATGCGGCGAGGCCGCGCTCGCCCCCCGGGTCGTGCCCTCCTGGG GGATGGCCGCCGCATTGCCGCTGAGCAGCACCGTGGCCGCCGCGATCCGA CTGGGCGACCTGGACCGGGCGGAGCGGTGGCTCGCCGAGCCGTTGCCGGA GGAGACCTCCGACAGCCTCTTCGGACTGCACATGGTCTGGGCCCGTGGGC AACACCATCTCGCGGCCGGGCGGTACCGGGCGGCGTACAACGCGTTCCGG GACTGCGGGGAGCGGATGCGACGCTGGTCCGTCGACGTGCCGGGCCTGGC CCTGTGGCGGGTCGACGCCGCCGAAGCGCTTCTGCTGCTCGGCCGCGGCC GTGACGAGGGGCTGAGGCTCATCTCCGAGCAGCTGTCCCGGCCGATGGGG TCCCGGGCGCGGGTGATGACGCTGCGGGTGCAGGCGGCCTACAGTCCGCC GGCCAAGCGGATCGAACTGCTCGACGAGGCCGCCGATCTGCTCATCATGT GCCGCGACCAGTACGAGCTGGCCCGCGTCCTCGCCGACATGGGCGAAGCG TGCGGCATGCTCCGGCGGCACAGCCGTGCGCGGGGACTGTTCCGCCGCGC ACGGCACCTCGCGACCCAGTGCGGAGCCGTGCCGCTCCTCCGGCGGCTCG GTGGGGAGTCCTCGGACGCGGACGGCACCCAGGACGTGACGCCGGCGCAG CGGATCACATCGCTGACCGAGGCGGAGCGGCGGGTGGCGTCGCACGCCGC GGTCGGGCGCACCAACAAGGAGATCGCCAGCCAGCTGTTCGTCACCTCCA GCACGGTGGAACAGCACCTCACCAACGTGTTCCGCAAGCTGGGGGTGAAG GGCCGTCAGCAACTGCCCAAGGAACTGTCCGACGCCGGCTGA SEQ ID NO: 186 GTGGAGTTTTACGACCTGGTCGCCCGCGATGACGAGCTCAGAAGGTTGGA CCAGGCCCTCGGCCGCGCCGCCGGCGGACGGGGTGTCGTGGTCACCGTCA CCGGACCGGTCGGCTGCGGCAAGACCGAACTGCTGGACGCGGCCGCGGCC GAGGAGGAATTCATCACGTTGCGTGCGGTCTGCTCGGCCGAGGAGCGGGC CCTGCCGTACGCCGTGATCGGCCAACTCCTCGACCATCCCGTACTCTCCG CACGCGCGCCCGACCTGGCCTGCGTGACGGCTCCGGGCCGGACGCTGCCG GCCGACACCGAGAACCGCCTGCGCCGCGACCTCACCCGGGCCCTGCTGGC CCTGGCCTCCGAACGACCGGTTCTGATCTGCATCGACGACGTGCACCAGG CCGACACCGCCTCGCTGAACTGCCTGCTGCACCTGGCCCGGCGGGTCGCC TCGGCCCGGATCGCCATGATCCTCACCGAGTTGCGCCGGCTCACCCCGGC TCACTCCCGGTTCGAGGCGGAACTGCTCAGCCTGCGGCACCGCCACGAGA TCGCGCTGCGTCCCCTCGGCCCGGCCGACACCGCCGAACTGGCCCGCGCC CGGCTCGGCGCCGGCGTCACCGCCGACGAGCTGGCCCAGGTCCACGAGGC CACCAGCGGGAACCCCAACCTGGTCGGAGGCCTGGTCAACGACGTGCGAG AGGCCTGGGCGGCCGGTGGCACGGGCATTGCGGCGGGGCGGGCGTACCGG CTGGCGTACCTCAGCTCCGTGTACCGCTGTGGTCCGGTCCCGTTGCGGAT CGCCCAGGCGGCGGCGGTGCTGGGTCCCAGCGCCACCGTCACGCTGGTGC GCCGGATCAGCGGGCTCGACGCCGAGACGGTGGACGAGGCGACCGCGATC CTCACCGAGGGCGGCCTGCTCCGGGACCACCGGTTCCCGCATCCGGCGGC CCGCTCGGTCGTACTCGACGACATGTCCGCGCAGGAACGCCGCCGCCTGC ACCGGTCCACGCTGGACGTGCTGGACGGCGTACCCGTCGACGTGCTCGCG CACCACCAGGCCGGCGCCGGTCTGCTGCACGGCCCGCAGGCGGCCGAGAT GTTCGCCCGGGCCAGCCAGGAGCTGCGGGTACGCGGCGAGCTGGACGCCG CGACCGAGTACCTGCAACTGGCCTACCGGGCCTCCGACGACGCCGGCGCC CGGGCCGCCCTGCAGGTGGAGACCGTGGCCGGCGAGCGCCGCCGCAACCC GCTGGCCGCCAGCCGGCACCTGGACGAGCTGGCCGCCGCCGCCCGGGCCG GCCTGCTGTCGGCCGAGCACGCCGCCCTGGTCGTGCACTGGCTGGCCGAC GCCGGACGACCCGGCGAGGCCGCCGAGGTGCTGGCGCTGCAGCGGGCGCT GGCCGTCACCGACCACGACCGGGCCCGCCTGCGGGCGGCCGAGGTGTCGC TCGCGCTGTTCCACCCCGGCGTCCCCGGTTCGGACCCGCGGCCCCTCGCG CCGGAGGAGCTCGCGAGCCTGTCCCTGTCGGCCCGGCACGGTGTGACCGC CGACAACGCGGTGCTGGCGGCGCTGCGCGGCCGTCCCGAGTCGGCCGCCG CCGAGGCGGAGAACGTGCTGCGCAACGCCGACGCCGCCGCGTCCGGCCCG ACCGCCCTGGCCGCGCTGACGGCCCTGCTCTACGCCGAGAACACCGACGC CGCCCAGCTCTGGGCGGACAAGCTGGCCGCGGGCATCGGGGCGGGGGAGG GGGAGGCCGGCTACGCGGGGCCGCGGACCGTGGCCGCCCTGCGTCGCGGC GACCTGACCACCGCGGTCCAGGCGGCCGGCGCGGTCCTGGACCGCGGCCG GCCGTCGTCGCTCGGCATCACCGCCGTGTTGCCGTTGAGCGGCGCGGTCG CCGCCGCGATCCGGCTGGGCGAGCTCGAGCGGGCCGAGAAGTGGCTGGCC GAGCCGCTGCCCGAAGCCGTCCACGACAGCCTGTTCGGCCTGCACCTGCT GATGGCGCGGGGCCGCTACAGCCTCGCGGTGGGCCGGCACGAGGCGGCGT ACGCCGCGTTCCGGGACTGCGGTGAACGGATGCGCCGGTGGGACGTCGAC GTGCCCGGGCTGGCCCTGTGGCGGGTGGACGCGGCCGAGGCGCTGCTGCC CGGCGATGACCGGGCGGAGGGCCGGCGGCTGATCGACGAGCAGCTCACCC GGCCGATGGGGCCCCGGTCACGAGCCCTGACCCTGCGGGTACGAGCGGCC TACGCCCCGCCGGCGAAACGGATCGACCTGCTCGACGAAGCGGCCGACCT GCTGCTCTCCAGCAACGACCAGTACGAGCGGGCACGGGTGCTGGCCGACC TGAGCGAGGCGTTCAGCGCGCTCCGGCAGAACGGCCGGGCGCGCGGCATC CTGCGGCAGGCCCGGCACCTGGCCGCCCAGTGCGGGGCGGTCCCCCTGCT GCGCCGGCTGGGCGTCAAGGCCGGCCGGTCCGGTCGGCTCGGCCGGCCGC CGCAGGGAATCCGCTCCCTGACCGAGGCCGAGCGCCGGGTGGCCACGCTG GCCGCCGCCGGGCAGACCAACCGGGAGATCGCCGACCAGCTCTTCGTCAC CGCCAGCACGGTCGAGCAGCACCTCACCAACGTGTTCCGCAAGCTCGGCG TGAAGGGCCGCCAGCAATTGCCGGCCGAGCTGGCCGACCTGCGGCCGCCG GGCTGA SEQ ID NO: 187 ATGGAGTTTTACGACCTGGTCGCCCGCGATGACGAGCTCAGAAGGTTGGA CCAGGCCCTCGGCCGCGCCGCCGGCGGACGGGGTGTCGTGGTCACCGTCA CCGGACCGGTCGGCTGCGGCAAGACCGAACTGCTGGACGCGGCCGCGGCC GAGGAGGAATTCATCACGTTGCGTGCGGTCTGCTCGGCCGAGGAGCGGGC CCTGCCGTACGCCGTGATCGGCCAACTCCTCGACCATCCCGTACTCTCCG CACGCGCGCCCGACCTGGCCTGCGTGACGGCTCCGGGCCGGACGCTGCCG GCCGACACCGAGAACCGCCTGCGCCGCGACCTCACCCGGGCCCTGCTGGC CCTGGCCTCCGAACGACCGGTTCTGATCTGCATCGACGACGTGCACCAGG CCGACACCGCCTCGCTGAACTGCCTGCTGCACCTGGCCCGGCGGGTCGCC TCGGCCCGGATCGCCATGATCCTCACCGAGTTGCGCCGGCTCACCCCGGC TCACTCCCGGTTCGAGGCGGAACTGCTCAGCCTGCGGCACCGCCACGAGA TCGCGCTGCGTCCCCTCGGCCCGGCCGACACCGCCGAACTGGCCCGCGCC CGGCTCGGCGCCGGCGTCACCGCCGACGAGCTGGCCCAGGTCCACGAGGC CACCAGCGGGAACCCCAACCTGGTCGGAGGCCTGGTCAACGACGTGCGAG AGGCCTGGGCGGCCGGTGGCACGGGCATTGCGGCGGGGCGGGCGTACCGG CTGGCGTACCTCAGCTCCGTGTACCGCTGTGGTCCGGTCCCGTTGCGGAT CGCCCAGGCGGCGGCGGTGCTGGGTCCCAGCGCCACCGTCACGCTGGTGC GCCGGATCAGCGGGCTCGACGCCGAGACGGTGGACGAGGCGACCGCGATC CTCACCGAGGGCGGCCTGCTCCGGGACCACCGGTTCCCGCATCCGGCGGC CCGCTCGGTCGTACTCGACGACATGTCCGCGCAGGAACGCCGCCGCCTGC ACCGGTCCACGCTGGACGTGCTGGACGGCGTACCCGTCGACGTGCTCGCG CACCACCAGGCCGGCGCCGGTCTGCTGCACGGCCCGCAGGCGGCCGAGAT GTTCGCCCGGGCCAGCCAGGAGCTGCGGGTACGCGGCGAGCTGGACGCCG CGACCGAGTACCTGCAACTGGCCTACCGGGCCTCCGACGACGCCGGCGCC CGGGCCGCCCTGCAGGTGGAGACCGTGGCCGGCGAGCGCCGCCGCAACCC GCTGGCCGCCAGCCGGCACCTGGACGAGCTGGCCGCCGCCGCCCGGGCCG GCCTGCTGTCGGCCGAGCACGCCGCCCTGGTCGTGCACTGGCTGGCCGAC GCCGGACGACCCGGCGAGGCCGCCGAGGTGCTGGCGCTGCAGCGGGCGCT GGCCGTCACCGACCACGACCGGGCCCGCCTGCGGGCGGCCGAGGTGTCGC TCGCGCTGTTCCACCCCGGCGTCCCCGGTTCGGACCCGCGGCCCCTCGCG CCGGAGGAGCTCGCGAGCCTGTCCCTGTCGGCCCGGCACGGTGTGACCGC CGACAACGCGGTGCTGGCGGCGCTGCGCGGCCGTCCCGAGTCGGCCGCCG CCGAGGCGGAGAACGTGCTGCGCAACGCCGACGCCGCCGCGTCCGGCCCG ACCGCCCTGGCCGCGCTGACGGCCCTGCTCTACGCCGAGAACACCGACGC CGCCCAGCTCTGGGCGGACAAGCTGGCCGCGGGCATCGGGGCGGGGGAGG GGGAGGCCGGCTACGCGGGGCCGCGGACCGTGGCCGCCCTGCGTCGCGGC GACCTGACCACCGCGGTCCAGGCGGCCGGCGCGGTCCTGGACCGCGGCCG GCCGTCGTCGCTCGGCATCACCGCCGTGTTGCCGTTGAGCGGCGCGGTCG CCGCCGCGATCCGGCTGGGCGAGCTCGAGCGGGCCGAGAAGTGGCTGGCC GAGCCGCTGCCCGAAGCCGTCCACGACAGCCTGTTCGGCCTGCACCTGCT GATGGCGCGGGGCCGCTACAGCCTCGCGGTGGGCCGGCACGAGGCGGCGT ACGCCGCGTTCCGGGACTGCGGTGAACGGATGCGCCGGTGGGACGTCGAC GTGCCCGGGCTGGCCCTGTGGCGGGTGGACGCGGCCGAGGCGCTGCTGCC CGGCGATGACCGGGCGGAGGGCCGGCGGCTGATCGACGAGCAGCTCACCC GGCCGATGGGGCCCCGGTCACGAGCCCTGACCCTGCGGGTACGAGCGGCC TACGCCCCGCCGGCGAAACGGATCGACCTGCTCGACGAAGCGGCCGACCT GCTGCTCTCCAGCAACGACCAGTACGAGCGGGCACGGGTGCTGGCCGACC TGAGCGAGGCGTTCAGCGCGCTCCGGCAGAACGGCCGGGCGCGCGGCATC CTGCGGCAGGCCCGGCACCTGGCCGCCCAGTGCGGGGCGGTCCCCCTGCT GCGCCGGCTGGGCGTCAAGGCCGGCCGGTCCGGTCGGCTCGGCCGGCCGC CGCAGGGAATCCGCTCCCTGACCGAGGCCGAGCGCCGGGTGGCCACGCTG GCCGCCGCCGGGCAGACCAACCGGGAGATCGCCGACCAGCTCTTCGTCAC CGCCAGCACGGTCGAGCAGCACCTCACCAACGTGTTCCGCAAGCTCGGCG TGAAGGGCCGCCAGCAATTGCCGGCCGAGCTGGCCGACCTGCGGCCGCCG GGCTGA SEQ ID NO: 188 GTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTGCTTGA CGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGGTCTGCGCGC CAGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGAC CCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCCCAGGG CGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCTCACCC GTGCCCTGCTGGCGCTTGCCGTCGACCGGCCTGTGCTGATCGGCGTCGAC GATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATTTGGC GCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCA GCCTCACCCCTACTCAGTCACGGTTCAAGGCGGAGCTGCTCAGCCTGCCG TACCACCACGAGATCGCGCTGCGTCCGTTCGGACCGGAGCAATCGGCGGA GCTGGCCCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCTCGTGG GGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGACTGATC AGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCCTTCGAGGCGGG CCGCGCGTTCCGGCTGGCGTACCTCGGCTCGCTCTACCGCTGTGGCCCGG TCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCGAGCGCCACC ACCACGCTGGTGCGCCGTCTAAGCGGGCTCAGCGCGGAGACGATAGACCG GGCAACCAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGCAGTTCC CGCACCCGGCCGCCCGCTCGGTGGTGCTTGATGACATGTCCGCCCAGGAA CGACGCGGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCGCCGGT TGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCA AGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTACGCAAC GAGTTGGGCGACGCGGCAGAATACCTGCAACTGGCTCACCGGGCCTCCGA CGATGTCTCCACCCGGGCCGCCTTACGGGTCGAGGCCGTGGCGATCGAGC GCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAGCTGAGCGCC GCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCCGTCTT CTGGCTGGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCTGGCGT CGGAACGCCCGCTAGCGACCACCGATCAGAACCGGGCCCACTTGCGATTT GTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGGATCGGACCG GCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAAGGCGG CCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGCACGGT CATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAGGCTGA TTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTACGCGG AGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCACGAAT GGCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGA GATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCA CCGTCCTGGACGACCGGTCGCTGCCGTCGCTCGGCATCACCGCCGCATTG CTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTCGAGCG TGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGACAGCC TTTTCGGTCTGCACCTGCTCTCGGCATACGGCCAGTACAGCCTCGCGATG GGCCGATATGAATCGGCTCTCCGGGCGTTTCACACCTGCGGAGAACGTAT GCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGCGTGTCGACG CCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATC GACGAACAACTCACCCGTCCGATGGGGCCTCGTTCCCGCGCGTTAACGCT GCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCTGCTCC ATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGCAAGCG CGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAG CCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCGCCCAGTGCG GTGCTGTCCCGCTGCTGCGCAGGCTCGGGGGCGAGCCCGGCCGGATCGAC GACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAGCGGCG GGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGCCAAAC AGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCGTCTTC CGCAAACTGGGGGTCAAGGGTCGCAAGCAGCTGCCGACCGCGCTGGCCGA CGTGGAACAGACCTGA SEQ ID NO: 189 ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGA CGAACTCGGCATTCTACAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGG GCGTCGTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTG CTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGGTCTG CGCGCCAGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCG ACGACCCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCC CAGGGCGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCT CACCCGTGCCCTGCTGGCGCTTGCCGTCGACCGGCCTGTGCTGATCGGCG TCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCAT TTGGCGCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTT GCGCAGCCTCACCCCTACTCAGTCACGGTTCAAGGCGGAGCTGCTCAGCC TGCCGTACCACCACGAGATCGCGCTGCGTCCGTTCGGACCGGAGCAATCG GCGGAGCTGGCCCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGTGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGAC TGATCAGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCCTTCGAG GCGGGCCGCGCGTTCCGGCTGGCGTACCTCGGCTCGCTCTACCGCTGTGG CCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCGAGCG CCACCACCACGCTGGTGCGCCGTCTAAGCGGGCTCAGCGCGGAGACGATA GACCGGGCAACCAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGCA GTTCCCGCACCCGGCCGCCCGCTCGGTGGTGCTTGATGACATGTCCGCCC AGGAACGACGCGGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCG CCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGG GCCCAAGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTAC GCAACGAGTTGGGCGACGCGGCAGAATACCTGCAACTGGCTCACCGGGCC TCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGTCGAGGCCGTGGCGAT CGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAGCTGA GCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCC GTCTTCTGGCTGGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCT GGCGTCGGAACGCCCGCTAGCGACCACCGATCAGAACCGGGCCCACTTGC GATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGGATCG GACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAA GGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGC ACGGTCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAG GCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTA CGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCA CGAATGGCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGC GCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGG TAGCACCGTCCTGGACGACCGGTCGCTGCCGTCGCTCGGCATCACCGCCG CATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGA CAGCCTTTTCGGTCTGCACCTGCTCTCGGCATACGGCCAGTACAGCCTCG CGATGGGCCGATATGAATCGGCTCTCCGGGCGTTTCACACCTGCGGAGAA CGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGCGTGT CGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGC TCATCGACGAACAACTCACCCGTCCGATGGGGCCTCGTTCCCGCGCGCTG ACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCT GCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGC AAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGC TATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCGCCCA GTGCGGTGCTGTCCCGCTGCTGCGCAGGCTCGGGGGCGAGCCCGGCCGGA TCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAG CGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CAAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCG TCTTCCGCAAACTGGGGGTCAAGGGTCGCAAGCAGCTGCCGACCGCGCTG GCCGACGTGGAACAGACCTGA SEQ ID NO: 190 ATGCCTGCCGTGGAGAGCTATGAACTGGACGCCCGCGATGACGAGCTCAG AAGACTGGAGGAGGCGGTAGGCCAGGCGGGCAACGGCCGGGGTGTGGTGG TCACCATCACCGGGCCGATCGCCTGCGGCAAGACCGAACTGCTCGACGCG GCCGCCGCGAAGAGCGACGCCATCACATTACGTGCGGTCTGCTCCGAGGA GGAACGGGCCCTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGG CGGTCGCCTCCCAGCTGCCGGATCCGGTCTCCATGGCCCTCCCGGGCGAG CACCTGTCGCCGGAGGCCGAGAACCGGCTGCGCGGCGACCTCACCCGTAC CCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCGGCATCGACGACA TGCACCACGCCGACACCGCCTCTTTGAACTGCCTGCTCCACCTGGCCCGG AGGGTCGGCCCGGCCCGGATCGCCATGGTCCTCACCGAGCTGCGCCGGCT CACCCCGGCCCACTCCCAGTTCCACGCCGAGCTGCTCAGCCTGGGGCACC ACCGCGAGATCGCGCTGCGCCCGCTCGGCCCGAAGCACATCGCCGAGCTG GCCCGCGCCGGCCTCGGTCCCGATGTCGACGAGGACGTGCTCACGGGGTT GTACCGGGCGACCGGCGGCAACCTGAACCTCGGCCACGGACTGATCAAGG ATGTGCGGGAGGCCTGGGCGACGGGCGGGACGGGCATCAACGCGGGCCGC GCGTACCGGCTGGCGTACCTCGGTTCCCTCTACCGCTGCGGCCCGGTCCC GTTGCGGGTCGCACGGGTGGCCGCCGTGCTGGGCCAGAGCGCCAACACCA CCCTGGTGCGCTGGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCG ACCGAGATCCTCACCGAGGGCGGCCTGCTGCACGACCTGCGGTTCCCGCA TCCGGCGGCCCGTTCGGTCGTACTCAACGACCTGTCCGCCCGGGAACGCC GCCGACTGCACCGGTCCGCTCTGGAAGTGCTGGATGACGTACCCGTTGAA GTGGTCGCGCACCACCAGGCCGGTGCCGGTTTCATCCACGGTCCCAAGGC CGCCGAGATCTTCGCCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGC TGGACGCCGCGTCCGACTATCTGCAACTGGCCCACCACGCCTCCGACGAC GCCGTCACCCGGGCCGCGCTGCGGGTCGAGGCCGTGGCGATCGAGCGCCG CCGCAACCCGCTGGCCTCCAGCCGCCACCTCGACGAGCTGACCGTCGCCG CCCGTGCCGGTCTGCTCTCCCTCGAGCACGCCGCGCTGATGATCCGCTGG CTGGCTCTCGGCGGGCGGTCCGGCGAGGCGGCCGAGGTGCTGGCCGCGCA GCGCCCGCGTGCGGTCACCGACCAGGACAGGGCCCACCTGCGGGCCGCCG AGGTATCGCTGGCGCTGGTCAGCCCGGGCGCGTCCGGCGTCAGCCCGGGT GCGTCCGGCCCGGATCGGCGGCCGCGTCCGCTCCCGCCGGATGAGCTCGC GAACCTGCCGAAGGCGGCCCGGCTTTGTGCGATCGCCGACAACGCCGTCA TATCGGCCCTGCACGGTCGTCCCGAGCTTGCCTCGGCCGAGGCGGAGAAC GTCCTGAAGCAGGCTGACTCGGCGGCGGACGGCGCCACCGCCCTCTCCGC GCTGACGGCCTTGCTGTACGCGGAGAACACCGACACCGCTCAGCTCTGGG CCGACAAGCTCGTCTCCGAGACCGGGGCGTCGAACGAGGAGGAAGGCGCG GGCTACGCGGGGCCGCGCGCCGAGACCGCGTTGCGCCGCGGCGACCTGGC CGCGGCGGTCGAGGCGGGCAGCGCCATTCTGGACCACCGGCGGGGGTCGT TGCTCGGCATCACCGCCGCGCTACCGCTGAGCAGCGCGGTAGCCGCCGCC ATCCGGCTGGGCGAGACCGAGCGGGCGGAGAAGTGGCTCGCCGAGCCGCT GCCGGAGGCCATTCGGGACAGCCTGTTCGGGCTGCACCTGCTCTCGGCGC GCGGCCAGTACTGCCTCGCGACGGGCCGGCACGAGTCGGCGTACACGGCG TTCCGCACCTGCGGGGAACGGATGCGGAACTGGGGCGTCGACGTGCCGGG TCTGTCCCTGTGGCGCGTCGACGCCGCCGAGGCGCTGCTGCACGGCCGCG ACCGGGACGAGGGCCGACGGCTCATCGACGAGCAGCTCACCCATGCGATG GGACCCCGTTCCCGCGCTTTGACGCTGCGGGTGCAGGCGGCGTACAGCCC GCAGGCGCAGCGGGTCGACCTGCTCGAAGAGGCGGCCGACCTGCTGCTCT CCTGCAACGACCAGTACGAGCGGGCGCGGGTGCTCGCCGATCTGAGCGAG GCGTTCAGCGCGCTCAGGCACCACAGCCGGGCGCGGGGACTGCTCCGGCA GGCCCGGCACCTGGCCGCCCAGTGCGGCGCGACCCCGCTGCTGCGCCGGC TCGGGGCCAAGCCCGGAGGCCCCGGCTGGCTGGAGGAATCCGGCCTGCCG CAGCGGATCAAGTCGCTGACCGACGCGGAGCGGCGGGTGGCGTCGCTGGC CGCCGGCGGCCAGACCAACCGCGTGATCGCCGACCAGCTCTTCGTCACGG CCAGCACGGTGGAGCAGCACCTCACGAACGTCTTCCGCAAGCTGGGCGTC AAGGGCCGCCAGCACCTGCCGGCCGAACTCGCCAACGCGGAATAG SEQ ID NO: 191 ATGCCTGCCGTGGAGAGCTATGAACTGGACGCCCGCGATGACGAGCTCAG AAGACTGGAGGAGGCGGTAGGCCAGGCGGGCAACGGCCGGGGTGTGGTGG TCACCATCACCGGGCCGATCGCCTGCGGCAAGACCGAACTGCTCGACGCG GCCGCCGCGAAGAGCGACGCCATCACACTGCGTGCGGTCTGCTCCGAGGA GGAACGGGCCCTCCCGTACGCCCTGATCGGGCAGCTCATCGACAACCCGG CGGTCGCCTCCCAGCTGCCGGATCCGGTCTCCATGGCCCTCCCGGGCGAG CACCTGTCGCCGGAGGCCGAGAACCGGCTGCGCGGCGACCTCACCCGTAC CCTGCTGGCGCTCGCCGCCGAACGGCCGGTGCTGATCGGCATCGACGACA TGCACCACGCCGACACCGCCTCTTTGAACTGCCTGCTCCACCTGGCCCGG AGGGTCGGCCCGGCCCGGATCGCCATGGTCCTCACCGAGCTGCGCCGGCT CACCCCGGCCCACTCCCAGTTCCACGCCGAGCTGCTCAGCCTGGGGCACC ACCGCGAGATCGCGCTGCGCCCGCTCGGCCCGAAGCACATCGCCGAGCTG GCCCGCGCCGGCCTCGGTCCCGATGTCGACGAGGACGTGCTCACGGGGTT GTACCGGGCGACCGGCGGCAACCTGAACCTCGGCCACGGACTGATCAAGG ATGTGCGGGAGGCCTGGGCGACGGGCGGGACGGGCATCAACGCGGGCCGC GCGTACCGGCTGGCGTACCTCGGTTCCCTCTACCGCTGCGGCCCGGTCCC GTTGCGGGTCGCACGGGTGGCCGCCGTGCTGGGCCAGAGCGCCAACACCA CCCTGGTGCGCTGGATCAGCGGGCTCAACGCGGACGCGGTGGGCGAGGCG ACCGAGATCCTCACCGAGGGCGGCCTGCTGCACGACCTGCGGTTCCCGCA TCCGGCGGCCCGTTCGGTCGTACTCAACGACCTGTCCGCCCGGGAACGCC GCCGACTGCACCGGTCCGCTCTGGAAGTGCTGGATGACGTACCCGTTGAA GTGGTCGCGCACCACCAGGCCGGTGCCGGTTTCATCCACGGTCCCAAGGC CGCCGAGATCTTCGCCAAGGCCGGCCAGGAGCTGCATGTGCGCGGCGAGC TGGACGCCGCGTCCGACTATCTGCAACTGGCCCACCACGCCTCCGACGAC GCCGTCACCCGGGCCGCGCTGCGGGTCGAGGCCGTGGCGATCGAGCGCCG CCGCAACCCGCTGGCCTCCAGCCGCCACCTCGACGAGCTGACCGTCGCCG CCCGTGCCGGTCTGCTCTCCCTCGAGCACGCCGCGCTGATGATCCGCTGG CTGGCTCTCGGCGGGCGGTCCGGCGAGGCGGCCGAGGTGCTGGCCGCGCA GCGCCCGCGTGCGGTCACCGACCAGGACAGGGCCCACCTGCGGGCCGCCG AGGTATCGCTGGCGCTGGTCAGCCCGGGCGCGTCCGGCGTCAGCCCGGGT GCGTCCGGCCCGGATCGGCGGCCGCGTCCGCTCCCGCCGGATGAGCTCGC GAACCTGCCGAAGGCGGCCCGGCTTTGTGCGATCGCCGACAACGCCGTCA TATCGGCCCTGCACGGTCGTCCCGAGCTTGCCTCGGCCGAGGCGGAGAAC GTCCTGAAGCAGGCTGACTCGGCGGCGGACGGCGCCACCGCCCTCTCCGC GCTGACGGCCTTGCTGTACGCGGAGAACACCGACACCGCTCAGCTCTGGG CCGACAAGCTCGTCTCCGAGACCGGGGCGTCGAACGAGGAGGAAGGCGCG GGCTACGCGGGGCCGCGCGCCGAGACCGCGTTGCGCCGCGGCGACCTGGC CGCGGCGGTCGAGGCGGGCAGCGCCATTCTGGACCACCGGCGGGGGTCGT TGCTCGGCATCACCGCCGCGCTACCGCTGAGCAGCGCGGTAGCCGCCGCC ATCCGGCTGGGCGAGACCGAGCGGGCGGAGAAGTGGCTCGCCGAGCCGCT GCCGGAGGCCATTCGGGACAGCCTGTTCGGGCTGCACCTGCTCTCGGCGC GCGGCCAGTACTGCCTCGCGACGGGCCGGCACGAGTCGGCGTACACGGCG TTCCGCACCTGCGGGGAACGGATGCGGAACTGGGGCGTCGACGTGCCGGG TCTGTCCCTGTGGCGCGTCGACGCCGCCGAGGCGCTGCTGCACGGCCGCG ACCGGGACGAGGGCCGACGGCTCATCGACGAGCAGCTCACCCATGCGATG GGACCCCGTTCCCGCGCTTTGACGCTGCGGGTGCAGGCGGCGTACAGCCC GCAGGCGCAGCGGGTCGACCTGCTCGAAGAGGCGGCCGACCTGCTGCTCT CCTGCAACGACCAGTACGAGCGGGCGCGGGTGCTCGCCGATCTGAGCGAG GCGTTCAGCGCGCTCAGGCACCACAGCCGGGCGCGGGGACTGCTCCGGCA GGCCCGGCACCTGGCCGCCCAGTGCGGCGCGACCCCGCTGCTGCGCCGGC TCGGGGCCAAGCCCGGAGGCCCCGGCTGGCTGGAGGAATCCGGCCTGCCG CAGCGGATCAAGTCGCTGACCGACGCGGAGCGGCGGGTGGCGTCGCTGGC CGCCGGCGGCCAGACCAACCGCGTGATCGCCGACCAGCTCTTCGTCACGG CCAGCACGGTGGAGCAGCACCTCACGAACGTCTTCCGCAAGCTGGGCGTC AAGGGCCGCCAGCACCTGCCGGCCGAACTCGCCAACGCGGAATAG SEQ ID NO: 192 GTGAAGCGCAACGATCTGGTTGCCCGCGATGGCGAGCTCAGGTGGATGCA AGAGATTCTCAGTCAGGCGAGCGAGGGCCGGGGGGCCGTGGTCACCATCA CGGGGGCGATCGCCTGTGGCAAGACGGTGCTGCTGGACGCCGCGGCAGCC AGTCAAGACGTGATCCAACTGCGTGCGGTCTGCTCGGCGGAGGAGCAGGA GCTGCCGTACGCGATGGTCGGACAACTACTCGACAATCCGGTGCTCGCCG CGCGAGTGCCGGCCCTGGGCAACCTGGCTGCGGCGGGCGAGCGGCTGCTG CCGGGCACCGAGAACAGGATCCGGCGGGAGCTCACCCGCACCCTGCTGGC TCTCGCCGACGAACGACCGGTGCTGATCGGCGTCGACGACATGCACCATG CGGACCCCGCCTCGCTGGACTGCCTGCTGCACCTGGCCCGGCGGGTCGGC CCGGCCCGCATCGCGATCGTTCTGACCGAGTTGCGCCGGCTCACCCCGGC TCACTCGCGCTTCCAGTCCGAGCTGCTCAGCCTGCGGTACCACCACGAGA TCGGGTTGCAGCCGCTCACCGCGGAGCACACCGCCGACCTGGCCCGCGTC GGCCTCGGTGCCGAGGTCGACGACGACGTGCTCACCGAGCTCTACGAGGC GACCGGCGGCAACCCGAGTCTGTGCTGCGGCCTGATCAGGGACGTGCGGC AGGACTGGGAGGCCGGGGTCACCGGTATCCACGTCGGCCGGGCGTACCGG CTGGCCTATCTCAGTTCGCTCTACCGCTGCGGCCCGGCGGCGCTGCGGAC CGCCCGCGCGGCCGCGGTGCTGGGCGACAGCGCCGACGCCTGCCTGATCC GCCGGGTCAGCGGCCTCGGTACGGAGGCCGTGGGCCAGGCGATCCAGCAG CTCACCGAGGGCGGCCTGCTGCGTGACCAGCAGTTCCCGCACCCGGCGGC CCGCTCGGTCGTGCTCGACGACATGTCCGCGCAGGAACGCCACGCGATGT ATCGCAGCGCCCGGGAGGCAGCCGCCGAAGGTCAGGCCGACCCCGGCACC CCGGGCGAGCCGCGGGCGGCTACGGCGTACGCCGGGTGTGGTGAGCAAGC CGGTGACTACCCGGAGCCGGCCGGCCGGGCCTGCGTGGACGGTGCCGGTC CGGCCGAGTACTGCGGCGACCCGCACGGCGCCGACGACGACCCGGACGAG CTGGTCGCCGCGCTGGGCGGGCTGCTGCCGAGCCGGCTCGTGGCGATGAA GATCCGGCGCCTGGCGGTGGCCGGGCGCCCCGGGGCGGCTGCCGAGCTGC TGACCTCGCAGCGGTTGCACGCGGTGACCAGCGAGGACCGGGCCAGCCTG CGGGCCGCCGAGGTGGCGCTCGCCACGCTGTGGCCGGGTGCGACCGGCCC GGACCGGCATCCGCTCACGGAGCAGGAGGCGGCGAGCCTGCCGGAGGGTC CGCGCCTGCTCGCTGCCGCCGACGATGCCGTCGGGGCCGCCCTGCGCGGT CGCGCCGAGTACGCCGCGGCCGAGGCGGAGAACGTCCTGCGGCACGCCGA TCCGGCAGCCGGTGGTGACGCCTACGCCGCCATGATCGCCCTGCTGTACA CGGAGCACCCCGAGAACGTGCTGTTCTGGGCCGACAAGCTCGACGCGGGC CGCCCCGACGAGGAGACCAGTTATCCCGGGCTGCGGGCCGAGACCGCGGT GCGGCTCGGTGACCTGGAAACGGCGATGGAGCTGGGCCGCACGGTGCTGG ACCAGCGGCGGCTGCCGTCCCTGGGTGTCGCCGCGGGCCTGCTCCTGGGC GGCGCGGTGACGGCCGCCATCCGGCTCGGCGACCTCGACCGGGCGGAGAA GTGGCTCGCCGAGCCGATCCCCGACGCCATCCGTACCAGCCTCTACGGCC TGCACGTGCTGGCCGCGCGGGGCCGGCTCGACCTGGCCGCGGGCCGCTAC GAGGCGGCGTACACGGCGTTCCGGCTGTGTGGCGAGCGGATGGCAGGCTG GGATGCCGATGTCTCCGGGCTGGCGCTGTGGCGCGTCGACGCCGCCGAGG CCCTGCTGTCCGCGGGCATCCGCCCGGACGAGGGCCGCAAGCTCATCGAC GACCAGCTCACCCGTGAGATGGGGGCCCGCTCCCGGGCGCTGACGCTGCG GGCGCAAGCGGCGTACAGCCTGCCGGTGCACCGGGTGGGCCTGCTCGACG AGGCGGCCGGCCTGCTGCTCGCCTGCCATGACGGGTACGAGCGGGCGCGG GTGCTCGCGGACCTGGGGGAGACCCTGCGCACGCTGCGGCACACCGACGC GGCCCAGCGGGTGCTCCGGCAGGCCGAGCAGGCGGCCGCGCGGTGCGGGT CGGTCCCGCTGCTGCGGCGGCTCGGGGCCGAACCCGTACGCATCGGCACC CGGCGTGGTGAACCCGGCCTGCCGCAGCGGATCAGGCTGCTGACCGATGC CGAGCGGCGGGTTGCCGCGATGGCCGCCGCCGGGCAGACCAACCGGGAGA TCGCCGGTCGGCTCTTCGTCACGGCCAGCACGGTGGAGCAGCACCTGACC AGCGTCTTCCGCAAGCTGGGCGTCAAGGGCCGCCGGTTCCTGCCGACCGA GCTCGCCCAAGCCGTCTGA SEQ ID NO: 193 ATGCCTGCCGTGAAGCGCAACGATCTGGTTGCCCGCGATGGCGAGCTCAG GTGGATGCAAGAGATTCTCAGTCAGGCGAGCGAGGGCCGGGGGGCCGTGG TCACCATCACGGGGGCGATCGCCTGTGGCAAGACGGTGCTGCTGGACGCC GCGGCAGCCAGTCAAGACGTGATCCAACTGCGTGCGGTCTGCTCGGCGGA GGAGCAGGAGCTGCCGTACGCGATGGTCGGACAACTACTCGACAATCCGG TGCTCGCCGCGCGAGTGCCGGCCCTGGGCAACCTGGCTGCGGCGGGCGAG CGGCTGCTGCCGGGCACCGAGAACAGGATCCGGCGGGAGCTCACCCGCAC CCTGCTGGCTCTCGCCGACGAACGACCGGTGCTGATCGGCGTCGACGACA TGCACCATGCGGACCCCGCCTCGCTGGACTGCCTGCTGCACCTGGCCCGG CGGGTCGGCCCGGCCCGCATCGCGATCGTTCTGACCGAGTTGCGCCGGCT CACCCCGGCTCACTCGCGCTTCCAGTCCGAGCTGCTCAGCCTGCGGTACC ACCACGAGATCGGGTTGCAGCCGCTCACCGCGGAGCACACCGCCGACCTG GCCCGCGTCGGCCTCGGTGCCGAGGTCGACGACGACGTGCTCACCGAGCT CTACGAGGCGACCGGCGGCAACCCGAGTCTGTGCTGCGGCCTGATCAGGG ACGTGCGGCAGGACTGGGAGGCCGGGGTCACCGGTATCCACGTCGGCCGG GCGTACCGGCTGGCCTATCTCAGTTCGCTCTACCGCTGCGGCCCGGCGGC GCTGCGGACCGCCCGCGCGGCCGCGGTGCTGGGCGACAGCGCCGACGCCT GCCTGATCCGCCGGGTCAGCGGCCTCGGTACGGAGGCCGTGGGCCAGGCG ATCCAGCAGCTCACCGAGGGCGGCCTGCTGCGTGACCAGCAGTTCCCGCA CCCGGCGGCCCGCTCGGTCGTGCTCGACGACATGTCCGCGCAGGAACGCC ACGCGATGTATCGCAGCGCCCGGGAGGCAGCCGCCGAAGGTCAGGCCGAC CCCGGCACCCCGGGCGAGCCGCGGGCGGCTACGGCGTACGCCGGGTGTGG TGAGCAAGCCGGTGACTACCCGGAGCCGGCCGGCCGGGCCTGCGTGGACG GTGCCGGTCCGGCCGAGTACTGCGGCGACCCGCACGGCGCCGACGACGAC CCGGACGAGCTGGTCGCCGCGCTGGGCGGGCTGCTGCCGAGCCGGCTCGT GGCGATGAAGATCCGGCGCCTGGCGGTGGCCGGGCGCCCCGGGGCGGCTG CCGAGCTGCTGACCTCGCAGCGGTTGCACGCGGTGACCAGCGAGGACCGG GCCAGCCTGCGGGCCGCCGAGGTGGCGCTCGCCACGCTGTGGCCGGGTGC GACCGGCCCGGACCGGCATCCGCTCACGGAGCAGGAGGCGGCGAGCCTGC CGGAGGGTCCGCGCCTGCTCGCTGCCGCCGACGATGCCGTCGGGGCCGCC CTGCGCGGTCGCGCCGAGTACGCCGCGGCCGAGGCGGAGAACGTCCTGCG GCACGCCGATCCGGCAGCCGGTGGTGACGCCTACGCCGCCATGATCGCCC TGCTGTACACGGAGCACCCCGAGAACGTGCTGTTCTGGGCCGACAAGCTC GACGCGGGCCGCCCCGACGAGGAGACCAGTTATCCCGGGCTGCGGGCCGA GACCGCGGTGCGGCTCGGTGACCTGGAAACGGCGATGGAGCTGGGCCGCA CGGTGCTGGACCAGCGGCGGCTGCCGTCCCTGGGTGTCGCCGCGGGCCTG CTCCTGGGCGGCGCGGTGACGGCCGCCATCCGGCTCGGCGACCTCGACCG GGCGGAGAAGTGGCTCGCCGAGCCGATCCCCGACGCCATCCGTACCAGCC TCTACGGCCTGCACGTGCTGGCCGCGCGGGGCCGGCTCGACCTGGCCGCG GGCCGCTACGAGGCGGCGTACACGGCGTTCCGGCTGTGTGGCGAGCGGAT GGCAGGCTGGGATGCCGATGTCTCCGGGCTGGCGCTGTGGCGCGTCGACG CCGCCGAGGCCCTGCTGTCCGCGGGCATCCGCCCGGACGAGGGCCGCAAG CTCATCGACGACCAGCTCACCCGTGAGATGGGGGCCCGCTCCCGGGCGCT GACGCTGCGGGCGCAAGCGGCGTACAGCCTGCCGGTGCACCGGGTGGGCC TGCTCGACGAGGCGGCCGGCCTGCTGCTCGCCTGCCATGACGGGTACGAG CGGGCGCGGGTGCTCGCGGACCTGGGGGAGACCCTGCGCACGCTGCGGCA CACCGACGCGGCCCAGCGGGTGCTCCGGCAGGCCGAGCAGGCGGCCGCGC GGTGCGGGTCGGTCCCGCTGCTGCGGCGGCTCGGGGCCGAACCCGTACGC ATCGGCACCCGGCGTGGTGAACCCGGCCTGCCGCAGCGGATCAGGCTGCT GACCGATGCCGAGCGGCGGGTTGCCGCGATGGCCGCCGCCGGGCAGACCA ACCGGGAGATCGCCGGTCGGCTCTTCGTCACGGCCAGCACGGTGGAGCAG CACCTGACCAGCGTCTTCCGCAAGCTGGGCGTCAAGGGCCGCCGGTTCCT GCCGACCGAGCTCGCCCAAGCCGTCTGA SEQ ID NO: 194 GTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTGCTTGA CGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGGTCTGCGCGC CAGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCGACGAC CCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCCCAGGG CGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCTCACCC GTGCCCTGCTGGCGCTTGCCGTGGACCGGCCTGTGCTGATCGGCGTCGAC GATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCATTTGGC CCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTTGCGCA GCCTCACCCCTACTCAGTCACGGTTCAAGGCGGAGCTGCTCAGCCTGCCA TACCACCACGAGATCGCGCTGCGTCCATTCGGACCGGAGCAATCGGCGGA GCTGGCTCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCTCGCGG GGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGACTGATC AGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCTTTCGAGGCGGG CCGCGCGTTCCGGCTGGCGTACCTCAGCTCGCTCTACCGCTGTGGCCCGG TCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAAGCGCCACC ACCACGCTGGTGCGCCGGCTAAGCGGGCTCAGCGCGGAGACGATAGACCG GGCAACCAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCAGCAGTTCC CGCACCCGGCCGCCCGCTCGGTGGTGCTCGATGACATGTCCGCCCAGGAA CGACGCAGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCGCCGGT TGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGGGCCCA AGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTACGCAAC GAGTTGGGCGACGCGGCCGAATACCTGCAACTGGCTCACCGGGCCTCCGA CGATGTCTCCACCCGGGCCGCCTTACGGGTCGAGGCCGTGGCCATCGAGC GCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAACTGAGCGCC GCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCCGTCTT CTGGCTAGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAAGTGCTGGCGT CGGAACGCCCGCTCGCGACCACCGATCAGAACCGGGCCCACCTGCGATTT GTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGGATCGGACCG GCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAAGGCGG CCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGCACGGC CATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAGGCTGA TTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTACGCGG AGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCACGAAT GCCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGCGCCGA GATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGGTAGCG CCGTCCTGGACGACCGGTCGCTGCCGTCGCTCGGCATCACCGCCGCATTG CTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTCGAGCG TGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGACAGCC TTTTCGGTCTGCACCTGCTCTCGGCGTACGGCCAGTACAGCCTCGCGATG GGCCGATATGAATCAGCTCACCGGGCGTTTCGCACCTGCGGAGAACGTAT GCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGCGTGTCGACG CCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGCTCATC GACGAACAACTCACCCGTCCGATGGGGCCTCGTTCCCACGCGTTAACGCT GCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCTGCTCC ATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGCAAGCG CGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGCTATAG CCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCAGTGCG GTGCTGTCCCGCTGCTGCGCAGGCTCGGGGGCGAGCCCGGCCGGATCGAC GACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAGCGGCG GGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGCCGAAC AGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCGTCTTC CGCAAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTGGCCGA CGTGGAACAGACCTGA SEQ ID NO: 195 ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGA CGAACTCGGTATTCTACAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGG GCGTCGTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTG CTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGGTCTG CGCGCCAGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCG ACGACCCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCC CAGGGCGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCT CACCCGTGCCCTGCTGGCGCTTGCCGTGGACCGGCCTGTGCTGATCGGCG TCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCAT TTGGCCCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTT GCGCAGCCTCACCCCTACTCAGTCACGGTTCAAGGCGGAGCTGCTCAGCC TGCCATACCACCACGAGATCGCGCTGCGTCCATTCGGACCGGAGCAATCG GCGGAGCTGGCTCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGCGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGAC TGATCAGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCTTTCGAG GCGGGCCGCGCGTTCCGGCTGGCGTACCTCAGCTCGCTCTACCGCTGTGG CCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAAGCG CCACCACCACGCTGGTGCGCCGGCTAAGCGGGCTCAGCGCGGAGACGATA GACCGGGCAACCAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCAGCA GTTCCCGCACCCGGCCGCCCGCTCGGTGGTGCTCGATGACATGTCCGCCC AGGAACGACGCAGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCG CCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGG GCCCAAGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTAC GCAACGAGTTGGGCGACGCGGCCGAATACCTGCAACTGGCTCACCGGGCC TCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGTCGAGGCCGTGGCCAT CGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAACTGA GCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCC GTCTTCTGGCTAGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAAGTGCT GGCGTCGGAACGCCCGCTCGCGACCACCGATCAGAACCGGGCCCACCTGC GATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGGATCG GACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAA GGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGC ACGGCCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAG GCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTA CGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCA CGAATGCCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGC GCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGG TAGCGCCGTCCTGGACGACCGGTCGCTGCCGTCGCTCGGCATCACCGCCG CATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGA CAGCCTTTTCGGTCTGCACCTGCTCTCGGCGTACGGCCAGTACAGCCTCG CGATGGGCCGATATGAATCAGCTCACCGGGCGTTTCGCACCTGCGGAGAA CGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGCGTGT CGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGC TCATCGACGAACAACTCACCCGTCCGATGGGGCCTCGTTCCCACGCGCTG ACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCT GCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGC AAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGC TATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCA GTGCGGTGCTGTCCCGCTGCTGCGCAGGCTCGGGGGCGAGCCCGGCCGGA TCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAG CGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CGAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCG TCTTCCGCAAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTG GCCGACGTGGAACAGACCTGA SEQ ID NO: 196 GTGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCCCGCGAGGA CGAACTCGGCATTCTGCAGAGGTCTCTGGAAGAAGCAGGCAGCGGCCAGG GCGCCGTGGTCACCGTCACCGGCCCGATCGCCTGCGGCAAGACAGAACTG CTTGACGCGGCTGCCGCGAAGGCTGACGCCATCATTCTGCGCGCGGTCTG CGCGCCCGAAGAGCGCGCTATGCCGTACGCCATGATCGGGCAGCTCATCG ACGACCCGGCGCTCGCGCATCGGGCGCCGGAGCTGGCTGATCGGATAGCC CAGGGCGGGCATCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCT CACCCGTGCCCTGCTGGCGCTTGCCGTCGACCGGCCTGTGCTGATCGGCG TCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCAT TTAGCCCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTT GCGCAGCCTCACCCCTACTCAGTCACGATTCAAGGCGGAGCTGCTCAGCC TGCCGTACCACCACGAGATCGCGCTGCGTCCACTCGGACCGGAGCAATCG GCGGAGCTGGCCCACGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGCGGGGTTGTATGGGATGACCAGGGGCAACCTGAGTCTCAGCCGTGGAC TGATCAGCGATGTGCGGGAGGCCCAGGCCAACGGAGAGAGCGCTTTCGAG GTGGGCCGCGCGTTCCGGCTGGCGTACCTCAGCTCGCTCTACCGCTGTGG CCCGATCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAAGCG CCACCACCACGCTGGTGCGCCGTCTAAGCGGGCTCAGCGCGGAGACGATA GACCGGGCAACCAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCACCA GTTCCCGCACCCGGCCGCCCGCTCGGTGGTGCTCGATGACATGTCCGCCC AGGAACGACGCAGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCG CCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGG GCCCAAGGCTGCGGAGATATTCGCCAGGGCTGGCCAGGCTCTGGTTGTAC GCAACGAGTTGGGCGACGCGGCCGAATACCTGCAACTGGCTCACCGAGCC TCCGACGATGTCTCCACCCGGGCCGCCTTACGGGTCGAGGCCGTGGCAAT CGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGTCACATGGACGAGCTGA GCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCAGCGCTGGCT GTCTTCTGGCTGGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCT GGCGTCGGAACACCCGCTCGCGACCACCGATCAGAACCGAGCACACCTGC GATTTGCCGAGGTGACTCTCGCGCTGTTCTGTCCCGGCGCCTTCGGGTCG GACCGGCGCCCACCTCCGCTGGCGCCGGACGAGCTCGCCAGCTTGCCGAA GGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGTCATGACAGCGTTGC ATGCTCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAG GCTGATTCGGCAGCCGACGCAATCCCCGCCGCACTGATCGCCCTGTTGTA CGCAGAGAACACCGAGTCCGCTCAGATCTGGGCCGACAAGCTGGGCAGCA CCAATGCCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGC GCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGG TGGCACCGTCCTGGACGACCGGCCGCTGCCGTCGCTCGGCATCACCGCCG CATTGCTGTTGAGCAGCAAGACGGCAGCCGCTGTCCGCCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCTGAGCCGCTTCCGAACGGTGTCCAGGA CAGCCTTTTCGGTCTGCACCTGCTCTCGGCGCACGGCCAGTACAGCCTCG CGATGGGCCGATATGAATCGGCTCACCGGGCGTTTCACACCTGCGGAGAA CGTATGCGCAGCTGGGGTGTTGACGTGCCTGGTCTAGCCCTGTGGCGTGT CGACGCCGCCGAGGCACTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGC TCATCGACGAACAACTCGCCCGTCCGATGGGACCTCGTTCCCGCGCATTA ACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCT GCTCCATGAGGCAGCTGAGCTGCTGCTCTCCTGCCCCGACCCGTACGAGC AAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGC TATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCA GTGCGGTGCTGTCCCGCTGCTGCGCCGACTCGGGGGCGAGCCCGGCCGGA TCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAG CGGCGGGTGTCGGCCCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CAAACAGCTATTCGTCACGGCCAGCACCGTGGAACAGCACCTCACAAGCG TCTTCCGCAAGCTGGGCGTTAAGGGCCGCAGGCAGCTACCGACCGCGCTG GCCGACGTGGAATAG SEQ ID NO: 197 ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCCCGCGAGGA CGAACTCGGCATTCTGCAGAGGTCTCTGGAAGAAGCAGGCAGCGGCCAGG GCGCCGTGGTCACCGTCACCGGCCCGATCGCCTGCGGCAAGACAGAACTG CTTGACGCGGCTGCCGCGAAGGCTGACGCCATCATTCTGCGCGCGGTCTG CGCGCCCGAAGAGCGCGCTATGCCGTACGCCATGATCGGGCAGCTCATCG ACGACCCGGCGCTCGCGCATCGGGCGCCGGAGCTGGCTGATCGGATAGCC CAGGGCGGGCATCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCT CACCCGTGCCCTGCTGGCGCTTGCCGTCGACCGGCCTGTGCTGATCGGCG TCGACGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCAT CTGGCCCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTT GCGCAGCCTCACCCCTACTCAGTCACGATTCAAGGCGGAGCTGCTCAGCC TGCCGTACCACCACGAGATCGCGCTGCGTCCACTCGGACCGGAGCAATCG GCGGAGCTGGCCCACGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGCGGGGTTGTATGGGATGACCAGGGGCAACCTGAGTCTCAGCCGTGGAC TGATCAGCGATGTGCGGGAGGCCCAGGCCAACGGAGAGAGCGCTTTCGAG GTGGGCCGCGCGTTCCGGCTGGCGTACCTCAGCTCGCTCTACCGCTGTGG CCCGATCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAAGCG CCACCACCACGCTGGTGCGCCGTCTAAGCGGGCTCAGCGCGGAGACGATA GACCGGGCAACCAAGATCCTCACTGAGGGCGGGCTGCTGCTCGACCACCA GTTCCCGCACCCGGCCGCCCGCTCGGTGGTGCTCGATGACATGTCCGCCC AGGAACGACGCAGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCG CCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGG GCCCAAGGCTGCGGAGATATTCGCCAGGGCTGGCCAGGCTCTGGTTGTAC GCAACGAGTTGGGCGACGCGGCCGAATACCTGCAACTGGCTCACCGAGCC TCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGTCGAGGCCGTGGCAAT CGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGTCACATGGACGAGCTGA GCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCAGCGCTGGCT GTCTTCTGGCTGGCCGACGGCGGGCGATCCGGCGAGGCAGCCGAGGTGCT GGCGTCGGAACACCCGCTCGCGACCACCGATCAGAACCGAGCACACCTGC GATTTGCCGAGGTGACTCTCGCGCTGTTCTGTCCCGGCGCCTTCGGGTCG GACCGGCGCCCACCTCCGCTGGCGCCGGACGAGCTCGCCAGCTTGCCGAA GGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGTCATGACAGCGTTGC ATGCTCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAG GCTGATTCGGCAGCCGACGCAATCCCCGCCGCACTGATCGCCCTGTTGTA CGCAGAGAACACCGAGTCCGCTCAGATCTGGGCCGACAAGCTGGGCAGCA CCAATGCCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGC GCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGG TGGCACCGTCCTGGACGACCGGCCGCTGCCGTCGCTCGGCATCACCGCCG CATTGCTGTTGAGCAGCAAGACGGCAGCCGCTGTCCGCCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCTGAGCCGCTTCCGAACGGTGTCCAGGA CAGCCTTTTCGGTCTGCACCTGCTCTCGGCGCACGGCCAGTACAGCCTCG CGATGGGCCGATATGAATCGGCTCACCGGGCGTTTCACACCTGCGGAGAA CGTATGCGCAGCTGGGGTGTTGACGTGCCTGGTCTAGCCCTGTGGCGTGT CGACGCCGCCGAGGCACTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGC TCATCGACGAACAACTCGCCCGTCCGATGGGACCTCGTTCCCGCGCACTG ACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCT GCTCCATGAGGCAGCTGAGCTGCTGCTCTCCTGCCCCGACCCGTACGAGC AAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGC TATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCA GTGCGGTGCTGTCCCGCTGCTGCGCCGACTCGGGGGCGAGCCCGGCCGGA TCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAG CGGCGGGTGTCGGCCCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CAAACAGCTATTCGTCACGGCCAGCACCGTGGAACAGCACCTCACAAGCG TCTTCCGCAAGCTGGGCGTTAAGGGCCGCAGGCAGCTACCGACCGCGCTG GCCGACGTGGAATAG SEQ ID NO: 198 GTGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGA CGAACTCGGCATTCTACAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGG GCGTCGTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTG CTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGGTCTG CGCGCCCGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCG ACGACCCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCC CAGGGCGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCT CACCCGTGCCCTGCTGGCGCTTGCCGTGCACCGGCCTGTGCTGATCGGCG TCGATGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCAT TTGGCGCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTT GCGCAGCCTCACCCCTACTCAGTCACGATTCAAGGCGGAGCTGCTCAGCC TGCCGTACCACCACGAGATCGCGCTGCGTCCATTCGGACCGGAGCAATCG GCGGAGCTGGCTCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGCGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGAC TGATCAGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCTTTCGAG GCGGGCCGCGCGTTCCGGCTGGCGTACCTCAGCTCGCTCTACCGCTGTGG CCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAAGCG CCACCACCACGCTGGTGCGCCGGCTAAGCGGGCTCAGCGCGGAGACGATA GACCGGGCAACCAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGCA GTTTCCGCACCCGGCCGCCCGCTCGGTGGTGCTCGATGACATGTCCGCCC AGGAACGACGCGGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCG CCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGG GCCCAAGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTAC GCAACGAGTTGGGCGACGCGGCCGAATACCTGCAACTGGCTCACCGGGCC TCCGACGATGTCTCCACCCGGGCCGCCTTACGGGTCGAGGCCGTGGCGAT CGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAGCTGA GCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCC GTCTTCTGGCTGGCCGACGGCGGGCGATCCGGCGAGGCAGCCCAGGTGCT GGCGTCGGAACGCCCGCTCGCGACCACCGATCAGAACCGGGCCCACCTGC GATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGGATCG GACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAA GGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGC ACGGCCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAG GCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTA CGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCA TGAATGCCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGC GCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGG TAGCACCGTCCTGGACGACCGGTCACTGCCGTCGCTCGGCATCACCGCCG CATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGA CAGCCTTTTCGGTCTGCACCTGCTCTCGGCGTACGGCCAGTACAGCCTCG CGATGGGCCGATATGAATCGGCTCACCGGGCGTTTCGCACCTGCGGAGAA CGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGCGTGT CGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGC TCATCGACGAACAACTCACCCGTCCGATGGGACCTCGTTCCCGCGCGTTA ACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCT GCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGC AAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGC TATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCA GTGCGGTGCTGTCCCGCTGCTGCGCCGACTCGGGGGCGAGCCCGGCCGGA TCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAG CGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CGAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCG TCTTCCGCAAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTG GCCGACGTGGAACAGACCTGA SEQ ID NO: 199 ATGTATAGCGGTACCTGCCGTGAAGGATACGAACTCGTCGCACGCGAGGA CGAACTCGGCATTCTACAGAGGTCTCTGGAACAAGCGAGCAGCGGCCAGG GCGTCGTGGTCACCGTCACCGGCCCAATCGCCTGCGGCAAGACAGAACTG CTTGACGCGGCTGCCGCGAAGGCTGAGGCCATCATTCTGCGCGCGGTCTG CGCGCCCGAAGAGCGGGCTATGCCGTACGCCATGATCGGGCAGCTCATCG ACGACCCGGCGCTCGCGCATCGGGCGCCGGGGCTGGCTGATCGGATAGCC CAGGGCGGGCAGCTGTCGCTGAGGGCCGAGAACCGACTGCGCAGGGATCT CACCCGTGCCCTGCTGGCGCTTGCCGTGCACCGGCCTGTGCTGATCGGCG TCGATGATGTGCATCACGCCGACACCGCCTCTTTGAACTGTCTGCTGCAT TTGGCGCGCCGGGTCCGTCCGGCCCGGATATCCATGATCTTCACCGAGTT GCGCAGCCTCACCCCTACTCAGTCACGATTCAAGGCGGAGCTGCTCAGCC TGCCGTACCACCACGAGATCGCGCTGCGTCCATTCGGACCGGAGCAATCG GCGGAGCTGGCTCGCGCCGCCTTCGGCCCGGGCCTCGCCGAGGATGTGCT CGCGGGGTTGTATAAAACGACCAGGGGCAATCTGAGTCTCAGCCGTGGAC TGATCAGCGATGTGCGGGAGGCCCTGGCCAACGGAGAGAGCGCTTTCGAG GCGGGCCGCGCGTTCCGGCTGGCGTACCTCAGCTCGCTCTACCGCTGTGG CCCGGTCGCGCTGCGGGTCGCCCGAGTGGCTGCCGTGCTGGGCCCAAGCG CCACCACCACGCTGGTGCGCCGGCTAAGCGGGCTCAGCGCGGAGACGATA GACCGGGCAACCAAGATCCTCACCGAGGGCGGGCTGCTGCTCGACCAGCA GTTTCCGCACCCGGCCGCCCGCTCGGTGGTGCTCGATGACATGTCCGCCC AGGAACGACGCGGCCTGCACACTCTCGCCCTGGAACTGCTGGACGAGGCG CCGGTTGAAGTGCTCGCGCACCACCAGGTCGGCGCCGGTCTCATACACGG GCCCAAGGCTGCGGAGATGTTCGCCAAGGCCGGCAAGGCTCTGGTCGTAC GCAACGAGTTGGGCGACGCGGCCGAATACCTGCAACTGGCTCACCGGGCC TCCGACGATGTCTCCACCCGGGCCGCCCTGCGGGTCGAGGCCGTGGCGAT CGAGCGCCGCCGCAATCCGCTGGCCTCCAGTCGGCACATGGACGAGCTGA GCGCCGCCGGCCGCGCCGGTCTGCTTTCCCCCAAGCATGCGGCGCTGGCC GTCTTCTGGCTGGCCGACGGCGGGCGATCCGGCGAGGCAGCCCAGGTGCT GGCGTCGGAACGCCCGCTCGCGACCACCGATCAGAACCGGGCCCACCTGC GATTTGTCGAGGTGACTCTCGCGCTGTTCTCTCCCGGCGCCTTCGGATCG GACCGGCGCCCACCTCCGCTGACGCCGGACGAACTCGCCAGCCTGCCGAA GGCGGCCTGGCAATGCGCGGTCGCCGACAACGCGGCCATGACCGCCTTGC ACGGCCATCCAGAACTTGCCACCGCTCAGGCGGAAACAGTTCTGCGGCAG GCTGATTCGGCAGCCGACGCGATCCCCGCCGCGCTGATCGCCCTGTTGTA CGCGGAGAACACCGAGTCCGCTCATATCTGGGCCGACAAGCTGGGCAGCA TGAATGCCGGGGTATCGAACGAGGCGGAAGCGGGCTACGCCGGCCCGTGC GCCGAGATCGCCCTGCGGCGCGGCGACCTGGCCACGGCGTTCGAGGCTGG TAGCACCGTCCTGGACGACCGGTCACTGCCGTCGCTCGGCATCACCGCCG CATTGCTGTTGAGCAGCAAGACGGCCGCCGCTGTCCGGCTGGGCGAACTC GAGCGTGCGGAGAAGCTGCTCGCCGAGCCGCTTCCGAACGGCGTCCAGGA CAGCCTTTTCGGTCTGCACCTGCTCTCGGCGTACGGCCAGTACAGCCTCG CGATGGGCCGATATGAATCGGCTCACCGGGCGTTTCGCACCTGCGGAGAA CGTATGCGCAGCTGGGATGTTGACGTGCCTGGTCTGGCCCTGTGGCGTGT CGACGCCGCCGAGGCGCTGCTCAGCCTCGACCGGAACGAGGGCCAGCGGC TCATCGACGAACAACTCACCCGTCCGATGGGACCTCGTTCCCGCGCGCTG ACGCTGCGGATCAAGGCGGCATACCTCCCGCGGACGAAGCGGATCCCCCT GCTCCATGAGGCGGCCGAGCTGCTGCTCCCCTGCCCCGACCCGTACGAGC AAGCGCGGGTGCTCGCCGATCTGGGCGACACGCTCAGCGCGCTCAGACGC TATAGCCGGGCGCGGGGAGTTCTCCGGCAGGCTCGTCACCTGGCCACCCA GTGCGGTGCTGTCCCGCTGCTGCGCCGACTCGGGGGCGAGCCCGGCCGGA TCGACGACGCCGGCCTGCCGCAGCGGAGCACATCGTTGACCGATGCGGAG CGGCGGGTGGCGGCGCTGGCCGCGGCCGGACAGACCAACCGGGAGATCGC CGAACAGCTGTTCGTCACGGCCAGCACAGTGGAACAGCACCTCACAAGCG TCTTCCGCAAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGACCGCGCTG GCCGACGTGGAACAGACCTGA SEQ ID NO: 200 GTGCGAGCTATTAATGCGTCCGACACCGGTCCTGAACTGGTCGCCCGCGA AGACGAACTGGGACGTGTACGAAGTGCCCTGAACCGAGCGAACGGCGGCC AAGGTGTCCTGATCTCCATTACCGGTCCGATCGCCTGCGGCAAGACCGAA CTGCTTGAGGCTGCCGCCTCGGAAGTTGACGCCATCACTCTGCGCGCGGT CTGTGCCGCCGAGGAACGGGCGATACCTTATGCCCTGATCGGGCAGCTTA TCGACAACCCCGCGCTCGGCATTCCGGTTCCGGATCCGGCCGGCCTGACC GCCCAGGGCGGACGACTGTCATCGAGCGCCGAGAACCGACTGCGTCGCGA CCTCACCCGTGCCCTGCTGACGCTCGCCACCGACCGGCTGGTGCTGATCT GTGTCGATGACGTGCAGCACGCCGACAACGCCTCGTTGAGCTGCCTTCTG TATCTGGCCCGACGGCTTGTCCCGGCTCGAATCGCTCTGGTATTCACCGA GTTGCGAGTCCTCACCTCGTCTCAGTTACGGTTCAACGCGGAGCTGCTCA GCTTGCGGAACCACTGCGAGATCGCGCTGCGCCCACTCGGCCCGGGGCAT GCGGCCGAGCTGGCCCGCGCCACCCTCGGCCCCGGCCTCTCCGACGAAAC ACTCACGGAGCTGTACCGGGTGACCGGAGGCAACCTGAGTCTCAGCCGCG GGCTGATCGACGATGTGCGGGACGCCTGGGCACGAGGGGAAACGGGCGTC CAGGTGGGCCGGGCGTTCCGGCTGGCCTACCTCGGTTCCCTCCACCGCTG TGGTCCGCTGGCGTTGCGGGTCGCCCGCGTAGCCGCCGTACTGGGCCCGA GCGCCACCAGCGTCCTGGTGCGCCGGATCAGTGGGCTCAGCGCGGAGGCC ATGGCCCAGGCGACCGATATCCTCGCTGACGGCGGCCTCCTGCGCGACCA GCGGTTCACACATCCAGCGGCCCGCTCGGTGGTGCTCGACGACATGTCCG CCGAGGAACGACGCAGCGTGCACAGCCTCGCCCTGGAACTGCTGGACGAG GCACCGGCCGAGATGCTCGCGCACCACCGGGTCGGCGCCGGTCTCGTGCA CGGGCCGAAGGCCGCGGAGACATTCACCGGGGCCGGCCGGGCACTGGCCG TTCGCGGCATGCTGGGCGAGGCAGCCGACTACCTGCAACTGGCGTACCGG GCCTCCGGCGACGCCGCTACCAAGGCCGCGATACGCGTCGAGTCCGTGGC GGTCGAGCGCCGACGCAATCCGCTGGTCGTCAGTCGCCATTGGGACGAGC TGAGCGTCGCGGCCCGCGCCGGTCTGCTCTCCTGCGAGCACGTGTCCAGG ACGGCCCGCTGGCTGACCGTCGGTGGGCGGCCCGGCGAGGCGGCCAGGGT GCTGGCGTCGCAACACCGACGGGTCGTCACCGATCAGGACCGGGCCCACC TGCGGGTCGCCGAGTTCTCGCTCGCGCTGCTGTACCCCGGTACGTCCGGC TCGGACCGGCGCCCGCACCCGCTCACGTCGGACGAACTCGCGGCCCTACC GACTGCGACCAGACACTGCGCGATCGCCGATAACGCTGTCATGGCTGCCT TGCGTGGTCATCCGGAGCTTGCCACCGCCGAGGCAGAAGCCGTTCTGCAG CAAGCCGACGCGGCGGACGGCGCTGCTCTCACCGCGCTGATGGCCCTGCT GTACGCGGAGAGCATCGAGGTCGCTGAAGTCTGGGCGGACAAGCTGGCGG CAGAGGCCGGAGCATCGAACGGGCAGGACGCGGAGTACGCCGGTATACGC GCCGAAATCGCCCTGCGGCGCGGCGATCTGACCGCGGCCGTCGAGACCGC CGGCATGGTCCTGGACGGCCGGCCGCTGCCGTCGCTCGACATCACCGCCA CGTTGCTGTTGGCCGGCAGGGCGTCCGTCGCCGTCCGGCTGGGCGAACTC GACCACGCGGAGGAGCTGTTCGCCGCGCCGCCGGAGGACGCCTTCCAGGA CAGCCTCTTCGGTCTGCATCTGCTCTCGGCGCACGGCCAGTACAGCCTCG CGACAGGCCGGCCCGAGTCGGCATACCGGGCCTTTCGTGCCTGCGGCGAA CGTATGCGCGATTGGGGCTTCGACGCGCCCGGTGTGGCCCTGTGGCGCGT CGGCGCCGCCGAGGCGCTGCTCGGCCTCGACCGGAACGAGGGCCGACGGC TCATCGACGAACAGCTGAGCCGGACGATGGCCCCCCGGTCCCACGCGTTG ACGCTGCGGATAAAAGCGGCGTACATGCCGGAGCCGAAGCGGGTCGACCT GCTCTACGAAGCGGCTGAGCTGCTGCTCTCCTGCCGGGACCAGTATGAGC GAGCGCGGGTGCTCGCCGATCTGGGCGAGGCGCTCAGCGCGCTCGGGAAC TACCGGCAGGCGCGAGGTGTGCTCCGGCAGGCTCGGCATCTGGCCATGCG AACCGGCGCGGACCCGCTGCTGCGCCGGCTCGGAATCAGGCCCGGCCGGC AGGACGACCCCGACCCGCAGCCGCGGAGCAGATCGCTGACCAACGCTGAG CGGCGTGCGGCGTCGCTGGCCGCGACCGGACTGACCAACCGGGAGATCGC CGACCGGCTCTTCGTCACCGCCAGCACCGTGGAGCAGCACCTCACCAACG TCTTCCGCAAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGGCCGAGTTG GACGACATGGAATAG SEQ ID NO: 201 ATGCGAGCTATTAATGCGTCCGACACCGGTCCTGAACTGGTCGCCCGCGA AGACGAACTGGGACGTGTACGAAGTGCCCTGAACCGAGCGAACGGCGGCC AAGGTGTCCTGATCTCCATTACCGGTCCGATCGCCTGCGGCAAGACCGAA CTGCTTGAGGCTGCCGCCTCGGAAGTTGACGCCATCACTCTGCGCGCGGT CTGTGCCGCCGAGGAACGGGCGATACCTTATGCCCTGATCGGGCAGCTTA TCGACAACCCCGCGCTCGGCATTCCGGTTCCGGATCCGGCCGGCCTGACC GCCCAGGGCGGACGACTGTCATCGAGCGCCGAGAACCGACTGCGTCGCGA CCTCACCCGTGCCCTGCTGACGCTCGCCACCGACCGGCTGGTGCTGATCT GTGTCGATGACGTGCAGCACGCCGACAACGCCTCGTTGAGCTGCCTTCTG TATCTGGCCCGACGGCTTGTCCCGGCTCGAATCGCTCTGGTATTCACCGA GTTGCGAGTCCTCACCTCGTCTCAGCTGCGGTTCAACGCGGAGCTGCTCA GCTTGCGGAACCACTGCGAGATCGCGCTGCGCCCACTCGGCCCGGGGCAT GCGGCCGAGCTGGCCCGCGCCACCCTCGGCCCCGGCCTCTCCGACGAAAC ACTCACGGAGCTGTACCGGGTGACCGGAGGCAACCTGAGTCTCAGCCGCG GGCTGATCGACGATGTGCGGGACGCCTGGGCACGAGGGGAAACGGGCGTC CAGGTGGGCCGGGCGTTCCGGCTGGCCTACCTCGGTTCCCTCCACCGCTG TGGTCCGCTGGCGTTGCGGGTCGCCCGCGTAGCCGCCGTACTGGGCCCGA GCGCCACCAGCGTCCTGGTGCGCCGGATCAGTGGGCTCAGCGCGGAGGCC ATGGCCCAGGCGACCGATATCCTCGCTGACGGCGGCCTCCTGCGCGACCA GCGGTTCACACATCCAGCGGCCCGCTCGGTGGTGCTCGACGACATGTCCG CCGAGGAACGACGCAGCGTGCACAGCCTCGCCCTGGAACTGCTGGACGAG GCACCGGCCGAGATGCTCGCGCACCACCGGGTCGGCGCCGGTCTCGTGCA CGGGCCGAAGGCCGCGGAGACATTCACCGGGGCCGGCCGGGCACTGGCCG TTCGCGGCATGCTGGGCGAGGCAGCCGACTACCTGCAACTGGCGTACCGG GCCTCCGGCGACGCCGCTACCAAGGCCGCGATACGCGTCGAGTCCGTGGC GGTCGAGCGCCGACGCAATCCGCTGGTCGTCAGTCGCCATTGGGACGAGC TGAGCGTCGCGGCCCGCGCCGGTCTGCTCTCCTGCGAGCACGTGTCCAGG ACGGCCCGCTGGCTGACCGTCGGTGGGCGGCCCGGCGAGGCGGCCAGGGT GCTGGCGTCGCAACACCGACGGGTCGTCACCGATCAGGACCGGGCCCACC TGCGGGTCGCCGAGTTCTCGCTCGCGCTGCTGTACCCCGGTACGTCCGGC TCGGACCGGCGCCCGCACCCGCTCACGTCGGACGAACTCGCGGCCCTACC GACTGCGACCAGACACTGCGCGATCGCCGATAACGCTGTCATGGCTGCCT TGCGTGGTCATCCGGAGCTTGCCACCGCCGAGGCAGAAGCCGTTCTGCAG CAAGCCGACGCGGCGGACGGCGCTGCTCTCACCGCGCTGATGGCCCTGCT GTACGCGGAGAGCATCGAGGTCGCTGAAGTCTGGGCGGACAAGCTGGCGG CAGAGGCCGGAGCATCGAACGGGCAGGACGCGGAGTACGCCGGTATACGC GCCGAAATCGCCCTGCGGCGCGGCGATCTGACCGCGGCCGTCGAGACCGC CGGCATGGTCCTGGACGGCCGGCCGCTGCCGTCGCTCGACATCACCGCCA CGTTGCTGTTGGCCGGCAGGGCGTCCGTCGCCGTCCGGCTGGGCGAACTC GACCACGCGGAGGAGCTGTTCGCCGCGCCGCCGGAGGACGCCTTCCAGGA CAGCCTCTTCGGTCTGCATCTGCTCTCGGCGCACGGCCAGTACAGCCTCG CGACAGGCCGGCCCGAGTCGGCATACCGGGCCTTTCGTGCCTGCGGCGAA CGTATGCGCGATTGGGGCTTCGACGCGCCCGGTGTGGCCCTGTGGCGCGT CGGCGCCGCCGAGGCGCTGCTCGGCCTCGACCGGAACGAGGGCCGACGGC TCATCGACGAACAGCTGAGCCGGACGATGGCCCCCCGGTCCCACGCGTTG ACGCTGCGGATAAAAGCGGCGTACATGCCGGAGCCGAAGCGGGTCGACCT GCTCTACGAAGCGGCTGAGCTGCTGCTCTCCTGCCGGGACCAGTATGAGC GAGCGCGGGTGCTCGCCGATCTGGGCGAGGCGCTCAGCGCGCTCGGGAAC TACCGGCAGGCGCGAGGTGTGCTCCGGCAGGCTCGGCATCTGGCCATGCG AACCGGCGCGGACCCGCTGCTGCGCCGGCTCGGAATCAGGCCCGGCCGGC AGGACGACCCCGACCCGCAGCCGCGGAGCAGATCGCTGACCAACGCTGAG CGGCGTGCGGCGTCGCTGGCCGCGACCGGACTGACCAACCGGGAGATCGC CGACCGGCTCTTCGTCACCGCCAGCACCGTGGAGCAGCACCTCACCAACG TCTTCCGCAAGCTGGGCGTCAAGGGCCGCAAGCAGCTGCCGGCCGAGTTG GACGACATGGAATAG SEQ ID NO: 202 MPAVECYELDARDDELRKLEEVVTGRANGRGVVVTITGPIACGKTELLDA AAAKADAITLRAVCSAEEQALPYALIGQLIDNPALASHALEPACPTLPGE HLSPEAENRLRSDLTRTLLALAAERPVLIGIDESHANALCLLHLARRVGS ARIAMVLTELRRLTPAHSQFQAELLSLGHHREIALRPLSPKHTAELVRAG LGPDVDEDVLTGLYRATGGNLNLTRGLINDVREAWETGGTGISAGRAYRL AYLGSLYRCGPVPLRVARVAAVLGQSANTTLVRWISGLNADAVGEATEIL TEGGLLHDLRFPHPAARSVVLNDMSAQERRRLHRSALEVLDDVPVEVVAH HQVGAGLLHGPKAAEIFAKAGQELHVRGELDTASDYLQLAHQASDDAVTG MRAEAVAIERRRNPLASSRHLDELTVVARAGLLFPEHTALMIRWLGVGGR SGEAAGLLASQRPRAVTDQDRAHMRAAEVSLALVSPGTSGPDRRPRPLTP DELANLPKAARLCAIADNAVMSALRGRPELAAAEAENVLQHADSAAAGTT ALAALTALLYAENTDTAQLWADKLVSETGASNEEEAGYAGPRAEAALRRG DLAAAVEAGSTVLDHRRLSTLGITAALPLSSAVAAAIRLGETERAEKWLA QPLPQAIQDGLFGLHLLSARGQYSLATGQHESAYTAFRTCGERMRNWGVD VPGLSLWRVDAAEALLHGRDRDEGRRLVDEQLTRAMGPRSRALTLRVQAA YSPPAKRVDLLDEAADLLLSCNDQYERARVLADLSETFSALRHHSRARGL LRQARHLAAQRGAIPLLRRLGAKPGGPGWLEESGLPQRIKSLTDAERRVA SLAAGGQTNRVIADQLFVTASTVEQHLTDVSTGSRPPAPAAELV SEQ ID NO: 203 MVPEVRAAPDELIARDDELSRLQRALTRAGSGRGGVVAITGPIASGKTAL LDAGAAKSGFVALRAVCSWEERTLPYGMLGQLFDHPELAAQAPDLAHFTA SCESPQAGTDNRLRAEFTRTLLALAADWPVLIGIDDVHHADAESLRCLLH LARRIGPARIAVVLTELRRPTPADSRFQAELLSLRSYQEIALRPLTEAQT GELVRRHLGAETHEDVSADTFRATGGNLLLGHGLINDIREARTAGRPGVV AGRAYRLAYLSSLYRCGPSALRVARASAVLGASAEAVLVQRMTGLNKDAV EQVYEQLNEGRLLQGERFPHPAARSIVLDDLSALERRNLHESALELLRDH GVAGNVLARHQIGAGRVHGEEAVELFTGAAREHHLRGELDDAAGYLELAH RASDDPVTRAALRVGAAAIERLCNPVRAGRHLPELLTASRAGLLSSEHAV SLADWLAMGGRPGEAAEVLATQRPAADSEQHRALLRSGELSLALVHPGAW DPLRRTDRFAAGGLGSLPGPARHRAVADQAVIAALRGRLDRADANAESVL QHTDATADRTTAIMALLALLYAENTDAVQFWVDKLAGDEGTRTPADEAVH AGFNAEIALRRGDLMRAVEYGEAALGHRHLPTWGMAAALPLSSTVVAAIR LGDLDRAERWLAEPLPQQTPESLFGLHLLWARGQHHLATGRHGAAYTAFR ECGERMRRWAVDVPGLALWRVDAAESLLLLGRDRAEGLRLVSEQLSRPMR PRARVQTLRVQAAYSPPPQRIDLLEEAADLLVTCNDQYELANVLSDLAEA SSMVRQHSRARGLLRRARHLATQCGAVPLLRRLGAEPSDIGGAWDATLGQ RIASLTESERRVAALAAVGRTNREIAEQLFVTASTVEQHLTNVFRKLAVK GRQQLPKELADVGEPADRDRRCG SEQ ID NO: 204 MIARLSPPDLIARDDEFGSLHRALTRAGGGRGVVAAVTGPIACGKTELLD AAAAKAGFVTLRAVCSMEERALPYGMLGQLLDQPELAARTPELVRLTASC ENLPADVDNRLGTELTRTVLTLAAERPVLIGIDDVHHADAPSLRCLLHLA RRISRARVAIVLTELLRPTPAHSQFRAALLSLRHYQEIALRPLTEAQTTE LVRRHLGQDAHDDVVAQAFRATGGNLLLGHGLIDDIREARTRTSGCLEVV AGRAYRLAYLGSLYRCGPAALSVARASAVLGESVELTLVQRMTGLDTEAV EQAHEQLVEGRLLREGRFPHPAARSVVLDDLSAAERRGLHELALELLRDR GVASKVLARHQMGTGRVHGAEVAGLFTDAAREHHLRGELDEAVTYLEFAY RASDDPAVHAALRVDTAAIERLCDPARSGRHVPELLTASRERLLSSEHAV SLACWLAMDGRPGEAAEVLAAQRSAAPSEQGRAHLRVADLSLALIYPGAA DPPRPADPPAEDEVASFSGAVRHRAVADKALSNALRGWSEQAEAKAEYVL QHSRVTTDRTTTMMALLALLYAEDTDAVQSWVDKLAGDDNMRTPADEAVH AGFRAEAALRRGDLTAAVECGEAALAPRVVPSWGMAAALPLSSTVAAAIR LGDLDRAERWLAEPLPEETSDSLFGLHMVWARGQHHLAAGRYRAAYNAFR DCGERMRRWSVDVPGLALWRVDAAEALLLLGRGRDEGLRLISEQLSRPMG SRARVMTLRVQAAYSPPAKRIELLDEAADLLIMCRDQYELARVLADMGEA CGMLRRHSRARGLFRRARHLATQCGAVPLLRRLGGESSDADGTQDVTPAQ RITSLTEAERRVASHAAVGRTNKEIASQLFVTSSTVEQHLTNVFRKLGVK GRQQLPKELSDAG SEQ ID NO: 205 MEFYDLVARDDELRRLDQALGRAAGGRGVVVTVTGPVGCGKTELLDAAAA EEEFITLRAVCSAEERALPYAVIGQLLDHPVLSARAPDLACVTAPGRTLP ADTENRLRRDLTRALLALASERPVLICIDDVHQADTASLNCLLHLARRVA SARIAMILTELRRLTPAHSRFEAELLSLRHRHEIALRPLGPADTAELARA RLGAGVTADELAQVHEATSGNPNLVGGLVNDVREAWAAGGTGIAAGRAYR LAYLSSVYRCGPVPLRIAQAAAVLGPSATVTLVRRISGLDAETVDEATAI LTEGGLLRDHRFPHPAARSVVLDDMSAQERRRLHRSTLDVLDGVPVDVLA HHQAGAGLLHGPQAAEMFARASQELRVRGELDAATEYLQLAYRASDDAGA RAALQVETVAGERRRNPLAASRHLDELAAAARAGLLSAEHAALVVHWLAD AGRPGEAAEVLALQRALAVTDHDRARLRAAEVSLALFHPGVPGSDPRPLA PEELASLSLSARHGVTADNAVLAALRGRPESAAAEAENVLRNADAAASGP TALAALTALLYAENTDAAQLWADKLAAGIGAGEGEAGYAGPRTVAALRRG DLTTAVQAAGAVLDRGRPSSLGITAVLPLSGAVAAAIRLGELERAEKWLA EPLPEAVHDSLFGLHLLMARGRYSLAVGRHEAAYAAFRDCGERMRRWDVD VPGLALWRVDAAEALLPGDDRAEGRRLIDEQLTRPMGPRSRALTLRVRAA YAPPAKRIDLLDEAADLLLSSNDQYERARVLADLSEAFSALRQNGRARGI LRQARHLAAQCGAVPLLRRLGVKAGRSGRLGRPPQGIRSLTEAERRVATL AAAGQTNREIADQLFVTASTVEQHLTNVFRKLGVKGRQQLPAELADLRPP G SEQ ID NO: 206 MYSGTCREGYELVAREDELGILQRSLEQASSGQGVVVTVTGPIACGKTEL LDAAAAKAEAIILRAVCAPEERAMPYAMIGQLIDDPALAHRAPGLADRIA QGGQLSLRAENRLRRDLTRALLALAVDRPVLIGVDDVHHADTASLNCLLH LARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPFGPEQS AELARAAFGPGLAEDVLVGLYKTTRGNLSLSRGLISDVREALANGESAFE AGRAFRLAYLGSLYRCGPVALRVARVAAVLGPSATTTLVRRLSGLSAETI DRATKILTEGGLLLDQQFPHPAARSVVLDDMSAQERRGLHTLALELLDEA PVEVLAHHQVGAGLIHGPKAAEMFAKAGKALVVRNELGDAAEYLQLAHRA SDDVSTRAALRVEAVAIERRRNPLASSRHMDELSAAGRAGLLSPKHAALA VFWLADGGRSGEAAEVLASERPLATTDQNRAHLRFVEVTLALFSPGAFGS DRRPPPLTPDELASLPKAAWQCAVADNAAMTALHGHPELATAQAETVLRQ ADSAADAIPAALIALLYAENTESAHIWADKLGSTNGGVSNEAEAGYAGPC AEIALRRGDLATAFEAGSTVLDDRSLPSLGITAALLLSSKTAAAVRLGEL ERAEKLLAEPLPNGVQDSLFGLHLLSAYGQYSLAMGRYESALRAFHTCGE RMRSWDVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLTRPMGPRSRAL TLRIKAAYLPRTKRIPLLHEAAELLLPCPDPYEQARVLADLGDTLSALRR YSRARGVLRQARHLAAQCGAVPLLRRLGGEPGRIDDAGLPQRSTSLTDAE RRVAALAAAGQTNREIAKQLFVTASTVEQHLTSVFRKLGVKGRKQLPTAL ADVEQT SEQ ID NO: 207 MPAVESYELDARDDELRRLEEAVGQAGNGRGVVVTITGPIACGKTELLDA AAAKSDAITLRAVCSEEERALPYALIGQLIDNPAVASQLPDPVSMALPGE HLSPEAENRLRGDLTRTLLALAAERPVLIGIDDMHHADTASLNCLLHLAR RVGPARIAMVLTELRRLTPAHSQFHAELLSLGHHREIALRPLGPKHIAEL ARAGLGPDVDEDVLTGLYRATGGNLNLGHGLIKDVREAWATGGTGINAGR AYRLAYLGSLYRCGPVPLRVARVAAVLGQSANTTLVRWISGLNADAVGEA TEILTEGGLLHDLRFPHPAARSVVLNDLSARERRRLHRSALEVLDDVPVE VVAHHQAGAGFIHGPKAAEIFAKAGQELHVRGELDAASDYLQLAHHASDD AVTRAALRVEAVAIERRRNPLASSRHLDELTVAARAGLLSLEHAALMIRW LALGGRSGEAAEVLAAQRPRAVTDQDRAHLRAAEVSLALVSPGASGVSPG ASGPDRRPRPLPPDELANLPKAARLCAIADNAVISALHGRPELASAEAEN VLKQADSAADGATALSALTALLYAENTDTAQLWADKLVSETGASNEEEGA GYAGPRAETALRRGDLAAAVEAGSAILDHRRGSLLGITAALPLSSAVAAA IRLGETERAEKWLAEPLPEAIRDSLFGLHLLSARGQYCLATGRHESAYTA FRTCGERMRNWGVDVPGLSLWRVDAAEALLHGRDRDEGRRLIDEQLTHAM GPRSRALTLRVQAAYSPQAQRVDLLEEAADLLLSCNDQYERARVLADLSE AFSALRHHSRARGLLRQARHLAAQCGATPLLRRLGAKPGGPGWLEESGLP QRIKSLTDAERRVASLAAGGQTNRVIADQLFVTASTVEQHLTNVFRKLGV KGRQHLPAELANAE SEQ ID NO: 208 MPAVKRNDLVARDGELRWMQEILSQASEGRGAVVTITGAIACGKTVLLDA AAASQDVIQLRAVCSAEEQELPYAMVGQLLDNPVLAARVPALGNLAAAGE RLLPGTENRIRRELTRTLLALADERPVLIGVDDMHHADPASLDCLLHLAR RVGPARIAIVLTELRRLTPAHSRFQSELLSLRYHHEIGLQPLTAEHTADL ARVGLGAEVDDDVLTELYEATGGNPSLCCGLIRDVRQDWEAGVTGIHVGR AYRLAYLSSLYRCGPAALRTARAAAVLGDSADACLIRRVSGLGTEAVGQA IQQLTEGGLLRDQQFPHPAARSVVLDDMSAQERHAMYRSAREAAAEGQAD PGTPGEPRAATAYAGCGEQAGDYPEPAGRACVDGAGPAEYCGDPHGADDD PDELVAALGGLLPSRLVAMKIRRLAVAGRPGAAAELLTSQRLHAVTSEDR ASLRAAEVALATLWPGATGPDRHPLTEQEAASLPEGPRLLAAADDAVGAA LRGRAEYAAAEAENVLRHADPAAGGDAYAAMIALLYTEHPENVLFWADKL DAGRPDEETSYPGLRAETAVRLGDLETAMELGRTVLDQRRLPSLGVAAGL LLGGAVTAAIRLGDLDRAEKWLAEPIPDAIRTSLYGLHVLAARGRLDLAA GRYEAAYTAFRLCGERMAGWDADVSGLALWRVDAAEALLSAGIRPDEGRK LIDDQLTREMGARSRALTLRAQAAYSLPVHRVGLLDEAAGLLLACHDGYE RARVLADLGETLRTLRHTDAAQRVLRQAEQAAARCGSVPLLRRLGAEPVR IGTRRGEPGLPQRIRLLTDAERRVAAMAAAGQTNREIAGRLFVTASTVEQ HLTSVFRKLGVKGRRFLPTELAQAV SEQ ID NO: 209 MYSGTCREGYELVAREDELGILQRSLEQASSGQGVVVTVTGPIACGKTEL LDAAAAKAEAIILRAVCAPEERAMPYAMIGQLIDDPALAHRAPGLADRIA QGGQLSLRAENRLRRDLTRALLALAVDRPVLIGVDDVHHADTASLNCLLH LARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPFGPEQS AELARAAFGPGLAEDVLAGLYKTTRGNLSLSRGLISDVREALANGESAFE AGRAFRLAYLSSLYRCGPVALRVARVAAVLGPSATTTLVRRLSGLSAETI DRATKILTEGGLLLDQQFPHPAARSVVLDDMSAQERRSLHTLALELLDEA PVEVLAHHQVGAGLIHGPKAAEMFAKAGKALVVRNELGDAAEYLQLAHRA SDDVSTRAALRVEAVAIERRRNPLASSRHMDELSAAGRAGLLSPKHAALA VFWLADGGRSGEAAEVLASERPLATTDQNRAHLRFVEVTLALFSPGAFGS DRRPPPLTPDELASLPKAAWQCAVADNAAMTALHGHPELATAQAETVLRQ ADSAADAIPAALIALLYAENTESAHIWADKLGSTNAGVSNEAEAGYAGPC AEIALRRGDLATAFEAGSAVLDDRSLPSLGITAALLLSSKTAAAVRLGEL ERAEKLLAEPLPNGVQDSLFGLHLLSAYGQYSLAMGRYESAHRAFRTCGE RMRSWDVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLTRPMGPRSHAL TLRIKAAYLPRTKRIPLLHEAAELLLPCPDPYEQARVLADLGDTLSALRR YSRARGVLRQARHLATQCGAVPLLRRLGGEPGRIDDAGLPQRSTSLTDAE RRVAALAAAGQTNREIAEQLFVTASTVEQHLTSVFRKLGVKGRKQLPTAL ADVEQT SEQ ID NO: 210 MYSGTCREGYELVAREDELGILQRSLEEAGSGQGAVVTVTGPIACGKTEL LDAAAAKADAIILRAVCAPEERAMPYAMIGQLIDDPALAHRAPELADRIA QGGHLSLRAENRLRRDLTRALLALAVDRPVLIGVDDVHHADTASLNCLLH LARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPLGPEQS AELAHAAFGPGLAEDVLAGLYGMTRGNLSLSRGLISDVREAQANGESAFE VGRAFRLAYLSSLYRCGPIALRVARVAAVLGPSATTTLVRRLSGLSAETI DRATKILTEGGLLLDHQFPHPAARSVVLDDMSAQERRSLHTLALELLDEA PVEVLAHHQVGAGLIHGPKAAEIFARAGQALVVRNELGDAAEYLQLAHRA SDDVSTRAALRVEAVAIERRRNPLASSRHMDELSAAGRAGLLSPKHAALA VFWLADGGRSGEAAEVLASEHPLATTDQNRAHLRFAEVTLALFCPGAFGS DRRPPPLAPDELASLPKAAWQCAVADNAVMTALHAHPELATAQAETVLRQ ADSAADAIPAALIALLYAENTESAQIWADKLGSTNAGVSNEAEAGYAGPC AEIALRRGDLATAFEAGGTVLDDRPLPSLGITAALLLSSKTAAAVRLGEL ERAEKLLAEPLPNGVQDSLFGLHLLSAHGQYSLAMGRYESAHRAFHTCGE RMRSWGVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLARPMGPRSRAL TLRIKAAYLPRTKRIPLLHEAAELLLSCPDPYEQARVLADLGDTLSALRR YSRARGVLRQARHLATQCGAVPLLRRLGGEPGRIDDAGLPQRSTSLTDAE RRVSALAAAGQTNREIAKQLFVTASTVEQHLTSVFRKLGVKGRRQLPTAL ADVE SEQ ID NO: 211 MYSGTCREGYELVAREDELGILQRSLEQASSGQGVVVTVTGPIACGKTEL LDAAAAKAEAIILRAVCAPEERAMPYAMIGQLIDDPALAHRAPGLADRIA QGGQLSLRAENRLRRDLTRALLALAVHRPVLIGVDDVHHADTASLNCLLH LARRVRPARISMIFTELRSLTPTQSRFKAELLSLPYHHEIALRPFGPEQS AELARAAFGPGLAEDVLAGLYKTTRGNLSLSRGLISDVREALANGESAFE AGRAFRLAYLSSLYRCGPVALRVARVAAVLGPSATTTLVRRLSGLSAETI DRATKILTEGGLLLDQQFPHPAARSVVLDDMSAQERRGLHTLALELLDEA PVEVLAHHQVGAGLIHGPKAAEMFAKAGKALVVRNELGDAAEYLQLAHRA SDDVSTRAALRVEAVAIERRRNPLASSRHMDELSAAGRAGLLSPKHAALA VFWLADGGRSGEAAQVLASERPLATTDQNRAHLRFVEVTLALFSPGAFGS DRRPPPLTPDELASLPKAAWQCAVADNAAMTALHGHPELATAQAETVLRQ ADSAADAIPAALIALLYAENTESAHIWADKLGSMNAGVSNEAEAGYAGPC AEIALRRGDLATAFEAGSTVLDDRSLPSLGITAALLLSSKTAAAVRLGEL ERAEKLLAEPLPNGVQDSLFGLHLLSAYGQYSLAMGRYESAHRAFRTCGE RMRSWDVDVPGLALWRVDAAEALLSLDRNEGQRLIDEQLTRPMGPRSRAL TLRIKAAYLPRTKRIPLLHEAAELLLPCPDPYEQARVLADLGDTLSALRR YSRARGVLRQARHLATQCGAVPLLRRLGGEPGRIDDAGLPQRSTSLTDAE RRVAALAAAGQTNREIAEQLFVTASTVEQHLTSVFRKLGVKGRKQLPTAL ADVEQT SEQ ID NO: 212 MRAINASDTGPELVAREDELGRVRSALNRANGGQGVLISITGPIACGKTE LLEAAASEVDAITLRAVCAAEERAIPYALIGQLIDNPALGIPVPDPAGLT AQGGRLSSSAENRLRRDLTRALLTLATDRLVLICVDDVQHADNASLSCLL YLARRLVPARIALVFTELRVLTSSQLRFNAELLSLRNHCEIALRPLGPGH AAELARATLGPGLSDETLTELYRVTGGNLSLSRGLIDDVRDAWARGETGV QVGRAFRLAYLGSLHRCGPLALRVARVAAVLGPSATSVLVRRISGLSAEA MAQATDILADGGLLRDQRFTHPAARSVVLDDMSAEERRSVHSLALELLDE APAEMLAHHRVGAGLVHGPKAAETFTGAGRALAVRGMLGEAADYLQLAYR ASGDAATKAAIRVESVAVERRRNPLVVSRHWDELSVAARAGLLSCEHVSR TARWLTVGGRPGEAARVLASQHRRVVTDQDRAHLRVAEFSLALLYPGTSG SDRRPHPLTSDELAALPTATRHCAIADNAVMAALRGHPELATAEAEAVLQ QADAADGAALTALMALLYAESIEVAEVWADKLAAEAGASNGQDAEYAGIR AEIALRRGDLTAAVETAGMVLDGRPLPSLDITATLLLAGRASVAVRLGEL DHAEELFAAPPEDAFQDSLFGLHLLSAHGQYSLATGRPESAYRAFRACGE RMRDWGFDAPGVALWRVGAAEALLGLDRNEGRRLIDEQLSRTMAPRSHAL TLRIKAAYMPEPKRVDLLYEAAELLLSCRDQYERARVLADLGEALSALGN YRQARGVLRQARHLAMRTGADPLLRRLGIRPGRQDDPDPQPRSRSLTNAE RRAASLAATGLTNREIADRLFVTASTVEQHLTNVFRKLGVKGRKQLPAEL DDME

LAL Binding Sites

[0097] 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).

[0098] 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.

Constitutively Active LALs

[0099] 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).

Engineering Unidirectional LALs

[0100] 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

Host Cells

[0101] 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.

EXAMPLES

Example 1. Single Module Swapping to Produce an Engineered PKS

[0102] 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 (FIG. 3A). Seven of the 10 predicted donor modules, ranging in length from 4-6 kb, were selectively amplified in their entirety using a GC-rich long PCR method. In parallel, a bacterial artificial chromosome (BAC) that harbored the PKS that produces Compound 1 was converted to a module swap acceptor for heterologous donor modules by introducing the restriction sites AflII and SpeI to the flanking intermodule sequence of module 3. The modified acceptor BAC was linearized by digestion with AflII and SpeI, and the 7 donor modules were gel-purified and subcloned by Gibson cloning. The resulting constructs were subjected to Sanger sequencing of region of interest, PCR-based analysis to confirm cluster integrity, and Illumina NGS to sequence the entire BAC. The PCR-mediated error rate of the module amplification protocol was determined to be approximately 1 bp per 5000 bp, or approximately 1 mutation per module.

[0103] 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 (FIG. 3B). The resulting clone was conjugated into a Streptomyces expression host and fermented. Production of compound was analyzed by LC-TOF mass spectrometry analysis by co-injecting purified native FKBP12, the protein to which both compounds are expected to bind, with either the product of the native PKS, Compound 1, or the compound produced by the engineered PKS cluster, Compound 2. Comparative LC-TOF analysis of indicated that Compound 2 had the expected mass of 611.38, corresponding to the conversion of the module 3 alkene to a fully reduced module at that position. Compound 2 was re-fermented at large scale, purified to homogeneity and the structure was confirmed by NMR spectroscopy.

[0104] 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.

[0105] Single module swaps of either module 3 or module 4 in the PKS that produces Compound 1 generated novel Compounds 2-5 (FIG. 3C). Therefore, single module swapping was used to introduce a range of module encoded chemistries and generate novel compounds. LC-TOF mass spectrometry analysis indicated that of the module swaps at module 3 and module 4, the resulting hybrid clusters yielded a range of compound expression.

Example 2. Library Construction by Combinatorial Dimodule Swapping

[0106] 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 (FIG. 4A). A total of 31 modules were amplified for transfer to the module 3 position and 25 modules for the module 4 position. To optimize Gibson dimodule assembly cloning, phosphothiorate-modified DNA oligos were synthesized for PCR amplification of the donor modules. Phosphothiorate-capped module ends function by constraining the exonuclease step of the Gibson cloning protocol, which resulted in a dramatic increase in Gibson capture of GC-rich DNA (FIG. 4B). An intermediate plasmid-based dimodule capture protocol was developed to assemble, capture, amplify, and enrich the dimodule units (FIG. 4C). Pooled module 3 and module 4 amplicons were mixed with a linear backbone amplicon based on pBR322 for a 3-part Gibson assembly reaction. Shuttle vectors containing dimodule assemblies could be resolved from empty vector by fractionating on a preparative 0.4% agarose gel. After dimodule capture, the assembled dimodule fragments were released from the shuttle vector by digestion with AflII and XbaI and subcloned by direct ligation to an expression vector containing the PKS that produces Compound 1, in which the PKS lacked the native module 3 and module 4.

[0107] 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 (FIG. 4D). As a representative example, Compound 6 was generated by dimodule swapping of a module encoding mDEK chemistry at module 3 and K chemistry at module 4 of the PKS tha produced Compound 1. The expected mass of Compound 6 was observed by LC-TOF analysis, confirming that the dimodule assembly protocol yields engineered derivatives Compound 1.

[0108] 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 (FIG. 4E). Clusters were cloned onto BACs, and the cloned BACs were subsequently used as templates to PCR modules of diverse sources from multiple heterologous donors.

[0109] 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 1515 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 (FIG. 4F). 650 clones were prosecuted and subjected to LC-TOF mass spectrometry analysis. 115 of the 650 sampled clones expressed compounds with novel masses.

Example 3. Characterization of a Combinatorial Dimodule Library by Single-Molecule Long-Read Sequencing

[0110] A library corresponding to 15 different donor modules at the module 3 position and 15 different donor modules at the module 4 position (the 1515 dimodule library), produced according to the methods of Example 2, was characterized by Nanopore sequencing (FIG. 4G). The dimodules present in the 1515 dimodule library were excised from the PKS clusters using CRISPR/Cas9 (NEB). The resulting excised dimodules each had a length of approximately 7-12 kilobases. The dimodules were purified by 96-well column purification, and well-specific adaptors were ligated to the dimodules. The resulting dimodules were normalized and pooled and prepared for sequencing according to the standard ligation preparation protocol for Nanopore sequencing of oligonucleotides. Nine 96-well plates (864 dimodule clones total) were sequenced by Nanopore and the resulting sequencing data was analyzed according to the informatics workflow provided in FIG. 4H, with 73.1% of clones being called. The comparison of the resulting sequencing data against the table of input of the donor modules allows the deconvolution of the resulting combinatorial library by identification of the resulting dimodules. The results of Nanopore sequencing of the 1515 dimodule library are provided in Table 1.

TABLE-US-00005 TABLE 1 Library Single Grand Plate IDs NoCall Ambiguous Read Called Total 163846 45 4 11 36 96 163848 14 10 14 58 96 163851 16 80 96 163896 5 8 5 78 96 163897 21 1 74 96 163898 3 10 11 72 96 163899 4 6 2 84 96 163900 1 26 3 66 96 50066321 12 84 96 Grand Total 72 113 47 632 864 % 8.3% 13.1% 5.4% 73.1%

Example 4. Library Construction by Combinatorial Trimodule Swapping

[0111] The combinatorial module swap protocols were modified to generate trimodule assemblies in the PKS that produces Compound 7 (FIG. 5A). Increasing the number of module swaps increases the size, and therefore diversity, of a PKS library. For example, given a collection of 13 different module-encoded chemistries, increasing size and diversity is based on the number of modules that are swapped such that the maximal library size of a single mod swap is 13; with a dimodule swap the maximal library size is 13.sup.2=169; and for a trimodule swap, the maximal library is 13.sup.3=2197.

[0112] 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 (FIG. 5B). As before, phosphorothioate chemistry was used to constrain the ssDNA intermediate for the first round of Gibson cloning into a shuttle vector. Shuttle vector clones harboring trimodule assemblies were enriched by preparative gel fractionation and isolation. Finally, Gibson-mediated error correction was used to trim restriction sites for scarless cloning in the expression vector. First, flanking PmeI restriction sites were introduced within the linker regions between Module 3 and Module 4, as well as between Module 6 and Module7. Sites with reduced GC content and secondary structure (as predicted by DNAfold; <8 kcal/ml) were selected for optimal Gibson homology arms. A Gibson Assembly Ultra Kit (SGI-DNA) was used to clone the trimodule assembly into the PKS that produces Compound 7 enabling the replacement of Modules 4, 5, and 6 and simultaneously removal of the additional extraneous PmeI sequence retained after digestion. This resulted in >95% correct assembly for the industrial scale production of compounds produced by trimodule swapped PKS clusters (>200 per week).

Example 5. Ring Expansion by Swapping a Single Module Acceptor with a Dimodule Donor

[0113] 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 (FIG. 6A). The compound produced by engineered PKS, Compound 8, was observed in high yield and had a mass of 655.41, as determined by LC-TOF analysis (FIG. 6B). This corresponds to a ring-expanded compound product in which Compound 8 contains an additional 2-carbon extender unit. Thus reprogramming PKS biosynthesis via module swapping by insertion of a dimodule assembly to replace a single module may produce functional PKS expression.

Example 6. Module Swapping of a PKS Loading Module

[0114] 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 (FIG. 7A). Loading modules may possess similar domain structure as conventional elongation PKS modules, including ketoreductase-like domains and an enoyl-reductase domain, which may or may not be catalytically active. The final chemistry of the starter unit depends on the presence and the sequence of the domains in the loading module, so the resulting starter unit can be engineered by swapping the loading module

[0115] The X23 PKS cluster produces Compound 9 and Compound 10 (FIG. 7B). The Rapamycin loading module from Streptomyces stain S303 was swapped into the X23 cluster by the methods described previously for a single module swap. The engineered PKS produced Compounds 11 and 12, in which the starter unit is replaced with the starter unit of Rapamycin. Additional single elongation module swaps of Module 2 and Module 7 of X23 produced Compounds 13 and 14, respectively.

Other Embodiments

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.